CA2226299A1 - Hyper comb-branched polymer conjugates - Google Patents

Hyper comb-branched polymer conjugates Download PDF

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Publication number
CA2226299A1
CA2226299A1 CA002226299A CA2226299A CA2226299A1 CA 2226299 A1 CA2226299 A1 CA 2226299A1 CA 002226299 A CA002226299 A CA 002226299A CA 2226299 A CA2226299 A CA 2226299A CA 2226299 A1 CA2226299 A1 CA 2226299A1
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Canada
Prior art keywords
agents
branched polymer
hyper comb
conjugate
comb
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Abandoned
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CA002226299A
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French (fr)
Inventor
Rui Yin
Donald A. Tomalia
David M. Hedstrand
Douglas R. Swanson
James R. Baker, Jr.
Jolanta F. Kukowska-Latallo
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University of Michigan
Dendritic Nanotechnologies Inc
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Individual
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Publication of CA2226299A1 publication Critical patent/CA2226299A1/en
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    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
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    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/641—Branched, dendritic or hypercomb peptides
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
    • A61K47/6885—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy the conjugate or the polymer being a starburst, a dendrimer, a cascade
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    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract

A novel class of hyper comb-branched polymers conjugated with carried materials are disclosed.

Description

CA 02226299 1998-02-lO

~lY~;~ COMB-BI~NCHED POIIYMER CONJUGATES
BACKGROUND OF THE INVENTION
It is believed that there is no art defined field to which the present inventionrelates. The closest which one might come to categorizing the present invention would be 5 to analogize it to the emerging experim~nt~tion involving the ~tt~chm~nt of a carried material to a macromolecule.
For example, branched polyethyleneimine (PEI), polylysine, DEAE-dextran and polyvinylpyriclinillm salts have been used to carry genetic material into cells. Recently issued United States Patent 5,338,532, entitled STARBURST~ CONJUGATES, discloses10 conjugating a variety of carried materials, including genetic materials, with dense star polymer lllal ~;l . lllolecules .
Beyond these examples, Applicants know of no other types of macromolecules which are being used to carry materials.
Further, these types of macromolecules are so distinctively ~lirrer~lL that the 15 a~lv~ ten~oss of categorizing them in the same field is questionable. The present invention relates to the use of yet another skikingly ~lirrelt ..- type of macromolecule to carry materials for a variety of purposes.
SUMMARY OF THE INVENTION
Surprising and nonobvious advantages are achieved by conjugating carried materials with a unique class of macromolecules referred to herein as hyper comb-branched polymers. The present invention is directed to polymer conjugate m~teri~l~
comprising hyper comb-branched polymers associated with desired carried materials, processes for pl~:pa-ing those conjugates, compositions cont~ining the conjugates, and methods of using the conjugates in compositions.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates a p-~r~ d embodiment hyper comb-branched polymer used in the conjugates of the present invention;
Fig. 2 illustrates the constituents of hyper comb-branched polymers comprising acore and four successive br~n~hing generations;
Fig. 3 illustrates a ~l~r~lled embodiment hyper comb-branched polymer having a relatively high grafting density;
Fig. 4 illustrates a pl~rell~d embodiment hyper comb-branched polymer having a relatively low grafting density;

CA 02226299 1998-02-lO

Fig. 5 illustrates a plerelled embodiment hyper comb-branched polymer having a relatively large interior void volume;
Fig. 6 illustrates a plefelled embodiment hyper comb-branched polymer having a relatively small interior void volume;
S Fig. 7 illustrates a pl~fellc:d embodiment hyper comb-branched polymer having relatively close spacing between first generation branches and relatively distant spacing between second ge-l~ldLion branches;
Fig. 8 illustrates a plt:r~ d embodiment hyper comb-branched polymer having relatively distant spacing between first generation branches and relatively close spacing 10 between secondary branches;
Fig. 9 illustrates a prer~ d embodiment hyper comb-branched polymer having NH~ groups as terminal moieties;
Fig. 10 illustrates a ~lerellc:d embodiment hyper comb-branched polymer having COOH groups as terminal moieties;
Fig. 11 illustrates a pl~r~ d embodiment hyper comb-branched polymer of relatively small outer ~1imPn~ions;
Fig. 12 illustrates a ~lert;ll~d embodiment hyper comb-branched polymer of relatively large outer dimensions;
Fig. 13 illustrates several embodiments of hyper comb-branched polymers having various combinations of hydrophilic and hydrophobic br~nrhing generations;
Fig. 14 illustrates a synthesis for the plc~r~ d hyper comb-br~nt~ht-~l polymers of the present invention;
Fig. 15 illustrates a hyper comb-branched polymer conjugate having carried material disposed within the interior of the polymer;
Fig. 16 illustrates a hyper comb-branched polymer conjugate having carried material disposed throughout a particular layer or generation;
Fig. 17 illustrates a hyper comb-branched polymer conjugate having carried material disposed at the surface of the polymer;
Fig. 18 illustrates several process sch.-ml s for modifying hyper comb-branched polymers;
Fig. 19 illustrates several additional process schemes for modifying hyper comb-branched polymers;
Fig. 20 illustrates a process for producing a monoclonal antibody bioconjugate;

CA 02226299 l998-02-lO

Fig. 21 illustrates a synthesis for producing hyper comb-branched polymers via ablock copolymerization method;
Fig. 22 illustrates a hyper comb-branched polymer conjugate comprising carried drug agents associated in the interior of the polymer;
'' S Fig. 23 illustrates a hyper comb-branched polymer conjugate comprising carried drug agents linked within the interior of the polymer;
~, Fig. 24 illustrates a hyper comb-branched polymer conjugate comprising carried drug agents linked to the polymer via ester linkages;
Fig. 25 illustrates a hyper comb-branched polymer conjugate comprising carried drug agents linked to the polymer via amide linkages;
Fig. 26 is a schematic in two flimen~ions of a polymer configuration of the polymers of the instant invention wherein 1 is the initiator core (initiator core molecule);
2 is the first grafting and first br~nrhing and generation 0;
lS 3 is second grafting and second br~nrhing and generation 1;
4 is third grafting and third br:~nrhing and gen~,.dLion 2;
5 is fourth grafting and fourth br~nrhing and ~ Lion 3;
6 is (i + 1)~ grafting and (i + l)th br~nrhing and generation i; and 7 is (i + 2)'h and all iterative grafting and (i + 2)~ and all iLel~,liv~
br~nrhing, and generation (i + 1) and all subsequent generations;
Fig. 27 illll~tr~tt s the grafting of oligomer branches to cyclen, and the subsequent grafting of branches upon branches; and Fig. 28 shows the grafting of oligomer branches onto a polyethylen~minr dendrimer core, and the subsequent grafting of branches upon branches.
Fig. 29 illustrates luciferase activity in Cosl cells after transfection using hyper comb-branched polymer conjugates;
Fig. 30 illustrates luciferase activity in Cosl cells after transfection using hyper comb-branched polymer conjugates;
Fig. 31 illustrates luciferase activity in Cosl cells after transfection using hyper comb-branched polymer conjugates;
Fig. 32 illustrates luciferase activity in Cosl cells after transfection using branched PEI conjugates;
Fig. 33 illustrates luciferase activity in Rat2 cells after transfection using hyper comb-branched polymer conjugates;
Fig. 34 illustrates luciferase activity in Rat2 cells after transfection using hyper comb-branched polymer conjugates;
Fig. 35 illustrates luciferase activity in Rat2 cells after transfection using hyper 5 comb-branched polymer conjugates, Fig. 36 illustrates luciferase activity in Rat2 cells after Ll~l~r~;lion using branched PEI conjugates; and Fig. 37 illustrates release of salicylic acid from hyper comb-breached polymer.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
lo The Hyper Comb-Branched Polymers A hyper comb-branched polymer of the plc:r~ d embodiment as illustrated in Fig.
1 comprises a generally linear core chain, s~lccçs~ive gt;n~ldlions of oligomers br~nrlling off the core and prior generations of branches, an exterior surface formed by the termini of the last generation of branches, and interior voids within the polymer molecule.
A comb-branched polymer comprises an elongated core with a plurality of arms br~n~lling th~.erl~lll to give the appearance of a comb. "Hyper comb-branched"
polymers comprise successive ~nel~tions of branches br~nrlling off of prior g~neld~ions of branches. The resulting structure has an exreerlingly high degree of bl;.~ i..g, and so is referred to as "hyper comb-branched. " Hyper comb-branched polymers are somPtimPS
20 referred to herein by their tr~lçm~rk: hyper comb-branched polymerTM.
Hyper comb-branched polymers are defined based upon the number of generations or grafting steps. As illustrated in Fig. 2, a hyper comb-branched polymer is constructed by grafting or ~tt~ ling a series of br~nrhing arrays to a core, or predecessor branches.
The first grafting array is defined as generation 0 (G0), the second grafting array is 25 generation 1 (Gl), the third grafting array is ~ellel~Lion 2 (G2), the fourth array is gellel~Lion 3 (G3), and so on. This novel design not only significantly reduces the synthetic effort for obtaining higher molecular weight (i.e. greater than 10 million, with a molecular weight distribution of about 1.2) or larger size (i.e. a diameter greater than about 100 nm) dendritic polymers, but also enables the designer to tailor particular 30 structural attributes of the hyper comb-branched polymers.
Structural aspects that may be varied include, but are not limited to, interior grafting density, interior void volume or "cargo space," spacing between generations, WO 97/06833 PCTrUS96/13080 types of branches utilized in each generation, the number and types of terminal moieties or functional outer groups disposed at or near the surface of the polymer molecule, and lateral cross-sectional diameter or geometric configuration of the polymer.
Figs. 3 and 4 illustrate two ~lerelled hyper comb-branched polymers having 5 different grafting ~len~iti~os. Fig. 3 depicts a polymer having a relatively high grafting density in which adjacent branches are tightly packed or closely ~itll~te-l to one another.
In contrast, Fig. 4 illustrates a polymer having a relatively low grafting density, in which the polymer has a much looser structure. Grafting density can be controlled by the choice of br~nl~hing or repeating unit type employed at each generation, number of 10 reactive sites of the core and branches, and reaction conditions utilized during assembly of the hyper comb-branched polymers. The hyper comb-branched polymers utilized in the present invention can have a grafting density of from about 0.1% to about 90% or higher such as 100%. Grafting density as referred to herein is defined as the number of ~tt~(~hme,nt or graft sites on a con~tit~-~nt, i.e. core or branch, of the hyper comb-15 branched polymer expressed as a percentage of the total number of sites in the polymeravailable for grafting or ~tt~chm~nt thereto during assembly of the polymer. Generally, relatively high grafting densities can be achieved by ~tili7ing small core and branch structures.
Figs. 5 and 6 illustrate two pl~r~ d hyper comb-branched polymers having 20 ~ignifir~ntly different interior void volumes. Fig. 5 depicts a polymer having a relatively large interior volume while Fig. 6 illustrates a polymer having a relatively small interior volume. Dirr~ interior void spaces can be achieved by varying grafting densities, core and branch chain length, number of reactive sites on the core and early generation branches, spacing between branches, and combinations of these parameters. The hyper 25 comb-branched polymers utilized in the present invention can have an interior void volume of about 10 angstroms to about 500 angstroms or more. Void volume as referred to herein generally refers to the void size, and in most instances the interior cavity m.ot~?r.
Figs. 7 and 8 illustrate hyper comb-branched polymers having dirr~ spacing 30 configurations between s-lccec~ive gellcl~lions. The polymer depicted in Fig. 7 has a much closer spacing between first generation branches than the polymer illustrated in Fig.
8. However, the polymer of Fig. 8 has a much closer spacing between second generation branches than the polymer illustrated in Fig. 7. Successive br~nrhing generations can be specifically tailored as previously described with respect to adjusting grafting density and interior void volume, to produce particular spacing configurations at desired regions within the hyper comb-branched polymer. In one embodiment, a hyper comb-branchedpolymer comprises multiple regions of dirr~,le,l~ grafting densities. Thus, such a polymer could comprise alternating regions of different grafting ~lPn~i~itps such as a first region having a grafting density less than about 50%, and another region having a grafting density of more than about 50%.
Figs. 9 and 10 illustrate hyper comb-branched polymers having particular chrmir~l moieties disposed generally about the periphery of each molecule. Various physical and 10 functional characteristics can be imparted to the hyper comb-branched polymer depending upon the types, combinations, and/or degree of surface congestion or number of tr~nin~l moieties disposed about the periphery of the polymer. Function~ tion of the polymer surface can be achieved either by direct modification of hyper comb-branched polymers with various organic reagents (i.e. alkylene oxide), or by forming hyper comb-br~nrhr(l 15 polymers having -NH2, -COOH, -COOMe, -NH4, -PEOX, -PEG, -PEO, or combin~tion~thereof, followed by functionalization with the desired surface molecules (Figs. 8 and 9).
In addition to functionalization of the polymer surface, a wide variety of chPmir~l moieties may be disposed at different regions within a polymer molecule by incorporating the desired moieties in particular br~nr-hin~ generations.
The overall size of a hyper comb-branched polymer may be varied as shown in Figs. 11 and 12. Relatively large hyper comb-branched polymers, such as that depicted in Fig. 12 can be ~lepaled, as disclosed below, by lltili7ing relatively long core and br~nrhing chains as constituents for building the hyper comb-branched polymers. That is, the maximum ~ mrt~r for such large polymers is generally about 100 nm. The inventors 25 contemplate however that even larger polymers could be formed. Alternatively, relatively small hyper comb-branched polymers as shown in Fig. 11 can be formed by ~1tili7in~
short core and br~nrhing chains. Hyper comb-branched polymers having particular geometries and shapes may be constructed by a~ ,L.liate selection of core and bldllcl~ing components.
In addition to controlling structural aspects of the hyper comb-branched polymers described herein, particular functional characteristics may be incorporated in the polymer during its assembly. For example, as illustrated in Fig. 13(a), a hyper comb-branched polymer with a hydrophobic interior and a hydrophilic exterior can be obtained by W O 97/06833 PCT~US96/13080 sequential synthesis of an initial hydrophobic interior and then a hydrophilic exterior.
~Altern~tively, such a polymer may be produced by function~li7ing the exterior or last generation branching array with hydrophilic polymers, followed by modification of the interior with hydrophobic monomers. Hydrophilic functionalization can be achieved by S grafting hydrophilic polymers such as poly(2-ethyloxazoline), poly(2-methyloxazoline), polyethylene glycol, polyethylene oxide, polyacrylic acid, polyacrylic amide, polyvinyl pyrrolidone, and combinations thereof at the desired grafting step. Conversely, a hyper comb-branched polymer with hydrophobic outer surfaces as shown in Fig. 13(b) can be prepared by grafting hydrophobic polymers such as polyethylene, polydimethylsiloxane, 10 polyb~lt~ n~, poly~Lylclle, polymethyl-methacrylate, perfluoropolymer, and poly(2-alkyl or phenyl oxazolines) etc. at the last gr~fting step. In applications employing poly(2-alkyl oxazolines), hydrophobicity is achieved when the alkyl group has about 4 or more carbon atoms. In addition, a hyper comb-branched polymer with alternating hydrophobic-hydrophilic layers as depicted in Fig. 13(c) may be prepared by using amphiphilic 15 polymers between each grafting step.
Hyper comb-branched polymers have also been referred to as non-cro~linked, polybranched polymers. They have the general formula:
C
G~
{(A0)(l y~0 (go~ o} o R~
G' {(Al)(l y)l~~(BI)yl}
G
(Ai)(l y)i~~(Bi)yi}ni Ri wherein: C is a core molecule; each R is the residual moiety of an initiator selected from a group consisting of free radical initiators, cationic initiators, anionic initiators, coordination polymerization initiators and group transfer initiators;
35 A and B are polymerizable monomers or comonomers capable of with~t~n-ling theconditions required for br:~n~hing thclerl~ or grafting thereto, at least during the polymerization of the {(A)-(B)} linear polymer chain and during its grafting to a prior CA 02226299 1998-02-lO

{(A)-(B)} branch or the {(A)-(B)} core branch; each G is a grafting component, and the d~sign~tion ZG
{(A)(l y)-(B)y} inflic~tes that G can attach to either an (A) unit or a (B) unit;
n is the degree of polymrri7~tion of the in~lir~t~l generation comb branches, y is the fraction of B units in the in~lir~t~d generation branch, and has a value of 0.001 to 1;
the superscripts 0, 1 and i desi~n~te the comb-branch ~,cllcl~lion level, with i beginning at "2" and co~ for the number of lciLclaLi\~e branch set generations in the polymer;
and 10 at least n~ and nl are > 2.
One process for producing hyper comb-branched polymers uses the following scheme:
(1) forming a first set of branches by initi~ting the polymerization of a first set of monomers which are either protected against or non-reactive to br~nr-hing and grafting 15 during said polymeri_ation, each of said branches having a reactive end unit upon completion of said polymerization, said reactive end units being incapable of reacting with each other; (2) grafting said branches to a core (preferably polymeric) having a plurality of reactive sites capable of reacting with said reactive end groups on said branches; (3) either deprotecting or activating a plurality of monomeric units on each of said br~nrh~s 20 to create reactive sites; (4) separately forming a second set of branches by repeating step (1) above with a second set of monomers; (5) ~tt~ching said second set of branches to said first set of branches by reacting said reactive end groups of said second set of branches with said reactive sites on said first set of branches.
Stated another way, the basic process for forming hyper comb-branched polymers 25 comprises:
(I) forming a core having at least one reactive site;
(II) reacting essentially all of the reactive sites of said core with a reactive polymer having the unit formula G~
Z
{(A~)ll y)~~~(B~)yo}no-Ro to form multiple branches which contain reactive (B~) sites on each branch, using a reactive scheme such that the reactive monomer units (B~) are capable of with~t~nrlin~ the conditions required for br~n~hin~ th~l~rlulll or grafting thereto tû ensure that said reactive polymer IG~
{(A~)(l y)~~~(B~)yo}no-Ro reacts with said reactive sites of said core, but that no reactions occur at said (B~) sites;
(III) repeating step (II) seq~lenti~lly by reacting reactive polymer having the unit formula G
{(A~ y)i--(Bi)yi}ni-Ri with the reactive sites of said polymerizable B(i-l) monomers or comonomers of the previous generation to form successive generation of branches to give the desired non-cro~linkP-l poly-branched polymer.
There does not seem to be any limit to the size of the hyper comb-branched polymers except that ~liCt~t~ by practicality and/or sterochPmi~try of the molecules 20 formed. Molecular weights preferably range from about lO,ûO0 to about 100,000,000 though there may be applications where molecular weights outside of this range would be approl,liate. More preferable are those having molecular weights of 10,000,000 or less.
~or most applications those molecules having a molecular weight of 500,000 or less are especially preferred.
The value of n~ can have a range of 2 to a value of in excess of 100, but the plefell~d value is from 2 to 100. In addition, values of n', *, and * can be in the range of 1 to a value in excess of 100, such as up to about 10,000 or even higher, but the er~ d range is from 1 to 100. The core itself can be a linear polymer having a degree of polymerization nC, whose value can be from 2 to a value in excess of 300, but a pler~lled range for the value of nc is from 2 to 300.
As in-lir~tPtl above, each of R~, R', R2, R3, and Ri in these inventive polymers is selected as a residual moiety from a radical initiator, a moiety from a cationic initiator, a moiety from an anionic initiator, a coordination polymerization hliLialur~ or a group LldlL~r~l initiator. R~-R' can be for example hydrogen, an alkyl group, Lewis acids, or the like, such materials being known in the art.

WO 97/06833 PCT~US96/13080 The Gi group is the grafting component formed by the reaction of the living end,or a derivative of the living end, of the i~ gell~.dLion oligomer with the reactive groups of the (i-1) generation material. Thus, an anionic oligomer may be reacted directly with an eleckophilic precursor ~ dLion, or it may be ~ r(l by, for example, a halogen 5 such as chlorine, bromine, or iodine, to create an electrophilic end group for grafting to a nucleophilic precursor. Similarly, a cationic oligomer may be reacted directly with a nucleophilic precursor generation, or ltllllil~lrCl with, for example, water, hydrogen sulfide, or an amine to give a nucleophilic end group for reaction with an electrophilic precursor.
As illustrated in Fig. 26, the core may itself be a polymer having the formula RC-{(AC)--(Bc)}ncGc. In the case of GC, the "graft" is to a monofunctional molecule, which may be as simple as quenching the active end with a proton or hydroxide, as would be the case with normal t~ ion of ionic oligomers with water, or trapping with aspecific molecule in order to introduce a single desired functional group to the molecule.
15 Other telechelic groups suitable for grafting purposes may be found in Goethals, "Telecheic Polymers: Synthesis and Applications," CRC Press (1989).
The oligomeric and polymeric segments of these materials can be homopolymers or copolymers, it being understood that the formulae herein represent bonding of the grafting G groups to either segment A, if it is present, or to segment B, and it being 20 further understood that the grafting to any A segment is at the terminal end of the molecule, any other segm~nt A grafting would result in the potential for cro~clinking the polymers. Each A segment can be monomeric or, oligomers or polymers formed from polymerizable monomers, the only condition being that the said monomers, oligomers and polymers must be capable of with~t~n~ing the conditions required for pl~al~Lion of 25 subsequent graft jun~:Lulc~s. As illustrated in the formulae, the bond from G to the next generation is in~ te~ by a vertical line about halfway between the A segments and the B
segments to illustrate that G can be bonded to either A, if it is present, or to B, which is always present in the molecule.
An example of a G group that fits this description would be a urea formed by the30 reaction of an isocyanate with an amine group. This is formed by the activation of the amines of a poly(vinyl amine) segment with phosgene to create a polyisocyanate precursor molecule which, then, is reacted with an amine t~ tr~l poly(vinyl ~cef~mi.le). The same G group can be formed by treating the poly(vinyl ~cet~mitle) with phosgene to form CA 02226299 1998-02-lO
W O 97/06833 PCT~US96/13080 the telechelic oligomer with isocyanate end group, followed by reaction with thepoly(vinyl amine) precursor molecule.
An example using the A group bonded to the G group would be the use of a copolymer of ethyl oxazoline and ethylene oxide. The hydroxyl group on the oxyethylene 5 is the termin~l group on the reactive oligomer segment. Activation of the hydroxyl group with phosgene gives a chloroformate which is reacted with the amine of poly(ethyleneimine) segment on the precursor generation to form a urethane. Thus, the A
group of the reactive oligomer is the "unreactive" oxyethylene and the B group is the m~ P-l iminoethylene, N-propionyl iminoethylene.
The range of possible G groups is limited only by the types of coupling reactions that are possible. In addition to ureas and urethanes, imide, thiourea, thiocarbamate, and anhydride linkages are readily available from similar reagents. Precursor molecules cont~ining olefins that result from polymerization or copolymerization of butadiene or ring opening m~t~thPsi~ polymerization of cyclic olefins can be activated by halogenation 15 for subsequent reaction with a nucleophilic end group, or reacted directly with me~ ls via radical addition, or be coupled with a silane end group via catalyzed hydrosilylation methods. Ether and ester linkages can be derived from hydroxyl groups on either the precursor molecule or the reactive oligomer end group.

