WO2000069942A1 - Stabilization and acoustic activation of polymeric micelles for drug delivery - Google Patents
Stabilization and acoustic activation of polymeric micelles for drug deliveryInfo
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- WO2000069942A1 WO2000069942A1 PCT/US2000/014081 US0014081W WO0069942A1 WO 2000069942 A1 WO2000069942 A1 WO 2000069942A1 US 0014081 W US0014081 W US 0014081W WO 0069942 A1 WO0069942 A1 WO 0069942A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
- A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
- C08L71/02—Polyalkylene oxides
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G2650/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G2650/28—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterised by the polymer type
- C08G2650/58—Ethylene oxide or propylene oxide copolymers, e.g. pluronics
Definitions
- This invention relates to stabilization of micelles, and activation of micelles for delivery of substances such as drugs.
- the most attractive drug carriers are polymeric micelles formed by hydrophobic-hydrophilic block copolymers, with the hydrophilic blocks including PEO chains. These micelles have a spherical, core-shell structure, with the hydrophobic block forming the core of the micelle, while the hydrophilic PEO block (or blocks) forms the shell.
- Block copolymer micelles have promising properties as drug carriers in terms of their size and architecture.
- polymeric micellar drug delivery systems over other types of drug carriers include: 1) long circulation time in blood; 2) appropriate size (10 to 30 nm) to escape renal excretion but to allow for the extravasation at the tumor site; 3) simplicity in drug inco ⁇ oration, compared to covalent bonding of the drug to the polymeric carrier and 4) drug delivery independent of drug character.
- PEO-coated particles The ability of PEO-coated particles to prohibit adsorption of proteins and cells depends on the surface density of PEO chains, their length and dynamics. [1,3] However, only a few known block copolymers form micelles in aqueous solutions. Among them, AB-type block copolymers, e.g. poly(L-aminoacid)-block-poly(ethylene oxide) [2,3-13] and ABA-type triblock copolymers. Triblock copolymers of this class are available under the name PLURONICTM, which shall be referred to generically herein as "P-triblock".
- P- triblocks are block polymers of PEO and PPO, usually triblock PEO-PPO-PEO copolymers, where PPO stands for poly(propylene oxide); the hydrophobic central PPO blocks form micelle cores, whereas the flanking PEO blocks form the shell, or corona which protects micelles from the recognition by RES.
- P-triblock copolymers are commercially available from BASF Corp. and ICI.
- P-triblock polymers are also disclosed in United States Patent 5,516,703 to Caldwell et al, issued 14 May 1996, which is hereby inco ⁇ orated by reference. P-triblock structure in aqueous solution have been extensively investigated by many authors and have been recently reviewed by Alexandridis [22], see also [16].
- phase state of P-triblock micelles can be controlled by choosing members of the P-triblock family with appropriate molecular weight, PPO PEO block length ratio, and concentration.
- the hydrodynamic radii of P-triblock micelles at physiological temperatures range between 10 and 20 nm, which makes them prospects as potential drug carriers.
- the inco ⁇ oration of drugs into block copolymer micelles may be achieved through chemical and physical routes.
- Chemical routes involve covalent coupling of the drug to the hydrophobic block of the copolymer leading to micelle-forming, block copolymer-drug conjugates.
- this approach involved complex synthetic steps and purification procedures. This concept is disclosed in Rigsdorf, et al. [24] and Kataoka, et al. [7-10, 25-27] Physical entrapment is a better way of loading drugs into micellar systems.
- P-triblock molecules in the unimeric form were found to sensitize multi-drug resistant (MDR) cancerous cells.
- MDR multi-drug resistant
- Kabanov and Alakhov [20, 28, 29] have found that there is a dramatic increase in Daunorubicin and DOX cytotoxic activity toward the multi-drug resistant cell lines while in the presence of 0.01 to 1% of PLURONIC P85 or L61.
- the drop in the efficacy of drug/P-triblock systems above the CMC may be due to the substantial decrease in the intracellular drug uptake from dense P-triblock micelles.
- the drug inco ⁇ orated into the micelle core is masked from the external media by the corona composed of PEO chains.
- This phenomenon may be used advantageously to prevent the unwanted drug interactions with healthy cells.
- the challenge is to ensure drug uptake at the tumor site.
