CN1927400B - Biomagnetism nano target anti-cancer drug and its preparation - Google Patents
Biomagnetism nano target anti-cancer drug and its preparation Download PDFInfo
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- CN1927400B CN1927400B CN2006100964093A CN200610096409A CN1927400B CN 1927400 B CN1927400 B CN 1927400B CN 2006100964093 A CN2006100964093 A CN 2006100964093A CN 200610096409 A CN200610096409 A CN 200610096409A CN 1927400 B CN1927400 B CN 1927400B
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Abstract
The invention relates to a biological nanometer magnetic target anti-cancer drug, which is formed by coupled biological nanometer magnetic smalls and anti-cancer chemical drug. Wherein, the biological nanometer magnetic small is nanometer magnetic particle formed in magnetic bacterial cell, at 35-120nm diameter, while its main component is Fe3O4 or Fe3S4; single magnetic particle is packed by film; and the coupled anti-cancer chemical drug comprises chemotherapy drug, nucleic acid drug, radioactive nuclide or antibody drug. The invention also provides relative production that using physical adsorption couple or based on the active function group contained by smalls and anti-cancer drug, to select right coupler via chemical reaction to couple them. And the smalls has better bioavailability, less side effect, therefore, the anti-cancer drug can be accurately transmitted to ill part via external magnetic field, to reduce the contact between drug and normal organism., to realize target positioning treatment.
Description
Technical field
The present invention relates to a kind of Biomagnetism nano target anti-cancer drug, is to be a kind of magnetic target medicine of carrier with biological nano magnetic corpusculum.
Background technology
At present, cancer is the pertinacious disease that human mortality rate ranks first, so far, traditional generalization therapy is still adopted in treatment of cancer mostly, because chemicals distributes at patient's whole body, patient's normal tissue cell is produced poison, there are many side effect in the clinical use, for example alopecia, spinal cord toxicity, liver, renal failure etc., cause untoward reaction, cause the cancer patient agonizing, a lot of patients die from cancer itself, but die from the side effect that chemotherapy causes.
In order to improve the chemotherapy of tumors effect, alleviate the toxic and side effects of medicine to normal structure, in recent decades, be that the various newtype drug delivery systems of purpose become the research focus with local targeting locating therapy.Wherein magnetic target therapy shows great application prospect in the local targeting locating therapy of cancer, is expected to progressively replace systemic chemotherapy, becomes a kind of efficient, safe cancer treatment method.Magnetic target therapy is to be pharmaceutical carrier with the magnetic nanoparticle, utilize the special superparamagnetism energy of magnetic grain, outside magnetic field promotes down, make medicine have accurately initiatively targeting, the penetration power that medicine cell membrane and blood brain barrier are powerful, directly act on tumor cell, thereby improve the curative effect of medicine, and reduce effectively to Normocellular toxic and side effects.
At present, the research of the magnetic target therapy preparation that mainly concentrates on synthetic ferrite magnetic nano medicine-carried system and magnetic liquid material with and correlation technique to the early diagnosis and therapy of malignant tumor.Studies show that the magnetic nanoparticle of synthetic can be connected with multiple cancer therapy drug, have stronger targeting positioning function, and be successfully applied to the early diagnosis and therapy of certain cancers.Yet owing to technical reason, the magnetic granule particle shape difference of synthetic is big at present, and size is inhomogeneous, and particle size distribution is wide, and the ratio that reaches nano-scale particle only accounts for fraction; In addition, also because inorganic non-metallic nano grain surface charge density is on the low side, easily assemble, the reason of difficult degradation causes the medicine useful load low, instability, and toxicity is bigger, and range of application is limited to.
Biological nano magnetic corpusculum claims bacillus nanometer magnetosome (magnetosome) again, it is a kind of novel nano biological magnet of just finding the nearest more than ten years, it is the nano magnetic particle that forms in having the gram negative bacteria-magnetotactic bacteria of magnetotaxis (magnetotactic bacteria) cell, magnetic grain size is in the 35-120nm scope, within the brilliant size range of permanent single magnetic domain, main component is Fe
3O
4Or Fe
3S
4, each magnetic particle surface is by the phospholipid lipid bilayer bag quilt of chimeric protein, the good dispersion in solution of the bacillus nanometer magnetosome after the separation and purification, and stability is high.At present, through fixed enzyme vector, immune detection, gene transfer, the result of study of aspects such as the separation of DNA, RNA and labelling shows, bacillus nanometer magnetosome immobilized enzyme amount big (but intelligent worker's synthesizing magnetic nano-particle 100 times), antibody connection amount is big, the sensitivity of immune detection height, the DNA adsorbance is big, and application prospect is boundless.But because magnetotactic bacteria is to the requirement harshness of nutrition, and have characteristic such as little aerobic, anaerobism, and its cultivation level is very low always, and being difficult to obtain q.s high-purity magnetic corpusculum provides related experiment to use, up to now, Shang Weijian adopts the report that biological nano magnetic corpusculum carries out the research of medicine carrying aspect.Recently, China Agricultural University has set up the method for a large amount of cultivation magnetotactic spirillums (M.gryphiswaldense) MSR-1, the easy magnetic corpusculum recovery and the technology path of purification have been found out, by broken magnetotactic bacteria cell, extract and the interior synthetic nanometer magnetic granule of purification magnetotactic bacteria cell, and obtained national patent (patent No. 02125920.8,03153488.0).