Segments of A include for example, -CH2CH2-, -CH2CH=CHCH2-, -CH2C(CH3)2--CH2CH(CN)-,-CH2CH-,-CH2CH-,-CH2CH-, -NCH2CH2-,-NCH2CH2CH2-,-CH2CH-S
C=O O O C=O C=O C=O
NH2 R C=O R R

10 -OCH2CH2-, -SCH2CH2-, -R'2SiO-, -CH2CH-, -OCH2CH-, where R' is an alkyl group, C6Hs ICH2 OR
aryls, arylalkyl, hydrogen, or carboalkoxy and R is an alkyl group, aryls, or hydrogen;
R
-CH2C-, CO2R"
whelcill R has the same mt~nin~ as set forth above, and wh~ill R" can be an alkyl 25 group.
Preferred as A segments are -CH2CH2-, -NCH2CH2-, R=O

-CH2CH-, -CH2C(CH3)2-, -CH2CH-,-CH2CH20-, -CH2CH2S-, NH-C =O O =C-NH2 R
-CH2CH=CHCH2-, -R'2SiO-, -CH2CH-, and -CH2C-. Most pler~ d are C6Hs R2 the A segments -NCH2CH2-, -CH2CH-, and -CH2CH-C = ~ C6Hs C6H4CH3 R
Examples of the B segment can be monomers, or oligomers or polymers formed CA 02226299 1998-02-lO
WO 97/06833 PCT~US96/13080 from polymerizable monomers, wherein said monomers, oligomers and polymers must be capable of wi~ ling the conditions required for ~lcpal~lLion of a graft polymer and further, the B segmP-nt~ must contain at least one unit which is nucleophilic orelectrophilic in character.
The groups B contain the reactive sites to which the oligomers may be grafted.
Indeed, there are embo-limPnt~ in which the only difference between A and B groups is that by definition, the A or B group from a sllccee~ling generation branch is grafted to the "B" group. The A groups have the same reactive sites, but they do not get grafted to.
In many cases, these B groups may need to be present in latent or m~k~(l form if10 they would otherwise be incompatible with the oligomerization process. For example, polymPri7~tion of aziridine leads to randomly branched polyethyleneilllil~e oligomers which are not useful for this invention because the secondary amines formed are also reactive under the polymerization conditions. Thus it is ~limrlllt to control the interior void size, grafting density, size, shape, and nature of surface groups. Oxazoline lS polymerization leads to linear polyethyleneimine in a protected form, and the secondary amines can be lmm~kP~d for grafting by hydrolysis. For vinyl acetate oligomerizations, hydroxyl groups inten-lP~l for use as future graft sites would need to be m~kefl as, for example, esters to ensure polymerization. Latent reactive sites can then be formed by hydrolyzing the ester groups of the polymerized vinyl acetate into alcohol groups.
Thus, B as a nucleophile can be selected from such groups as -NCH2CH2-, -CH2CH-, -CH2CH(OH)-, -CH2CH(SH)-, H NHR

-OCH2CH(CH2OH)-, and -NCH2CH2CH2-, while B as an H

electrophile can be selectt--l from such groups as -CH2CH-, -CH2CH-, -CH2CH-, -OCH2CH-, 5CH2Cl CH2 (tosylate) CO2R" CH2Cl R R~0 -CHzCH2CH-, -SiO-, and -SiO-, wherein R and R" have the mP~ningc CH2Cl Cl CH2Cl set forth above.
It should be understood that homopolymers consist of only the B segment, while copolymers can be had by combining the B segments with the A segments. Copolymers can also be prepared by using different monomers for the B segment of dirr~
gel~l~lions, for example Bl being dirre,~"l from B~. There must be at least one B
segment and therefore the ratio of A segments to B segments ranges from 0 to 1 to 100 to 20 1.
The core may or may not be produced by "living" polymerization, l~tili7ing "living polymers" or "living oligomers," which oligomers and/or polymers are generally known to those skilled in the art. "Living systems" are preferred in order to control polydispersity of the hyper comb-branched polymers. Using specific ~ mictry, this is 25 illustrated by lere,~llce to "Polymeric Amines And Ammonium Salts," edited by E.J.
Goethals, Pergamon Press, (1980), with particular reference to pages 55 et seq. wherein there is taught one method of producing living polymers in a paper entitled "Linear Polyalkylenimin~c," Saegusa, T. and Kobayashi, S.
Using the example of Saegusa, page 58, one can observe that an initiator such as30 methyl iodide is first reacted with an oxazoline in the following sequence to give an oligomeric "living oligomer" having, in this case, two protected reactive sites desi~n~t~-1 WO 97/06833 PCT~US96/13080 as -NCH2CH2-:
C=O
H

N ~,H Me-N ~,H I 2 MeNCH CH I
< o 0 1 2 2 Me HC=O

2 Z N ~, H NCH CH
HC=O HC=O

that is, Me(NCH2CH2)2 - I.
HC=O
Referring to Fig. 2G, the initiator core in the specific case described just above would be shown in Fig. 26 as Rc (Bc) " c GC; where Rc is methyl and Gc is as described above.
Reaction sequences are then chosen to deE~rotect the nitrogen groups so that each of the two reactive sites adds a reactant possessing its own, new reactive site, or sites, which introduces multiplicity, to obtain a comb polymer -{(A~)--(B~)}no~R~ of generation O (see Fig. 1). As can be observed from the reaction sequence set forth above, this process requires that protection-deprotection strategies are used to ensure that the reactant reacts with all reactive (BC) sites, but does not react any (B~) sites. Protection-deprotection strategies are generally known to those skilled in the art and great detail does not have to be set forth herein. Suffice it to suggest that the living oligomer set forth above has the protective group l on each nitrogen of the oligomer whereupon the oligomer is then HC =O
hydrolyzed with an acid to give polymeric units having reactive amine groups i.e.
Me-(NCH2 CH2 -)l2 -I
H

WO 97/06833 PCT~US96/13080 which are then used as the reactive sites to form the next generation, it being understood that the reactive sites of the polymer being grafted to the amine groups are protected before this reaction takes place, and that they too are hydrolyzed after the grafting reaction to give additional reactive sites for the next generation of b~ lg. Additional S iL~ldLivc sequences involving addition of new re~rt~nt~ having reactive sites is then undertaken in order to add branches onto branches to form the poly-branched polymer of this invention until the polymers will not form due to steric hinderance referred to as dense packing.
One of the processes used to prepare hyper comb-branched polymers used in this 10 invention relies on the polymeriz~tion of 2-ethyl-2-oxazoline. Methyl p-tol~ n~?s~llfonate has been shown to polymerize oxazolines and the polymerization mech~ni~m has been ~letermin~ to be cationic, producing a "living polymer." This allows the ~lc~cudtion of polymer samples with well defined molecular weight and low polydispersity. The end of the growing polymer chain contains an oxazolinium ion as disclosed above, that can be 15 termin~te~l or quenched by a variety of nucleophiles. For example, to graft the living poly(2-ethyl-2-oxazoline) chains during the first reaction step, the living chain ends are termin~t~cl with the secondary amine groups contained on linear poly(ethyleneimine)(LPEI). After grafting onto the linear poly(ethyleneimine) has been accomplished, hydrolysis of the poly(2-ethyl-2-oxazoline) grafts will gellcl~tc 20 poly(ethylc,leh,,i,,e) branches. This allows further living poly(2-ethyl-2-oxazoline) chains to be grafted onto the poly(ethyleneimine) branches. Repetition of the grafting and hydrolysis forms the inventive polymers with the structures shown herein.
Figs. 27 and 28 and Examples EE and FF below illustrate 1)1A~ g "0"
generation branches onto cores comprising ring compounds and dendrimers respectively, 25 wh~,~cill "dendrimer" has the same or similar m.ozlning as that used by Tomalia et al., in Angewandte Chemie, 29/2 (1990), pages 138 to 175. In Fig. 27, branches which can be generated in the manner described above are ~tt~h.o~l to the four nitrogens in the ring compound 1,4,7,10-tetraazacyclododecane (cyclen), much as they are grafted to the nitrogens of a linear polyethyleneimine core molecule as ~ cu~se~ above. First 30 geneldLion branches are then grafted upon the "0" generation branches, second generation branches are grafted upon the first generation branches, etc. as discussed above.
In Fig. 28, "0" ~,eneldLion branches are grafted to the surface nitrogens of a hyper-termin~3l-branched or dendrimer core molecule, specifically, a second g~;lleld~ion polyethy!en~o~min~ . At the generation 2 level (~esign~ting the first ~ lGldLion as ~m;ldLion 0), such hyper-terminally-branched molecules are typically referred to as "dendrimers." Hyper-termin~l-branched or dendrimer cores can be prepared in various manners known to those skilled in the art including without limitation by the techniques disclosed in United States Patents 4,507,466 entitled "DENSE STAR POLYMER
BRANCHES HAVING CORE, CORE BRANCHES, TERMINAL GROUPS," 4,558,120 entitled "DENSE STAR POLYMER," 4,568,737 entitled "DENSE STAR POLYMERS
AND DENDRIMER," 4,587,329 entitled "DENSE STAR POLYMER HAVING TWO-DIMENSIONAL MOLECULAR DIAMETER," 4,631,337 entitled 10 "HYDROLYTICALLY-STABLE DENSE STAR POLYAMINE," 4,737,550 entitled "BRIDGED DENSE STAR POLYMER," 4,599,400 entitled "STAR/COMB-BRANCH
POLYAMIDE," 4,690,985 entitled "STAR/COMB-BRANCHED POLYAMINE,"
4,694,064 entitled "ROD-SHAPED DENDRIMER," and 4,857,599 entitled "MODIFIED
DENSE STAR POLYMERS." Similarly, any of the clen~lrimer molecules described in 15 said patents could be used as the hyper-branched dendrimer core to which oligomer branches are grafted in reiterative fashion in accordance with the present invention. One need only develop an appr-~liate strategy for ~tt~rhing the oligomer bldllclles to the surface moieties of such hyper-branched cores, and various alternatives will be d~,al~ L
to those of ordinary skill in the art.
For purposes of clarifying terminology, it should be noted that the hyper-tt rmin~l-branched core molecule disclosed in Fig. 28 and in Example FF, and those disclosed in the United States patents tli~c-l~se-l above are built by reiL~ldLive terminal br~nr-hing rather than reiterative comb-br~nrhing. That is to say, one ~tt~rh~s subsequent gell~laLion branches to the terminal moieties of a previous generation, thus 25 limiting the degree of br~nr-hing to the functionality of the previous generation terminal moiety, which would typically be two or three. In contrast by br~nrhing oligomers upon prior generation oligomer branches in accordance with the present invention, one can dr~m~tir,~lly increase the degree of br~nrhin~ from ~,~neldLion to gel~ldLion, and indeed can vary the degree of brzlnrhing from generation to gelleldLion.
In another process, the non-cro~link~fl poly-branched polymers, or hyper comb-branched polymers, are produced in a rem~rk~bly low number of iterations by lltili7ing a particular combination of process parameters and re~ct~nt~ having certain characteristics.
It has been surprisingly discovered that hyper comb-branched polymers having a molecular weight of about 1 million and up to about 10 million or even higher can be produced in only several reaction iterations by this pier~ ed embodiment process. A
hyper comb-branched polymer product having a molecular weight e~cee~ling 10 million was formed in only 4 iterations from a core of linear PEI 20, and side chains of PEOX
10 for the first iteration and PEOX 100 for the next 3 iterations, by the pl~fell~d embodiment process described below. It is contemplated that hyper comb-br~nrhrclpolymers having a molecular weight ranging from about 10 million to about 50 million could be produced in about 4 iterations. It is further contemplated that even higher molecular weight products could be formed such as products having a molecular weight 10 of about 100 million or more by co~ l;llg the iterations. Such rern~rk~hly high molecular weight polymers are produced in a surprisingly few number of iterations primarily by ~ltili7ing longer side chains, a particular grafting ratio, shorter reaction time periods, and ntili7.ing a proton trap to increase grafting yields and prevent chain scission of the comb-branched int~rmPtli~trs and resnlting hyper comb-branched polymer product.
15 In another aspect of the process, a novel separation technique is provided for ~7~ala~ g a hyper comb-branched polymer product from a reaction mixture, that is both economical and rapid.
The present inventors have discovered that grafting yields may be ~ignifir~ntly increased by lltili7ing a particular grafting ratio of living chain ends to secondary amines, 20 and in some in~t~nres, by also employing a proton scavenger during grafting operations.
Prior to the present discovery, when producing comb-branched polymers from PEI cores and PEOX 5 to PEOX 10 as grafting chains at a grafting ratio of 0.3 living chain ends per secondary amine, grafting yields typically ranged from about 10% to about 15%. In the present plef~ d process, it is pl~fell~d to utili7.ing a grafting ratio of from about 0.8 25 to about 1.2 living chain ends per secondary amine, and most ~l~f~lled to lltili7.ing a grafting ratio of about 1:1 of living chain ends to secondary amines. These grafting ratios result in ~ignifir~ntly improved grafting yields.
At these grafting ratios, i.e. about 0.8 to about 1.2:1, it has been found that it is also beneficial to utilize a proton scavenger during grafting to trap or scavenge protons 30 which are generated during grafting, such as when a living PEOX chain is grafted onto a secondary amine such as PEI. Without such scavengers, expelled protons are lld~r~ d to basic secondary amine sites along the PEI polymer backbone, thereby blocking and thus rendering those sites in~rces~ihle for further grafting. In the ~lerell~d embodiment WO 97/06833 PCTrUS96/13080 process, the use of a proton scavenger and a grafting ratio of about 1:1 has been found to .cignifit~ntly increase grafting efficiency, such as up to about 75% to 95% when grafting PEOX 5 or PEOX 10 branches onto a PEI core.
Proton scavengers may comprise nearly any suitable base that is c(>mp~tihle withS the core and side chain re~t~nt~. A pl~ert;ll~d proton scavenger for use when grafting PEOX chains onto PEI is a relatively hindered, tertiary amine such as i-Pr2NEt.
However, it is conternplated that a wide array of suitable bases could be utilized instead of, or in addition to i-Pr2NEt, such as triisobutylamine, triisooctylamine and triethylamine. The proton scavenger is preferably utili_ed in the grafting llli~LUlc~ in a 10 concentration of from at least about 1 to about 2 equivalents of the proton scavenger for every living or reactive chain end. It is envisioned that even higher ratios may be utili_ed in certain in~t~nePs.
As previously noted, the rem~rkzlhly high molecular weight polymeric products are produced in a surprisingly few number of iterations by increasing grafting yield, and by 15 ~ lLillg chain scission of the comb-branched interm~di~tPs and resl-lting hyper comb-br~n-~htod polymer products. In the case of lltili7in~ PEOX and PEI to produce a hyper comb-branrhP~l polymer, chain scission often occurs when there exists an excess of chain ends to secondary amines in the reaction environment. An excess of chain ends tosecondary amines promotes the formation of quaternary amines along the polymer 20 backbone, which readily undergo Hofmann degradation to produce undesirable lower molecular weight fr~gment~ upon heating.
It has been discovered that chain scission may be essentially prevented or ~ignifir~ntly minimi7P-l by employing one or more of the following practices: (a) ntili7ing shorter reaction periods, (b) lltili7ing relatively long chains for grafting onto 25 polymer backbones, (c) ensuring that NaOH or other salts are completely removed, or nearly so, from the reaction mixture(s) throughout the various stages of the process, and (d) ensuring that the reslllting hyper comb-branched product is m~int~inPd at relatively low temperatures and not exposed to high tempcld~ules. It is ~lerellc;d to employ all of these practices to prevent chain scission, and most pl~fell~d to employ all of these 30 practices in conjunction with lltili7in~ the previously described grafting ratios and proton scavenger during grafting operations to increase grafting yields.
Shorter reaction periods are utili_ed for both polymerization of the re~rt~nt~, e.g.
core and branches, and grafting operations in the pl~relled embodiment process since WO 97/06833 PCTrUS96/13080 shorter reaction periods have been found to reduce the tendency for quaternary amines to be forTned. Quaternary amines, as previou~ly noted, are prone to undergo Hofmanndegradation and thereby cause chain scission. When forming PEOX side chains fromPEOX 10 or PEOX 20, for later use in l~reparing hyper comb-branched polymers, it is 5 preferred to utilize a time period of less than about 5 hours for the polyll,eli~tion of PEOX. When forming PEOX side chains from PEOX 100, longer time periods may be required such as up to about 10 hours. It is particularly preferred to employ relatively short time periods during grafting operations, such as a grafting reaction time of less than about 1 hour for grafting polymerized PEOX chains onto a PEI core.
In addition to forming side chains from PEOX, it is possible to utilize a wide array of monomer units such as, but not limited to, any 2-4-, or 5-substituted oxazoline;

N 4 N_~4 N 4 lS~0 2~ 10~5 ~ R

any 2-unsubstituted 5,6-dihydro-4H-1,3-oxazines;

203 N----~5 any 2-substituted 5,6-dihydro-4H-1,3-oxazines;

3 N ::~
~6 R

or any block copolymers containing 5,6-dihydro-4H-1,3-oxazines and 2-alkyloxazolines.
Hyper comb-branched r~olypropyleneimine polymer obtained by hydrolysis of poly(S,6-dihydro-4H-1,3-oxazines), both 2-substituted and 2-unsubstituted, were found to exhibit a WO 97/06833 PCT~US96/13080 relatively high degree of thermal stability as compared to those having PEI side chains;
The tendency for chain scission is further reduced by lltili7ing relatively longchains for grafting onto a polymer backbone. Once a long side chain is grafted onto a secondary amine to generate a tertiary amine site along the polymer backbone, it is nearly 5 impossible to introduce another chain, particularly another long chain, at this tertiary amine site due to steric inhibition. In the case of forming hyper comb-branched polymers from PEOX and PEI, it has been discovered that the plt:r~ d length for PEOX sidechains or branches are at least about 50 monomer units, and most preferably at least about 100 monomer units.
In another embo~limpnt~ relatively short chains are utilized during the early stages in f~ hlg the hyper comb-branched product, i.e., gelleldLion 0, (G0) and relatively long side chains are utilized during later stages, i.e., gell~.ations 1 (Gl) and above. This practice has been found to increase interior br~nr'ning density, and reduce the previously described tendency for chain scission to occur at higher gell~ldLions.
Chain scission may also occur after initial formation of the poly-branched polymer or hyper comb-branched polymer, such as during or after neutralization of the hydrolyzed polymer product. After formation of the comb-branched polymer product and addition of acid to hydrolyze the product, the polymer product is neutralized and separated from the reaction llli~Lule by adding base to form an oily layer cont:3ining the polymer product.
20 This is typically accomplished by adding a base such as NaOH followed by heating until an oily layer separates from the mixture, that layer cont~ining the high molecular weight product. The oily layer is then cooled to harden or solidify it, wherein it can be readily removed. It has been discovered that after nP~ltr~li7~tion with NaOH, the PEI moieties in the polymer product tend to chelate the sodium cations, thereby freeing hydroxyl ions and 25 increasing the pH of the environment, and further promoting Hofmann degradation of any quaternary amines present upon heating, which in turn leads to chain scission.
Additionally, unwanted amounts of NaOH or other salts may cont~min~tr the rP~rt~nt~
used in forming the polymer product, such as linear PEI. Such cont~min~tion can later promote chain scission. Removal of NaOH or other salts from the poly-branched 30 polymer, and/or from the components used to form such product, has been discovered to reduce the tendency of chain scission of the poly-branched or hyper comb-branched polymer. NaOH or other salts may be removed from the reaction llli~LLll~e at various points of the process by a wide variety of techniques such as exhaustive washing with CA 02226299 1998-02-lO

water of precipitated polymer product or of the re~ct~ntc used to form the polymer product which are believed to contain NaOH, and then dissolving the polymer product in toluene, in which NaOH is insoluble, heating to remove water by azeotropic ~lictill~tion~
then filtering or otherwise separating the hot polymer product from the NaOH and/or S other salts. The various points of the process in which it is desirable to remove NaOH or any other salts include the stage in which the rç~ct~ntc are polymerized to form chains for subsequent grafting onto the polymer core or backbone, and the stage in which the grafting occurs.
Chain scission is also ",i"i...i,~l in accordance with the plcrcllcd embodiment 10 process by not exposing the res~ ing poly-branched or hyper comb-branched polymer product to Ic~ cldtures that are cignifir~ntly above room telllpeldLulc, such as when drying by ovens in which case, temperatures of 100~C or more are often reached.
Conventional drying procedures in which PEI polymer was dried at 80~C in an oil bath and under vacuum overnight were found to degrade the comb-branched polymer into 15 undesirable numerous smaller fragments. Thus, it is ~lcrcllcd that PEI poly-branched or hyper comb-branched polymer product is dried at temperatures less than about 60~C, and it is most pLcrt:llcd that the PEI product be stored at room tempcldlulcs, i.e. about 20~C.
It has been found that PEOX-PEI comb-branched polymers exhibit greater thermal stability than PEI comb-branched polymers. Thus, it is preferred to store hyper comb-20 branched polymers in the PEOX-PEI stage and hydrolyze the polymers to PEI hyper comb-branched polymers prior to use.
In another aspect of the process, a novel separation technique is provided for Sc~-dldl.-lg a poly-branched or hyper comb-branched polymer product from a reaction mixture cont~ining lower molecular weight products that is both econnmir~l and rapid.
25 Cullcll~ly k~nown techniques for separating high molecular weight, highly branched polymers from reaction mixtures generally involve some type of ultrafiltration process.
Ultrafiltration, although s~ricf~rtory in many respects, is undesirable in view of the relative high cost of ultrafiltration equipment and the inefficiencies associated with separating high molecular weight products from undesired low molecular weight products.
30 The present inventors have discovered a separation technique, whereby ultrafiltration is avoided and the polymer product is separated by a polymer refractionation technique.
The ~lcfellcd polymer refractionation technique is performed by separation of hyper comb-branched polymer product from a reaction mixture Colllyli~illg the product WO 97/06833 PCT~US96/13080 and ull~culLcd lower molecular weight components at the PEOX-PEI stage, by a first addition of an alcohol solvent in which both high molecular weight and low molecular weight products are soluble, and a second increment~l addition of a poor solvent in which the high molecular weight product is less soluble than the ~ wallLed low molecular 5 components. Addition of the poor solvent to the alcohol and dissolved components causes the high molecular weight polymer product to precipitate from solution. Examples of suitable poor solvents include, but are not limited to, diethyl ether or other ether-based solvents and hexane.
An example of the preferred refractionation technique is as follows. An alcohol 10 solvent such as methanol is added to the reaction mixture, until all of the components in the llli~LUlC, including unwanted low molecular weight components and the high molecular weight polymer product, are dissolved and are in solution. Then, a poor solvent such as diethyl ether, is incrementally added to plcrcrcllLially precipitate the desired high molecular weight components from the alcohol phase cont~ining the low 15 molecular weight products. Poor solvent is added until all, or substantially all, of the high molecular weight product is in the ~lcci~ilate. Periodically, the res llting oil sludge bottom product, i.e. precipitate, and/or top layer cont~ining the dissolved low molecular weight products are analyzed for the presence of the high molecular weight polymer product. Analysis may be performed by SEC (size exclusion chromatography) methods.
20 Once the high molecular weight polymer product no longer precipitates from the resulting -li~lure of alcohol solvent, poor solvent, and low molecular weight components, and thus is in the precipitate, addition of the poor solvent is halted. The mixture rem~ining above the precipitate and cont~ining the low molecular weight product, is then removed. The high molecular weight poly-branched or hyper comb-branched product remains in the 25 bottom precipitate layer and can be redissolved in water and subsequently dried by lyophilization .
The present invention also provides hyper comb-branched polymers that are joinedto one another by cro~.~linking or by other types of bridging. Very large macromolecules can also be formed without cross-linking or bridging between polymer molecules. Either 30 type of macromolecule can be suitable as a carrier, and are all included within the class of hyper comb-branched polymers described herein.
The Conjn~s~tf~
The conjugates of the present invention comprise the previously described hyper CA 02226299 1998-02-lO
W 097/06833 PCTrUS96/13080 comb-branched polymers conjugated with one or more carried materials. The pl~r~ d embodiment conjugates have the formula:
Mv - Mb wherein H is a hyper comb-branched polymer as described herein that may comprise one or more functional groups disposed generally about the periphery of the polymer; M is a carried material, v is an integer of 1 or greater; and b is an integer of 1 or greater.
The most ~ r~lled hyper comb-branched polymer conjugates are those in which 10 the carried material M, may be any one or more of a 1) ~ gnostic agent, 2) agricultural agent, 3) bioactive agent, 4) industrial agent, 5) environm~ont~l agent, and 6) consumer product agent. Examples of diagnostic agents include, but are not limited to, metal ions, radioactive drugs, radioactive tracers, radio-opaques, radionuclides, signal ~t:n~ldL~
signal reflectors, signal absorbers, diagnostic opacifier agents, fluorescent moieties, and 15 dye moieties. Agricultural agents may include, but are not limited to, ~griclllh-r~l materials, pheromones, pesticides, herbicides, and bioactive agents suited for agricultural uses. Examples of bioactive agents include, but are not limited to, ph~rm~(~ellti~ ~l agents or drugs, ph~rm~c~elltir~l interm~ ries, radioprotective agents, toxins, antibodies or fragments thereof, hormones, biological response modifiers, scavenging agents, imuno-20 pote~ g agents, genetic materials, antigens, and polypeptides. Examples of industrial agents include, but are not limited to, scavenging agents, agents for material modifiers such as adhesives and colloid dispersants, stabilizing agents, and chromophores.Examples of ~llvilolllllental agents include, but are not limited to, radioprotective agents, scavenging agents, pollutants, and agents for agricultural materials. Examples of 25 consumer product agents include, but are not limited to, fragrance moieties, stabilizing agents, material modifiers and chromophores.
The present invention also provides derivatives and variations of the preferred embodiment conjugates. The pl~r~ d embodiment conjugate variations include target directors and have the formula:
Hv - Mb - T
where T is a target director and z is an integer of 1 or greater. Examples of T include agents that target the carried m~t~ri~l, M, such as a bioactive agent, to, for example, a plant or pest or a particular factor in a target organism. Other examples of target CA 02226299 1998-02-lO