- micellar systems are structurally stable (these are micelles with solid-like cores that dissociate slowly at levels below their CMC, e.g. micelles formed by poly(L- aminoacid)-block-poly(ethylene oxide) copolymers [2, 5, 26]). As indicated by NMR data, molecular motion in the core of these micelles is substantially frozen. In contrast, P- triblock micelles or those formed by poly(ethylene oxide-b/oc&-isoprene-b/ocA:-ethylene oxide) triblock copolymer dissociate very fast upon dilution [16].
- micelles have "soft" cores, which means that at room temperature theft molecular segments are above corresponding glass transition temperature, T g and move relatively fast. Since upon TV injections, the concentration of the polymeric drug carrier can drop to levels below the CMC, non-stable micelles require additional stabilization to be used in micellar form.
- an object of the invention to provide a system for the stabilization of micelles.
- Another object of the invention is to provide a system for activation of stabilized micelles.
- the present invention involves various routes of micelle stabilization against degradation upon dilution.
- the invention also involves the effect of ultrasound on drug release from micelles and drug uptake by cancerous cells.
- a drug can be encapsulated in a long-circulating micelle. Extravasation proceed only at tumor sites due higher permeability of blood vessels, and is enhanced by ultrasound.
- the micelle-encapsulated drug is accumulated at the tumor site.
- the uptake of the micelle-encapsulated drug is enhanced by ultrasound.
- the advantages of the micelle drug carriers of the invention included long circulation time in the blood, appropriate size to escape renal excretion, appropriate size to allow for extravasation at the tumor site, and simplicity of drug loading.
- sterilization is possible by filtration, and micelles can be introduced by intravenous injections.
- the micelles are formed from any suitable micelle forming block copolymer, including AB-type, and ABA-type.
- Exemplary micelle forming block polymers are the polymers of the P-triblock family.
- P-triblock micelles require stabilization to prevent degradation caused by significant dilution accompanying IV injection.
- Three routes of P- triblock micelle stabilization are included in the present invention The first route is direct radical crosslinking of micelles cores which results in micelle stabilization.
- a small concentration of an oil such vegetable oil (about 0.0005 percent) is introduced into diluted P-triblock solutions.
- the amount oil used is much small than that required to form an emulsion, which is about 1 percent.
- the third route is a technique based on polymerization of the temperature- responsive LCST hydrogel in the core of P-triblock micelles. The hydrogel phase is in a swollen state at room temperature, which provided for a high drug loading capacity of the system.
- P-gel Phase transitions in P-gel caused by variations in temperature or concentration were studied by the EPR.
- the p-gel micelles are stable for drug delivery, but not so stable that they cannot be degraded by the body. After a matter of weeks, the stabilized p- gels will gradually destabilize. This allows sufficient time to function effectively as a drug delivery system, but the degradation will allow eventual removal from the body. This is unlike many drug delivery systems that involve stable components that are slow to be removed from the body.
- the thermodynamics of the p-gel system direct the system toward dissolution, and instability, but the kinetics are very slow.
- Another aspect of this embodiment is the use of hydrogels that are stimuli responsive to other environmental states, such as pH.
- Other substances that are introduced into the body, other than drugs, can be encapsulated and delivered by the stabilized micelle system of the invention.
- Another aspect of the invention is the use of pulsed ultrasound to release an encapsulated drug.
- the release of drug by ultrasound is reversible, with allows a highly controlled release of drug using a pulsed ultrasound system.
- FIGS la, lb, lc. 3-dimensional size exclusion chromatograms of P-triblock samples: x-axis - retention time, mm; y axis - wave length, nm; z-axis - abso ⁇ tion. Concentration of the injected polymer 1 wt%; eluent - water, detector: UV diode array; injection volume 10 ⁇ l; temperature 30°C. (a) control P-triblock sample, (b) and (c) crosslinked samples using 5 wt% (b) and 10 wt% (c) of benzoyl peroxide in P-triblock Micelles.
- FIG. 1 The concentration of a solubilized 16-DS vs. P-triblock PLURONIC P405 concentration in aqueous solutions.
- the EPR spectra are shown for 16-DS in a 0.01 wt% (lower spectrum) and 10 wt% P-triblock solution (upper spectrum) at room temperature.
- Figure 6. A schematic diagram showing the inte ⁇ enetrating network of PPO blocks and hydrogel molecules.