Summary of the invention
The objective of the invention is demand at the targeted therapy tumor, solve that the ubiquitous drug loading of present synthetic nano-magnetic medicinal carrier is low, problem such as poor stability, toxicity height and targeting are not strong, utilize the natural resources of this nanoscale of biological nano magnetic corpusculum, stable crystal form to be pharmaceutical carrier, prepare a kind of have suitable particle diameter and particle shape, high drug load and entrapment efficiency, biocompatibility is good, easily degraded, toxicity is low, nano magnetic target medicine with anti-cancer function and biological nano magnetic corpusculum medicine-carrying method that stability is high.
Biomagnetism nano target anti-cancer drug of the present invention is formed by physical absorption or chemical coupling agent coupling by biological nano magnetic corpusculum and cancer therapy drug, said biological nano magnetic corpusculum is the nano magnetic particle that forms in the magnetotactic bacteria cell, particle diameter 35-120nm, main component is Fe
3O
4Or Fe
3S
4, single magnetic granule has adipose membrane bag quilt, and said anticancer chemicals comprises: chemotherapeutics, antibody drug, nucleic acid drug, radionuclide.Chemotherapeutics commonly used as: amycin, epirubicin, daunorubicin, darubicin, pirarubicin, mitomycin, Bleomycin A5, peplomycin, Yi Li are for health, paclitaxel etc.; Commonly used nucleic acid drug is as cytosine arabinoside, methotrexate, hydroxyurea, hypoxanthine etc.; Radionuclide commonly used as:
99mTc,
131I,
123I,
111In etc.); Antibody drug commonly used as: Herceptin (He Saiting), Rituxan (Rituximab), U.S. appropriate former times monoclonal antibody, Zevalin, Bexxar, Edrecolomab, muromonab, Trastuzumab, Alemtuzumab etc., comprise polytypes such as anti-IgG1, anti-CEA, anti-CD3, anti-CD 33, anti-CD52, anti-CD20, anti-ERBB2, anti-IGE Fc, anti-EpCAM.
Biomagnetism nano target anti-cancer drug of the present invention can be prepared by following method respectively:
One. physisorphtion
Step is as follows:
The pure water or the buffer solution of anticancer chemicals, biological nano magnetic corpusculum are mixed, stir or impulse ultrasound reaction down discontinuously; With Magnet sucking-off magnetic corpusculum, with pure water or phosphate buffer solution washing several, reuse Magnet sucking-off magnetic corpusculum gets product.
The method of this law by physical absorption is adsorbed on the surface of magnetic corpusculum with cancer therapy drug, is applicable to nearly all anticancer chemicals.
Two. chemical coupling method
The adipose membrane of biological nano magnetic corpusculum expoeridium is made up of the phospholipid bilayer of chimeric protein, and its biochemical property is consistent with cell membrane with function.The at present existing three kinds of synthetic magnetic corpusculum of magnetotactic bacteria bacterial strain adipose membrane components are proved: the peplos lipid of the magnetic corpusculum of Magnetospirillum.Magnetotacticum MS-1 comprises three parts: neutral lipid and free fatty, glycolipid and thioester, phosphoric acid fat, their shared percentage by weights in total fat are respectively 8%, 30%, 62%, and wherein the main component of phosphoric acid fat is Phosphatidylserine and PHOSPHATIDYL ETHANOLAMINE; The phospholipid bilayer of the magnetic corpusculum peplos of M.magneticum AMB-1 contains 98% the lipid and the complex of 2% other components, every milligram of magnetic corpusculum contains the fatty acid of 31.79 micrograms, wherein palmitoleic acid and oleic acid account for 90%, phospholipid accounts for 58% of total lipid, and PHOSPHATIDYL ETHANOLAMINE then accounts for 50% of phospholipid.Biochemical analysis to the magnetic corpusculum peplos of M.gryphiswaldense MSR-1 shows, similar on phospholipid that it contains and fatty acid and the subcellular organelle adventitia all exists PHOSPHATIDYL ETHANOLAMINE and phosphatidyl glycerol in a large number.
The present invention makes full use of the said components and wherein abundant amino, adjacent glycol, the carboxyl isoreactivity group of biological nano magnetic corpusculum surface adipose membrane, adopt different methods of attachment and specific coupling agent, be connected the preparation Biomagnetism nano target anti-cancer drug with various cancer therapy drugs at different chemical groups.
The chemical coupling method that the present invention adopts has following several:
1, directly coupling method is realized amino with amino being connected
Because containing important component in the adipose membrane of magnetic corpusculum expoeridium is PHOSPHATIDYL ETHANOLAMINE, have a large amount of primary amino radicals, on the other hand, common cancer therapy drug almost all contains amino, as chemotherapeutics such as amycin, epirubicin, daunorubicin, darubicin, pirarubicin and cytosine arabinoside, methotrexate, hydroxyurea, hypoxanthine etc.) etc. nucleic acid drug, all contain an amino in the chemical formula; Chemotherapeutics such as mitomycin, Bleomycin A5, peplomycin and Herceptin (He Saiting), Rituxan (Rituximab), U.S. appropriate former times, the medicines such as radionuclide of monoclonal antibody, Zevalin, Bexxar, Edrecol omab, muromonab, antibody drug, albumen or antibody labelings such as Trastuzumab, Alemtuzumab then contained a plurality of amino.The present invention selects for use specific coupling agent to be connected with amino according to these characteristics, realizes the coupling of nano biological magnetic corpusculum adipose membrane and chemicals.