directors include, but are not limited to, entities which when used in the conjugates of the present invention result in at least a portion of the hyper comb-branched conjugate being delivered to a desired target, for example, a protein, lipid, a targeted cell, a targeted organ, antibodies, preferably monoclonal antibodies, antibody fragments, hormones, 5 biological response modifiers, and the like. Target director T includes ploLeillS, antibodies, antibody fragments, saccharides, and oligo~cçh~rides.
The plerelled embodiment conjugate derivatives also include derivatives having more than one carried material M', different than the primary conjugated carried material M, and/or target material T, of the formula:
T~ - Hv - Mb - M a where a is an integer of 1 or more. Examples of M' include all of the previouslydescribed agents for M.
There are numerous ways in which the carried material may be conjugated with the hyper comb-branched polymer. The carried material may be disposed generally 15 within the polymer, such as depicted in Fig. 15. This is accomplished by incorporating the carried material in the core or one or more of the beginning br~n~hing gelle.~Lions.
~ltPrn~tively, or in addition, carried material may be disposed between layers, as illustrated in Fig. 16. Carried material may also be disposed on the surface of the hyper comb-branched polymer, as illustrated in Fig. 17. In this embo-liment, the carried 20 material is incorporated in one or more of the latter br~nrhing generations. The carried material can also be ~tt~ hP~l to the outer periphery as a termin~l moiety. In all of these embodiments, the carried material may be conjugated with the hyper comb-branchedpolymers by physically encapsulating or e~Ll~L,illg the carried material within the core of the polymer, dispersing the carried material partially or fully throughout the polymer, or 25 ~tt~hing or otherwise linking the carried material to the polymer, whereby the ~tt~chmPnt or linkage is by means of covalent bonding, hydrogen bonding, adsorption, absorption, mPt~llic bondirlg, van der Waals forces, ionic bonding, or any combination thereof.
Moreover, since the size, shape and functional group density of the hyper comb-branched polymers can be precisely controlled, there are numerous other ways in which 30 the carrier material can be conjugated or associated with the polymer. For example, conjugation may be effected by covalent, coulombic, hydrophobic, or chelation type association between the carried materials and entities, typically functional groups, located at or near the surface of the hyper comb-branched polymer. There can be covalent, WO 97/06833 PCT~US96/13080 coulombic, hydrophobic or chelation type association between the carried materials and moieties located within the interior of the hyper comb-branched polymer. The polymer can be prepared to have an interior which is pre~ min~nfly hollow allowing for physical ~ ld~lllent of the carried materials within the interior, i.e. void volume, wherein the release of the carried material can optionally be controlled by congesting the surface of the polymer with diffusing controlling moieties.
There may be in~t~nres in which it is desirable, or even n.ocÇcs~ry to control the t~nre between the hyper comb-branched polymer and the carried material. This can be accomplished by lltili7ing a spacer group between the polymer and carried material. The 10 plt;rellc:d lengths for these spacers generally range from about 2 angstroms to about 20 angstroms or higher. Typically, the use of such spacers results in o~ ion of thereactivity of the conjugate.
As described below, there are numerous applications for hyper comb-br~nrhr~l polymers conjugated with carried materials. Examples of such applications include, but 15 are not limited to, protein conjugation, drug encapsulation, signal amplification, metal chelation, and gene transfection.
Protein Conjn~tion Generally, the stability of proteins in solution is very poor. The hyper comb-branched polymers described herein can conjugate proteins and thereby stabilize the 20 protein by minimi7ing structural tran~rolll,aLion of the protein such as refolding and m~int~in protein activity. As described in Examples 1-8 set forth below, water soluble hyper comb-branched polymers with PEOX, COOH, COONa, and -NH2 surfaces were obtained by grafting water soluble polymers (i.e. PEOX) or small organic molecules onto the outermost ge~ d~ions of hyper comb-branched polymers, as illustrated in Figs. 18 25 and 19. In all cases, hyper comb-branched polymers with superior water solubility as compared to that of unmodified hyper comb-branched PEI polymers were obtained. Since both NH2 and COOH are naturally occl-rring functionalities in ~loL~illS and peptides, the bioconjugation of such hyper comb-branched polymers can be achieved under physiological conditions without denaturing the proteins.
As described in Example 9 set forth below, and illustrated in Fig. 20, a hyper comb-branched polymer was first derivatized with sulfo-SIAB, a reagent, to generate an iodoacetyl modified hyper comb-branched polymer. The reslllting polymer product was then reacted with sulfhydryl functionalized monoclonal antibody formed by the antibody with reducing dithiothreitol (DTT) at pH 7.6. The conjugate was characterized by UV
radiation at 280 nm, and the activity of the antibody after conjugation was m~int~in~rl as in~lir~ted by creatin kinase monoclonal antibody (CKMB) assay. This monoclonal antibody bioconjugate could be utilized in tli~gnostiC assays and targeted drug deliveries 5 both in vitro and in vivo.
It is envisioned that a wide array of enzymes and proteins can be stabilized by conjugating such with the hyper comb-branched polymers described herein. Examples of such enzymes include, but are not limited to, ghlt~m~te ~ylllvdt~ LlA~ in~e (GTP), ~lk~lin.o phosphatase, acid phosphatase, gl~ t~ et~te Ll,.~ ...in~e (GOT), 10 c~ P kinase, and lactate dehydrogenase. Examples of such proteins include, but are not limited to, troponin, ferritin, prolactin, gastrin, and calcitonin.
Example 1:
Synthesis of Hyper Comb-Br~nrh~l Polymers ~ICB G0]
The synthesis of PEOX100-g-PEI20 is provided as a general procedure for the 15 ~lc~dldlion of hyper comb-branched PEOX-PEI and PEI polymers. A l~ ule of MeOTs (0.1845 g, 0.9906 mmol) in 150 ml of toluene was azeotroped to remove water with a till~tinn head under Ar for 10 mimltes. After cooling to about 90~C, 2-ethyloxazoline (10 ml, 99.06 mmol) was c~nmll~t~d in and the mixture was allowed to reflux for 5-10 hours. To this llli~LUlC was added morpholine termin~t~d LPEI 20 (49.5 mg, 0.9906 20 mmol of NH), which was dried by azeotropic ~ till~tion from toluene, followed by imm.o~ te addition of i-Pr2NEt (1 to 2 eq.). The llli~lUl~ was refluxed for 1 hour, and then cooled. The top layer was ~l~c~nt.otl and the bottom layer ~polymer product) was redissolved in mPth~nol, followed by fractionation by methanol/diethyl ether llli~luic to remove the unreacted monomers, oligomers and catalysts. The entire separation process 25 was monitored by SEC. The purified product was rotary evaporated and lyophili7~d to produce a hyper comb-branched G0 PEOX-PEI polymer as a white powder. This white powder was further hydrolyzed in 50% H2SO4 at 100~C and then neutralized by 50%
NaOH solution. This solution (pH of about 10 to 11) was heated to boil under N2, and the product (G0 PEI) floated on top as an oil layer. After cooling to room L~ eldture, 30 the top layer became a solid cake on the surface which was subsequently removed and redissolved in 600 ml deionized, boiling water. After slow se~liment~tion overnight, the white precipitate was filtered by suction funnel. The pure hyper comb-branched PEI
polymer was obtained by azeotropic removal of water from a toluene solution of the polymer, followed by a gravity filtration and then rotary evaporation of the toluene at 60~C. Both hyper comb-branched PEOX-PEI and PEI polymers were characterized by size exclusion chromatography (SEC), nuclear m~gnPtic resonance (NMR), th~rm~l gravimetric analysis (TGA), and dirrelcllLial sc~nnin~ coulimetry (DSC). Other hyper 5 comb-branched polymers with dirr~l~llL grafting chain lengths, branched cores, and generations were prepared in a similar manner.
FY~mrle 2:
Fun~ n~ ti~n of Hyper Comb-Br~n-he~l PEI Polymers with 50% PEOX10 [HCB G1 (50% PEOX)]
The reaction procedure is similar to the ~ a.dLion of hyper comb-branched PEOX-PEI polymers described in Example 1 except the ratio of the living chain end of PEOX10 to NH was 0.5:1.
Example 3:
Hyper Comb-Branched Polymer-COOMe [HCB G3 (COOMe)]
To a 100 ml round bottom flask cont~ining 1.0256 g of hyper comb-branched G3 PEI polymer was added 4.709 g of methyl acrylate solution in MeOH (50%) at 0~C. The res--lting llli~Lul~ was then diluted with 20 ml MeOH. The lll~Lul~ was reacted at 37~C
for 24 hours, and then dried by rotary evaporation and N~ bubbling (80% yield). The 20 product was analyzed by NMR.
F,Y ~nrle 4:
Hyper Comb-Br~n(~h~(l Polymer-NH2 (PAMAM Modified) [HCB G3 (PAMAM)]
To a 1 L polyethylene jug cont~ining 740 ml ethylene tli~min~ (EDA)/mPth~n~ll 25 (MeOH) mixture (78 wt. % of EDA) was added ester functionalized hyper comb-br~nrh~l polymer (from Example 3) at a ratio of EDA/ester of about 300:1. The mixture was allowed to react at -5~C for 5 days, and the pure product was obtained by rotary evaporation of EDA and MeOH, followed by ultrafiltration with Amicon 10,000 molecular weight cut off membrane. The reslllting product was characterized by NMR
30 and SEC (molecular weight of about 29,000, molecular weight distribution of about 1.11). Other generations of hyper comb-branched polymers with such p~ aly amine modifications were prepared in a similar manner.

~nnr~ 5:
Hyper Comb-Br~n~h~l Polymer-NH2 (Chlo~b~ll.ylamine Modified) [HCB G3 (CEA)]
To a 100 ml round bottom flask cont~ining 0.44 g hyper comb-branched G3 PEI
polymer dissolved in 30 ml MeOH was added 0.48 g chloroethylamine hydrochloride (dissolved in S ml of MeOH), and 2 eq. of NaOH dissolved in MeOH. The reaction mixture was refluxed at 60~C for 1 to 3 hours, the solution was filtered and dried by rotary evaporation. The crude product was redissolved in MeOH and precipitated out from the solution by addition to diethyl ether. The pure product was characterized by NMR and SEC (molecular weight of about 35,000). Other generations of hyper comb-branched polymers with such ~lhllaly amine modifications were prepared in a similar manner.
F,Y~nP~ 6:
Hyper Comb-Br~n~he~l Polymer-NH2 (Chain Tern~inal Modified) tHCB G2 (NEI2 Termir.~e~l)]
A llli~lUl~ of p-tol len~sl~lfonic acid (0.754 g) in 150 ml of toluene was azeotroped to dryness with a (li~till~tion head. To this lllix.Lulc~ was added 2-ethyloxazoline (39.28 g), 20 and the mixture was allowed to reflux for 5 to 10 hours. To this mixture was added G1 PEI polymer (0.20 g, molecular weight of about 130,000) dried by azeotropic riictill~tion from toluene, followed by imm~ te addition of i-Pr2NEt (1 to 2 eq.). The llliX.IUl~ was refluxed for 1 hour, and then cooled to room temperature. The pure product (G2) was obtained by fractionation with a methanol/diethyl ether mixture, and was characterized by 25 NMR, DSC, TGA, and SEC (molecular weight of about 3,900,000, molecular weight distribution of about 1.33).
To a 250 ml round bottom flask cont~ining hyper comb-branched G2 PEOX
polymer (S g in 150 ml of H2O) was added 1 eq. 50% H2SO4. The mixture was allowed to reflux for 3 hours, and then was neutralized to a pH of from about 10 to about 11.
30 After heating, an oil layer was formed on the bottom and was separated imm~ t~ly.
The pure product (white solid) was dried by rotary evaporation and high vacuum, and was characterized by NMR, DSC, TGA, titration, and SEC (molecular weight of about 2,600,000, molecular weight distribution of about 1.50). Other gell~ldlions of hyper comb-branched polymers with primary amines at the polymer chain ends were prepared in 35 a similar manner.

F,Y~ )le 7:
Hyper Comb-Branched Polymer-COOH [HCB G0 (Propionic Acid)]
To a 100 ml round bottom flask cont~ining ester-functionalized hyper comb-branched G0 polymer was added lN NaOH (about 2 eq.) and water. The reaction 5 mixture was refluxed for 1 to 5 hours. The solution was then cooled and neutralized by lN HCl until white precipitate was formed (pH about 1). The pure product was obtained by filtration, followed by dryness with vacuum drying (>60% yield).
F,Y~mrl~ 8:
Hyper Comb-Branched Polymer-COOH [HCB G0 (Acetic Acid)]
To a 100 ml round bottom flask cont~ining hyper comb-branched G0 PEI polymer in water and MeOH mixture was added bromoacetic acid (1 eq.) and about 2 eq. NaOH.
The mixture was refluxed for 6 hours. The solution was cooled and then neutralized by 1 N HCl until a white precipitate was formed. The solution was filtered and the solid was dried in a vacuum oven at 45~C for 6 hours. The product was analyzed by NMR and 15 SEC.
F,Y~TnP'e 9:
~ ntibody - Hyper Comb-Branched Polymer Conjugate Iodoacetyl Hyper Comb-branched Polymer To a test tube CO~.I;.iJ~ g 1 ml of 10 to 50 mg/ml of hyper comb-branched polymer 20 functionalized with -NH2 terminal groups, in water was added 0.2 ml of 0.5 M sodium phosphate (pH 7.0), and the solution pH adjusted up to 7.6 using lN HCl. This solution was then added to freshly dissolved sulfo-SIAB (20 mg/ml in water), and gently vortexed.
After incubation at 30~C for one hour (or at room temperature for 2 hours), the pure product was obtained by passing the reaction mixture through a G-25 Sephadex colurnn.
25 The concentration of the polymer was de~ermin~d using a fluorescamine assay monitored with a fluorimeter and the iodo content was qll~ntifiPd with DTT and 4,4'-dithiodipyridine .
Preparation of protein with SH groups An anti-CKMB IgG protein was buffer exchanged into a reduction buffer (0. lM
30 sodium phosphate~ 5 mM EDTA, (pH 6)) and the resulting concentration was adjusted to 5 mg/ml. To this solution was added a solution of 11.4 mg/ml of DTT equal to 1/9 of the volume of the protein solution. After incubation at 37~C for one hour, the free sulfhydryl groups were formed and the product was purified from low molecular reagents , -WO 97/06833 PCTAJS96tl3080 by a passage through a G-25 Sephadex column. The protein concentration was determined by UV absorption at 280 nm.
Antibo~y Hyper Comb-Branched Polymer C~onjugate To a test tube was added iodoacetyl modified hyper comb-branched polymer and 5 IgG-SH at a challenge ratio of 3:1 (pH of 7.6, protein concentration of 5 mg/ml). After inr~1b~tion at 2 to 8~C for 16 to 24 hours, the reaction was quenched by addition of 20 mg/ml N-ethyl maleimide in dimethyl fonn~mi-l~ at 2 to 8% for 2 hours. The pure conjugate was obtained by gel filtration (Ultrogel AcA, Pharmacia Sephadex or Sepharose gels) or by ultrafiltration with YM-100 Amicon Membrane.
0 Drll~ Encapsulation Since drugs, both human and agricultural, are generally hydrophobic, a hydrophobic interior of a hyper comb-branched polymer performs like a well-defined, covalently-fixed micelle. Associating or linking a hydrophobic drug within that region provides a novel m.ocll~ni~m for solubilizing or delivering such drugs.
Interior hydrophobically modified hyper comb-branched polymers can be produced through three general synthetic methods. The first method is to utilize a functionalized hydrophobic, linear or branched polymeric core (i.e. chloroethyl or bromoethyl functionalized linear or branched polystyrene) as an initiator to polymerize 2-ethyloxazoline and form an amphiphilic hyper comb-branched (PS-PEOX) polymer. The second method is to first functionalize hyper comb-branched PEI polymers with ana~ fi~te percentage of linear water soluble polymers. Due to steric effects, thegrafting polymeric chains can only react with the readily available exterior secondary amines, thereby shielding the interior secondary amines. The resulting water soluble polymer is then reacted with hydrophobic monomers in the interior to provide an amphiphilic hyper comb-branched polymer. The third approach involves a combination of the above methods, in which an amphiphilic block copolymer is grafted onto a functionalized linear or branched polymeric core. In all three schemes, higher generation hyper comb-branched amphiphilic polymers can be produced by the same iteration steps utilized in the synthesis of hyper comb-branched PEOX and PEI polymers.
As described in Examples l0 and ll, hyper comb-branched polymers having hydrophobic interiors were formed by modifying the interior of exterior-function~1i7~
water soluble hyper comb-branched polymers with hydrophobic monomers such as epoxy hexane and methyl acrylate. In addition to the NMR evidence, where characteristic resonance of the alkyl chain or ester group could easily be identifito(1, SEC measurements also in-lir~tt~-l that the modified hyper comb-branched polymers shrunk in size when dissolved in water after the hydrophobic modification. This is primarily due to the shrinking of the hydrophobic interior.
An interior hydrophobically-modified hyper comb-branched polymer was also obtained through block copolymerization. In this case, homo poly(2-phenyloxazoline) (PPOX) is utilized as a hydrophobic segment, while homo PEOX is utilized as a hydrophilic segment, as depicted in Fig. 21. The block copolymer of PEOX-PPOX is, therefore, an amphiphilic polymer. By grafting such a block copolymer onto a linear or a 10 branched PEI core, a hyper comb-branched polymer with a hydrophobic interior and a hydrophilic exterior can readily be obtained (Example 12). As measured by SEC, the a~a~ L molecular weight of the polymer product increased upon addition of the second monomer. This increase in~ tes the formation of the desired block copolymer. Thesize of the hyper comb-branched polymer formed by lltili7.ing block copolymer side chains 15 is, however, smaller than that obtained with homo PEOX side chains, suggesting the formation of the hydrophobic interior.
If the last grafting PEOX side chains are produced with para-toluenesulfonic acid as an initiator, the reslllting amphiphilic PEOX HCB polymer can be partially or fully hydrolyzed to g~ t~ an amphiphilic PEOX/PEI or PEI HCB polymer with primary 20 amines at the chain ends of the exterior layer. These primary amines can then be utilized to conjugate with targeting moieties such as monoclonal antibodies while the hydrophobic interior is still able to carry a large amount of drugs. If the number of surface functional groups produced by this method is not large enough, the amphiphilic PEOX/PEI or PEI
HCB polymer can then be further modified through standard PAMAM dendrimer 25 synthesis techniques (i.e. the addition of methyl acrylate and DEA to generate amphiphilic HCB polymers with more ~lhlldly amines or carboxylates at the surface).
Since acid hydrolysis of PEOX is much easier than that of PPOX, functional secondary amines can be pl~relcllLially formed at the exterior of the hyper comb-branched polymers, while the hydrophobic interior remains intact. The grafting of additional layers 30 of the PEOX-PPOX block copolymers at higher generations can generate hyper comb-branched polymers with a PEOX-PPOX-PEI-PPOX-PEI- -PEOX multilayered dendritic architt-cl~lre. The intermediate PEI layers can then be conveniently converted into more hydrophilic layers by reaction with hydrophilic monomers such as ethylene oxide and CA 02226299 1998-02-lO
WO 97/06833 PCTrUS96/13080 bromoacetic acid. Thus, a multilayered amphiphilic hyper comb-branched polymer can be prepared by the same iteration steps described in Example 12.
In the case of ntili7ing hyper comb-branched polymers, drugs can be ~tt~ch~-1 inthe interior of the polymer through both physical and chemical methods, as illllctr~t~-l in S Figs. 22 and 23. Fig. 22 illustrates carried drugs, or drug agents or interm~ tPs, associated or ellcap~ulated within the interior of a hyper comb-branched polymer. Fig. 23 illustrates such carried material chemically linked to the interior of a hyper comb-branched polymer. The release rate of the drug or carried material may be controlled by the thickn~ of the exterior layer for physically en~apsul~te-l drugs or by cleavage of 10 ch-?micz~l bonds for chemic~lly linked drugs.
In Examples 13 and 14, it can be seen that hydrophobic drugs (i.e. 4-~cet~miclophenol) can be physically encapsulated or solubilized by hydrophobic interior modified hyper comb-branched polymers (i.e. C6 and methyl acrylate modified) when dissolved in tris buffer (pH 7.4). In contrast, in the case of unmodified interior hyper 15 comb-branched polymers (hyper comb-branched PEOX or hyper comb-branched terrnin~l NH2 polymers) or tris buffer alone, the drug was completely insoluble. These results suggest that the encapsulation is based on hydrophobic integration instead of an acid-base reaction or ionic interaction. The inventors contemplate that a wide array of ph~nn~cel~tic~l agents could be encapsulated within, or otherwise associated with hyper 20 comb-branched polymers, such as antibiotics, analgesics, antihypertensives, cardiotonics, sedatives, antiepileptics, antipyretics, stimnl~nt~, immllnnsuppressives, and the like;
examples are acetaminophen, acyclovir, alkeran, amikacin, ampicillin, amphotericin B, aspirin, bisa~ e, bleomycin, neocardiostatin, chloroambucil, chloramphenicol, cytarabine, daunomycin, ~ ntin, doxorubicin, fluorouracil, g~ lycin, ibu~lofe~l,25 kan~lllycin, meprobamate, methotrexate, novantrone, nystatin, oncovin, phenobarbital, polyllly~iu, probucol, procarbazine, rifampin, streptomycin, spectinomycin, symmetrel, thioguanine, tobramycin, trimethoprim, and valban.
~ ltern~tively, drugs can be linked inside the hyper comb-branched polymers through ch~mi~l reactions. In Example 15, hydrophobic moieties such as fluorescein 30 isothiocyanate (FITC) were ch~-mic~lly reacted with interior secondary or primary amines of hyper comb-branched polymers (Figs. 18 and 19). Drug attachment can also be achieved through other chemical linkages such as ester, anhydride, or amide bonds.
Therefore, t_e drug releasing rate can be controlled by hydrolysis of these ch~mir~l bonds CA 02226299 l998-02-lO
WO 97/06833 PCTrUS96/13080 under physiological conditions, as illustrated in Figs. 24 and 25.
~,Y~mrl~ 10:
Hydrophilic - Hydrophobic Hyper Comb-Branched Polymer (C6 Interior) [HCB G0 (50% PEOX-C6)]
To a mixture of 50% functionalized PEOX-G0 (0.50 g in 20 ml MeOH) was added 1 g of epoxyhexane. The mixture was heated at 40~C for 5 days. The solvent and unreacted monomers were removed by rotary evaporation and high vacuum at 80~C. The product was characterized by NMR and SEC.
10 ~Yq~nplE 11:
Hydrophilic - Hydrophobic Hyper Comb-Br~n~h~-l Polymers (Ester Interior) IHCB G0 (50% PEOX Methyl Acrylate)]
To a mixture of 50% functionalized PEOX-G0 (0.475 g, 20 ml MeOH) was added 15 methyl acrylate solution (0.5 g MA in 0.5 g MeOH) at 0~C. The mixture was allowed to react at 40~C for 3 days. The pure product was obtained by rotary evaporation, dried by bubbling with N2, and then characterized by NMR and SEC.
Example 12:
Hydrophilic - Hydrophobic Hyper Comb-Branched Block Copolymers [HCB G0 (PEOX-b-PPOX)]
The synthesis of PEOX100-b-polyphenyloxazoline (PPOX10)-g-PEI20 is provided as a general procedure for the preparation of hyper comb-branched PEOX-b-PPOX-g-PEI
copolymers. A mixture of MeOTs (0.1845 g, 0.9906 mmol) in 150 ml of toluene was 25 azeotroped to remove water with a ~1ictill~tion head under Ar for 10 minlltPc. After cooling to about 90~C, 2-ethyloxazoline (10 ml, 99.06 mmol) was c~nmll~tP(l in and the lulc was allowed to reflux for 5-10 hours. 2-phenyloxazoline (1.8 ml, 7.6 mmol) was then added and the reaction mixture was refluxed for 5 to 10 hours. To this mixture was added morpholine- tprminz~tptl LPEI 20 (49.5 mg, 0.9906 mmol of NH), which was dried 30 by azeotropic rlictill~tion from toluene, followed by immediate addition of i-Pr2NEt (1 to 2 eq.). The llli~Lul~ was refluxed for 1 hour, cooled, and then dissolved in methanol.
After rotary evaporation of the solvents, the crude product was purified by fractionation by m~th~nol/diethyl ether mixture to remove the unreacted monomers, oligomers and catalysts. The entire separation process was monitored by SEC. The purified product 35 was rotary evaporated and lyorhili7P~l to produce a hyper comb-branched PEOX-b-PPOX-g-PEI polymer as a white powder. Such polymers were characterized by SEC and NMR.