- Figure 7. A schematic diagram showing the reversible collapse and reswelling of a hydrogel stabilized micelle.
- FIG. 8 The EPR spectra of 16-DS solubilized in 10% P-105/1% ⁇ oly(NiPAAm) nanoparticles.
- the probe At room temperature (upper spectrum), the probe is localized predominantly in the hydrophilic environment of a swollen hydrogel; heating the P-gel solution to 37°C (above hydrogel's LCST) results in probe re-distribution: 94% of the probe is transferred into the hydrophobic environment (lower spectrum).
- FIG. 9 The EPR spectra of 16-DS in a 10-fold diluted initial P-gel solution (final concentration 1% P-triblock/0.1% poly(NlPAAm). In a diluted solution, the hydrogel phase has much lower microviscosity than in the initial P-gel solution (compare upper spectrum of this Figure to that of Figure 8). Spectral changes with increasing temperature indicate progressive micellization of a diluted P-gel solution.
- FIG. 10 The EPR spectrum at room temperature of Rb (0.1 mM) solubilized in 10%) P-triblock solution showing the supe ⁇ osition of signals arising from two drug populations differing in motion intensity.
- Figure 12. Is a chromatogram showing the molecular weight of a NNDEA P-gel.
- Figure 13 Is graph showing preservation of hydrophobic cores of micelles at very high dilutions of P-gel samples.
- Figure 14 A scheme showing conjugation of nitroxide radical (1 -oxo-2,2,6,6- piperidone-4-hydrazone) to DOX molecule to form Rb.
- Figure 15 A graph showing polymerized NNDEA light scattering.
- Figure 16 A graph showing particle sizes of NNDEA stabilized micelles at 37°C
- Figure 18 Experimental arrangement for fiberoptic detection of fluorescence of drug under ultrasound exposure. For 20 kHz exposure, the transducer was controlled by different electronics and was inserted into the exposure bath from above.
- Figure 19 Example of release profiles of DOX from a 10%> P-triblock solution and from PBS.
- Raw and Fourier-filtered data are presented for the 10% P-triblock solution.
- ultrasound was turned on at 60s and off at 120s; there was a negligible change of DOX fluorescence under sonication.
- Figure 20 Rb release profile from 10% P-triblock P-105 micelles at CW and pulsed sonication; Rb concentration 20 ⁇ g/ml; ultrasound frequency 47 kHz, power density 3.5 W/cm 2 .
- Figure 21 DOX release profiles from 10% P-triblock P-105 micelles at CW (a) and pulsed (b-e) sonication; DOX concentration 6.7 ⁇ g/ml; ultrasound frequency 20 kHz, power density 0.058 W/cm 2 ; pulse sequence: (b)- 0.1 s "on” : 0.1 s "off; (c)- 0.5 s : 0.5 s; (d)- 1 s : 1 s; e- 1 s : 3 s.
- P-triblock PLURONIC P-105 micelles were chosen as a model polymeric micellar system.
- the rational behind the choice of P-triblock was that P-triblock can form unimers, loose water penetrated aggregates, and micelles with hydrophobic cores, and the phase state of the drug carrier can be controlled by choosing members of the P-triblock family with appropriate molecular weight, PPO/PEO block length ratio, and concentration.
- the phase state of P-triblock solutions can be characterized by the Electron Paramagnetic Resonance technique (EPR) [16].
- EPR Electron Paramagnetic Resonance technique
- reports have indicated that P-triblock solutions in concentration below 1 wt% are non-toxic [14].
- the hydrodynamic radii of P-triblock micelles at physiological temperatures range between 10 and 30 nm, which makes them prospective drug carriers.
- P-triblock P-105 with an average molecular weight of 6,500, the number of monomer units in PEO and PPO segments being respectively 37 and 56, was supplied by BASF Co ⁇ oration and used as received.
- Spin probe 16-doxylstearic acid (16-DS) was purchased from Sigma, St. Louis, MO.
- Monomers, crosslinkers, radical initiators were purchased from Polysciences, Inc., Warrington, PA.
- HL-60 promyelocytic cell line was provided by Dr. Murray (Brigham Young University, Provo, Utah), They were cultured in RPMI 1640 medium supplemented with 20%) fetal calf serum, 2mM L-glutamine, 0.2% sodium bicarbonate and 50 ⁇ g/ml gentamicin at 37°C in humidified air containing 5%> CO 2 .