Coupling method is as follows:
Raw material cancer therapy drug and biological nano magnetic corpusculum adipose membrane contain amino respectively, adopting coupling agent, said coupling agent is the chemical compound that contains following at least two identical or groups inequality: aldehyde radical, carboxyl/anhydride, N-butanimide ester group, N-butanimide sodium sulfonate ester group or carbodiimides ester group; The coupling agent that wherein contains N-butanimide ester group, N-butanimide sodium sulfonate ester group or carbodiimides ester group also can and get by carboxylic reagent and the reaction of N-maloyl imines, N-maloyl imines sodium sulfonate or 1-ethyl-3-(3-diformazan ammonia n-pro-pyl) carbodiimides (EDC).Can the two reacts simultaneously with coupling agent and biological nano magnetic corpusculum and cancer therapy drug, a step coupling, also can with coupling agent earlier with the little precursor reactant of biological nano magnetic, and then with cancer therapy drug reaction, two step couplings.
Coupling agent is preferably from glutaraldehyde, Biformyl, malonaldehyde, disuccinimidyl suberate (N, N '-Disuccinimidyl suberate, DSS), N, N '-two succinimidyl carbonate (N, N '-Disuccinimidyl carbonate, DSC), 3-butanimide-amino triacetate, succinic acid/acid anhydride or 1,3-propanedicarboxylic acid/acid anhydride.
With the coupling agent glutaraldehyde is example, and the coupling reaction general formula is as follows:
Black formula ball represents to be surrounded by the living nanometer magnetic corpusculum of adipose membrane in the formula, and rectangle frame is represented cancer therapy drug, down together.
Above coupling agent also can be earlier and biological nano magnetic corpusculum and polyamino acid, Polyethylene Glycol or polysaccharide base polymer one step or stepwise reaction, biological nano magnetic corpusculum is connected with polymer earlier, biological nano magnetic corpusculum and N-maloyl imines or 1-ethyl-3-(3-diformazan ammonia n-pro-pyl) carbodiimides 9 (being called for short EDC) reaction of polymer will have been connected again, make the carboxyl on the polymer be converted into N-butanimide ester group or carbodiimides ester group, with the reaction of raw material cancer therapy drug, get product then.
With polyglutamic acid (be called for short PLGA, down with) is example, the coupling process as shown in the formula:
Polymer P LGA molecule contains amino and a plurality of carboxyls, in the following formula, make biological nano magnetic corpusculum and polymer P LGA coupling by the coupling agent glutaraldehyde, it is connected go up a plurality of carboxyl functional groups, under the effect of EDC, make each carboxyl be converted into butanimide ester group or carbodiimides ester group then, be connected with the amino of cancer therapy drug then, thus the coupling of realization nanometer magnetic corpusculum and cancer therapy drug.This law utilizes polymer to contain the characteristic of a plurality of carboxyls, can make the more cancer therapy drug of magnetic corpusculum coupling.
2. oxidizing process realizes adjacent diol structure and amino being connected
The biological nano magnetic corpusculum that contains adjacent glycol group for adipose membrane, the present invention oxidant sodium metaperiodate or Potassium metaperiodate. and the little precursor reactant of biological nano magnetic, adjacent diol structure in the adipose membrane molecule of magnetic corpusculum surface is oxidized to aldehyde radical, with the cancer therapy drug reaction that contains amino, gets product then.
Reaction expression is as follows:
3. indirect method realizes carboxyl and amino being connected
Contain carboxyl for the side in raw material cancer therapy drug and the biological nano magnetic corpusculum, the opposing party is contained amino situation, the present invention is earlier with N-maloyl imines or 1-ethyl-3-(3-diformazan ammonia n-pro-pyl) carbodiimides (being called for short EDC) and a carboxylic side reactor, carboxyl is converted into butanimide ester group or carbodiimides ester group, with a side reactor that contains amino, get product then.
Reaction scheme is as follows:
4. realize being connected of amino and sulfydryl or disulfide bond with special-shaped coupling agent
In view of the adipose membrane of biological magnetic corpusculum expoeridium contains a large amount of primary amino radicals, on the other hand, nearly allly contain the characteristics that antibody and protein medicaments (as the radionuclide of antibody drug, albumen or antibody labeling, part chemotherapeutics etc.) all have disulfide bond or sulfydryl, the present invention selects special-shaped coupling agent amido modified to biological nano magnetic corpusculum for use, make it connect disulfide bond or dimaleoyl imino, be connected with the medicine that contains sulfydryl then; Or the disulfide bond reduction that again the magnetic corpusculum has been connected becomes sulfydryl, is connected with the cancer therapy drug that contains disulfide bond again; This method makes the side in cancer therapy drug and the biological nano magnetic corpusculum be connected with disulfide bond by different approaches, and the opposing party is connected with sulfydryl, utilizes the exchange reaction realization magnetic corpusculum of disulfide bond and sulfydryl and the coupling of cancer therapy drug; Perhaps a side is connected with dimaleoyl imino, and the opposing party is connected with sulfydryl, realizes the coupling of magnetic corpusculum and cancer therapy drug with the sulfydryl substitution reaction.
