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CN110368500B - Amphiphilic copolymer prodrug, preparation method and calcipotriol-entrapped nanoparticles - Google Patents

Amphiphilic copolymer prodrug, preparation method and calcipotriol-entrapped nanoparticles Download PDF

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CN110368500B
CN110368500B CN201910630574.XA CN201910630574A CN110368500B CN 110368500 B CN110368500 B CN 110368500B CN 201910630574 A CN201910630574 A CN 201910630574A CN 110368500 B CN110368500 B CN 110368500B
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hydroxycamptothecin
calcipotriol
polyethylene glycol
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刘祥瑞
王砾莹
顾苏芳
唐建斌
周珠贤
申有青
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Shanghai Shawya Biotechnology Co ltd
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Zhejiang University ZJU
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Abstract

The invention discloses an amphiphilic copolymer prodrug containing 7-ethyl-10-hydroxycamptothecin, which comprises a section of polyethylene glycol as a hydrophilic section and poly (7-ethyl-10-hydroxycamptothecin) and cholesterol as a hydrophobic section; wherein the poly (7-ethyl-10-hydroxycamptothecin) and the cholesterol are connected by a degradable chemical bond formed by hydroxyl, and the poly (7-ethyl-10-hydroxycamptothecin) and the cholesterol are connected with the polyethylene glycol monomethyl ether skeleton by forming the degradable chemical bond. The invention also discloses a preparation method of the precursor and calcipotriol-entrapped nanoparticles. The invention can self-assemble in water to form nano particles and simultaneously entrap compound calcipotriol, improves the solubility of 7-ethyl-10-hydroxycamptothecin and calcipotriol in water, and the obtained nano particles can reduce extracellular matrix by inhibiting the activity of stellate cells, thereby increasing the permeation and treatment effects of chemotherapeutic drugs.