- P-triblock was dissolved at 0.1, 1.0, 10, or 20 wt% in RPMI media or PBS and the solutions obtained were sterilized by filtration through a 0.2 um filter.
- Size exclusion chromatography was performed on a Spherogel TSK 6000PW (Beckman Co), with water as a mobile phase (flow rate of 1 ml/mm at 30°C); injection volume was 10 ⁇ l.
- Hewlett Packard Series 1100 Liquid Chromatograph equipped with multi- wavelength detector was used, which allowed to measure abso ⁇ tion of analytes in the range of 190 - 400 nm. The column was calibrated using PEG standards. Before the experiment, control and crosslinked P-triblock samples were diluted to the final P-triblock concentration of 1 % w/v.
- a dry powder of the EPR probe (16-DS) was incubated with unstabilized or stabilized micellar solutions of P-triblock for 15 mm under constant shaking.
- Unsolubilized probe was removed by centrifugation. The concentration of a solubilized probe was measured by the EPR. The same technique of probe introduction was used for pyrene. Water solubility of 16-DS or pyrene is very low and can be neglected. Dissolved probes are associated with P-triblock molecules and thus reflect the loading capacity, dynamics and environment of the latter. For a fluorescent drug, Rb, the same concentration of Rb (20 ⁇ g/ml) was introduced into non-stabilized and stabilized micellar P-triblock solutions at 37°C from a stock solution.
- F mes a m F m + (l-a m )F s
- Fmes is a measured fluorescence intensity
- F m is Rb fluorescence intensity when all the drug is localized in the hydrophobic core of micelles (measured in a 20%> P-triblock solution at 37°C)
- Fs is fluorescence intensity in non-micellar solutions (i.e., in PBS)
- a m is the fraction of the drug located in the hydrophobic core of micelles.
- a 16-DS spin probe was introduced into P-triblock solutions in the following manner: an aliquot of a stock solution of 16-DS in ethyl alcohol was placed at the bottom of a test tube; alcohol was evaporated in the air stream, upon which P-triblock solutions of various concentrations were added. The samples were sonicated for 15 mm to enhance spin probe solubilization.
- ⁇ PR capillaries or flat ⁇ PR cells The samples were placed in the ⁇ PR capillaries or flat ⁇ PR cells (Wilmad Glass Co ⁇ oration, Buena, NJ). ⁇ PR spectra were acquired in a Bruker (Billerica, MA) ⁇ R-200 SRC X-band EPR spectrometer (installed at the University of Utah) or Varian (Palo Alto, CA) Century Series E- 112 X-band spectrometer in the
- EPR Research Center (University of Illinois at Urbana-Champaign). Incident microwave power was set to 0.5 -2 mW to avoid saturation. Modulation frequency was 100 kHz, modulation amplitude was a quarter of a line width. EPR spectra were recorded at room temperature and at 37°C. The EPR spectra were taken sequentially, digitized, and stored with the aid of a commercial EPR software/hardware package (Scientific Software Services, Bloomington, IL). Double integrals of individual EPR signals are proportional to the corresponding spin concentrations.
- Hyperfine splitting of spectral lines characterizes the hydrophobicity of a probe environment; signal shape (pick-to-pick line width, low-field to high-field line intensity ratio) is used to measure probe motion parameters (rotational correlation time, t r , anisotropy of motion etc.).
- Lorenzian component of the line shape and double integrals of the spectra were measured by fitting of each spectrum to the inhomogeneous line shape model using a computer program described in [35]. This program provides for the separation of overlapping EPR signals. All spectra were processed in automatic mode, in which the best-fit parameters for the spectrum were used as an initial approximation for the Levenberg - Marquard optimization of the next spectrum in the sequence. Dynamic light scattering.
- Dynamic light scattering was measured using a BI 200 Spectrometer from Brookhaven Instruments equipped with a BI 2030 AT 72-channel autocorrelator. DSC data were analyzed using a BI30AT program.
- Intracellular uptake of DOX and Rb was measured using a fluorescence technique, in which compounds were excited at 488 nm and technical emission spectra were recorded between 510 - 700 nm.
- Two sets of samples were studied, one incubated and another sonicated.