The special-shaped coupling agent that is adopted is meant that an end contains aldehyde radical, carboxyl/anhydride, N-butanimide ester group, N-butanimide sodium sulfonate ester group or carbodiimides ester group, and the other end contains the chemical compound of disulfide bond or N-succinimido.Wherein the end coupling agent that contains N-butanimide ester group, N-butanimide sodium sulfonate ester group or carbodiimides ester group also can and get by carboxylic reagent and the reaction of N-maloyl imines, N-maloyl imines sodium sulfonate or 1-ethyl-3-(3-diformazan ammonia n-pro-pyl) carbodiimides (EDC).
Special-shaped coupling agent commonly used has 3-(2-pyridine dimercapto) propanoic acid N-hydroxy-succinamide ester (SPDP), N-(6-dimaleoyl imino hexylyloxy) butanimide (EMCS), N-(4-dimaleoyl imino butyryl acyloxy) butanimide (GMBS), N-(4-dimaleoyl imino acyloxy in the last of the ten Heavenly stems) butanimide (HMCS), N-(4-dimaleoyl imino butyryl acyloxy) butanimide (GMBS), N-(4-dimaleoyl imino undecanoyl oxygen base) butanimide (KMUS), N-(6-dimaleoyl imino hexylyloxy) butanimide sodium sulfonate (Sulfo-EMCS), N-(4-dimaleoyl imino butyryl acyloxy) butanimide sodium sulfonate (Sulfo-GMBS), N-(4-dimaleoyl imino acyloxy in the last of the ten Heavenly stems) butanimide sodium sulfonate (Sulfo-HMCS), N-(4-dimaleoyl imino butyryl acyloxy) butanimide sodium sulfonate (Sulfo-GMBS), N-(4-dimaleoyl imino undecanoyl oxygen base) butanimide sodium sulfonate (Sulfo-KMUS) etc.
Contain sulfydryl or disulfide bond for cancer therapy drug, biological nano magnetic corpusculum contains amino situation, employing this law is as follows: with special-shaped coupling agent and the little precursor reactant of biological nano magnetic, make the magnetic corpusculum connect disulfide bond or N-succinimido, get product by one of following three process routes then: the magnetic corpusculum that (1) will connect disulfide bond or N-succinimido directly reacts with the cancer therapy drug that contains sulfydryl; (2) will contain the cancer therapy drug of disulfide bond and dithiothreitol, DTT (being called for short DTT) reaction and make disulfide bond be converted into sulfydryl, again with the little precursor reactant of magnetic that has been connected disulfide bond or N-succinimido; (3) connected the magnetic corpusculum of disulfide bond and DTT reaction and made disulfide bond be converted into sulfydryl, then with the cancer therapy drug reaction that contains disulfide bond.
Process route (1) and (2) are contained in following formula: (representing in the formula that the medicine that contains sulfydryl can get by containing the reaction of disulfide bond medicine and dithiothreitol, DTT)
With 3-(2-pyridine dimercapto) propanoic acid N-hydroxy-succinamide ester (SPDP) is special-shaped coupling agent:
Be special-shaped coupling agent perhaps with N-(6-dimaleoyl imino hexylyloxy) butanimide (EMCS):
Process route (3) is as shown in the formula expression:
All contain amino situation for cancer therapy drug and biological nano magnetic corpusculum, adopt this law as follows:
Biological nano magnetic corpusculum and cancer therapy drug are reacted with special-shaped coupling agent respectively, making magnetic corpusculum and cancer therapy drug connect disulfide bond or a side respectively connects disulfide bond the opposing party and connects the N-succinimido, to wherein connect the reaction of side of disulfide bond and dithiothreitol, DTT again and be reduced into sulfydryl, with unreduced the opposing party's reaction, get product then.
When cancer therapy drug and magnetic corpusculum react with same special-shaped coupling agent 3-(2-pyridine dimercapto) propanoic acid N-hydroxy-succinamide ester (SPDP) respectively, this law such as reaction scheme (a).
When cancer therapy drug and magnetic corpusculum during respectively with different special-shaped coupling agent N-(6-dimaleoyl imino hexylyloxy) butanimide (EMCS) and magnetic corpusculum and the reaction of 3-(2-pyridine dimercapto) propanoic acid N-hydroxy-succinamide ester (SPDP), this law such as reaction scheme (b).
Route (a): route (b):
This method also can be before a side reactor of above special-shaped coupling agent and cancer therapy drug or nanometer magnetic corpusculum, earlier with polyamino acid, Polyethylene Glycol or the reaction of polysaccharide base polymer, coupling agent is connected with polymer, again with N-maloyl imines or the reaction of 1-ethyl-3-(3-diformazan ammonia n-pro-pyl) carbodiimides, make the carboxyl on the polymer be converted into N-butanimide ester group or carbodiimides ester group, then with this side's raw material reaction.