Description

Amphiphilic copolymer prodrug, preparation method and calcipotriol-entrapped nanoparticles
Technical Field
The invention relates to a prodrug and the preparation field thereof, in particular to a 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug and a preparation method and application thereof.
Background
7-ethyl-10-hydroxycamptothecin (abbreviated as SN-38) is one of important derivatives of camptothecin, and has a molecular formula of C22H20N2O5, a molecular weight of 392.31, CAS. NO: 86639-52-3, the structural formula is as follows:
Figure BDA0002128588210000011
SN-38 is one of camptothecin anticancer drugs. Clinically, irinotecan and topotecan are approved to be used in camptothecin medicaments, and the two medicaments are obtained by respectively modifying SN-38 and 10-hydroxycamptothecin through functional groups, so that the solubility of the camptothecin medicaments in water is improved. SN-38 is an active product obtained by metabolizing irinotecan in vivo, and the drug property of the product can reach 1000 times that of irinotecan in some cell strains, but the direct application of the product in vivo is limited due to the poor water solubility of the product.
Cancer is an important disease which endangers human health, and many cancers with high fatality rate are characterized by rich and compact collagen matrix (Sermour, A.B.; Hruban, R.H.; Redston, M.et al. Allelotype of Pancretic Adenoc Cancer [ J ]. Cancer Res,1994,54(10):2761-4.), and growth factors, osteopontin, periostin, acidic serine protein and cysteine existing in the colloid play an important role in regulating and controlling the interaction between tumor cells and extracellular matrix, thereby not only promoting the metastasis of the tumor cells and enhancing the drug resistance of the tumor cells, but also preventing the penetration of drugs by the thick collagen matrix.
Among the numerous cells in the tumor microenvironment, astrocytes are the most deeply studied and play a crucial role in the glial secretion in the tumor microenvironment (Hanahan, d.; Coussens, l.m. accesories to the crime functions of cells recovering to the tumor microorganism [ J ]. cancer, 2012,21(3): 309-22.). Whereas stellate cells exist in two states, resting and activated: in normal human pancreatic tissue, the pancreatic stellate cells are usually in a resting state, accounting for approximately 4% -7% of normal human pancreatic parenchymal cells; the dense collagen matrix produced by activated astrocytes reduces the penetration of the drug (Netti, P.A.; Berk, D.A.; Swartz, M.A.et al.role of extracellular matrix in organic proteins J. Cancer Res,2000,60(9): 2497) 503.).
Calcipotriol is an important vitamin D derivative, can remarkably reverse the activation state of stellate cells, but has the advantages of strong toxicity, large side effect, extremely low water solubility, strong compound crystallinity, difficult entrapment, extremely short half-life of calcipotriol per se, only 4 minutes, very easy inactivation in human bodies and extremely poor metabolism into two metabolites (Kragballe, K.Calcipotriol-a New Drug for clinical Psorasis Treatment [ J ]. PharmacolToxicol,1995,77(4): 241-6.). Studies have shown that calcipotriol in combination with chemotherapeutic drugs can increase the efficacy of chemotherapeutic drugs (Sherman, M.H.; Yu, R.T.; Engle, D.D.et al.vitamin D Receptor-medial structural replication supressures and Enhances Pancreatic Cancer Therapy [ J ] Cell,2014,159(1): 80-93.).
The specification of application No. CN200410048016 discloses a branched polyethylene glycol-amino acid oligopeptide and a drug conjugate thereof, wherein the polymer comprises a plurality of active groups, linear polyethylene glycol is derived from the groups, and finally, multi-branched polyethylene glycol is formed.
Publication No. CN102060991A discloses a drug in which SN-38 is linked to short-chain PEG, and the drug has very high cytotoxicity. Publication No. CN103251596A discloses an amphiphilic polymer prodrug of 7-ethyl-10-hydroxycamptothecin, which comprises a section of polyethylene glycol and a section of poly (7-ethyl-10-hydroxycamptothecin); the poly (7-ethyl-10-hydroxycamptothecin) is formed by connecting 10-position or/and 20-position hydroxyl groups on 7-ethyl-10-hydroxycamptothecin with a polymer skeleton through degradable chemical bonds, and by introducing polyethylene glycols with different lengths, the drug precursor is self-assembled in water to form nanoparticles, so that the solubility of 7-ethyl-10-hydroxycamptothecin in water is improved.
Disclosure of Invention
The invention provides a 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug, which is easily dissolved in water by introducing cholesterol, so that calcipotriol is efficiently entrapped, and the prodrug has high tumor toxicity and low toxicity to normal tissues.
The 7-ethyl-10-hydroxycamptothecine amphiphilic copolymer prodrug comprises a section of polyethylene glycol monomethyl ether as a hydrophilic section and a hydrophobic section consisting of poly (7-ethyl-10-hydroxycamptothecine) and cholesterol, wherein the poly (7-ethyl-10-hydroxycamptothecine) and the cholesterol are connected with a polyethylene glycol monomethyl ether framework through forming degradable chemical bonds.
The number average molecular weight of the polyethylene glycol monomethyl ether in the 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug is 550-20000, the number average molecular weight of the poly (7-ethyl-10-hydroxycamptothecin) is 600-60000, and the number average molecular weight of the cholesterol is 600-60000.
The degradable chemical bond is selected from one or two of urea bond, ester bond, carbonate bond, carbamate bond, phosphate bond and other in vivo degradable chemical bonds.
When the degradable chemical bond is an ester bond, the-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug is obtained by reacting 7-ethyl-10-hydroxycamptothecin with polyethylene glycol taking one or more carboxyl groups as end groups under the action of a condensing agent; the condensing agent is one selected from N, N '-Dicyclohexylcarbodiimide (DCC), 1- (3-dimethylamino) -3-Ethylcarbodiimide (EDC), N' -Diisopropylcarbodiimide (DIPC) and the like.
Preferably, the 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug (PEG-PSN-38-PCHL for short) is a compound with a structure shown in the following formula I:
Figure BDA0002128588210000051
in the formula I, the number average molecular weight of the polyethylene glycol monomethyl ether is 550-20000(x is 10-500), the number average molecular weight of the poly (7-ethyl-10-hydroxycamptothecin) is 600-60000(y is 1-100), and the number average molecular weight of the cholesterol is 600-60000(z is 1-100).
The 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug can be self-assembled to form nanoparticles (or micelles or vesicles). Due to the addition of the cholesterol segment, calcipotriol can be efficiently entrapped, so that calcipotriol-entrapped nanoparticles can be self-assembled in an aqueous solvent, and the preparation method comprises a dialysis method, a film method, a solvent evaporation method and the like.