- the cells were incubated at 37°C with DOX or Rb, which were either dissolved in the RPMI medium (or PBS), or the drugs were solubilized in P-triblock PLURONIC P-105 solutions of various concentrations.
- the cells were sonicated by 70 kHz ultrasound at 37°C up to 1 hour in the presence of drug to assess the effect of ultrasound on the drug uptake from molecular and micellar solutions.
- Calibration experiments showed a linear dependence of Rb or DOX fluorescence intensity on concentration in 1% SDS solutions in the concentration range of interest. Upon the completion of cell lysis, fluorescence spectra of the lysates were recorded. To quantify the concentration of lysed cells, cell lysates were filtered through 0.2 mm filters, and their optical density was measured by protein abso ⁇ tion at 280 nm (OD 280 nm). Calibration experiments showed a linear dependence of OD 280 nm on the concentration of lysed cells. The fluorescence intensity of lysates was normalized by OD 280 nm. In parallel, the depletion of the drug from the incubation medium was measured by the decrease of supernatants' fluorescence.
- P-triblock exists in aqueous solutions as individual coils, or unimers, with a size of approximately 1-2 nm [36].
- the transition proceeds from unimers to loose, water penetrated aggregates to micelles with hydrophobic cores.
- Micelle cores consist of PPO blocks.
- these micelles dissociate into loose multimolecular aggregates or unimers within minutes. (See Rolland [23].)
- This experiment shows that radical crosslinking of P-triblock micelle cores will prevent micelle dissociation.
- the crosslinking procedure is designed to confine the crosslinking to micelle cores without compromising the structure and dynamics of the PEO side chains.
- a hydrophobic radical initiator was chosen, benzoyl peroxide, that dissolves predominately in the micelle core.
- the initiator at the concentration range of 0.5 to 20 mg/ml (which corresponds to 0.25 wt% - 10 wt%o in respect to P-triblock) was introduced into 20% P-triblock PLURONIC P-105 solution under sonication (30 sec, 70 kHz).
- the solution was degassed and the crosslinking was performed by heating at 60°C for 24 hours. Micelle stabilization upon dilution was tested by size exclusion chromatography.
- This peak presumably belongs to unimers and characterizes the molecular weight distribution of the initial polymer that is found rather broad (corresponding to molecular weights from 600 D to 23,000 D, with a maximum at 7,000 D, which is close to 6,500 D reported for P-triblock PLURONIC PI 05 by the manufacturer).
- the concentration of benzoyl peroxide was 5 wt%> of P-triblock for a crosslinked- 1 sample and 10 wt% of P-triblock for a crosslinked-2 sample. Introduction of low concentrations of vegetable oil.
- the spin-probe EPR technique provides the following information on the system: solubilization efficiency (the concentration of a solubilized probe measured by the double integral of the spectral line), polarity of the probe environment (characterized by the hyperfine splitting constant, a>j), the microviscosity of the probe environment (characterized by the rotational correlation time, t rot ), and local concentrations of the probe (characterized by the line width or a shape of the spectrum at 77K).
- EPR spectra of solubilized nitroxide probes are presented by three-line signals; line shape (hyperfine splitting constant a N , peak-to-peak amplitude ratio, line width) reports hydrophobicity and microviscosity of the probe environment. Differences in the EPR spectra of a 16-DS probe solubilized in P-triblock P-105 solutions of various concentrations indicated probe transition from the hydrophilic environment of P-triblock unimers ( Figure 3, lower spectrum) to the hydrophobic environment of cores of P-triblock micelles ( Figure 3, upper spectrum; see also Figure 2).
- P-triblock PLURONIC P-105 concentration corresponding to P-triblock unimers (0.001 wt%)
- one P-triblock molecule was associated with several 16-DS molecules, which were located in the hydrophilic environment.
- P-triblock concentration up to 0.1% at room temperature
- several P-triblock molecules corresponded to the solubilization of one probe molecule; the probe was still located in the hydrophilic environment; which system is called "loose aggregates”.
- P-triblock concentrations of 1 wt%> or higher EPR spectra showed probe transfer into the hydrophobic environment indicating the formation of P-triblock micelles with hydrophobic cores [16].