With polyglutamic acid (being called for short PLGA) is polymer, and 3-(2-pyridine dimercapto) propanoic acid N-hydroxy-succinamide ester (SPDP) is a coupling agent, and reaction scheme is as follows:
In the above various coupling methods of the present invention, each step reaction is all stirred in pure water or phosphate buffer solution or is carried out under the impulse ultrasound discontinuously, and each reaction afterproduct of biological nano magnetic corpusculum is all with Magnet sucking-off and pure water or buffer solution washing.
The present invention provides multiple selection for the magnetic corpusculum with coupling method between the medicine with different physicochemical properties, and various coupling methods all carry out under room temperature (0-45 ℃) normal pressure, and are simple for process.
In sum, the present invention utilizes outer adipose membrane particular structure feature of biological nano magnetic corpusculum and active function groups, adopts various physics and chemical method with biological nano magnetic corpusculum and cancer therapy drug coupling, makes Biomagnetism nano target anti-cancer drug.The biological nano magnetic corpusculum that is loaded with cancer therapy drug can be realized the release of medicine by degraded magnetic corpusculum in patient's body.By externally-applied magnetic field, Biomagnetism nano target anti-cancer drug can be transported to lesions position exactly, and the minimizing medicine contacts with normal structure, reduces side effect, improves drug effect, realizes the local targeting locating therapy at tumor.Because biological nano magnetic corpusculum from active somatic cell, has superior biocompatibility, toxic and side effects is little; Biological nano magnetic corpusculum grain type unanimity, uniform particles, stable crystal form does not have other impurity; Its size has bigger specific surface area all at nanoscale; The big and scalable of electronegativity is difficult for assembling; Because the monomer of each magnetic corpusculum all has adipose membrane bag quilt, active group is abundant simultaneously, and various chemical modifications can be carried out in the surface, are connected with multiple medicine easily.Therefore, this biomagnetism targeted anticancer medicine is showing tempting application prospect aspect the targeting locating therapy tumor.
The specific embodiment
Below the reagent and the instrument that adopt among each embodiment be described as follows:
Biological nano magnetic corpusculum: got by MSR-1 magnetotactic bacteria strain culturing, particle diameter is 40-50nm, is provided by Biology College, Chinese Agriculture Univ.'s department of microbiology.
Anticancer chemotherapeutic agent amycin (ADM): purchase in Pharmaceutical Technology Co., Ltd of Beijing Yun Di Tontru.
Water: secondary deionized water.
Solvent for use such as chloroform, ethanol, dioxane, hydrochloric acid, sodium hydroxide and reagent are analytical pure.
Checkout equipment: day island proper Tianjin UV-3100 type ultraviolet-visible spectrometer, thick 1cm of quartz cell and 0.5mm;
KQ-100 type ultrasonic oscillator;
The biological magnetic corpusculum of embodiment 1 physical absorption legal system carries amycin
Experimental procedure:
Anticancer drugs, doxorubicin solution and biological nano magnetic corpusculum suspension are mixed, stir or impulse ultrasound reaction 0.5-24 hour down discontinuously, 4 ℃ of placements are spent the night; With Magnet absorption magnetic corpusculum, with pure water or buffer solution washing several, reuse Magnet absorption magnetic corpusculum gets product.
Experimental result:
By table one result as can be seen:
(1) adopt the direct physical absorption method, biological nano magnetic corpusculum (BMP) is to amycin (AMS) drug loading height, and this experiment reaches as high as 11.07 * 10
3μ gADM/mgBMP, and the content 57.5 μ g of amycin in the common synthetic ferrite magnetic nano granule, visible biological nano magnetic corpusculum drug loading is higher nearly 192 times than it.
(2) adopt the direct physical absorption method, the drug loading size is closely related with both concentration and concentration ratio, with C
AMS/ C
BMPThe increase of value, the unit drug loading of BMP also significantly increases.
(3) the BMP absolute concentration also influences its drug loading strongly.Under the lower condition of BMP concentration (0.018mg/ml), at C
AMS/ C
BMPValue is that 1: 1 o'clock its unit drug loading is 431.1 μ gADM/mgBMP, is particulate 7.5 times of synthetic ferrite magnetic nano.But when BMP concentration is increased to 0.2mg/ml, even at C
AMS/ C
BMPValue is that 1.6: 1 o'clock its unit drug loading only are 101.6 μ gADM/mgBMP, is about particulate 1.8 times of synthetic ferrite magnetic nano.
Table one, variable concentrations than biological nano magnetic corpusculum (BMP) under the condition to amycin (AMS) drug loading data:
| Medicine-carrying method | C ADM (mg/ml) | C BMP (mg/ml) | C ADM/C BMP | Drug loading (μ gADM/mgBMP) |
| The direct direct absorption method of the direct absorption method of the direct absorption method of the direct absorption method of the direct absorption method of the direct absorption method of the direct absorption method of absorption method | 0.018 0.018 0.018 0.018 0.018 0.018 0.018 0.32 | 0.0006 0.0012 0.0024 0.018 0.135 0.27 0.54 0.2 | 30∶1 15∶1 7.5∶1 1∶1 1∶7.5 1∶15 1∶30 1.6∶1 | 11.07×10 3 7.529×10 3 3.724×10 3 431.1 48.5 26.4 11.4 101.6 |
C wherein
ADM, C
BMPEach represents amycin, the biological nano magnetic corpusculum concentration of back in mixed system that feeds intake, C
ADM/ C
BMPBe the concentration ratio of amycin and biological nano magnetic corpusculum, below each the table with.