WO 97/06833 PCTrUS96/13080 Other hyper comb-branched polymers with dirr~ lL grafting chain lengths, branched cores, and generations (i.e., multi hydrophilic-hydrophobic copolymers) can be prepared in a similar manner.
~y~Tnple 13:
S Drug Solubili7~ti~m with C6-Modified Hyper Comb-Branched Polymers To a vial cont~ining 19.7 mg 4-~cet~mi(lophenol in 1 ml tris buffer (pH = 7.4) was added 0.12 ml of 7.7% (9 mg) C6 modified hydrophobic interior hyper comb-br~nf~h~l polymer of Example 10. The solution became clear imm~ t~ly, in~licating that drug was solubilized completely. In the control vial, the white solid 4-~ret~mi(lophenol 10 remzlinlotl as a precipitate in tris buffer at the same concentration. Other water soluble hyper comb-branched polymers without hydrophobic interior modifications (i.e. hyper comb-branched G2 PEOX and termin~l modified G2-NH2 polymers) could only disperse, but not solubilize 4-~et~mi-lophenol.
FY~-npl~ 14:
Drug Solubi~ ti~n with Methyl Acrylate-Modified Hyper Comb-B~ ed Polymers To a vial cont~ining 18.7 mg 4-acet~mic~Qphenol in 1 ml tris buffer (pH = 7.4) was added 0.11 ml of 6.3% (6.9 mg) methyl acrylate-modified hydrophobic interior hyper 20 comb-branched polymer of Example 11. The solution became clear imm~ tely, in~ ting that the drug was solubilized completely. In the control vial, the white solid 4-~cet~mi~ phenol remained as a precipitate in tris buffer at the same concentration. Other water soluble hyper comb-branched polymers without hydrophobic interior modifications (i.e. hyper comb-branched G2 PEOX and terminal modified G2-NH2 polymers) could 25 only disperse, but not solubilize 4-~cet~mi(lophenol.
Another aspect of the present invention relating to encapsulated conjugates is the encapsulation of fragrances. One of the benefits in encapsulating fragrant compounds is that their release rate can be ~ignifirantly decreased and the time period of release can be signific~ntly extended as described in Example lS. Furthermore, it is contemplated that 30 specific release time periods and release rates can be provided by use of the hyper comb-branched polymers described herein.
Example 15:
Encaps~ n and R~ e of Fragrances To a glass vial was added 73.4 mg of fragrance (Lagerfeld) and 0.15 mg of C6 WO 97/06833 PCT~US96/13080 interior modified hyper comb-branched polymer in 0.2 ml of water or pH 7.4 buffer.
The control solution was form~ t.o-1 by adding 74.6 mg of fragrance in 0.2 mg of water or pH 7.4 buffer. These solutions (5 ,ul) were then deposted onto AccuWipe~ papers or -into open vials to allow slow evaporation in air. In all cases, the fragrance release rates 5 of fragrances encapsulated by hypdrophobically modified hyper comb-branched polymers were much slower than the controls, as evi~ nre-l by longer lasting odors (3-4 weeks for hydrophobically modified HCBPs and less than 5 days for controls). The experiments were also performed in an ethanol/water or buffer mixture. The release rates were observed to increase with the ethanol contents.
0 Si~ mrlif;c~tion Signal amplification is very important for many in vitro and in vivo ~ gnosti~
applications, since carried materials can greatly enhance detection capabilities of nearly all types of measurement instrumentation that employ some type of measuring signal, such as light. In this aspect of the present invention, light reflecting or light absorbing moieties are preferably ~tt~rhlod to the periphery of a hyper comb-branched polymer. The FITC
,-ent (Example 16) is an example of signal amplification, where a large number of chromophores were ~tt~rhed onto a hyper comb-branched polymer. As a result, the signal was greatly enh~nred Example 16:
Signal ~mrliffr~tionlDrug ~tt~hmPnt A mixture of 10 mg of HCB G0 (50% PEOX) in 5 ml sodium borate buffer (pH
= 9.5) was slowly added, dropwise, to a FITC/DMSO solution (7 mg FITC in 1 ml DMSO). The reaction mixture was shaken for 12 hours in the dark. After extensiveultrafiltration to remove the unreacted FITC, the solution rem~in.od yellow-red in color.
The formation of the product could also be conveniently monitored by thin layer chromatography (TLC) to check for the formation of dye-modified polymer. Similarcovalent chemi.~try could be used to covalently bond a drug to a hyper comb-branched polymer.
Metal Chelation A variety of surface modification reactions can be performed upon the hyper comb-branched polymer system. Chelating groups such as -NH2 and COONa were ~, attached onto hyper comb-branched PEI polymers. In Example 17, C*+ and Co2+ were CA 02226299 1998-02-lO
W O 97/06833 PCT~US96/13080 chelated with hyper comb-branched polymers, such as hyper comb-branched PEI
polymers, and -CQONa, or -NH2 modified hyper comb-branched polymers (Figs. 18 and 19). All of the hyper comb-branched polymers were able to strongly complex thesemetals as evi~ n~e(l by the change of the solution color and/or solubility. The formation S of the complexes was also supported by UV measurements, where .~ignific~nt wavelength changes were observed. In addition to Cu2+ and Co2+, it is contemplated that a wide array of other metal ions could be chelated lltili7.ing the hyper comb-branched polymers described herein. Examples of such metal ions include, but are not limited to the metals in the Periodic Table Groups VIIIA (Fe, Ni, Ru, Rh, Pd, Os, Ir, Pt), IVB (Pb, Sn, Ge), 10 IIIA (Sc, Y, l~nth~ni~l~s and ~tini-les), IIIB (B, Al, Ga, In, Tl), IA alkali metals (Li, Na, K, Rb, Cs, Fr), and IIA :~lk~lin~-earth metals (Be, Mg, Ca, Sr, Ba, Ra) and other transition metals. This chelating property is useful not only in biome~ l applications such as m~gnPtic resonance im~3~ing (MRI), protein separation, in vitro diagnosis, but also in industrial applications such as metal separation and nuclear waste recovery.
15 FY~nr'e 17:
Metal Ch~
A stock solution cont~ining 1.25 wt. % of Cu2+ cupric sulflate in water was divided into five vials. To each of these solutions was added 5 mg each of HCB G0 (propionic acid), HCB G0 (50% acetic acid), HCB G0, HCB Gl (50% PEOX) and HCB
20 G1 (PEOX). The solutions changed from light blue to blue or even deep blue, in(lir~ting the formation of the Cu2+ / hyper comb-branched polymer complexes. These complexes were also characterized by UV. The Co2+ / hyper comb-branched polymer complexes were prepared and characterized in a similar manner. In this case, solutions from light brown to dark brown were formed.
Gene Transfection The hyper comb-branched polymers of the present invention may be complexed with genetic material and used for gene therapy in m~mm~ n org~ni~m~, e.g., hnm~n~.
Genetic materials are nucleotide based materials, including without limit~tinn, viruses and viral fragments, pl~mi(lc, phages, cosmids, genes and gene fragments (i.e., exons, introns), deoxyribonucleic acid (DNA) both single and double stranded, ribonucleic acid (RNA), ribosomal RNA (rRNA), catalytic RNA (cRNA), small nuclear RNA (snRNA), mt-ssenger RNA (mRNA), transfer RNA (tRNA), DNA and RNA oligonucleotides (both single and double stranded) or oligomers and (anti-sense) oligonucleotides, protein nucleic WO 97/06833 PCTrUS96/13080 acids (PNA), and substituted nucleic acid oligonucleotides.
A method for preventing or treating a disease comprises transfecting a m~mm~ n cell with hyper comb-branched polymer complexed with genetic material. Genetic material may be tr~ncf~ctç(l into cells for a variety of reasons including the production of 5 proteins within cells, altering cell function, correcting genetic defects, function as drugs, and the like. Thus, genetic diseases or conditions, in particular, may be prevented or treated using the complex of the hyper comb-branched polymer and genetic material of the present invention.
The amount of genetic material used in the genetic material:hypes comb-branched 10 polymer complex solution is sufficient to achieve the desired prophylactic, therapeutic or diagnostic effect. This amount will vary as a function of the effect sought, the ease with which target cells are s~lcceccfully transfected, the efficiency of any target director zltt~-~h~od to the ~lçn-lrimer, and the mode of zl~lminictration of the complex, i.e., in vitro, ex vivo, in vivo, and, if in vivo, intravenous, topical or direct injection into a particular 15 tumor, organ, gland or other tissue.
Once the amount of genetic material and its charge has been dt:tt~ r1 the amount of hypes conb-hr~nrh~l polymer used is then determined as a function of the genetic material:hypes comb-branched polymer charge ratio selected. Sufficient hypes comb-branched polymer is used in the solution to give the desired charge ratio. The 20 charge ratio selected will vary as a function of the same variables which affect the solution concentration of genetic material, as well as with whether DEA-dextran or glycerol is used to enh~n~e transfection. The "charge ratio" refers to the number of unit positive charges on the hyper comb-branched polymer carrier relative to the number of unit negative charges on the carried genetic material. For purposes of dt:L~ ;.lg this 25 ratio, only the positive charges on the last generation of added branches are included, where the genetic material is to be carried on the surface of the polymer. If the genetic m~tçri~l were to be carried on an interior generation of branches, only the positive charges on that interior generation of branches would be included. The generation of branches primarily responsible for carrying the genetic material are sometimes referred to 30 herein as the "carrier generation branches."
The number of charges on the carrier generation branches in one molecule of a hyper comb-branched polymer can be obtained by measuring the absolute molecular weights of the hyper comb-branched polymers with their carrier generation branches, and CA 02226299 1998-02-lO
WO 97/06833 PCT~US96/13080 without their carrier generation branches, and the absolute molecular weight of the carrier generation linear branches by multi angle laser light scattering. The number of charges were calculated based upon the following equations:
N = number of charges = MW(G)--MW(G--1) x DP (br) (1) MW(br) Charge density (charges/,ug) = N x 6.02 x 10l7 charges /~g (2) MW(G) MW(G) is the absolute molecular weight of hyper comb-branched polymer generation G
polymer i.e. with the carrier ~ d~ion branches ~tt~rhf r1 MW(G--1) is the absolute molecular weight of hyper comb-branched polymer generation G--1 polymer i.e. without the carrier generation branches ~tt~Ched MW(br) is the absolute molecular weight of the linear carrier generation branches.
DP(br) is the degree of polymerization of the linear carrier generation branches. It is ~nmed in this equation that each polymerization unit on each carrier generation branch has or will be modified to have a unit positive charge. Where the chemi~try used is otherwise, the formula has to be modified accordingly.
Generally speaking, the genetic material:hypes comb-branched polymer charge ratio may be from about 1:10 to about 1:10,000 (possibly even lower), but more preferably from about 1:10 to 1:1,000, as a function of the above variables. Even more plert:lled are charge ratios of from about 1:100 to about 1:1000, with a charge ratio of about 1:200 being most pl~rell~d.
A complex or conjugate of a hyper comb-branched polymer and genetic material is prepared by reacting the hyper comb-branched polymer with the desired genetic material in a suitable solvent at a temperature which facilitates the complexing of the genetic material with the polymer. This method may further include placing the complex in a solution with DEAE-dextran, chloroquine, or glycerol as an en~,h~n~,ing agent. The plefelled temperatures to facilitate complexing range from about 20~C to about 40~C, however preparation techniques at both higher and lower temperatures are encompaese-1 within the present invention. The pl~fe-l~d pH for the complexing solution range from about S to about 10, but higher and lower values may be ap~ ,.iate. The hyper comb-branched polymer preferably has an outer periphery of positively charged sites to facilitate electrostatic ~tt~chment of genetic material.

In an alternate embodiment, a complex or conjugate of a hyper comb-branched polymer and genetic material that can be readily diluted for subsequent use, is prepared by mixing genetic material and hyper comb-branched polymer having a positive surface functionality, in water. The concentration of genetic material is about 1 ~g to about S 10 ,ug per 20 ,uL of mixture. The amount of polymer is primarily dependent upon the number of its available positively charge sites, but should be added to effect a genetic material: polymer charge ratio of from about 1:10 to about 1:10,000. The method preferably further comprises mixing the above described mixture at a pH of from about S
to about 10 and a temperature of from about 20~C to about 40~C.
A method for introducing human genes into m~mm~ n cells to avoid substantial gene rearrangement or other alterations that may affect gene expression may be con-lllcte(l by transfecting a m:~mm~ n cell with a hyper comb-branched polymer complexed with genetic material.
Gene L,dl~r~l can be effected by Lldll~re-;~illg a variety of cell types such ashematopoietic cells, skin fibroblasts, hepatocytes and the like. Thus, a method for preventing or treating a genetic disease may comprise transfecting a hyper comb-branched polymer complexed with genetic material into a hematopoietic stem cell, skin fibroblast cell, hepatocyte, or the like, ~lmini~tPring the transfected cell into a m~mm~ n ol~al~i~
and expressing said cell to obtain a prophylactic or therapeutic effect.
Cell transfecting can be effected in a variety of fashions. In one method, a complex of a hyper comb-branched polymer and genetic material is simply made available to cells to be transfected. This technique may be employed to transport genetic material through a cellular membrane and into a cellular nucleus.
In another embo-limenf, a method for protecting genetic material from digestion during transit to and transfection into a cell may be provided by complexing genetic m~t~ri~l with hyper comb-branched polymer prior to exposing the genetic material to digestive enzymes.
Moreover, the hyper comb-branched polymers of the present invention can be utilized to stabilize and compact genetic material by complexing genetic material with the hyper comb-branched polymers described herein.
The transfection as ~ cll~serl in the present invention can be used for a variety of purposes, including in vitro, in vivo and ex vivo uses. Further, the in vitro use of the complex of hyper comb-branched polymers and genetic material of the present invention CA 02226299 1998-02-lO

can be useful in ~et~cting or ~ gn~ing various conditions. A method for diagnosing a disease or condition in a m~mm~ n organism may be detected or diagnosed using the complex of the hyper comb-branched polymer and genetic material of the present invention.
S Due to the convenient design and synthesis of hyper comb-branched polymers having dirr~ lL sizes, shapes, and functionalities, a variety of surface modified hyper comb-branched polymers were prepared and evaluated for gene transfection. Experiments were conrlnrt~-l in which the ability of hyper comb-branched polymers to transport carried genetic material was evaluated and compared to several known carriers, i.e. the controls, 10 SLalbul~L~ polymers, which are the subject of U.S. Patents 4,507,466 entitled DENSE
STAR POLYMER BRANCHES HAVING CORE, CORE BRANCHES, TERMINAL
GROUPS, 4,558,120 entitled DENSE STAR POLYMER, and 4,568,737 entitled DENSE
STAR POLYMERS AND DENDRIMERS, and Lipofect~min~.
In all experiment~, samples of carrier agent, i.e. hyper comb-branched polymers,15 Lipofect~min~TM, or Sl~lbul~L~ polymers, were complexed with genetic material. The hyper comb-branched polymers included -NH2, -NH-, 100% PEOX, 50% PEOX, and chloroethylamine modified hyper comb-br~nf~h.od polymers. The inventors also contemplate that a wide array of amino acids, could be ~tf~h~Qd to the periphery of the polymer, such as but not limited to lysine or arginine. The conjugated samples included a 20 large range of ratios of negatively charged genetic material to positively charged carrier agent, i.e. about 1:10 up to about 1:1000, respectively (ratios based upon the number of charge sites).
Testing also included the use of other known carrying agents to assist either the hyper comb-branched polymers of the present invention, or the control samples which 25 utilized S~all,ul~L~ polymers or Lipofect~min~. The m~linm utilized in all experiments was DMEM, Dulbecco's modified eagles meflillm Ratios of genetic material to hyper comb-branched polymer were based on the calculation of the electrostic charge present on each component; the number of phosphate groups in the nucleic acid ratioed to the number of charges on the carrier branches in one molecule of a hyper comb-branched 30 polymer.
The transfection efficiency, or genetic material carrying ability of the samples was qll~nti~led by measuring relative light units or RLU per 10 ~g protein. In order to quantity the transfection efficiency, a reporter gene (pCMV LUC) was complexed to the hyper comb-branched polymer and tr~n~fecte~l into the cells. The cells were lysed and the amount of light produced after addition of Luciferin~ was ~n~ntifi~d in a lllminnmeter. The experiments were performed on Cosl and Rat2 cell lines. The CMV-luceferase gene was utilized as a reporter gene. The total protein concentration in the cell 5 lysate was measured in a standard protein assay.
Due to the lower charge densities in the 100% PEOX and 50% PEOX modified hyper comb-branched samples, the binding between negatively charged DNA and positively charged hyper comb-branched polymers was very weak. Therefore, the transfection efficiency observed was the lowest for that system, as illustrated in Figs. 29 10 and 33. Referring to both figures, it can be seen that the measured RLU was barely tletect~ble for hyper comb-branched polymers (designated as HCB in the acc~ ally.llg figures) HCB Gl PEOX 100GO at charge ratios of 1:10 and 1:70; and HCB G1 50%
PEOX 50GO at charge ratios of 1:10; 1:100; and 1:200.
In contrast, when modified with primary amines, hyper comb-branched polymers 15 were found to Lldl~irtcl genes very efficiently. As shown in Figs. 29 and 30, the transfection efficiency of polyz~mic~o~min~ (PAMAM) modified hyper comb-branchedpolymer in Cosl cells was better than not only the SLdlbul~tG G5 and G10 ~le-mlrim~rs, but also Lipofecr~minf ~, which is recognized as a premier standard for gene transfection.
That is, the RLU measurements for HCB G3 (PAMAM) in Figs. 29 and 30 (including 20 conjugation ratios of 1:10, 1:100, 1:200, and 1:500), were ~ignific~ntly greater than the RLU measurement for the corresponding control samples presented in those figures.
Since S~dllJUl j~ en-lrim~-rs are formed by a series of reiterative or generational reactions, they are typically identifi.--l by the number of ~t;lleldlions to which they have been reacted, e.g. G5, G10, etc. Under current nom.-nrl~tllre, a Sldlbul~L~ ~len-lriml-r 25 core with a first set of branches ~tt~-'h~l thereto is referred to as a "zero geneldtion" or G0 ~len~lrimer~ Once the second set of branches it ~tt~rhecl to the first set of branches, it is a first generation or G1 SL~1bU1~LC dendrimer. S~dlbUl~i~oe) dendrimers are identified herein in accordance with this ~llel~ional nomenclahure scheme.
Much of the prior patent literahure involving SL~llbUl:jt~ dendrimers uses a 30 variation on this nrlmenf lz~nlre in which a core with a first set of branches em~n~ting th~l~rlolll is referred to as a first generation or G1 dendrimer, instead of a zero gellel~tion or G0 dendrimer. Thus, the same SLdllJul~it~ dendrimer will have a dirr.,l~llL
"G" number, depending upon whether the whole nomenclahure liLeldLul~; is followed, or ~2--WO 97/06833 PCT~US96/13080 whether the current nomenclature is utilized. The current nomenclature, in which the core and first set of branches are referred to a "G0" SLalbul~L~ ~~P.n-lriml r, is used herein.
In addition, it is even more remarkable that in one of the experiments, illustrated in Fig. 30, the highest transfection utili7ing the hyper comb-branched polymers was S obtained with Dulbecco's modified eagles m~inm (DMEM) alone without the help of "boosters" such as DEAE-dextran which is cytotoxic, and chloroquine, which blocks endosomal loc~li7~tion of the complexes and subsequent DNA degradation. This suggests that hyper comb-branched polymer polymers enh~nre transfection in certain cells by a~palclllly blocking endosomal uptake of complexed DNA; this is a unique char~ct~ri~tic of the hyper comb-branched polymer conjugated system not seen with SL~1bU1~L~
dendrimer mP~ tr(l transfection.
Hyper comb-branched polymers have another important advantage in transfection.
These polymers are minim~lly cytotoxic, at charge ratios of DNA:polymer up to 1: 100.
This is in contrast to randomly branched PEI polymers, which transfect cells but show marked toxicity at charge ratios as low as 1:50 for both the Rat2 and Cosl cell lines.
Figs. 32 and 36 show these results in comparison to the G5 and G10 SL~lbul~tC
dendrimers. Also by c~-mp~ri~on, the level of transfection efficiency is very low with respect to the SLdlbul~L~ dendrimer and therefore to the hyper comb-branched polymers of the present invention. This data demonstrates that it will not likely be feasible to utilize branched PEI polymers for gene transfection nor to safely deliver genetic material for therapeutic interest due to the relatively high cytotoxicity of the polymers.
Chloroethylamine modified hyper comb-branched polymers were also found to transfect genes reasonably well (Figs. 31 and 35). Terminal primary amine modified hyper comb-branched polymers could not transfect genes efficiently, even though their sizes are larger than that of G10 dendrimers. This may be the result of an inability of these polymers to bind either the DNA or negatively charged phospholipids on the surface of cells (thereby failing to trigger transfection). Secondary amine modified hyper comb-br~nrhr~l polymers did show some transfection. However, the overall solubility of these types of polymers in physiological condition was very low.
In Rat2 cell line, primary amine modified (i.e. PAMAM modified) hyper comb-branched polymer (molecular weight of about 29,000) in DMEM also showed much higher gene Lldl~re~;Lion efficiency than that of the Stalbul~L~ G5 and G10 with DMEM
alone. Referring to Figs. 33 and 34, the RLU measurements for HCB G3 (PAMAM) at WO 97/06833 PCTAUS96/1308~

charge ratios of 1:10, 1:100, 1:200, 1:500, all in DMEM, were ~ignifi~ntly higher than any of the control SL~.bU1~l~ samples in DMEM.
Example 18:
Hyper Comb-Br~nrhe~l Polymers as Gene Transfer Carriers A hyper comb-branched polymer diluting buffer cont~ining 20 mM HEPES (pH
7.9), 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 20% (v/v) glycerol, was formed.
A binding buffer including 10 mM EDTA, 40% (v/v) glycerol, 50 mM DTT, 100 mM
TRIS HCl (pH 7.5), and 1000 mM NaCl was formed. To a sterile EppendorfrM tube cont7~ining an a~,yl~liate volume of binding buffer was added ap~lop.iate amounts of hyper comb-branched polymers followed by CMV-Luc plasmid DNA at the desired ratios (i.e. from 1:1 to 1:1000). The complex spontaneously formed within about 15 ,.,i,lll~e~.
The complex was applied to the cells at the conditions such as with Dulbecco's modified eagles m~ lm (DMEM) alone, or with DEAE-dextran, or with chloroquine, or with both DEAE-dextran and chloroquine. The transfection efficiency was ~l~t~rmin~.rl by luciferase assay. The assay reagent contained 2 mM TRIS glycine, 10.7 mM (MgCO3) Mg(OH)2 5H20, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT, 270 ,uM Coenzyme A, 40 ,uM Luciferin~, 530 ~M ATP, at a total solution pH of 7.8.
Hyper comb-branched polymers with different surfaces (i.e. PEOX, PEI, 50%
PEOX, -NH2, etc.) and sizes (i.e. from 3 to 50 mn) were utilized in the gene transfer experiments. The transfection experiment was performed on dirre~e~lt cell lines. The optimized transfection results were observed with primary amine modified hyper comb-branched polymers in both Rat2 and Cosl cell lines. Among the primary amine modified hyper comb-branched polymers, the PAMAM modified hyper comb-branched polymers gave the best result, particularly in the Cosl cell line, where efficient transfection efficiency was obtained without the requirement for additional agents.