- oil 1% v/v
- a 20% (w/v) P- triblock solution together with a spin probe, 16-DS; the sample was sonicated for 15 mm, upon which sequential dilutions were done to the final P-triblock concentrations of 10%, 2%, 1%, 0.1%) and 0.01%.
- sequential dilutions were made to the control sample that did not comprise oil.
- the concentration ratio of the probe in the hydrophobic and hydrophilic environment reflects the ratio of micelle-encapsulated and unimer-associated (or loose aggregates-associated) probe.
- This experiment shows a novel synthetic pathway to stabilize P-triblock micelles by polymerizing a temperature-responsive low critical solution temperature (LCST) hydrogel in the micelle core.
- the hydrogel-forming polymer produced the inte ⁇ enetrating network inside the core of P-triblock micelles.
- the rational behind this approach was that at room temperature the LCST hydrogel was in a swollen state, which provided for a very high drug loading capacity for lipophilic and hydrophilic drugs. At physiological temperatures, the micelle-encapsulated gel collapsed, "locking" the core of the micelle thus preventing micelles from rapid degradation upon dilution.
- This new drug delivery system is known under the trademark PLUROGEL, which will be generically referred to herein as P-gel.
- P-gel is a sterically protected nano-dispersed hydrogel.
- P-gel particles were measured by dynamic light scattering.
- P-gel particles were larger than P-triblock micelles (particle diameter ranged between 30 nm and 400 nm depending on P-triblock concentration, temperature, and type of a gel- forming monomer. This is to be compared to 12-15 nm for P-triblock micelles at 37°C).
- An example of particle sizes for P-gel particles is shown in Figure 16.
- P-gel particle size is advantageous for drug delivery applications.
- An example of a protocol of P-gel polymerization is given below.
- NiPAAm N- isopropylacrylamide (NiPAAm) (NI was carried out in inert atmosphere at 70°C for 24 hours.
- N,N-Diethylacrylamide (NNDEA) was polymerized in the presence of P-triblock P-105 micelles. The polymerizations resulted in an inte ⁇ enetrating network of NNDEA and P-105 that stabilizes the micelles at concentrations below the critical micellar concentration of free P-105.
- the NNDEA was crosslinked with N,N'-Bis(acryoyl)cystamine (BAC) and the degree of micellar stability was determined using dynamic light scattering and the fluorescent probe diphenylhexatriene (DPH). The increased micellar stability was not permanent and disappeared over a time period of days to weeks.
- BAC N,N'-Bis(acryoyl)cystamine
- N,N-diethylacrylamide (NNDEA) monomer was added to give concentrations ranging from 0 to 1 wt%> monomer.
- BAC was added as a crosslinking agent to give BAC:NNDEA mole ratios ranging from zero to 1 :20.
- AIBN was added as an initiator and the flask was connected to a water condenser and purged with nitrogen for at least one hour. The system was then allowed to polymerize for 24 hours at 65° C with magnetic stirring and a continuous nitrogen purge.
- the molecular weight of un-crosslinked p(NNDEA) was investigated by gel permeation chromatography using a Waters GPC system (Milford, MA) (model 515 pump with styragel columns and a model 2410 refractive index detector). Polymerization samples were dried, dissolved in tetrahydrofuran, and filtered through a 0.22 ⁇ m teflon filter before being injected into the GPC system. Molecular weights were determined using polystyrene standards and Water's Millennium 32 software.
- micellar concentration CMC
- micellar stability were studied for
- NNDEA has polymerized totally within the micellar cores and does not aggregate with other particles upon thermally induced collapse.
- the EPR and fluorescence techniques were used with 16-DS as a spin probe and DPH as a fluorescent probe, respectively.
- the probe was solubilized in the initial P-gel solution (10% P-triblock/1% poly(NiPAAm)) or 10% P-triblock 1% poly (NNDEA) at room temperature.
- the transition temperature of a gel may be set slightly above the physiological temperature; the repeated heating/cooling cycles of the tumor volume (e.g. by pulsed ultrasound) would result in a controlled drug release from nanoparticles. Effect of a P-gel dilution.
- Rotational correlation time t corr hyperfine splitting constant a N and a fraction of a probe in the hydrophobic environment for a 16-DS spin probe.
- Percentage indicates P-triblock weight concentration. A concentration of a gel- forming polymer is 10-fold lower.