The biological magnetic corpusculum of embodiment 2 direct coupling substep legal systems carries amycin
Experimental procedure:
Add coupling agent in the little liquid suspension of magnetic, mixing stirs or impulse ultrasound reaction 0.5-24 hour discontinuously, product Magnet sucking-off, and the washing several is removed unreacted coupling agent, and then is adsorbed with Magnet.Amycin solution is mixed with above-mentioned magnetic corpusculum after coupling agent is modified, stir or impulse ultrasound discontinuously repeatedly, reacted 0.5-24 hour, product is adsorbed with Magnet, remove unreacted anticancer chemicals, after pure water or the phosphate buffer solution washing for several times, reuse Magnet sucking-off magnetic corpusculum gets product.
Experimental result:
From table two result as can be seen:
(1) adopts the direct coupling method of fractional steps, biological nano magnetic corpusculum (BMP) is higher to amycin (AMS) drug loading, make coupling agent with glutaraldehyde in this experiment and can reach 536.4 μ gADM/mgBMP, usually high nearly 10 times of the content of amycin in the synthetic ferrite magnetic nano granule.
Directly coupling method of fractional steps drug loading size is relevant with both concentration and concentration ratio, with C
AMS/ C
BMPThe increase of value, the corresponding increase of unit drug loading of BMP.But compare with direct absorption method, directly coupling method of fractional steps drug loading is more stable relatively, and changing does not have the remarkable of direct physical absorption method.
(2) with directly absorption method is different, and directly the absolute concentration of the drug loading of the coupling method of fractional steps and BMP relation does not have direct relation.BMP concentration raises, and its drug loading unit of unit drug loading changes not obvious 40 times the time.
(3) different coupling agents is very big to the drug loading influence.The drug loading of the drug loading of BMP when adopting DSC when adopting glutaraldehyde and succinic acid as coupling agent as coupling agent.
Biological nano magnetic corpusculum (BMP) amycin (ADM) drug loading data under table two, the direct coupling method of fractional steps different condition of employing:
| Medicine-carrying method | Coupling agent | Coupling agent concentration | C BMP (mg/ml) | C ADM (mg/ml) | C ADM/C BMP | Drug loading (μ gADM/mgBMP) |
| The direct direct coupling method of fractional steps of the direct coupling method of fractional steps of the direct coupling method of fractional steps of the direct coupling method of fractional steps of the direct coupling method of fractional steps of the direct coupling method of fractional steps of the direct coupling method of fractional steps of the direct coupling method of fractional steps of the coupling method of fractional steps | Glutaraldehyde glutaraldehyde glutaraldehyde glutaraldehyde glutaraldehyde glutaraldehyde glutaraldehyde succinic acid DSC | 2.5％ 2.5％ 2.5％ 2.5％ 2.5％ 2.5％ 2.5％ 0.08mol/L 0.1mol/L | 0.02 0.05 0.1 0.2 0.05 0.16 0.1 0.02 0.2 | 0.2 0.2 0.2 0.32 0.05 0.2 0.05 0.04 0.4 | 10∶1 4∶1 2∶1 1.6∶1 1∶1 0.8∶1 1∶2 2∶1 2∶1 | 536.4 252.3 234.1 176.3 144.6 158.4 138.8 145.7 36.4 |
The biological magnetic corpusculum of embodiment 3 direct coupling one-step method systems carries amycin
Experimental procedure:
The little liquid suspension of biological nano magnetic is mixed with the solution of coupling agent aqueous solution and anticancer chemicals, stir or impulse ultrasound reaction 0.5-24 hour down discontinuously,, remove unreacted reactant with Magnet adsorption reaction product.After pure water or the phosphate buffer solution washing for several times, reuse Magnet sucking-off magnetic corpusculum gets product.
Experimental result:
Table three, adopt under the direct coupling one-step method different condition biological nano magnetic corpusculum (BMP) to amycin (ADM) drug loading data:
| Medicine-carrying method | Coupling agent | Coupling agent concentration | C BMP(mg/ml) | C ADM(mg/ml) | C ADM/C BMP | Drug loading (μ gADM/mgBMP) |
| The direct direct coupling one-step method of the direct coupling method of fractional steps of the direct coupling one-step method of the direct coupling one-step method of the direct coupling one-step method of the direct coupling one-step method of coupling one-step method | Glutaraldehyde glutaraldehyde glutaraldehyde glutaraldehyde glutaraldehyde succinic acid DSC | 2.5％1.5％0.5％0.15％0.015％0.08mol/L15μmol/ml | 0.2 0.2 0.2 0.2 0.2 0.02 0.2 | 0.32 0.4 0.4 0.4 0.4 0.04 0.4 | 1.6∶1 2∶1 2∶1 2∶1 2∶1 2∶1 2∶1 | 1201 466 469.8 467.9 239.2 152.8 25.58 |
Above result shows:
(1) with direct coupling one-step method, biological nano magnetic corpusculum (BMP) can reach 1201 μ g ADM/mgBMP to amycin (AMS) drug loading height in this experiment, and the amount of carrying amycin than common synthetic ferrite magnetic nano granule exceeds more than 20 times.