Con~l~t~s And Other Vehicles The present invention is also directed to hyper comb-branched polymer conjugate compositions in which the conjugates are form~ te~1 with other suitable vehicles. The hyper comb-branched polymer conjugate compositions may optionally contain other active ingredients, additives and/or diluents. Injectable compositions of the present invention may be either in suspension or solution form. In solution form the complex is dissolved in a physiologically acceptable carrier. Such carriers comprise a suitable solvent, WO 97/06833 PCTrUS96/13080 preservatives such as benzyl alcohol, if needed, and buffers. Useful solvents include, for example, water, aqueous alcohols, glycols, and phosphonate or carbonate esters. The hyper comb-branched polymer drug conjugate also could be incorporated in vesicles or -liposomes. Also the conjugate could be encapsulated into a polymeric host system that 5 could either be degradable (i.e., lactic-glycolic acid copooymers or a polyanhydride polymer) or nondegradable (ethylene-vinylacetate copolymer). Also the conjugate could be incorporated into a hydrogel matrix comprising either poly(hydroxylethylmt-fh~crylate) or poly(vinylalcohol). A variety of enteric coating systems could be employed to help the hyper comb-branched polymer drug conjugate pass through the stomach.
The hyper comb-branched polymer drug conjugate could be form~ te~l into a tablet using binders known to those skilled in the art. Such dosage forms are described in Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publishing C~ .ally, Easton, Pennsylvania. Suitable tablets include compressed tablets, sugar coated tablets, film-coated tablets, enteric-coated tablets, multiple compressed tablets, controlled-release tablets, and the like.
Enteric-coated tablets are particularly advantageous in the practice of the present invention. Enteric co~tingc are those which remain intact in the stomach, but will dissolve and release the contents of the dosage form once it reached the small int~stin~
The purpose of an enteric coating is to delay the release of drugs which are inactivated by the stomach contents, or may cause nausea or bleeding by irritating the gastric mllcQc~, In addition, such coatings can be used to give a simple repeat-action effect where additional drug that has been applied over the enteric coat is released in the stomach, while the remainder, being protected by the coating, is released further down the gastroint~stin~l tract.
Useful polymers for the preparation of enteric coated tablets includes celluloseacetate phth~l~te, polyvinyl acetate phth~l~t~, hydro~y~ pyl methylcellulose phth~l~t~
methacrylic acid ester copolymers and the like.
In the agricultural materials embodiment of the invention, the hyper comb-branched polymer conjugates can be formnl~t~ with suitable vehicles useful in agriculture such as in treatment of crops or fallow land, or as pesticides, or in treatment of in vivo or in vitro testing of ~nim~l.c. An agriculturally acceptable carrier or diluent which may also be present with one or more hyper comb-branched polymer conjugates of the present invention includes those carriers or diluents customarily used in granular W O 97/06833 PCT~US96/13080 formulations, emnl~ifi:lble concentrates, solutions, or suspensions such as, for example, toluene, xylene, benzene, phenol, water, mf th~n~, hydrocarbons, n~phth~lene and the like.
~ml~les Rel~in~ To Pro~ in~ Hyper Comb-branched Polymers 5 ~Y~.nrl~ A
A 250 ml one-necked round-bottomed flask equipped with a m~gn~ti~ stirring bar and a Dean-Stark trap that was surmounted with a reflux condenser was charged with 2.84 gm (15.3 mmole) of methyl tosylate and 125 ml of toluene. The lllixlure was heated at reflux and solvent was collected until all water had been removed. At this time, 30.0 10 gm (303 mmoles) of freshly distilled 2-ethyl-2-oxazoline was added all at once and the mixture was refluxed for approximately 4 hours. During this time, in a separate flask, 1.64 gm (38.1 mmole of repeat units) of linear poly(ethyleneimine) (LPEI) was azeotropically dried with toluene. When the poly(ethyl~llehllille) was dry it was added to the round-bottomed flask cont~inin~ the oxazoline oligomer and then allowed to reflux for 15 an additional 3 hours. Any ungrafted living poly(2-ethyl-2-oxazoline) chains were neutralized by the addition of 20 ml of water with l~nu~ing for an additional 1 hour.
Toluene was removed under reduced pressure to leave a yellowish oily solid that was dissolved in chloroform and precipitated dropwise into diethyl ether. The yellow solid was filtered from solution and dried overnight in a vacuum oven to yield 29.7 gm (94%
20 yield) of grafted poly(2-ethyl-2-oxazoline) (PEOX) as a yellow powder.
Example B
Into a 500 ml one-necked round-bottomed flask was placed 21.6 gm of the PEOX
from Example A and 350 ml of water. When the polymer had completely dissolved, 35 ml of concentr~tt-~l sulfuric acid was added. The flask was equipped with a fli~till~tion 25 head and the mixture was heated at reflux and t1ictill~t~ was collected until propionic acid could not be (letect~d Water was added to the 11istilling pot when the volume was reduced to less than approximately 75 ml. Upon removal of the propionic acid thetill~tion head was replaced with a reflux condenser surmounted with a pressure equalized addition funnel charged with SN NaOH. The base was slowly dripped into the 30 reaction mixture m~int~in.ofl at reflux. When the pH of the reaction mixture was approximately 12, heating was discontinued. While st~n-ling at room t~ dLul~ a solid formed at the surface of the aqueous mixture. This solid was removed and placed in a 250 ml round-bottomed flask with 175 ml of toluene. The water was removed from the WO 97/06833 PCTrUS96tl3080 water-toluene azeotrope by t1i.~till~tion. When water removal was complete, the solid became soluble in the lellw~ g toluene. The hot toluene solution was poured into a 250 ml round-bottomed flask leaving behind insoluble salts. Toluene was removed under reduced pressure to leave a brownish, waxy solid. The sample was dried for 5 approximately 24 hours under vacuum to give 9.14 gm (97% yield) of polymer sample.
~,Y~rl~ C
Using the general method of Example B, hydrolysis of the graft polymers, was carried out on a separate batch of the graft polymers in the following manner. Five grams (5.0 gm) of the graft copolymer were placed in a 250 ml round-bottomed flask 10 with 100 ml of water and 10 gm of sulfuric acid. The flask was heated with a heating mantle to give a slow ~lictill~tion of the propionic acid/water azeotrope. The ~ till~tion was continued for 2 days, with water being added as nl~cess~ry to m~int~in the reaction volume. Approximately 200 ml of (1i~till~t.- was collected over the course of the hydrolysis. The heating was discontinued and 50% NaOH was added slowly to bring the 15 pH to 10. The free polyamine was insoluble in the saturated salt solution, giving a ~aldt~ phase on top of the aqueous solution. The phases were separated and the polyamine was placed in a 250 ml round-bottomed flask. One hundred fifty ml of toluene was added and a Dean-Stark trap was ~tt~htoc~. After reflux overnight (about 16 hours), no more water was being removed and the polyamine had dissolved in the hot toluene.
20 The hot solution was filtered and the solvent was removed from the filtrate using vacuum and agitation to give branched poly(ethyleneimine) weighing 2.2 gm (100% of theory) as an orange oil. The l3C-NMR spectrum showed a peak for linear poly(ethyleneimine)(49.4 ppm/intensity 8075), residual unhydrolyzed propionamide (9.5 ppm/intensity 156), (26.3 ppm/hlLellsily 180), and primary amine end group (41.7 ppm/hlL~ iLy 61). No 25 peak for a hydroxy terminal group was observed. While the intensities may not be hlL~ d as a qll~ntitzltive measure of the groups present, qualitatively, hydrolysis was 80% to 90% complete and grafting was complete within the limits of detection.
FY~rl~ D
A 2-liter, 3-necked, round-bottomed, glass flask was used with a shaft driven r 30 stirrer, instead of m~gn~ti~ stirring. The initial loading was: water - 250 ml, material prepared e~sçnti~lly by the method of example 3 - 125 gm, sulfuric acid - 150 gm.
Additional sulfuric acid, 100 gm was added halfway through the hydrolysis to improve solubility. Internal flask temperature was monitored and a solenoid valve was rigged to add water whenever the Le~ el~Lulc~ rose above 107~C. Thus, constant attention was not nf~ce~ ry and the ~ till~ti~n could be left unattended overnight. The heating mantile was also set to shut off at the same temperature so that the flask would not overheat if the water reservoir ran out of water. After 2 days of continuous tli~till~tion, 1.6 liters of 5 ~ till~te was collected. The reaction llPL~I.LUlC~ was neutralized and the polymer phase was se~aldlt:d. The crude polymer was purified by dissolving in hot water (1 liter) and precipitated by slow addition to cold water. After two precipitations, the ~u~ellla~llL
solution was neutral to Hydrion~ paper. The reslllting hydrated polymer was dehydrated via toluene azeotrope as described above to give LPEI (51 gm 94% yield). The l3C-NMR
10 spectrum showed LPEI with residual amide carbon illL~:llsilies 0.5% of the LPEI illLel~iLy.
Primary amine end group hlLel~iLy was 0.4% of the LPEI inLel~iLy.
Example E:
Into a 250 ml round-bottomed glass flask was placed p-toll-en~s -lfonic acid monohydrate (2.0 gm, 11 mmole) and toluene (100 ml). A Dean-Stark trap was ~tt~ch 15 and the llli~Lulc~ was heated at reflux until water removal was complete. Ethyl oxazoline (10 gm, 100 mmole) was added all at once and the reflux was continued for 2 hours.
LPEI (1.0 gm, 23 meq.) was placed in toluene (25 ml) and the mixture was heated to boiling to dissolve the polymer and azeotropically remove trace water in the polymer.
The hot LPEI solution was added all at once, to the cloudy oligomer suspension. An 20 orange oil began to precipitate imm~ tely. After 1 hour at reflux, the lll~Lulc: was cooled and the solvent stripped using vacuum. The residue was dissolved in CH2 C12 (40 ml) and precipitated by a slow addition to ether (500 ml). The solid was collected by filtration and dried in a vacuum oven at 40~C to 50~C to give the grafted polymer (12 gm, 92% yield) as a yellow powder. At higher M/I ratios, the oligomerization time had 25 to be increased to allow complete conversion of the ethyl oxazoline. For example, interm.o~ te(l degree of polymerization runs (M/I = 200, olig. time = 3 hours. or M/I
= 400, olig. time = 6 hours) had low yields due to incomplete conversion. Increasing the reaction time to 12 hours and 24 hours respectively, gave higher conversions and yields. The highest M/I (1000) run, had an oligomerization time of 36 hours, which was 30 not long enough for complete conversion. This gave a material with actual oligomer dp of 700. The l3C-NMR spectrum of the poly-branched polymer derived from this material showed a peak for primary amine end groups which was approaching the limits of detection for the signal/noise ratio. No hydroxyl termin~l group was rletect~hle.

WO 97/06833 PCT~US96/13080 l~,Y~Inrl~ F I~alion of Morph~lin~ Termin~t~-l Linear Polyethyl~n~imin~
Having a Degree of Poly~ lion (dp) of 20 A mixture of methyl tosylate (7.46 g, 40 mmol) in 200 ml of toluene wasS azeotroped to dryness with a Dean-Stark trap for about 10 to 15 mimlt~s. To this ~ Lu which had cooled to about 90~C was added ethyl oxazoline (79.3 g, 800 mmol) and the mixture was refluxed for 18 hours. After this time, morpholine (14g, 161 mmol) was added. This mixture was refluxed for 16 hours. This mixture was evaporated of volatiles on a rotary evaporator. This crude mixture was hydrolyzed with 400 ml of 50% H2SO4 10 by azeotroping the water-propionic acid mixture with a Dean-Stark trap until about 500 ml were collected or until the pH of the ~1ictill~t~ was neutral. This hot l~ Lul~ was slowly poured into a 50% KOH llli~Lul~ under an atmosphere of N2. The reslll~ingheterogenous llli~Lul~ was made homogeneous by heating to reflux. The product floated to the top of this mixture as a clear liquid. This hot mixture was allowed to cool under 15 N2 to room temperature. The solid cake that formed on the surface of this mixture was dissolved in 600 ml of deionized water by heating to reflux, allowed to cool andultracentrifuge (8000 rpm) for 10 mimltec. The clear liquid was fl~c~nt~l and the rem~inin~ white solid-water llli~Lul~ was mixed with toluene. This llli~Lule wasazeotroped of water to form a dry toluene-LPEI mixture. The toluene was removed from 20 this mixture by a rotary evaporator followed by high vacuum (0.2 mm Hg) at 80~C for 2 hours to give 34 g (88% yield) of the title compound.
~npl~? G Preparation of Comb-Branched PEI Wherein Nc is 20, Nb is 5 and G is 0 A mixture of methyl tosylate (MeTOs) (3.7 g 20 mmol) in 50 ml of toluene was azeotroped to dryness with a Dean-Stark trap under nitrogen for 10 ~ S. To this ixLul~ cooled to 90~C was added ethyl oxazoline (10 g, 100 mmol). This mixture was stirred for 10 hours at 90~C. To this llli~Lulc: was added N-morpholine termin~t~o-l LPEI
(dp of 20) (0.53 g, 0.55 mmol, 11 mmol NH) dissolved in 20 ml of hot (90~C) toluene which had been dried by azeotropic ~lictill~rion for about 15 minutes. This was imm~ tely followed by the addition of diisopropylethylamine (12 g, 93 mmol, 8 equivalents of amine per NH). This mixture was refluxed for 48 hours. The volatiles were removed from this mixture and the resulting residue dissolved in deionized water.
After ultrafiltration (MW > 1000), the retentate was refluxed in 400 ml of 50% H2SO4 for 18 hours. The cooled reaction mixture was made basic to a pH ~ 14 with KOH to CA 02226299 1998-02-lO
WO 97/06833 PCTrUS96/13080 produce a clear colorless liquid that floated to the top of the mixture. Upon cooling the liquid solidified. The solid was removed from the mixture and dissolved in 500 ml of hot deionized water. This mixture was allowed to cool forming a white suspension. This resllltin~ mixture was ultracentrifuge at about 8000 rpm for about 10 mimltes. The clear S liquid was ~lec~nt~d from the white precipitate. The white precipitate was refluxed with toluene with an ~tt~h~-l Dean-Stark trap to dry the product. The toluene ~ Luie was evaporated of volatiles on a rotary evaporator. The rem~ining volatiles were removed at 0.1 mm Hg at 50~C to give 1.8 g (70%) of the title compound. A l3C-NMR spectrum of this mixture in CDC13 in-lic~tt-A at 65% grafting of PEOX onto LPEI as shown by 10 integration of the termin:ll methyl signals versus the methylene carbon signals.
Example H F~l,alalion of a hyper comb-branched polymer PEI Wherein Nc is 20, Nh is S and G is 1 The compound dendrimer was prepared in the same manner as in Example G
15 using MeOTs (3.7 g, 20 mmol), 300 ml of toluene, ethyl oxazoline, (10 g, 100 mmol), diis~ opylethylamine (12 g, 93 mmol) and comb-branched PEI where Nc is 20, Nb is 10 and G is zero (1.0 g, 23 mmol NH m~ximllm). Ultrafiltration, hydrolysis and drying gave 5.0 g (80% yield) of the title compound. The l3C-NMR spectrum was consistent with the proposed structure.
20 ~Y~npl~ I Preparation of a hyper comb-branched polymer PEI Wherein Nc is 20, Nb is 5 and G is 2 This example shows the use of the material formed in Example H which was refluxed with two equivalents of PEOX having a dp of 5 per NH and 11 equivalents of 25 diisopropylethylamine for two days. This mixture was worked up dirr~cllLly than the previous example. The crude PEOX-hyper comb-branched polymer PEI was hydrolyzed directly, without ultrafiltration, to give 8.6 g of a hyper comb-branched polymer and linear Polyethyleneimine (PEI). This mixture was dissolved in hot deionized water.
Upon cooling the product cryst~lli7~1 from the mixture. The mixture was 30 ultracentrifuged at 8000 rpm and the white precipitate was azeotropically dried with toluene to give 4.5 g for a yield of 72%.

WO 97/06833 PCT~US96/13080 Example J Preparation of a hyper comb-br~n~h~-l polymer PEI Wherein Nc is 20, Nb is 5 and G is 3 The plc~ lion of hyper comb-branched polymer PEI where G is 3 incorporated S illlplov~lllents in the grafting step by using two equivalents of PEOX per NH and 26 mmols of diisopropylethylamine per NH. The crude material was hydrolyzed as before and the resulting mixture precipitated from PEI by making basic with KOH.
Recryst~11i7z-tion of the cake of product floating on the KOH mixture from deionized water followed by ultracentrifugation at 8000 rpm and azeotropic drying of the white 10 solid with toluene gave 5.6 g for a 90% yield.
Example K Preparation of a hyper comb-br~ he-l polymer PEI Wherein Nc is 20, Nb is 5 and G is 4 The hyper comb-branched polymer PEI was prepared in a manner similar to the 15 previous example, using two equivalents of PEOX per NH, and 23 equivalents ofdiisopropylethylamine per NH and l~nu~illg two days. The crude mixture was not ultrafiltered by hydrolyzed with H2SO4, removed from solution by KOH and recrystallized twice from deionized water. Each recryst~lli7~tion involved dissolving the product in hot water, allowing the llli~ Lul~ to cool to 25~C and ultracentrifugation at 8000 rmp 10 20 minutes. The clear supt;l~ ll was ~l~c~ntt-d from the white solid and the white solid was azeotropically dried with toluene. The isolated yield from the second recryst~11i7~tion came to 4.9 g for a yield of 78%. The first recryst~11i7~tion gave 5.5 g, for a yield of 87%.
~Y~mp'e L Preparation of a hyper comb-branched polymer PEI Wherein Nc is 20, Nb is 5 and G is 5 The next generation (G) was prepared at twice the scale of all the other grafting clilllents (2.0 g starting material versus l.0 g of starting material). Only 4 to 5 equivalents of diisopropylethylamine per NH were used along with two equivalents of 30 PEOX per NH and lc:~lu~illg 2 days. After evaporating the volatiles the crude mixture was dissolved in deionized water and ultrafiltered using a spiral wound cartridge Amicon SlY3 (3000 MWCO). Hydrolysis of the retentate gave an 85% yield of the title compound. The ultrafiltration with this membrane was not tried on earlier generations of G = lto4.