- P-gel particles are expected to have a long circulation time in blood since they have protective poly(ethylene oxide) chains on their surface.
- the properties of P-gel nanoparticles described above make them excellent prospects as carriers of lipophilic drugs.
- DOX DNA intercalating anti-cancer drugs
- Rb DNA intercalating anti-cancer drugs
- DOX is widely used in clinical practice as a chemotherapeutic agent.
- DOX is cardiotoxic due to the induced production of active oxygen radicals [38, 39].
- a paramagnetic Tempo-type nitroxide radical (1 -oxo-2,2,6,6-piperidone-4-hydrazone) was conjugated to DOX molecule to form Rb ( Figure 14) [40].
- the nitroxide moiety in position 14 served as a radical trap.
- a unique Rb molecule is both fluorescent and paramagnetic, which allows fluorescence and EPR spectroscopy to be used independently in investigations of drug uptake, distribution and metabolism. This makes Rb a powerful research tool.
- Rb was used as a spin- and fluorescent probe to monitor drug distribution in P- triblock micelles and P-gel nanoparticles. Drug localization in P-triblock micelles in the absence of a hydrogel.
- Rb fluorescence is quenched in collisions with water molecules; when Rb molecules are screened from collisions with water, theft fluorescence increases manifold. This phenomenon was used to study P-triblock P-105 micellization [31]: As illustrated in Figure 23, Rb fluorescence increased sha ⁇ ly upon the onset of micelle formation in P-triblock solutions; as could be expected (see, e.g.[22]), P-triblock concentration corresponding to the onset of micelle formation decreased with increasing temperature.
- p-gel micelles are stable for drug delivery, but not so stable that they cannot be degraded by the body. After a matter of weeks, the stabilized p- gels will gradually destabilize. This allows sufficient time to function effectively as a drug delivery system, but the degradation will allow eventual removal from the body.
- thermodynamics of the p-gel system direct the system toward dissolution, and instability, but the kinetics are very slow.
- HL-60 cells were incubated at 37°C with DOX or Rb.
- the drugs were either dissolved in the RPMI medium (or PBS), or they were solubilized in P- triblock PLURONIC PI 05 solutions of various concentrations.
- FIG. 18 The apparatus employed a single-line argon-ion laser (Ion Laser Technology, Model 5500 A) whose beam was divided by a variable beam splitter (a graded metal-film neutral density filter). One portion of the beam was sent directly to a silicon photodetector (Newport Model 818-SL with 835 display) to monitor the laser power. The other portion was directed into the glass cuvette containing the trial solution to be sonicated. It was designed in collaboration with Dr. Christensen (University of Utah); details of the design will be described elsewhere.
- pulsed ultrasound appears to be superior to CW since pulse, and pulse duration and sequence can be carefully controlled. Also, heating and burning of skin can be prevented by the application of pulsed ultrasound with appropriate pulse sequences.
- a custom ultrasonic exposure chamber with real-time fluorescence detection was used to measure acoustically-triggered drug release from P-triblock PLURONIC P-105 micelles under continuous wave (CW) or pulsed ultrasound in the frequency range of 20 kHz to 90 kHz .
- the measurements were based on the decrease in fluorescence intensity when drug was transferred from the micelle core to the aqueous environment.
- Two fluorescent drugs were used: doxorubicin (DOX) and its paramagnetic analogue, ruboxyl (Rb).
- DOX doxorubicin
- Rb ruboxyl
- P-triblock PLURONIC P-105 at various concentrations in aqueous solutions was used as a micelle-forming polymer. Drug release was highest at 20 kHz ultrasound and dropped with increasing ultrasonic frequency despite much higher power densities.
- Fluorescence of the drug was excited at an excitation wavelength of 488 nm with an optical power of approximately 0.5 mW in a 2 mm diameter beam. At this light intensity no photobleaching was observed, based on the constant level of drug fluorescence during continuing irradiation for 8 hours.
- the drug release was quantified by measuring the changes in fluorescence emissions before, during, and after the ultrasound exposure.
- a fiberoptic probe (a sheathed bundle of multimode glass fibers, 3 mm entrance diameter, 0.6 numerical aperture, and 90 cm in length) was used to collect the fluorescence emission.
- the light passed through a dielectric bandpass filter with a 35 nm bandwidth centered at 535 nm (Omega Optical Model 535DF35) to a sensitive silicon detector (EG&G Model 450-1).