(2) it is stable to adopt direct coupling one-step method BMP to carry the amycin dose, and glutaraldehyde concentration changes has certain influence to drug loading.Suitably increase glutaraldehyde concentration and can improve drug loading.
(3) in the direct coupling one-step method, different coupling agents is very big to the drug loading influence.The drug loading of the drug loading of BMP when adopting succinic acid and DSC when adopting glutaraldehyde in this experiment as coupling agent as coupling agent.
Embodiment 4 sodium periodate oxidation system biological nano magnetic corpusculums carry amycin
Experimental procedure:
In the suspension of biological nano magnetic corpusculum grain, add the oxidant sodium metaperiodate, stir or impulse ultrasound reaction 0.5-24 hour down discontinuously; With Magnet sucking-off magnetic corpusculum,, remove unreacted oxidant with pure water or buffer solution washing several.Magnetic corpusculum after the oxidation is mixed with anticancer chemicals solution, stir or impulse ultrasound reaction 0.5-24 hour down discontinuously, reactant is removed unreacted anticancer chemicals with Magnet absorption: after pure water or phosphate buffer solution washing for several times, reuse Magnet sucking-off magnetic corpusculum gets product.
Experimental result:
Table four, adopt under the sodium periodate oxidation different condition biological nano magnetic corpusculum (BMP) to amycin (ADM) drug loading data:
| Medicine-carrying method | NaIO 4Concentration (mol/L) | C BMP (mg/ml) | C ADM (mg/ml) | C ADM/C BMP | Drug loading (μ gADM/mgBMP) |
| Sodium periodate method sodium periodate method sodium periodate method sodium periodate method | 0.15 0.05 0.0175 0.005 | 0.02 0.02 0.02 0.02 | 0.06 0.06 0.06 0.06 | 3∶1 3∶1 3∶1 3∶1 | 234.5 117.2 465.6 45.54 |
Above result shows: adopt sodium periodate method, biological nano magnetic corpusculum (BMP) is to reaching 465.6 μ gADM/mgBMP in this experiment of amycin (AMS) drug loading, and the amount of carrying amycin than common synthetic ferrite magnetic nano granule is high 8 times.Simultaneously, NaIO
4Concentration is influential to drug loading, at NaIO
4Concentration is about 0.0175, and BMP is to the drug loading maximum of ADM.
Embodiment 5 special-shaped bifunctional coupling agent 3-(2-pyridine dimercapto) propanoic acid N-hydroxy-succinamide ester (calling SPDP in the following text) legal system biological nano magnetic corpusculum carries amycin
Experimental procedure:
Biological nano magnetic corpusculum suspends with phosphate buffer solution, dissolve in special-shaped bifunctional coupling agent 3-(2-pyridine dimercapto) propanoic acid N-hydroxy-succinamide ester rapidly, stirring or impulse ultrasound reacted 0.5-24 hour down, with Magnet sucking-off magnetic corpusculum, removed unreacted coupling agent and by-product.Behind phosphate buffer solution washing several, add the solid dithiothreitol, DTT in the little liquid suspension of magnetic after SPDP handles, stir, impulse ultrasound reacted 0.5-24 hour down, with Magnet sucking-off magnetic corpusculum, with the buffer solution washing for several times; Add the reaction of solid dithiothreitol, DTT after the reaction of anticancer chemicals and 3-(2-pyridine dimercapto) propanoic acid N-hydroxy-succinamide ester again; The pure water or the buffer solution of the magnetic corpusculum of above-mentioned processing and anticancer chemicals are mixed, stir or impulse ultrasound reaction 0.5-24 hour repeatedly,, wash with pure water or buffer solution with Magnet sucking-off magnetic corpusculum, with Magnet sucking-off magnetic corpusculum, product.
Experimental result:
Table five, adopt under the SPDP different condition biological nano magnetic corpusculum (BMP) to amycin (ADM) drug loading data:
| Medicine-carrying method | SPDP concentration (mmol/L) | C BMP (mg/ml) | C ADM (mg/ml) | C ADM/C BMP | Drug loading (μ gADM/mgBMP) |
| SPDP method SPDP method | 0.08 0.4 | 0.2 0.2 | 0.4 0.4 | 2∶1 2∶1 | 297.4 118.2 |
By the result of table five as can be known, adopt the SPDP method, biological nano magnetic corpusculum (BMP) is stable to amycin (AMS) drug loading, reaches 297.4 μ gADM/mgBMP in this experiment, and the amount of carrying amycin than common synthetic ferrite magnetic nano granule exceeds more than 5 times.
Embodiment 6 biological nano magnetic corpusculum medicine carrying stability experiments
Experimental procedure:
The biological nano magnetic corpusculum that respectively embodiment 1-5 is made carries amycin medicine particle, adds 0.8% normal saline solution, and ultrasonic 10min behind the mixing is adsorbed on container bottom with Magnet with the magnetic corpusculum, gets supernatant and surveys ultraviolet spectra with spectrogrph.