W O 97/06833 PCT~US96/13080 F.Yqmp'~ M I~~,ardLion of a hyper comb-branched polymer PEI Wherein Nc is 20, Nb is 5 and G is 6 This generation was prepared in a similar manner as before using two equivalents5 of PEOX per NH, six equivalents of diisopropylethylamine per NH and lenu~h~g 2 days.
The workup again was done by ultrafiltration in deionized water using a spiral wound SlY3 membrane. The isolated yield of PEOX-hyper comb-br~nrh~-~l polymer after ultrafiltration came to one-half the amount normally obtained from an 80% to 90%grafting experiment. Hydrolysis of the mixture as before followed by tre~tm~nt with 10 NaOH and azeotropic drying with toluene gave only a 32% yield of the G = 6 product.
A repeat of this sarne experiment except with two recryst~lli7~tions in water instead of an ultrafiltration gave a 38% yield of the title compound.
~,Y~-np'~ N Preparation of a hyper comb-branched polymer PEI Wherein Nc is 20, Nb is 10 and G is 0 A lllixLule of MeOTs (7.4 g, 40 mmol) in 100 ml of toluene was azeotroped to dryness with a Dean-Stark trap under nitrogen for 10 to 15 mimltes. To this l~ Lu~c, cooled to about 90~C, was added ethyl oxazoline (39.7 g, 400 mmol). This mixture was refluxed under nitrogen for 18 hours. To this llli~Lulc~ was added N-morpholine 20 termin~te~l LPEI having a dp of 20 (1.0 g, 1.1 mmol, 23 mmol of NH) dissolved in 50 ml of hot (100~C) toluene which had been dried by azeotropic (1i~till~tion for 15 minutes.
This was imm~ t~ly followed by the addition of diisopropylethylamine (24 g, 186 mmol, 8 equivalents amine per NH). This mixture was refluxed for 48 hours. The mixture was cooled, dissolved in m.ofh~nol and evaporated of volatiles on a rotary 25 evaporator and the resllltingllliYlul~ was dissolved in deionized water (about 60 ml).
This mi~Lule was ultrafiltered using an Amicon spiral wound cartridge SlY3 with the above volume as a retentate until 12 liters of permeate had been obtained (20 recirculations). This l~ was refluxed in 400 ml of 50% H2SO4 with a Dean-Stark trap collecting about 400 to 500 ml of distillate (replenishing the equivalent water) until 30 the ~ till~r~- was neutral to pH paper. This hot mixture was made basic by pouring slowly into a 50% KOH mixture under a blanket of nitrogen. The heterogenous mixture was heated to a homogeneous mixture that produces a liquid that floats to the top of the mixture. Upon cooling the liquid solidified. The solid was removed from the mixture and dissolved in 500 ml of hot deionized water. This ll~ Lule was allowed to cool 35 forming a white suspension. This resulting lllix.Lulc~ was ultracentrifuged at 8000 rpm for WO 97/06833 PCTrUS96/13080 about 10 mimlt(~s. The clear liquid was ~lec~ntt?~l from the white precipitate. The white precipitate was refluxed with toluene with an attached Dean-Stark trap to dry the product.
The toluene mixture was evaporated of volatiles on a rotary evaporator. The rem~inin~
volatiles were removed at 0.1 mm Hg at 50~C to give 1.8 g (70%) of the title compound.
S A l3C-NMR spectrum of this lllb~Lul~ in CDC13 in~ ted a 65% grafting of PEOX onto LPEI as shown by integration of the terminal methyl signals versus the methylene carbon slgnals.
F,Y~ ,1e O Preparation of a Comb-Branched PEI Wherein Nc is 20, Nb is 10 and G is 0 A mixture of morpholine-terrnin~tPd LPEI having a dp of 20 (1.04 g, 22 mmol), PEOX oligomers having a dp of 10 (47.5 g, 40 mmol) and diisopropylethylamine (20 g, 6 to 7 equivalents per NH) were refluxed under nitrogen obtained from a nitrogen cylinder (constant pressure and no flow) and a Hg bubbler for 48 hours. The volatiles were 15 removed from the mixture and the resl-lting yellow orange residue was dissolved in 1 liter of deionized water. The mixture was ultrafiltered with an Amicon spiral wound cartridge using 700 ml of leLe~ and 8.5 liters of permeate to give 24 grams of the PEOX-Comb-Branched PEI copolymer. The material was hydrolyzed with 50% H2SO4 and the reslllting~ Luie added to an excess of 50% KOH. The cake floating on the KOH was 20 mixed with toluene and azeotropically dried under nitrogen to give 10.1 g (90%) of the Comb-Branched PEI dendrimer.
e p ne~a dtion of a hyper comb-br~n~h~-l polymer PEI Wherein Nc is 20, Nb is 10 and G is 1 The pl~aldLion of G= 1 of this hyper comb-branched polymer PEI series was illrntir~l in all respects to the preparation of G=0. The isolated yield of the title compound from 1.1 g of G=0 Comb-Branched PEI was 10.5 g (84%). The l3C-NMR
system showed a little more of the carbinol signal at 60.1 ppm than before, plus a signal at 59.46 ppm.
~mp'e Q l'le~a.~ion of a hyper comb-branched polymer PEI Wherein Nc is 20, Nb is 10 and G is 2 This material was prepared as described in the previous preparations lltili7ing an Amicon SlY10 spiral wound ultrafiltration cartridge (10,000 MWCO) (600 ml l~lr~ r/g liters of permeate). From 1.1 g of hyper comb-branched polymer PEI wherein G = 1, there was obtained 10.8 g (86%) of the title product. The l3C-NMR spectrum inrlir:lted more of the signal at 60.1 ppm than at 59.67 ppm.
Example R Preparation of a hyper comb-branched polymer PEI Wherein Nc is 20, Nb is 10 and G is 3 S The material was plc~al~d as described before using an Amicon SlY10 spiral wound ultrafiltration cartridge (10,000 MWCO) and filtration volumes as described before. From 1.1 g (25 mmol NH) of hyper comb-branched polymer PEI dendrimer wherein Nc is 20, Nb is 10 and G is 2 there was obtained 10.3 g (82%) of the hyper comb-branched polymer PEI dendrimer wherein Nc is 20, N~, is 10, and G is 3. The 13C-NMR spectrum of the material again int1ir~te~1 carbinol signals at 60.1 ppm and 57 ppm.
Example S Preparation of a hyper comb-br~nrh~l polymer PEI Wherein Nc is 20, Nb is 10 and G is 4 This material was prepared as described above using an Amicon SlY10 spiral wound ultrafiltration cartridge with the volumes infli~terl above. From 1.1 g (25 mmol NH m~ximllm) of hyper comb-branched polymer PEI dendrimer wh~ l Nc is 20, Nb is 10 and G is 3, there was obtained 10.1 g of the title compound (80% yield).
Example T I~el,al ~lion of a hyper comb-br~n~ hefl polymer PEI Wherein Nc is 20, Nb is 10 and G is 5 The material was prepared as described above ntili7ing 1.1 g (25.5 mmol NH) of hyper comb-branched polymer PEI wherein Nc is 20, Nb is 10 and G is 4, 47.5 g (40 mmol) of PEOX oligomer, and 25 g (8 equivalents of amine per NH) of diisopropylethylamine. Workup as before using an Amicon SlY10 spiral wound cartridge (700 ml of lc~t~ , and 9 liters of perrn.?~te) gave 18 g of the PEOX-hyper comb-branched polymer copolymer. Hydrolysis with 50% H2SO4 and treatment with excess NaOH gave a cake of material that floated on the caustic mixture with a lot of trapped NaOH and sodium sulfate salts. The cake was heated in 300 ml of deionized water to boiling and allowed to cool giving a white precipitate. This mixture was ultracentrifuged at 8000 rpm for 10 mimlr~c and the res-llting clear liquid was poured from the settled white solid. This white solid was mixed with toluene and dried by azeotropic ~lictill~tion to give 7.0 g (56%) of the title compound.
E2~ 1c U ~l~aldlion of a hyper comb-branched polymer PEI Wherein Nc is 20, Nb is 20 and G is 0 A PEOX oligomer having a dp of 20 was prepared from MeOTs (7.5 g, 40 mmol) and ethyl oxazoline (80 g, 800 mmol) by l~nu~illg under tank nitrogen using a Hg -54-W O 97/06833 PCT~US96/13080 bubbler. The LPEI (0.5 g, 0.52 mmol, 10 mmol per NH) in hot toluene was added tothe PEOX oligomer followed by diisopropylethylaimine (74 g, 574 mmol, 29 mmol per NH). This mixture was refluxed for 72 hours. The volatiles were removed and the resulting residue was dissolved in deionized water. This mixture was ultrafiltered using a 5 SlY3 cartridge. Workup as before gave 9.8 g of a PEI product (theory 9.1 g). The 13C-NMR spectrum of this material in~ te~ a si~ni~ nt amount of a carbinol signal at 60.2 ppm.
li'Yq~nrle V Preparation of a Comb-Branched PEI Polymer Wherein Nc is 20, Nb is 20andGisO
In this experiment, two equivalents of PEOX oligomer per NH of the PEI and diisopropylethylamine (30 equivalents per NH of PEI) were refluxed for five days. A
very large stir bar was used to get more efficient stirring of the mixture than was obtained in the above experiment. The mixture was stripped of volatiles and the res lhing residue 15 dissolved in deionized water. Ultrafiltration of this mixture using the SlY3 spiral wound cartridge gave no separations as cle~terminPd by SEC. The SEC plot in~ t~orl two peaks.
Upon co-injection with ~llthPnti-~ PEOX oligomer having a dp of 20, one of the peaks was enh~n-e-l. The ultrafiltration was then carried out on a SlY10 (10,000 MWCO) spiral wound cartridge. The SEC plot of the l~t~llLaL~ was i~1enti~1 to the SlY3 cartridge 20 l~ t~"~
The ultrafiltration was switched to an Amicon flat stock stirred cell system using YM10 (10,000 MWCO) cartridge. After 1.5 liters of permeate only a small amount of the presumed PEOX oligomer having a dp of 20 had been ultrafiltered.
The material was then ultrafiltered with the flate stock stirred cell using a YM 30 25 membrane (30,000 MWCO) (100 ml, retentate; 2000 ml, permeate) to give a good separation by SEC. The lel~ al~ evaporated to 18 g (42%) of the PEOX-Comb-Branched PEI copolymer. This material hydrolyzed to 7.0 g (38%) of the Comb-Branched PEI. The l3C-NMR spectrum of the Comb-Branched PEI in~ t~-l only a minor amount (about 10%) of the carbinol signal at 60.1 ppm relative to the methyl 30 tt~rrnin~te signal at 36.5 ppm.
~n~pl~ W Preparation of a hyper comb-branched polymer PEI Polyrner Wherein Nc is 20, Nb is 20 and G is 1 This material was prepared with two equivalents of PEOX oligomer having a dp of 35 20 and renu~ g with diisopropylethylamine for three days. The reaction parameters WO 97/06833 PCTrUS96/13080 were to be held constant to permit a reasonable analysis of the cht-mi.ctry. An analysis of the crude reaction mixture by SEC at 48 hours, 72 hours and 96 hours in~ t.od a progressive increase in molecular weight. Ultrafiltration of the crude material in water with the Amicon flat stock stirred cell using a YM30 (30,000 MWCO) membrane as 5 before (100 ml, retentate; 2000 ml permeate) gave a 74% yield of the PEOX-hyper comb-branched polymer PEI copolymer. Hydrolysis and tre~tment with NaOH, recryst~lli7~tion from water, and azeotropic drying in toluene, gave a 68% yield of the title compound.
FY~mp~ X Preparation of a Comb-Branched PEI Polymer Wherein Nc is 20, Nb is 100 and G is 0 Further exploration of the PEOX chain length on the grafting efficiency was done.
A PEOX having a dp of 100 was prepared (24 hrs at reflux) and refluxed 65 hour with PEI (1 equivalent PEOX per NH) with 11 equivalents of diisopropylethylamine per NH.
The mixture was evaporated of volatiles, dissolved in deionized water and ultrafiltered 15 with an SlY30 (30,000 MWCO) cartridge. Hydrolysis of the reLell~dt~ and workup gave a 31% yield of a white amorphous powder. Hydrolysis of the permP~l~ gave a whitecrystalline material, LPEI having a dp of 100.
liY~mp~e Y r~Jiion of a Styrene Core Polymer The styrene core polymer precursor was prepared by polymerization of 20 g (192 20 mmol) of styrene in benzene (20 ml), initi~t.od by s-butyl lithium (4 mmol). After 4 hours, the reaction was te....i..~trd by addition of methanol (1 ml). Chloromethylation of the product polymer (10 g poly~,Lylclle, 60 ml chloromethyl methyl ether, and 1 ml stannic chloride in 500 ml of carbon tetrachloride for 48 hours) gave the chloromethylated core polymer.
25 FY~n~rl~ Z n~ dlion of a Comb-Branched Poly~ly~ e Wherein G is 0 Living polystyrene oligomer was generated by initiation of 20 g of styrene by 4 mmol of s-butyl lithillm, as in Example AA. After 4 hours at room t~ cldlul~, 6 mmol of diphenylethylene in 350 ml of tetrahydrofuran was added. The chloromethylatedpoly~ylelle core was added portionwise, over 30 mimltPs, until most of the orange color 30 of the carbanion had disappeared. After an additional 30 mimlttos, residual carbanions were termin~te-l by the addition of 1 ml of m~th~nnl. Evaporation of the solvent and ,.
fractionation in toluene/m~th~nol gave an 80% yield of the title compound.

WO 97/06833 PCT~US96/13080 Example AA I~ ~dlion of a hyper comb-br~n~hP-l polymer Poly~lyl~lle Polymer Wherein G is Equal to 1 The product of Example Z was chloromethylated as described in Example Y.
Grafting was carried out as described in Example Z, sllhstitllting the chloromethylated-comb-branched material for the linear-chloromethylated-polystyrene core.
Ex~l~lc BB ~udlion of a hyper comb-br~nrhP~l polyrner Poly~ly~lle Polymer Wherein G is 2 The product of Example AA was chloromethylated as described in Example Y.
Grafting was carried out as described in Example Z, sub~ uLhlg the chloromethylated-10 comb-burst material for the linear-chloromethylated-poly~lylelle core.
Example CC Preparation of Rod-Shaped hyper comb-br~nrhp~l polymer PEI
Wherein Nc is 200, Nb is 5 and G is 3 This material was prepared as described above using N-morpholine terrnin~t~d PEIas an initiator core. Repeated grafting (4 times) with methyl tosylate (3.7 g, 20 mmol) 15 and ethyl oxazoline (10 g, 100 mmol) in 100 ml of toluene, followed by hydrolysis with 150 ml of 50% H2SO4 gave the dendrimers in a 70% to 80% yields. These products were characterized by l3C-NMR spectroscopy, titration and electrophoresis and shown to be the titled material.
~Y~nrhP DD Preparation of Spherically-Shaped hyper comb-br~n~hP-l polymer PEI
Wherein Nc is 10, Nb is 100 and G is 3 This material was prepared in the same manner as the rod-shaped dendrimer using LPEI (dp of 10) as an initiator core. The branches were constructed with PEOX (dp of 100), initi~te~l as shown in the examples above.
25 li ~mrl~. EE Synthesis of Ring Core Hyper Comb-Branched Polymers AZACROWNTM (1,4,7,10-tetraazacyclododecane, cyclen) was obtained from The Dow Chrtnir~l Company, and was further recryst~lli7~1 from toluene. The purifiedAZACROWN~ is a white needle-like crystal.
A mixture of methyl tosylate (MeOTs)(0.922 g, 4.95 mmol) in 100 ml of toluene 30 was azeotroped to remove water with a ~lict~ tion head under Ar for 10 mim-trs. After cooling to ~90~C, 2-ethyloxazoline (10 ml, 99.06 mmol) was c~nn~ to~l in and themixture was allowed to reflux for 5 hours. To this mixture was added AZACROWNTM
core (0.214 g, 4.95 mmol of NH), which was dried by azeotropic ~ till~tion from toluene, followed by immediate addition of diiso~ ylethylamine (i-Pr2NEt)(2-4 eq.).

WO 97/06833 PCT~US96/13080 The mixture was refluxed for 1 hour, cooled, and then dissolved in methanol (~ 100%
grafting yield as fiPt~ . ,"i~Prl by SEC). After rotary-evaporation of the solvents, the crude product was either purified by ultrafiltration with Amicon spiral wound cartridges SlY3 (3000 MWCO), or fractionated by mPth~nol/diethyl ether mixture to remove the 5 unreacted monomers, oligomers, and catalysts. The entire separation process was monitored by size exclusion chromatography (SEC). The purified product was rotary-evaporated and Iyophilized to give a ring-branched polyethyloxazoline-polyethyleneimine (PEOX-PEI) polymer as a white powder. The higher generations of the ring core comb-burst polymers can be prepared in a similar manner as described in the linear core case as 10 described above. All the products were analyzed by size exclusion chromatography (SEC), capillary electrophoresis (CE), nuclear m~gnPtic resonance (NMR), and ele~;LLu~7~ldy mass spectroscopy (ES-MS).
l~Y~-nrl~ FF Synthesis of Hyper-Terminally Branched Core Hyper Comb-Br~n~h~
Polymers A mixture of MeOTs (0.39 g, 1.98 mmol) in 100 ml of toluene was azeotroped to remove water with a ~ till~tion head under Ar for 10 mimltl~s. After cooling to ~90~C, 2-ethyloxazoline (10 ml, 99.06 rnmol) was c~nn--l~tP~l in and the llli~LUlc~ was allowed to reflux for 5 hours. To this mixture was added a hyper-branched polyethylene amine core 20 (0.214 g, 4.95 mmol of NH), which was dried by azeotropic ~ tills~tit~n from toluene, followed by immP~ tP. addition of i-Pr2NEt (large excess). The mixture was refluxed for 3 hours, cooled, and the top toluene solution was ~lec~nt~ off. The rem~inin~ viscous oil was redissolved in a small amount of MeOH and ~ ci~ ted out in diethyl ether(Et20). After the top Et20 solution was ~lec~ntPcl~ the bottom precipitate was redissolved 25 in mPth~nol (MeOH) and dried over rotary evaporator and high vacuum to give a light yellow polyethyloxazoline-polyethylenP~minP (PEOX-PEA) polymer. The higher generations of the hyper-branched core comb-burst polymers can be prepared in a similar manner as described in the linear core case described above. All the products were analyzed by SEC and NMR.
30 I~ nt~l for ~mp'~ EE and FF
SEC measurements were performed on a series of Beckman TSK 4000 PW (or POLY-OH, Polymer Laboratory), 3000 PW, and 2000 PW columns using Waters 510 HPLC pump, Thermo Separation Products AS 3000 Allto~"mI-ler, Wyatt DAWN DSP-F
Multi Angle Laser Light Scattering Detector, and Wyatt illLelrel~,llleter refr~ct-mPtPr W097/06833 PCT~US96t13080 (Optilab 903). IH and l3C-NMR spectra were obtained on Brucker 360 MHz or VarianUnity 300 MHz NMR spectrometer using either CDC13 or MeOD as solvents. Purity ofmonomers was checked by GC (HP 5890, He as carrier gas~. Ultrafiltration was achieved using either an Amicon 3,000 or 10,000 molecular weight cutoff (MWCO) 5 membrane. CE was peRormed on Beckman P/ACE System 2050 (Software System Gold). The polymer MWs were also measured by ES-MS (Finnigan Mat TSQ 700).
F,Y~ GG Preparation of Poly (2-Ethyloxazoline) and Polyethylf~n~imin~
Linear Polymers (DP=10, 20, 50, 100, and 200) Methyl p-toll~en~slllfonate, 2-ethyloxazoline, morpholine and diisopropylethylamine were purchased from Aldrich. Methyl p-tolnPnt-s llfonate was purified by ~lictill~tion~
while 2-ethyloxazoline, morpholine, diisopropylethylamine and toluene were stirred over CaH2 and ~ tilled prior to use. All the reactions were peRormed under an ultra pure Ar atmosphere.
The synthesis of poly(2-ethyloxazoline), (PEOX20, DP=20) is described to illustrate the general procedure for the preparation of linear PEOX. To a 250 ml two-neck round- bottom flask was added methyl p-toluenesulfonate (7.45 g, 40 mmol) and dry toluene (150 ml). A ~li.ctill~tion head (vacuum type) was ~tt~ .o(l and trace amounts of water in the ~ L~ were removed by azeotroping with toluene for 10-15 mimltes. After~0 cooling to about 90~C, ethyloxazoline (80.8 ml, 800 mmol) was r~nn~ t~--l in, and the Lu~ was allowed to reflux for 10 hours before termination with excess morpholine.
During the polymerization, a cloudy PEOX suspension was formed. After the lr..,,i,,~lion with morpholine, the solution became clear again. The crude ",i~Lul~ was rotary-evaporated and then hydrolyzed with 500 ml of 50% HzSO4~ followed by 25 azeotroping the water-propionic acid mixture with a Dean-Stark trap until the pH of the .li.ctill~t~ was neutral. This hot acidic solution was slowly added (with a separatory funnel) into a 50% NaOH solution cooled by an ice bath. This solution (pH 2 11) was heated to boil under N2, and the product (linear PEI) floated on top as an oily layer.
After cooling to room temperature, the top layer became a solid cake on the suRace 30 which was subsequently removed and redissolved in 600 ml deionized, boiling water.
After slow sedimentation overnight, the white precipitate was filtered by suction funnel.
In order to completely remove excess NaOH, cold water was used to exh~nctively wash the precipitate until pH of the filtrate solution was neutral. Pure polymer was obtained by azeotropic removal of water from a toluene solution of the polymer, followed WO 97/06833 PCT~US96/13080 by a gravity filtration and then rotary evaporating the toluene at 60~C. Such polymer was further dried under high vacuum overnight (33 g, 85.8% yield, MW=1,130, MWD=1.05). Linear PEOX and PEI 10, 50, 100, and 200 were ple~alcd in a similar manner (PEOX yield 2 90%, PEI yield 2 80%). All the polymers were analyzed by 5 SEC, ES-MS, NMR, CE, and PAGE.
~mrl~ HH Synthesis of Comb-Branched Poly-mers (GO) The synthesis of PEOX10-g-PEI20 is provided as a general procedure for the pl~al~lion of Comb-Branched PEOX-PEI and PEI polymers. A mixture of MeOTs (7.38 g, 39.62 mmol) in 150 ml of toluene was azeotroped to remove water with a 10 (1i~till~tion head under Ar for 10 minutes. After cooling to about 90~C, 2-ethyloxazoline (40 ml, 396.24 mmol) was c~nmll~t~-l in and the mixture was allowed to reflux for 5 hours. To this llliX.~UlC~ was added morpholine termin~ted LPEI 20 (1.90 g, 39.62 mmol of NH), which was dried by azeotropic ~ till~tion from toluene, followed by immP~ te addition of i-Pr2NEt (1 to 2 eq.). The mixture was refluxed for 1 hour, cooled, and then 15 dissolved in m~th~nnl (about 75% grafting yield as lleterminPfl by SEC). After rotary-evaporation of the solvents, the crude product was either purified by ultrafiltration with Amicon spiral wound cartridges SlY3 (3,000 MWCO), or fractionated by m~thzln~l/diethyl ether mixture to remove the unreacted monomers, oligomers, andcatalysts. The entire separation process was monitored by SEC. The purified product 20 was rotary-evaporated and lyophilized to give a comb-branched PEOX-PEI polymers as a white powder. This white powder was further hydrolyzed in 50% H2SO4 at 100~C andpurified as described before to provide a PEI comb-branched polymer as a white viscous oil (MW=2,500, MWD=1.22). Comb-branched PEOX20-g-PEI20, PEOX10-g-PEI50, PEOX50-g-PEI20, PEOX100-g-PEI50, PEOX200-g-PEI50, and PEOX20-g-1,4,7,10-25 Tetraazacylododecane) (PEOX20-g-AZACROWNT~ were also prepared in a similar manner. All the products were analyzed by SEC-multi angle laser light scattering, CE, NMR, ES-MS and PAGE.
liY~np'e II Sy--Ll-e~is of Comb-Branched Polymers (G1) A mixture of MeOTs (0.738 g, 3.962 mmol) in 150 ml of toluene was azeotroped 30 to dryness with a ~ till~tion head under Ar for 10 mimltes. After cooling to about 90~C, 2-ethyloxazoline (40 ml, 396.24 mmol) was c~nmll~re~l in and the mixture was allowed to reflux for 10 hours. To this ~ lul~ was added Comb-branched PEI (0.209 g, about 3.962 mmol of NH) dried by azeotropic ~i~till~tion from toluene, followed by imm.o~ t~

WO 97/06833 PCTrUS96/13080 addition of i-Pr2NEt (1-2 eq.). The ~ lul~ was refluxed for 1 hour, and then cooled to room temperature. The top toluene solution was ~1ec~nt~-1 off and the bottom polymer product was redissolved in methanol. This crude product was purified by refractionation with a m~th~nol/diethyl ether mixture to remove the unreacted monomers, oligomers, and catalysts. The entire separation was monitored by SEC. The purified product was rotary-evaporated and lyophilized to give the Comb-branched PEOX-PEI polymer as a white powder (MW=260,000, MWD= 1.10). The grafting yield depends on the length of the side chains (normally around 40%-80% as determined by SEC). Shorter side chains give a higher grafting yield. This white powder was further hydrolyzed in 50%
10 H2SO4 at 100~C and purified as described before to provide a PEI Comb-branched polymer as a white solid (80% yield, MW=138,800, MWD=1.34).
Example JJ Synthesis of Comb-branched (G2) A mixture of MeOTs (0.738 g, 3.962 mmol) in 150 ml of toluene was azeotroped to dryness with a ~ till~tion head under Ar for 10 minutes. After cooling to about 90~C, 15 2-ethyloxazoline (40 ml, 396.24 mrnol) was c~nmll~f~c~ in and the mixture was allowed to reflux for 10 hours. To this mixture was added Comb-branched PEI polymer (0.200 g, about 3.962 mmol of NH) dried by azeotropic ~ till~tion from toluene, followed by immPrli~te addition of i-Pr2NEt (1-2 eq.). The mixture was refluxed for 1 hour, and then cooled to room temperature. The top toluene solution was ~l( ç~nttol1 off and the bottom 20 polymer product was redissolved in methanol. This crude product was purified by refractionation with a methanol/diethyl ether mixture to remove the unreacted monomers, oligomers, and catalysts. The entire separation was monitored by SEC. The purified product was rotary-evaporated and lyophilized to give the Comb-branched PEOX-PEIpolymer as a white powder (MW=2,182,000, MWD=1.50). This white powder was 25 further hydrolyzed in 50% H2SO4 at 100~C and purified as described before to provide PEI Comb-branched polymers as a white solid (85% yield, MW=1~078,000, MWD= 1.47). All the products were analyzed by SEC, CE, NMR and PAGE, viscometry, TGA, and DSC.
Example KK Synthesis of Comb-Branched (G3) The G3 Comb-Branched polymer was synthesized in a similar manner as above.
The molecular weight of the reslllting product was 10,400,000 and the molecular weight distribution was 1.20. The higher generation Comb-Branched polymers and other Comb-Branched polymers with l;lirrelclll shapes due to the dirr~ ll side chains and initiator cores used were p~ d in a similar manner.
L~ 1 For F,Y~mrl~.c GG-KK
SEC measurements were performed on a series of Rerkm~n TSK 4000 PW (or POLY-OH Polymer Laboratory) 3000 PW and 2000 PW columns using Waters 510 5 HPLC pump Thermo Separation Products AS 3000 Autosampler Wyatt DAWN DSP-F
Multi Angle Laser Light Scattering Detector and Wyatt h~ relc""eter refr~rtr,mrter (Optilab 903). lH and l3C-NMR spectra were obtained on Brucker 360 MHz or VarianUnity 300 MHz NMR spectrometer using either CDCl3 or MeOD as solvents. Purity ofmonomers was rl-rr~t-cl by GC (HP 5890 He as carrier gas). Ultrafiltration was 10 achieved using an Amicon 3 000 10 000 or 100 000 molecular weight cutoff (MWCO) membrane. Thermal analysis was l!elrol~lled on DuPont Thermal Gravimetric Analyzer (Model 951) with TA InstrllmPnt~l Software (2000 Series). CE was performed on Beckman P/ACE System 2050 (Software System Gold). The polymer MWs were also measured by ES-MS (Finnigan Mat TSQ 700). PAGE analysis was performed on 15 Gradipore gradient microgel (5-40% T) with a BioRad 500/200 power supply. Theviscosity measurements were achieved on a Cannon-Ubbelohde semi-micro viscometer.
le LL Rri~ce of salicylic acid from hyper comb-breached polymer In these ~ entc, the interaction of salicylic acid with hyper comb-breached polymer was investigated. Since salicylic acid contains a carboxylic acid group and hyper 20 comb-branched polymers contain secondary amines the charge interaction between the two could slow down the transport of the acid across a dialysis membrane. This would demol~LldL~ that hyper comb-branched polymers could be used as slow-release or drug delivery agents.
The experiments were carried out at room temperature using equilibrium static 25 dialysis cell methodology. The dialysis apparatus consisted of a Spectrum Spectra/Por MacroDialyzer (half cell volume of 10 mL; part no. 132 377) with a pre-soaked Spectra/Por 1 membrane disk (molecular weight cutoff-6-8000~ se~a~dLi"g the two half-cells.
Ten mL of a solution cont~ining salicylic acid (1.0 mg/mL) and hyper comb-30 branched polymer (7.5 mg/mL; 50% PEOX and PEI comb G 1.0 lot #9124193) andadjusted to pH 6.65 with HCI solution was placed in the donor co~pa~L~ent of the dialysis cell. An equal volume of pure water adjusted to pH 6.65 was placed in the receptor co~ a~L"~ent. Transport of salicylic acid into the receptor Colll~a,L.llent was -CA 02226299 l998-02-lO

monitored at various time intervals by removing a small aliquot from the receptor phase and assaying the concentration by absorption at 296 nm.
The control experiment was carried out with an identical procedure to that described above, except that the donor solution contained no hyper comb-branched5 polymer. The results are shown in Figure 37. The hyper comb-branched polymer release data is ~ st:llL~d by squares, while the control experiment data is represented by diamonds. Note that although the absorbance values are mostly greater than one, the aliquots were ~plopliately diluted (to bring the absorbance below one) before their absorbances were measured. The absorbance of the lln(lilnted aliquot could then be 10 calculated from the dilution factor.
It can be seen from Figure 37 that the presence of hyper comb-branched polymer caused the salicylic acid to be released more slowly (i.e., the control had a steeper slope) and less total salicylic acid was transported within the time frame of the experiments (3 days). Also, there appears to be sl-et~inPd release characteristics caused by the presence 15 of hyper comb-branched polymer, since the levels of released salicylic acid continued to slowly rise after the approximate 12-hour equilibrium point seen in the control study.
These experiments therefore demonstrate that hyper comb-branched polymer has thepotential to be used as a slow-release or drug delivery agent.
Of course, it is understood that the foregoing are merely pl~rtll~d embo-liment~ of 20 the invention and that various changes and alterations can be made without departing from the spirit and broader aspects thereof as set forth in the appended claims, which are to be h~ ed in accordance with the principals of patent law including the Doctrine of Equivalents.