- the filter effectively cuts off emissions below 500 nm, including any Rayleigh-scattered laser light.
- the detector signal was digitized using a 12- bit A/D converter (National Instruments) and sent, along with the digitized monitor photodetector signal, to a Macintosh computer for storage and processing.
- the analog outputs of both photodetectors were also plotted on a stripchart recorder.
- the temperature of the ultrasonic exposure chamber was maintained at 37°C by circulating thermostated water throughout the sonicating bath.
- the glass cuvette used to measure drug release had two open tubes for filling or removing drug solution and one sealed tube in the middle to allow the excitation beam to enter the solution through a flat stationary surface. This prevents any distortions that could otherwise arise from waves on the surface of the sonicated liquid.
- the main chamber of the cuvette was completely filled with the solution, and the excess liquid partially filled the side tubes.
- Digitized data were analyzed, to calculate the percentage of drug release from micelles. To reduce the noise, the data were Fourier transformed, and a small magnitude narrow band noise of unknown origin and its next three harmonics were filtered out. After Fourier filtering, the data were smoothed using a 10 point moving average. An example of the raw and filtered data is presented in Figure 19 for DOX release from 10% P- triblock micelles.
- F us is fluorescence during exposure to ultrasound.
- the data also showed the time required to release the drug from micelles and the time required for drug re-encapsulation once the ultrasound was turned off.
- Drug release as a function of ultrasound frequency was explored in a low- frequency range, from 20 to 90 kHz; both CW and pulsed ultrasound was investigated.
- the ultrasound power density was varied from 0 to 3 W/cm 2 as measured by a hydrophone as described earlier [34].
- the 20-kHz ultrasound was generated by a probe transducer (Sonics and Materials, Newton, CT) inserted into the water bath; sonication at 47 kHz was performed in a Cole-Parmer sonication bath (Cole-Parmer Inc., Mount Vernon, EL); sonication at 67 and 90 kHz was performed in two different Sonicor SC 100 sonication baths (Sonicor Instruments, Copaique, NY).
- the power density was controlled by adjusting the a.c. input voltage with a Variac.
- the 20-kHz ultrasound probe was programmed to generate continuous wave (CW) or pulsed ultrasound of varying power densities and duty cycles; in the pulsed experiments both "ultrasound on” and “ultrasound off durations were varied. For the sonication baths, pulses were generated by turning the instruments on and off manually.
- DMPO 5,5- dimethyl- 1-pyrroline-N-oxide
- drug release was measured from micelles under CW or pulsed ultrasound in the frequency range of 20 kHz to 90 kHz.
- DOX release was 10%) ⁇ 1% (mean and s.d.), while at a concentration of 30 ⁇ g/ml, the release was 5.5%) + 1% (at 67 kHz and 2.8 W/cm power density).
- the lower drug release at the lower drug concentration could be attributed to a higher ratio of PPO to DOX in the hydrophobic core of P-triblock micelles, which favors hydrophobic interaction. It is postulated that increased hydrophobic interaction reduces percentage of drug that can be released from micelle core upon the application of ultrasound. This is confirmed by the above mentioned lower release of Rb in comparison to DOX.
- drug/PPO hydrophobic interactions are replaced by weaker drug/drug interactions, which facilitates drug release. Radical formation under sonication
- the threshold for transient cavitation was measured by trapping radicals that were produced upon the collapse of cavitation bubbles. Cavitation threshold increased with increasing ultrasound frequency; at 20 kHz, radicals were observed even at a power density as low as 0.01 W/cm 2 , which is consistent with the relatively high efficiency of 20-kHz ultrasound for drug release from micelles. At 67 kHz, no radicals and no drug release were observed below a power density of 1.0 W/cm .
- transient cavitation plays an important role in triggering drug release from micelles. It is hypothesized that shock waves produced by transient cavitation events disrupt micelles and release drug into aqueous environment. During the "ultrasound off phase, the micelles are restored and drug is re-encapsulated, which takes less than 1 s at 37°C.
- micellar drug delivery with acoustic activation of micelles may be developed into a new technique of drug targeting to tumors.
- micellar drug delivery is believed to be an effective therapeutic technology for targeted delivery of drugs to solid tumors.
Abstract
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Claims
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