Experimental result:
Table six. different medicine-carrying methods carry the data that influence of amycin stability to biological nano magnetic corpusculum:
| Medicine-carrying method | Coupling agent | C ADM/C BMP | Free amycin percentage composition (%) |
| The special-shaped bifunctional reagent method of the direct coupling one-step method of the direct coupling one-step method of the direct coupling method of fractional steps of the direct coupling method of fractional steps of direct physical absorption method oxidizing process | -glutaraldehyde DSC glutaraldehyde DSC NaIO 4 SPDP | 1.6∶1 1.6∶1 2∶1 1.6∶1 2∶1 3∶1 2∶1 | 5.7％ 4.5％ 21.54％ 0.26％ 18.68％ 5.6％ 1.8％ |
Free amycin percentage composition reflects the medicine carrying stability of magnetic corpusculum in the supernatant in the table six, and this value is low more, and then stability is high more.Medicine carrying stability is relevant with the kind and the coupling method of coupling agent.Experimental result shows, adopts glutaraldehyde the highest as the direct coupling one-step method stability of coupling agent, the amycin ratio that is unstable bonding state with the magnetic corpusculum only 0.26%.
Medicine and the bonded stability of nanometer magnetic corpusculum are related to the side effect and the rate of releasing drug of targeted anticancer medicine, and the amycin ratio that is unstable bonding state with the magnetic corpusculum is more little, enter the also corresponding minimizing of free drug of systemic circulation, and targeting efficient is higher.
Embodiment 7 biological nano magnetic corpusculums detect the safety of rat
With the high-purity magnetic corpusculum that extracts in the magnetotactic bacteria, carry out toxicological experiment by sublingual vein injection SD rat, the result is 62.7mg/kg for the rat median lethal dose(LD 50).Get blood from the survival rats abdominal vein and carry out the blood biochemical detection, its routine blood test (24), heart kinases and hepatic and renal function there is no unusually.
The heart, liver, spleen, lung, kidney, brain, intestinal and adrenal gland to rat carry out the common pathology cut sections for microscopic examination, and the Electronic Speculum ultrathin section is made in sampling simultaneously, observes the distribution situation of magnetic corpusculum in internal organs.The magnetic corpusculum does not cause that tissue stops up and the infarction phenomenon in giving dosage range; Only the hepatocyte of liver but not assemble in the macrophage, find no the magnetic corpusculum in other internal organs.
Need to prove: above embodiment only is used to illustrate technical scheme of the present invention, although the present invention is had been described in detail with reference to the foregoing description, those of ordinary skill in the art is to be understood that: still can make amendment or be equal to replacement the present invention, and not breaking away from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (3)
1. the preparation method of a Biomagnetism nano target anti-cancer drug, it is characterized in that raw material cancer therapy drug and biological nano magnetic corpusculum contain amino respectively, adopt the reaction of coupling agent and biological nano magnetic corpusculum and cancer therapy drug, the contained amino of magnetic corpusculum and cancer therapy drug is connected with coupling agent; Magnetic corpusculum, cancer therapy drug and coupling agent three react simultaneously, and a step coupling gets product, react maybe with this coupling agent elder generation and the little precursor reactant of biological nano magnetic, and then with cancer therapy drug, and the substep coupling gets product; Said coupling agent is the chemical compound that contains following at least two identical or groups inequality: aldehyde radical, carboxyl/anhydride, N-butanimide ester group, N-butanimide sodium sulfonate ester group or carbodiimides ester group.
2. the preparation method of Biomagnetism nano target anti-cancer drug according to claim 1, it is characterized in that said coupling agent elder generation and biological nano magnetic corpusculum and polyamino acid, Polyethylene Glycol or polysaccharide base polymer one step or stepwise reaction, biological nano magnetic corpusculum is connected with polymer earlier, biological nano magnetic corpusculum and the N-maloyl imines or the reaction of 1-ethyl-3-(3-diformazan ammonia n-pro-pyl) carbodiimides of polymer will have been connected again, make the carboxyl on the polymer be converted into N-butanimide ester group or carbodiimides ester group, with the reaction of raw material cancer therapy drug, get product then.
3. the preparation method of Biomagnetism nano target anti-cancer drug according to claim 1 and 2, it is characterized in that respectively the step reaction is all stirred in pure water or phosphate buffer or carried out under the impulse ultrasound discontinuously, each reaction afterproduct of biological nano magnetic corpusculum is all with Magnet sucking-off and pure water or buffer solution washing.
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| CN112439080B (en) * | 2019-08-30 | 2023-06-27 | 深圳先进技术研究院 | Diagnosis and treatment magnetic bacteria and preparation method thereof |
| CN111569064B (en) * | 2020-05-15 | 2022-12-16 | 北京国科融智生物技术有限公司 | Efficient targeted antitumor drug nano-carrier system and application thereof |
| CN112661500B (en) * | 2021-01-07 | 2022-06-14 | 中国科学院上海硅酸盐研究所 | Biological ceramic bracket with micro-nano structure on surface and preparation method and application thereof |
| CN115254069A (en) * | 2022-06-18 | 2022-11-01 | 太古宙基因科技(深圳)有限公司 | Preparation and application of high-magnetism nano magnetic beads |
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