Claims (129)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows.
1. A hyper comb-branched polymer conjugate of the formula:
Hv - Mb wherein H is a hyper comb-branched polymer;
M is a carried material;
v is an integer of at least 1; and b is an integer of at least 1.
2. The conjugate of claim 1 wherein M is selected from the group consisting of diagnostic agents, agricultural agents, bioactive agents, industrial agents, environmental agents, consumer product agents, and combinations thereof.
3. The conjugate of claim 2 wherein M is a diagnostic agent selected from the group consisting of metal ions, radioactive drugs, radioactive tracers, radio-opaques,radionuclides, signal generators, signal reflectors, signal absorbers, diagnostic opacifier agents, fluorescent moieties, dye moieties, and combinations thereof.
4. The conjugate of claim 2 wherein M is an agricultural agent selected from thegroup consisting of biological response modifiers, scavenging agents, agricultural materials, pheromones, pesticides, herbicides, genetic materials, and combinations thereof.
5. The conjugate of claim 2 wherein M is a bioactive agent selected from the group consisting of pharmaceutical agents, drugs, pharmaceutical intermediaries, radioprotective agents, toxins, antibodies, antibody fragments, hormones, biological response modifiers, scavenging agents, imuno-potentiating agents, genetic materials, antigens, polypeptides, and combinations thereof.
6. The conjugate of claim 2 wherein M is an industrial agent selected from the group consisting of scavenging agents, agents for material modifiers, stabilizing agents, chromophores, and combinations thereof.
7. The conjugate of claim 2 wherein M is an environmental agent selected from the group consisting of radioprotective agents, scavenging agents, pollutants, agents for agricultural materials, and combinations thereof.
8. The conjugate of claim 2 wherein M is a consumer product agent selected from the group consisting of fragrance moieties, stabilizing agents, material modifiers, chromophores, and combinations thereof.
9. The conjugate of claim 1 wherein H has the formula:

wherein C is a core molecule; each R is the residual moiety of an initiator selected from a group consisting of free radical initiators, cationic initiators, anionic initiators, coordination polymerization initiators and group transfer initiators;
A and B are polymerizable monomers or comonomers capable of withstanding the conditions required for branching therefrom or grafting thereto, at least during the polymerization of the {(A)-(B)} linear polymer chain and during its grafting to a prior {(A)-(B)} branch or the {(A)-(B)} core branch; each G is a grafting component, and the designation indicates that G can attach to either an (A) unit or a (B) unit;
n is the degree of polymerization of the indicated generation comb branches; y is the fraction of B units in the indicated generation branch, and has a value of .01 to 1;
the superscripts 0, 1 and i designate the comb-branch generation level, with i beginning at "2" and continuing for the number of reiterative branch set generations in the polymer;

and at least n° and n1 are ~ 2.
10. The conjugate of claim 9 wherein M is selected from the group consisting of diagnostic agents, agricultural agents, bioactive agents, industrial agents, environmental agents, consumer product agents, and combinations thereof.
11. The conjugate of claim 9 wherein said hyper comb-branched polymer has a hydrophobic exterior formed by grafting hydrophobic polymers selected from the group consisting of polyethylene, polydimethylsiloxane, polybutadiene, polystyrene, polymethylmethacrylate, perfluoropolymer, poly(2-alkyl or phenyl oxazolines), and combinations thereof, at the last grafting step.
12. The conjugate of claim 9 wherein said hyper comb-branched polymer has a hydrophilic exterior formed by grafting hydrophilic polymers selected from the group consisting of poly(2-ethyloxazoline), poly(2-methyloxazoline), polyethylene glycol, polyethylene oxide, polyacrylic acid, polyacrylic amide, polyvinyl pyrrolidone, and combinations thereof.
13. The conjugate of claim 9 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 100,000,000.
14. The conjugate of claim 13 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 10,000,000.
15. The conjugate of claim 9 wherein n has a value of from about 2 to about 10,000.
16. The conjugate of claim 9 wherein A is selected from the group consisting of -CH2CH2-, -CH2CH=CHCH2-, -CH2C(CH3)2-, -CH2CH(CN)-, , , , , , ,-OCH2CH2-, -SCH2CH2-, -R'2SiO-, , -, , , , and combinations thereof, wherein R' is an alkyl group, aryls, arylalkyl, hydrogen, or carboalkoxy; R is an alkyl group, aryls, or hydrogen; and R" is an alkyl group.
17. The conjugate of claim 9 wherein B is selected from the group consisting of - , , -CH2CH(OH)-, -CH2CH(SH)-, -OCH2CH(CH2OH)-, , , , , , , , , and combinations thereof, wherein R is an alkyl group, aryls, or hydrogen, and R" is an alkyl group.
18. The conjugate of claim 1 wherein said hyper comb-branched polymer has an outer periphery functionalized with ethylene diamine or chloroethylamine.
19. A hyper comb-branched polymer conjugate of the formula:
Hv - Mb - Tz wherein H is a hyper comb-branched polymer;
M is a carried material director;
T is a target director;
v is an integer of at least 1;
b is an integer of at least 1; and z is an integer of at least 1.
20. The conjugate of claim 19 wherein M is selected from the group consisting ofdiagnostic agents, agricultural agents, bioactive agents, industrial agents, environmental agents, consumer product agents, and combinations thereof.
21. The conjugate of claim 20 wherein M is a diagnostic agent selected from the group consisting of metal ions, radioactive drugs, radioactive tracers, radio-opaques,radionuclides, signal generators, signal reflectors, signal absorbers, diagnostic opacifier agents, fluorescent moieties, dye moieties, and combinations thereof.
22. The conjugate of claim 20 wherein M is an agricultural agent selected from the group consisting of biological response modifiers, scavenging agents, agricultural materials, pheromones, pesticides, herbicides, genetic materials, and combinations thereof.
23. The conjugate of claim 20 wherein M is a bioactive agent selected from the group consisting of pharmaceutical agents, drugs, pharmaceutical intermediaries, radioprotective agents, toxins, antibodies, antibody fragments, hormones, biological response modifiers, scavenging agents, imuno-potentiating agents, genetic materials, antigens, polypeptides, and combinations thereof.
24. The conjugate of claim 20 wherein M is an industrial agent selected from the group consisting of scavenging agents, agents for material modifiers, stabilizing agents, chromophores, and combinations thereof.
25. The conjugate of claim 20 wherein M is an environmental agent selected from the group consisting of radioprotective agents, scavenging agents, pollutants, agents for agricultural materials, and combinations thereof.
26. The conjugate of claim 20 wherein M is a consumer product agent selected from the group consisting of fragrance moieties, stabilizing agents, material modifiers, chromophores, and combinations thereof.
27. The conjugate of claim 19 wherein H has the formula:
wherein C is a core molecule; each R is the residual moiety of an initiator selected from a group consisting of free radical initiators, cationic initiators, anionic initiators, coordination polymerization initiators and group transfer initiators;
A and B are polymerizable monomers or comonomers capable of withstanding the conditions required for branching therefrom or grafting thereto, at least during the polymerization of the {(A)-(B)} linear polymer chain and during its grafting to a prior {(A)-(B)} branch or the {(A)-(B)} core branch; each G is a grafting component, and the designation indicates that G can attach to either an (A) unit or a (B) unit;
n is the degree of polymerization of the indicated generation comb branches; y is the fraction of B units in the indicated generation branch, and has a value of .01 to 1;
the superscripts 0, 1 and i designate the comb-branch generation level, with i beginning at "2" and continuing for the number of reiterative branch set generations in the polymer;
and at least n° and n1 are ~ 2.
28. The conjugate of claim 27 wherein M is selected from the group consisting ofdiagnostic agents, agricultural agents, bioactive agents, industrial agents, environmental agents, consumer product agents, and combinations thereof.
29. The conjugate of claim 27 wherein said hyper comb-branched polymer has a hydrophobic exterior formed by grafting hydrophobic polymers selected from the group consisting of polyethylene, polydimethylsiloxane, polybutadiene, polystyrene, polymethylmethacrylate, perfluoropolymer, poly(2-alkyl or phenyl oxazolines), and combinations thereof, at the last grafting step, wherein the alkyl group of saidpoly(2-alkyl oxazoline) has about 4 or more carbon atoms.
30. The conjugate of claim 27 wherein said hyper comb-branched polymer has a hydrophilic exterior formed by grafting hydrophilic polymers selected from the group consisting of poly(2-ethyloxazoline), poly(2-methyloxazoline), polyethylene glycol, polyethylene oxide, polyacrylic acid, polyacrylic amide, polyvinyl pyrrolidone, and combinations thereof.
31. The conjugate of claim 27 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 100,000,000.
32. The conjugate of claim 31 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 10,000,000.
33. The conjugate of claim 27 wherein n has a value of from about 2 to about 10,000.
34. The conjugate of claim 27 wherein A is selected from the group consisting of -CH2CH2-,-CH2CH=CHCH2-,-CH2C(CH3)2-,-CH2CH(CN)-,,, , ,,,-OCH2CH2-,-SCH2CH2-,-R'2SiO-,, , , , , and combinations thereof, wherein R' is an alkyl group, aryls, arylalkyl, hydrogen, or carboalkoxy; R is an alkyl group, aryls, or hydrogen; and R" is an alkyl group.
35. The conjugate of claim 27 wherein B is selected from the group consisting of , , -CH2CH(OH)-, -CH2CH(SH)-, -OCH2CH(CH2OH)-, , , , , , , , , and combinations thereof, wherein R is an alkyl group, aryls, or hydrogen, and R" is an alkyl group.
36. The conjugate of claim 19 wherein T is selected from the group consisting ofbioactive agents, proteins, antibodies, antibody fragments, saccharides, and oligosaccharides.
37. A hyper comb-branched polymer conjugate of the formula:
Hv - Mb - M'a wherein H is a hyper comb-branched polymer;
M is a first carried material;
M' is a second carried material different than said first carried material;
v is an integer of at least 1;
b is an integer of at least 1; and a is an integer of at least 1.
38. The conjugate of claim 37 wherein one of M or M' is selected from the group consisting of diagnostic agents, agricultural agents, bioactive agents, industrial agents, environmental agents, consumer product agents, and combinations thereof.
39. The conjugate of claim 38 wherein one of M or M' is a diagnostic agent selected from the group consisting of metal ions, radioactive drugs, radioactive tracers,radio-opaques, radionuclides, signal generators, signal reflectors, signal absorbers, diagnostic opacifier agents, fluorescent moieties, dye moieties, and combinations thereof.
40. The conjugate of claim 38 wherein one of M or M' is an agricultural agent selected from the group consisting of biological response modifiers, scavenging agents, agricultural materials, pheromones, pesticides, herbicides, genetic materials, and combinations thereof.
41. The conjugate of claim 38 wherein one of M or M' is a bioactive agent selected from the group consisting of pharmaceutical agents, drugs, pharmaceutical intermediaries, radioprotective agents, toxins, antibodies, antibody fragments, hormones, biological response modifiers, scavenging agents, imuno-potentiating agents, genetic materials, antigens, polypeptides, and combinations thereof.
42. The conjugate of claim 38 wherein one of M or M' is an industrial agent selected from the group consisting of scavenging agents, agents for material modifiers, stabilizing agents, chromophores, and combinations thereof.
43. The conjugate of claim 38 wherein one of M or M' is an environmental agent selected from the group consisting of radioprotective agents, scavenging agents,pollutants, agents for agricultural materials, and combinations thereof.
44. The conjugate of claim 38 wherein one of M or M' is a consumer product agentselected from the group consisting of fragrance moieties, stabilizing agents, material modifiers, chromophores, and combinations thereof.
45. The conjugate of claim 37 further comprising a target director T.
46. The conjugate of claim 45 wherein T is selected from the group consisting ofbioactive agents, proteins, antibodies, antibody fragments, saccharides, and oligosaccarides.
47. The conjugate of claim 37 wherein H has the formula:

wherein C is a core molecule; each R is the residual moiety of an initiator selected from a group consisting of free radical initiators, cationic initiators, anionic initiators, coordination polymerization initiators and group transfer initiators;
A and B are polymerizable monomers or comonomers capable of withstanding the conditions required for branching therefrom or grafting thereto, at least during the polymerization of the {(A)-(B)} linear polymer chain and during its grafting to a prior {(A)-(B)} branch or the {(A)-(B)} core branch; each G is a grafting component, and the designation indicates that G can attach to either an (A) unit or a (B) unit;
n is the degree of polymerization of the indicated generation comb branches; y is the fraction of B units in the indicated generation branch, and has a value of .01 to 1;
the superscripts 0,1 and i designate the comb-branch generation level, with i beginning at "2" and continuing for the number of reiterative branch set generations in the polymer;
and at least n° and n1 are ~ 2.
48. The conjugate of claim 47 wherein one of M or M' is selected from the group consisting of diagnostic agents, agricultural agents, bioactive agents, industrial agents, environmental agents, consumer product agents, and combinations thereof.
49. The conjugate of claim 47 wherein said hyper comb-branched polymer has hydrophobic exterior formed by grafting hydrophobic polymers selected from the group consisting of polyethylene, polydimethylsiloxane, polybutadiene, polystyrene, polymethylmethacrylate, perfluoropolymer, poly(2-alkyl or phenyl oxazolines), and combinations thereof.
50. The conjugate of claim 47 wherein said hyper comb-branched polymer has a hydrophilic exterior formed by grafting hydrophilic polymers selected from the group consisting of poly(2-ethyloxazoline), poly(2-methyloxazoline), polyethylene glycol, polyethylene oxide, polyacrylic acid, polyacrylic amide, polyvinyl pyrrolidone, and combinations thereof.
51. The conjugate of claim 47 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 100,000,000.
52. The conjugate of claim 51 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 10,000,000.
53. The conjugate of claim 47 wherein n has a value of from about 2 to about 10,000.
54. The conjugate of claim 47 wherein A is selected from the group consisting of -CH2CH2-, -CH2CH=CHCH2-, -CH2C(CH3)2-, -CH2CH(CN)-, , , , , , , -OCH2CH2-, -SCH2CH2-, -R'2SiO-, , , , , , and combinations thereof, wherein R' is an alkyl group, aryls, arylalkyl, hydrogen, or carboalkoxy; R is an alkyl group, aryls, or hydrogen; and R" is an alkyl group.
55. The conjugate of claim 47 wherein B is selected from the group consisting of , , -CH2CH(OH)-, -CH2CH(SH)-, -OCH2CH(CH2OH)-, , , , , , , , , and combinations thereof, wherein R is an alkyl group, aryls, or hydrogen, and R" is an alkyl group.
56. A method of preparing a hyper comb-branched polymer conjugate, said method comprising:
(1) providing a first set of monomers which are either protected against or non-reactive to branching and grafting;
(2) forming a first set of branches by initiating polymerization of said first set of monomers, whereby each of said branches has a reactive end unit upon completion of said polymerization, said reactive end units being incapable of reacting with each other;
(3) providing a core having a plurality of reactive core sites capable of reacting with said reactive end units on said branches;
(4) grafting said first set of branches to said core;
(5) either deprotecting or activating a plurality of monomeric units on each of said branches to create reactive branch sites;
(6) forming a second set of branches by repeating steps (1) and (2) with a second set of monomers;
(7) attaching said second set of branches to said first set of branches by reacting said reactive end units of said second set of branches with said reactive end units of said first set of branches, to thereby form a hyper comb-branched polymer; and (8) conjugating at least one carried material with said hyper comb-branched polymer.
57. The method of claim 56 further comprising:
attaching a target director to said hyper comb-branched polymer conjugate.
58. The method of claim 56, wherein said hyper comb-branched polymer conjugate comprises at least two different carried materials.
59. The hyper comb-branched polymer conjugate produced by the method of claim 56.
60. The method of claim 56 further comprising:
reacting said hyper comb-branched polymer with chloroethylamine or chloroethylamine hydrochloride.
61. The method of claim 56 further comprising:
ester-functionalizing said hyper comb-branched polymer; and reacting said ester-functionalized hyper comb-branched polymer with ethylene diamine.
62. A method of preparing a hyper comb-branched polymer conjugate, said method comprising:
(I) forming a core having at least one reactive site;
(II) reacting essentially all of the reactive sites of said core with a reactivepolymer having the unit formula to form multiple branches which contain reactive (B°) sites on each branch, using a reactive scheme such that the reactive monomer units (B°) are capable of withstanding the conditions required for branching therefrom or grafting thereto to ensure that said reactive polymer reacts with said reactive sites of said core, but that no reactions occur at said (B°) sites;
(III) repeating step (II) sequentially by reacting reactive polymer having the unit formula with the reactive sites of said polymerizable B(i-1) monomers or comonomers of the previous generation to form successive generation of branches to give the desired hyper comb-branched polymer; and (IV) conjugating at least one carried material with said hyper comb-branched polymer.
63. The method of claim 62 further comprising:
reacting said hyper combo-branched polymer with at least one of ethylene diamine, chloroethylamine, and chloroethylamine hydrochloride.
64. The hyper comb-branched polymer conjugate produced by the method of claim 62.
65. A composition comprising a hyper comb-branched polymer conjugate of the formula Hv - Mb wherein H is a hyper comb-branched polymer;
M is a carried material;
v is an integer of at least 1; and b is an integer of at least 1.
66. The composition of claim 65 wherein said hyper comb-branched polymer H has the general formula:

wherein C is a core molecule; each R is the residual moiety of an initiator selected from a group consisting of free radical initiators, cationic initiators, anionic initiators, coordination polymerization initiators and group transfer initiators;
A and B are polymerizable monomers or comonomers capable of withstanding the conditions required for branching therefrom or grafting thereto, at least during the polymerization of the {(A)-(B)} linear polymer chain and during its grafting to a prior {(A)-(B)} branch or the {(A)-(B)} core branch; each G is a grafting component, and the designation indicates that G can attach to either an (A) unit or a (B) unit;
n is the degree of polymerization of the indicated generation comb branches; y is the fraction of B units in the indicated generation branch, and has a value of .01 to 1;
the superscripts 0,1 and i designate the comb-branch generation level, with i beginning at "2" and continuing for the number of reiterative branch set generations in the polymer;
and at least n° and n1 are ~ 2.
67. The composition of claim 66 wherein M is selected from the group consisting of diagnostic agents, agricultural agents, bioactive agents, industrial agents, environmental agents, consumer product agents, and combinations thereof.
68. The composition of claim 66 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 100,000,000.
69. The composition of claim 66 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 10,000,000.
70. A composition comprising a hyper comb-branched polymer conjugate of the formula Hv - Mb - Tz wherein H is a hyper comb-branched polymer;
M is a carried material;
T is a target director;
v is an integer of at least 1;
b is an integer of at least 1; and z is an integer of at least 1.
71. The composition of claim 70 wherein said hyper comb-branched polymer H has the general formula:

wherein C is a core molecule; each R is the residual moiety of an initiator selected from a group consisting of free radical initiators, cationic initiators, anionic initiators, coordination polymerization initiators and group transfer initiators;
A and B are polymerizable monomers or comonomers capable of withstanding the conditions required for branching therefrom or grafting thereto, at least during the polymerization of the {(A)-(B)} linear polymer chain and during its grafting to a prior {(A)-(B)} branch or the {(A)-(B)} core branch, each G is a grafting component, and the designation indicates that G can attach to either an (A) unit or a (B) unit;
n is the degree of polymerization of the indicated generation comb branches; y is the fraction of B units in the indicated generation branch, and has a value of .01 to 1;
the superscripts 0,1 and i designate the comb-branch generation level, with i beginning at "2" and continuing for the number of reiterative branch set generations in the polymer;
and at least n° and n1 are ~ 2.
72. The composition of claim 71 wherein M is selected from the group consisting of diagnostic agents, agricultural agents, bioactive agents, industrial agents, environmental agents, consumer product agents, and combinations thereof.
73. The composition of claim 71 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 100,000,000.
74. The composition of claim 71 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 10,000,000.
75. The conjugate of claim 70 wherein T is selected from the group consisting ofbioactive agents, proteins, antibodies, antibody fragments, saccharides, and oligosaccharides.
76. A composition comprising a hyper comb-branched polymer conjugate of the formula Hv - Mb - M'a wherein H is a hyper comb-branched polymer;
M is a first carried material;
M' is a second carried material different than said first carried material;
v is an integer of at least 1;

b is an integer of at least 1; and a is an integer of at least 1.
77. The composition of claim 76 wherein said hyper comb-branched polymer H has the general formula:

wherein C is a core molecule; each R is the residual moiety of an initiator selected from a group consisting of free radical initiators, cationic initiators, anionic initiators, coordination polymerization initiators and group transfer initiators;
A and B are polymerizable monomers or comonomers capable of withstanding the conditions required for branching therefrom or grafting thereto, at least during the polymerization of the {(A)-(B)} linear polymer chain and during its grafting to a prior {(A)-(B)} branch or the {(A)-(B)} core branch; each G is a grafting component, and the designation indicates that G can attach to either an (A) unit or a (B) unit;
n is the degree of polymerization of the indicated generation comb branches; y is the fraction of B units in the indicated generation branch, and has a value of .01 to 1;
the superscripts 0,1 and i designate the comb-branch generation level, with i beginning at "2" and continuing for the number of reiterative branch set generations in the polymer;
and at least n° and n1 are ~ 2.
78. The composition of claim 77 wherein M or M' is selected from the group consisting of diagnostic agents, agricultural agents, bioactive agents, industrial agents, environmental agents, consumer product agents, and combinations thereof.
79. The composition of claim 77 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 100,000,000.
80. The composition of claim 77 wherein said hyper comb-branched polymer has a molecular weight of from about 10,000 to about 10,000,000.
81. The composition of claim 76 wherein said conjugate further comprises a target director T.
82. The composition of claim 81 wherein T is selected from the group consisting of bioactive agents, proteins, antibodies, antibody fragments, saccharides, and oligosaccharides.
83. A method of stabilizing a protein to minimize structural transformations, said method comprising:
reacting said protein with a hyper comb-branched polymer to produce a hyper comb-branched polymer and protein conjugate.
84. The method of claim 83 wherein said protein comprises at least one of troponin, ferritin, prolactin, gastrin, calcitonin, and combinations thereof.
85. The stabilized conjugated protein of claim 83.
86. A method of stabilizing an enzyme to minimize structural transformations, said method comprising:
reacting said enzyme with a hyper comb-branched polymer to produce a hyper comb-branched polymer and enzyme conjugate.
87. The method of claim 86 wherein said enzyme comprises at least one of glutamate pyruvate transaminase, alkaline phosphatase, acid phosphatase, glutamate oxalacetate transaminase, creatinine kinase, lactate dehydrogenase, and combinations thereof.
88. The stabilized enzyme of claim 86.
89. A method of encapsulating a pharmaceutical agent, said method comprising:
providing a hyper comb-branched polymer comprising a generally hydrophobic interior;
exposing said hyper comb-branched polymer to one or more generally hydrophobic pharmaceutical agents for a time sufficient to allow said agents to migrate into the interior of said hyper comb-branched polymer, to thereby encapsulate said agent within said polymer.
90. The method of claim 89 wherein said hyper comb-branched polymer has a hydrophobic interior modified with monomers selected from the group consisting of epoxy hexane, methyl acrylate, and combinations thereof.
91. The method of claim 89 wherein said pharmaceutical agent is selected from the group consisting of antibiotics, analgesics, antihypertensives, cardiotonics, sedatives, antiepileptics, antipyretics, stimulants, immunosuppressives, and combinations thereof.
92. The method of claim 89 wherein said pharmaceutical agent is selected from the group consisting of acetaminophen, acyclovir, alkeran, amikacin, ampicillin, amphotericin B, aspirin, bisantrene, bleomycin, neocardistatin, chloroambucil, chloramphenicol, cytarabine, daunomycin, dilantin, doxorubicin, fluorouracil, gentamycin, ibuprofen, kanamycin, meprobamate, methotrexate, novantrone, nystatin, oncovin, phenobarbital, polymyxin, probucol, procarbazine, rifampin, streptomycin, spectinomycin, symmetrel, thioguanine, tobramycin, trimethoprim, valban, 4-acetamindophenol, and combinations thereof.
93. The encapsulated product of claim 89.
94. A method of increasing the detection capability of measuring instruments utilizing a measuring signal, said method comprising:

providing a hyper comb-branched polymer;
attaching signal reflecting or signal absorbing moieties to the periphery of said hyper comb-branched polymer.
95. The method claim 94 wherein said moieties are chromophores.
96. The product of claim 94.
97. A method of chelating metal ions, said method comprising:
providing a hyper comb-branched polymer capable of chelating one or more metal ions;
exposing said polymer to said metal ions for a time sufficient to chelate said ions to said polymer.
98. The method claim 97 wherein said metal ions are selected from the group consisting of Cu2+, Co2+, the metals in the Periodic Table Groups VIIIA (Fe, Ni, Ru, Rh, Pd, Os, Ir, Pt), IVB (Pb, Sn, Ge), IIIA (Sc, Y, lanthanides and actinides), IIIB (B, Al, Ga, In, Tl), IA alkali metals (Li, Na, K, Rb, Cs, Fr), and IIA alkaline-earth metals (Be, Mg, Ca, Sr, Ba, Ra), transition metals and combinations thereof.
99. The chelated product of claim 97.
100. A method of carrying genetic material, said method comprising:
providing a hyper comb-branched polymer having a plurality of positively chargedsites; and conjugating said polymer with genetic material having a plurality of negatively charged sites.
101. The method of claim 100 wherein the ratio of said negatively charged sites to said positively charged sites ranges from about 1:10 to about 1:1000.
102. The method claim 100 wherein said hyper comb-branched polymer is modified with a moiety selected from the group consisting of -NH2, -NH-, PEOX, 50% PEOX, chloroethylamine, and combinations thereof.
103. The conjugate product of claim 100.
104. A process for preparing a complex of hyper comb-branched polymer and genetic material comprising:
reacting said hyper comb-branched polymer with said genetic material in a suitable solvent at a temperature which facilitates the complexing of said genetic material with said hyper comb-branched polymer.
105. The process of claim 104 which includes attaching a target director to said hyper comb-branched polymer before complexing it with genetic material.
106. The process of claim 105 wherein said target director is selected from the group consisting of bioactive agents, proteins, antibodies, antibody fragments, saccharides, and oligosaccharides.
107. The process of clam 104 wherein said hyper comb-branched polymer has a predominantly cationic surface, said process comprising electrostatically attaching genetic material to said polymer to create said complex.
108. A process for forming a genetic material: hyper comb-branched polymer complex comprising:
mixing, in water, sufficient genetic material to yield a final concentration from about 1 to about 10 µg per mL, with sufficient hyper comb-branched polymer, having positive surface functionality, to yield a genetic material: polymer charge ratio from about 1:10 to about 1:10,000;
said mixing being done at a pH of about 5 to about 10 and at a temperature from about 20 to about 40°C.
109. The process of claim 108 wherein said charge ratio is from about 1:10 to about 1:1,000.
110. The process of claim 108 wherein said charge ratio is from about 1:100 to about 1:1000.
111. The process of claim 108 wherein said charge ratio is about 1:200.
112. The process of claim 108 wherein said hyper comb-branched polymer comprisesamino acids over a substantial portion of its surface.
113. The process of claim 112 wherein said amino acid is lysine or arginine.
114. The process of claim 108 wherein said polymer particles include target director moieties attached thereto.
115. A process for forming a concentrated genetic material: hyper comb-branched polymer complex which can be diluted for use comprising:
mixing, in water, sufficient genetic material to yield a concentration from about 1 to about 10 µg per 20 µL, with sufficient hyper comb-branched polymer, having positive surface functionality, to yield a genetic material: polymer charge ratio from about 1:10 to about 1:10,000;
said mixing being done at a pH from about 5 to about 10 and at a temperature from about 20 to about 40°C.
116. The process of claim 115 wherein said charge ratio is from about 1:10 to about 1:1,000.
117. The process of claim 115 wherein said charge ratio is from about 1:100 to about 1:1000.
118. The process of claim 115 wherein said charge ratio is about 1:200.
119. A method of effecting cell transfection and bioavailability of genetic material comprising providing a complex of a hyper comb-branched polymer and genetic material, and making said complex available to cells to be transfected.
120. A method for transporting genetic material through a cellular membrane and into a cellular nucleus comprising:
complexing said genetic material with hyper comb-branched polymer;
followed by making said complex available to cells to be transfected.
121. A method for protecting genetic material from digestion during transit to and transfection into a cell comprising:
complexing said genetic material with hyper comb-branched polymer prior to exposing said genetic material to digestive enzymes.
122. A method for stabilizing and compacting genetic material comprising complexing said genetic material with hyper comb-branched polymer.
123. A hyper comb-branched polymer conjugate comprising a hyper comb-branched polymer and a carried material, wherein said hyper comb-branched polymer has a grafting density of from about 0.1 % to about 90% .
124. A hyper comb-branched polymer conjugate comprising a hyper comb-branched polymer and a carried material, wherein said hyper comb-branched polymer has an interior void volume of from about 10 angstroms to about 500 angstroms.
125. A hyper comb-branched polymer conjugate comprising a hyper comb-branched polymer and a carried material, wherein said hyper comb-branched polymer comprises alternating regions of (i) a first region having a grafting density of less than about 50%, and (ii) a second region having a grafting density of more than about 50%.
126. A hyper comb-branched polymer conjugate comprising a hyper comb-branched polymer and a carried material, wherein said hyper comb-branch polymer has at least one chemical moiety disposed about the periphery of said polymer, said moiety selected from the group consisting of -NH2, -COOH, -COOMe, -NH4, -PEOX, -PEG, -PEO, and combinations thereof.
127. A hyper comb-branched polymer conjugate comprising a hyper comb-branched polymer and a carried material wherein said hyper comb-branched polymer has a maximum diameter of about 100 nm.
128. A hyper comb-branched polymer conjugate comprising a hyper comb-branched polymer and a carried material, wherein said hyper comb-branched polymer has a hydrophobically-modified region, said region being at least one of an interior region or an exterior region.
129. A hyper comb-branched polymer conjugate comprising a hyper comb-branched polymer and a carried material, wherein said hyper comb-branched polymer has a hydrophilically-modified region, said region being at least one of an interior region or an exterior region.
CA002226299A 1995-08-11 1996-08-09 Hyper comb-branched polymer conjugates Abandoned CA2226299A1 (en)

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Families Citing this family (99)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2744125B1 (en) * 1996-01-30 1998-06-05 Peintures Jefco GRAFT COPOLYMERS, THEIR MANUFACTURING METHOD, THE COMPOSITIONS CONTAINING THEM AND THEIR USE FOR THE PREPARATION OF PIGMENTAL DISPERSIONS IN AQUEOUS AND / OR ORGANIC MEDIA
US20060008494A1 (en) * 1997-02-21 2006-01-12 The Regents Of The University Of California Complete inactivation of infectious proteins
US6322802B1 (en) 1999-06-01 2001-11-27 The Regents Of The University Of California Method of sterilizing
US6214366B1 (en) * 1999-06-01 2001-04-10 The Regents Of The University Of California Clearance and inhibition of conformationally altered proteins
US6719988B2 (en) 1997-02-21 2004-04-13 The Regents Of The University Of California Antiseptic compositions for inactivating prions
US6720355B2 (en) 1997-02-21 2004-04-13 The Regents Of The University Of California Sodium dodecyl sulfate compositions for inactivating prions
US6419916B1 (en) 1999-06-01 2002-07-16 The Regents Of The University Of California Assay for compounds which affect conformationally altered proteins
US6331296B1 (en) * 1999-06-01 2001-12-18 Stanley B. Prusiner Food additives which affect conformationally altered proteins
JP2002515932A (en) * 1997-04-18 2002-05-28 カリフォルニア インスティチュート オブ テクノロジー Multifunctional polymeric tissue coating
DE19743135A1 (en) * 1997-09-30 1999-04-01 Hoechst Marion Roussel De Gmbh Biologically compatible low molecular weight polyethyleneimines
US6475994B2 (en) * 1998-01-07 2002-11-05 Donald A. Tomalia Method and articles for transfection of genetic material
FR2777016B1 (en) * 1998-04-06 2002-06-14 Rhone Poulenc Agrochimie NITROGEN SILICONE USEFUL FOR COMPACTING NUCLEIC ACID SEQUENCES AND USE FOR TRANSFECTION
DE69920198T2 (en) 1998-04-13 2005-09-22 Massachusetts Institute Of Technology, Cambridge CAMM POLYMERS FOR REGULATING CELL SURFACE INTERACTION
FR2791990A1 (en) * 1999-04-06 2000-10-13 Oreal Cosmetic composition comprising polymers having original star structure, for use in preparations for maintaining or fixing hairstyle, or for treatment of hair
FR2791989A1 (en) 1999-04-06 2000-10-13 Oreal COMPOSITION, IN PARTICULAR COSMETIC, COMPRISING POLYMERS HAVING A STAR STRUCTURE, SAID POLYMERS AND THEIR USE
US6946525B2 (en) 1999-04-06 2005-09-20 L'oreal S.A. Composition comprising polymers having a star structure, the polymers, and their use
FR2791987A1 (en) 1999-04-06 2000-10-13 Oreal COMPOSITION, IN PARTICULAR COSMETIC, COMPRISING POLYMERS HAVING A STAR STRUCTURE, SAID POLYMERS AND THEIR USE
FR2791988A1 (en) * 1999-04-06 2000-10-13 Oreal COMPOSITION, IN PARTICULAR COSMETIC OR PHARMACEUTICAL COMPOSITION COMPRISING POLYMERS HAVING A STAR STRUCTURE, SAID POLYMERS AND THEIR USE
FR2791986A1 (en) 1999-04-06 2000-10-13 Oreal COMPOSITION, IN PARTICULAR COSMETIC, COMPRISING POLYMERS HAVING A STAR STRUCTURE, SAID POLYMERS AND THEIR USE
NZ515607A (en) * 1999-06-01 2003-07-25 Univ California Method of sterilizing
KR20020057946A (en) 1999-07-29 2002-07-12 로퍼, 랜달 비. Targeting multimeric imaging agents through multilocus binding
DE19936997B4 (en) * 1999-08-02 2007-06-14 Biolitec Ag Method for the application of photosensitizers (PS) by means of multipliers in photodymic therapy
FI19991705A (en) * 1999-08-11 2001-02-12 Neste Chemicals Oy An antimicrobial hyperbranched polymer composition
CA2395636A1 (en) * 1999-12-30 2001-07-12 Novartis Ag Novel colloid synthetic vectors for gene therapy
JP2003520210A (en) * 2000-01-05 2003-07-02 イマレックス セラピューティクス， インコーポレイテッド Pharmaceutical formulations for delivery of drugs with low water solubility
US6616944B2 (en) * 2000-03-08 2003-09-09 Medinnova Gesellschaft Fur Medizinsche Innovationen Aus Adkademischer Forschung Mbh Self-assembling colloidal carriers for protein delivery
ATE466042T1 (en) * 2000-03-08 2010-05-15 Lab Internat Barbados Srl COLLOIDAL NANOPARTICULAR CARRIER CONTAINING CHARGED OR UNCHARGED WATER-SOLUBLE COMB POLYMERS AND THEIR USE FOR MUCOSAL APPLICATION
US6716450B1 (en) * 2000-05-18 2004-04-06 The United States Of America As Represented By The Secretary Of The Army Enhancing protein activity through nanoencapsulation
MXPA03000310A (en) * 2000-07-12 2004-12-13 Gryphon Therapeutics Inc Polymer-modified bioactive synthetic chemokines, and methods for their manufacture and use.
EP1318827A4 (en) * 2000-09-08 2005-09-14 Gryphon Therapeutics Inc Polymer-modified synthetic proteins
US7118737B2 (en) * 2000-09-08 2006-10-10 Amylin Pharmaceuticals, Inc. Polymer-modified synthetic proteins
WO2002067908A1 (en) * 2001-02-26 2002-09-06 Duke University Novel dendritic polymers and their biomedical uses
ES2441412T3 (en) * 2001-03-09 2014-02-04 Trovagene, Inc. Conjugated probes and optical analyte detection
AU2002303146A1 (en) * 2001-03-22 2002-10-08 The Administrators Of The Tulane Educational Fund Biaryl monomers and dendritic polymers derived therefrom
DE10114247A1 (en) * 2001-03-22 2002-10-10 Heraeus Kulzer Gmbh & Co Kg Antibiotic / antibiotics-polymer combination
US20030108585A1 (en) * 2001-05-04 2003-06-12 Roe R. Michael Polymer conjugates of insecticidal peptides or nucleic acids or insecticides and methods of use thereof
WO2002089830A1 (en) * 2001-05-04 2002-11-14 North Carolina State University Polymer conjugates of insecticidal peptides or nucleic acids and methods of use thereof
ES2525695T3 (en) * 2001-05-17 2014-12-29 Basf Se Polymer additives with improved permanence and surface affinity
US7101643B2 (en) * 2001-05-31 2006-09-05 The Regents Of The University Of California Polymeric electrolytes based on hydrosilyation reactions
US6956083B2 (en) * 2001-05-31 2005-10-18 The Regents Of The University Of California Single ion conductor cross-linked polymeric networks
US20040077835A1 (en) * 2001-07-12 2004-04-22 Robin Offord Chemokine receptor modulators, production and use
TWI221406B (en) 2001-07-30 2004-10-01 Epix Medical Inc Systems and methods for targeted magnetic resonance imaging of the vascular system
AU2002348163A1 (en) * 2001-11-02 2003-05-19 Intradigm Corporation Therapeutic methods for nucleic acid delivery vehicles
US20040009495A1 (en) * 2001-12-07 2004-01-15 Whitehead Institute For Biomedical Research Methods and products related to drug screening using gene expression patterns
US20040131582A1 (en) * 2002-02-26 2004-07-08 Grinstaff Mark W. Novel dendritic polymers and their biomedical uses
EP1616003A4 (en) 2002-12-30 2007-06-20 Gryphon Therapeutics Inc Water-soluble thioester and selenoester compounds and methods for making and using the same
WO2004083450A2 (en) * 2003-03-21 2004-09-30 The General Hospital Corporation D/B/A Massachusetts General Hospital Hyperbranched dendron and methods of synthesis and use thereof
FR2858766B1 (en) * 2003-08-11 2005-11-11 Oreal COSMETIC COMPOSITION COMPRISING STABILIZED METALLIC PARTICLES, POSSIBLY COATED
US6872505B1 (en) * 2003-09-16 2005-03-29 Intel Corporation Enabling chain scission of branched photoresist
US7754500B2 (en) * 2003-11-21 2010-07-13 Anp Technologies, Inc. Asymmetrically branched polymer conjugates and microarray assays
EP1696974B1 (en) * 2003-12-19 2007-05-23 Basf Aktiengesellschaft Swellable hydrogel-forming polymers having a low fine dust concentration
JP4510881B2 (en) * 2004-04-20 2010-07-28 デンドリティック ナノテクノロジーズ，インコーポレイテッド Dendritic polymers with enhanced expandability and internal functionality
US7985424B2 (en) * 2004-04-20 2011-07-26 Dendritic Nanotechnologies Inc. Dendritic polymers with enhanced amplification and interior functionality
US8153111B2 (en) * 2004-06-18 2012-04-10 Ceramoptec Industries, Inc. Photo-triggered release of active substances from dendrimer-photosensitizer complexes
DE102004039048A1 (en) * 2004-08-11 2006-02-23 Siemens Ag Method and apparatus for molecular imaging with a molecular probe
US7560520B1 (en) 2004-11-08 2009-07-14 The United States Of America As Represented By The Secretary Of The Army Interface-directed branched polymer transports and methods for producing same
WO2006102484A2 (en) * 2005-03-21 2006-09-28 Anp Technologies, Inc. Symmetrically branched polymer conjugates and microarray assays
US11457813B2 (en) 2005-03-29 2022-10-04 Martin W. Roche Method for detecting body parameters
EP1868652A2 (en) 2005-04-05 2007-12-26 Istituto di Richerche di Biologia Molecolare P. Angeletti S.p.A. Method for shielding functional sites or epitopes on proteins
AU2006242303A1 (en) 2005-05-02 2006-11-09 Anp Technologies, Inc. Polymer conjugate enhanced bioassays
DE102005051366A1 (en) * 2005-10-25 2007-04-26 Degussa Gmbh Drug delivery systems
US20070141134A1 (en) * 2005-12-16 2007-06-21 Kosak Matthew K Shielded micelles for polynucleotide delivery
PL1979407T3 (en) * 2006-01-19 2015-03-31 Allexcel Inc Solubilization and targeted delivery of drugs with self-assembling amphiphilic polymers
AU2012268917B2 (en) * 2006-01-19 2014-09-04 Allexcel, Inc. Solubilization and targeted delivery of drugs with self-assembling amphiphilic polymers
US20070224115A1 (en) * 2006-03-22 2007-09-27 The Regents Of The University Of California Polymeric chelators for radionuclide delivery systems
US7985783B2 (en) 2006-09-21 2011-07-26 The Regents Of The University Of California Aldehyde tags, uses thereof in site-specific protein modification
US20080221224A1 (en) * 2007-03-06 2008-09-11 Sheiko Sergei S Unimolecular tweezers
JP2010535264A (en) * 2007-07-31 2010-11-18 ポリプラス トランスフェクション Method for producing linear polyethyleneimine (PEI) for transfection purposes and linear PEI obtained by the method
WO2009108760A2 (en) 2008-02-26 2009-09-03 Board Of Regents, The University Of Texas System Dendritic macroporous hydrogels prepared by crystal templating
JP6042815B2 (en) 2010-10-08 2016-12-14 ザ ボード オブ リージェンツ オブ ザ ユニバーシティ オブ テキサス システム Anti-adhesion barrier membranes using alginate and hyaluronic acid for biomedical applications
US8946194B2 (en) 2010-10-08 2015-02-03 Board Of Regents, University Of Texas System One-step processing of hydrogels for mechanically robust and chemically desired features
US20140314664A1 (en) 2011-01-09 2014-10-23 Anp Technologies, Inc. Hydrophobic Molecule-Induced Branched Polymer Aggregates And Their Use
PT2717917T (en) 2011-07-05 2016-07-27 Bioasis Technologies Inc P97-antibody conjugates
US20130217023A1 (en) * 2012-02-22 2013-08-22 454 Life Sciences Corporation System And Method For Generation And Use Of Compact Clonally Amplified Products
AU2013296557B2 (en) 2012-07-31 2019-04-18 Bioasis Technologies Inc. Dephosphorylated lysosomal storage disease proteins and methods of use thereof
WO2014071285A1 (en) 2012-11-02 2014-05-08 Ab Specialty Silicones, Llc Silicone lubricant
EP2916835A4 (en) 2012-11-12 2016-07-27 Redwood Bioscience Inc Compounds and methods for producing a conjugate
WO2014078733A1 (en) 2012-11-16 2014-05-22 The Regents Of The University Of California Pictet-spengler ligation for protein chemical modification
US9310374B2 (en) 2012-11-16 2016-04-12 Redwood Bioscience, Inc. Hydrazinyl-indole compounds and methods for producing a conjugate
US11565027B2 (en) 2012-12-11 2023-01-31 Board Of Regents, The University Of Texas System Hydrogel membrane for adhesion prevention
CN105263528A (en) 2013-02-05 2016-01-20 Anp科技公司 Nanoparticles containing a taxane and their use
US20190015350A1 (en) 2013-02-05 2019-01-17 Anp Technologies, Inc. Nanoparticles Containing a Taxane and their Use
NZ711373A (en) 2013-03-13 2020-07-31 Bioasis Technologies Inc Fragments of p97 and uses thereof
CN104341590A (en) * 2013-08-01 2015-02-11 韩冰 A dendritic polymer compound, preparation thereof and uses of the dendritic polymer compound
AU2014312190A1 (en) 2013-08-28 2016-02-18 Bioasis Technologies Inc. CNS-targeted conjugates of antibodies
US9493413B2 (en) 2013-11-27 2016-11-15 Redwood Bioscience, Inc. Hydrazinyl-pyrrolo compounds and methods for producing a conjugate
EP3352800B1 (en) 2015-09-24 2022-01-05 The University of North Carolina at Chapel Hill Methods and compositions for reducing metastases
WO2017112852A1 (en) * 2015-12-23 2017-06-29 Preceres Inc. Modified polyethyleneimines and uses thereof
EP3732254A4 (en) 2017-12-26 2021-12-22 Becton, Dickinson and Company Deep ultraviolet-excitable water-solvated polymeric dyes
US10844228B2 (en) 2018-03-30 2020-11-24 Becton, Dickinson And Company Water-soluble polymeric dyes having pendant chromophores
CA3107332A1 (en) 2018-07-22 2020-01-30 Bioasis Technologies Inc. Treatment of lymphatic metastases
US20220002497A1 (en) * 2018-10-30 2022-01-06 University Of Notre Dame Du Lac Block polymer composite membranes
US20210355468A1 (en) 2020-05-18 2021-11-18 Bioasis Technologies, Inc. Compositions and methods for treating lewy body dementia
US20210393787A1 (en) 2020-06-17 2021-12-23 Bioasis Technologies, Inc. Compositions and methods for treating frontotemporal dementia
WO2022271183A1 (en) * 2021-06-25 2022-12-29 Allexcel Inc. Self-assembling amphphlic polymers as anti-covid-19 agents
EP4155349A1 (en) 2021-09-24 2023-03-29 Becton, Dickinson and Company Water-soluble yellow green absorbing dyes
WO2024007016A2 (en) 2022-07-01 2024-01-04 Beckman Coulter, Inc. Novel fluorescent dyes and polymers from dihydrophenanthrene derivatives
WO2024044327A1 (en) 2022-08-26 2024-02-29 Beckman Coulter, Inc. Dhnt monomers and polymer dyes with modified photophysical properties
CN116656745A (en) * 2023-08-01 2023-08-29 吉林大学第一医院 Non-viral gene vector and preparation method and application thereof

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3251664A (en) * 1960-08-04 1966-05-17 Petrolite Corp Fuel compositions containing branched polyalkylenepolyamines and derivatives thereof
US4289872A (en) * 1979-04-06 1981-09-15 Allied Corporation Macromolecular highly branched homogeneous compound based on lysine units
US4558120A (en) * 1983-01-07 1985-12-10 The Dow Chemical Company Dense star polymer
US4599400A (en) * 1984-12-18 1986-07-08 The Dow Chemical Company Star/comb-branched polyamide
US5527524A (en) * 1986-08-18 1996-06-18 The Dow Chemical Company Dense star polymer conjugates
US5631329A (en) * 1990-08-27 1997-05-20 Dendritech, Inc. Process for producing hyper-comb-branched polymers
IL99234A (en) * 1990-08-27 1994-05-30 Michigan Molecular Inst Non-crosslinked, poly-branched polymers and their preparation
US5266106A (en) * 1992-10-22 1993-11-30 Xerox Corporation Ink compositions with dendrimer grafts
TW320784B (en) * 1994-05-13 1997-11-21 Gould Electronics Inc

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