The invention also provides a preparation method of the 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug, which has the advantages of high yield, simple method and suitability for industrial production.
The preparation method of the 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug comprises the following steps:
(1) dissolving polyethylene glycol monomethyl ether and a micromolecular chain transfer agent PETTC into an organic solvent, adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine for catalytic reaction, washing, drying, and precipitating with anhydrous ether to obtain a polyethylene glycol monomethyl ether compound with a structure shown in a formula a;
Figure BDA0002128588210000061
whereinXIs 10-500
(2) Adding 7-ethyl-10-hydroxycamptothecin and succinic acid mono [2- [ (2-methyl-acryloyl) oxy ] ethyl ] ester into an organic solvent, reacting for more than 24h at room temperature under the action of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, washing, drying, concentrating, and passing through a column to obtain a 7-ethyl-10-hydroxycamptothecin derivative (MMESSN-38 for short) with the structure shown in formula b;
Figure BDA0002128588210000062
(3) dissolving cholesteryl chloroformate and hydroxyethyl methacrylate in organic solvent, reacting overnight under the catalysis of anhydrous pyridine, washing, drying, concentrating, and passing through column to obtain cholesterol derivative (MACHL);
Figure BDA0002128588210000063
(4) dissolving a polyethylene glycol monomethyl ether compound with a structure shown in a formula a, a poly (7-ethyl-10-hydroxycamptothecin) compound with a structure shown in a formula b and a cholesterol compound with a structure shown in a formula c in an organic solvent, heating and reacting for more than 24 hours under the catalysis of azodiisobutyronitrile and the anhydrous and anaerobic conditions, and dialyzing to remove small molecules to obtain the 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug shown in a formula I.
The organic solvent comprises one of reaction solvents such as dichloromethane, anisole, trichloromethane, tetrahydrofuran, pyridine, dimethyl sulfoxide, N' -dimethylformamide and the like.
The invention also provides a preparation method of the 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug assembly calcipotriol-loaded nanoparticles, which comprises a dialysis method, a film method and a solvent evaporation method.
Preferably, in a dialysis method as an example, the preparation method of the nanoparticle in which calcipotriol is entrapped in the amphiphilic copolymer prodrug of 7-ethyl-10-hydroxycamptothecin comprises the following steps: weighing 7-ethyl-10-hydroxycamptothecine amphiphilic copolymer prodrug powder, dissolving the powder in an organic solvent, adding calcipotriol, fully mixing, slowly dropwise adding deionized water into the solution, fully stirring, removing the organic solvent by using a semipermeable membrane for dialysis after dropwise adding is finished, centrifuging to remove free small molecular compounds, and distilling at normal temperature under reduced pressure to obtain the 7-ethyl-10-hydroxycamptothecine amphiphilic copolymer prodrug entrapped calcipotriol nanoparticle solution.
The organic solvent used in the method for preparing the nano-particles comprises one of tetrahydrofuran, dimethyl sulfoxide, N' -dimethylformamide and other solvents mixed with water.
The size range of the nanoparticle of the amphiphilic copolymer prodrug of the 7-ethyl-10-hydroxycamptothecin for encapsulating calcipotriol is 10nm-300nm, and the size of the nanoparticle and the amount of the encapsulated calcipotriol can be achieved by controlling the molecular weight of the poly (7-ethyl-10-hydroxycamptothecin) and the molecular weight of the cholesterol. Controlling the size of the nanoparticles and the amount of calcipotriol loaded allows the pharmacokinetic values within the drug body to be controlled, making possible personalized treatment of different cancers, patients, with the same formulation.
The invention has the following beneficial effects:
because the chemical bond bonding the 7-ethyl-10-hydroxycamptothecin in the 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug can be efficiently broken at the tumor, the polymer prodrug has high tumor toxicity and low toxicity to normal tissues.
Because the 7-ethyl-10-hydroxycamptothecin has strong hydrophobicity, the prodrug is self-assembled in water to form nano particles by introducing polyethylene glycol monomethyl ether, so that the solubility of the 7-ethyl-10-hydroxycamptothecin in water is greatly improved, and the in-vivo application of the 7-ethyl-10-hydroxycamptothecin is possible. Meanwhile, the drug precursor nano micelle has extremely high drug loading capacity, and can reduce the usage amount of the drug so as to reduce the side effect of the drug in healthy tissues.
Because calcipotriol can effectively inhibit the activity of stellate cells, the proliferation, drug resistance and metastasis of the stellate cells to tumors are reduced. By innovatively introducing cholesterol into the 7-ethyl-10-hydroxycamptothecin polymer prodrug, calcipotriol is efficiently encapsulated to form nanoparticles, so that the distribution of the calcipotriol in normal tissues is remarkably reduced, and the toxic and side effects of the calcipotriol are reduced.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a methoxypolyethylene glycol compound prepared in example 1;
FIG. 2 is a NMR spectrum of MMESSN-38 prepared in example 1;
FIG. 3 is a NMR spectrum of MACHL prepared in example 1;
FIG. 4 is a NMR chart of PEG-PSN-38-PCHL prepared in example 1;
FIG. 5 is a gel permeation chromatography characterization of PEG-PSN-38-PCHL prepared in example 1;
FIG. 6 is a CMC graph of the critical micelle concentration of PEG-PSN-38-PCHL prepared in example 1;
FIG. 7 is a dynamic light scattering diagram of nanoparticles formed by entrapment of calcipotriol in PEG-PSN-38-PCHL prepared in example 1;
FIG. 8A is a transmission electron micrograph of nanoparticles formed by entrapping calcipotriol in PEG-PSN-38-PCHL prepared in example 1, and FIG. 8B is an enlarged view of FIG. 8A;
FIG. 9 is a graph of the in vitro release rate of 7-ethyl-10-hydroxycamptothecin and calcipotriol from nanoparticles formed by entrapping calcipotriol in PEG-PSN-38-PCHL prepared in example 1.
Detailed Description
The present invention is described in further detail below with reference to specific figures and specific examples, but it should be noted that the following does not limit the scope of the present invention.
Example 1
Synthesizing the 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug shown as the formula I, wherein the number average molecular weight of polyethylene glycol is 5000, the number of 7-ethyl-10-hydroxycamptothecin residues in a poly (7-ethyl-10-hydroxycamptothecin) block is 12, the molecular weight is 7200, the number of cholesterol residues in a polycholesterol block is 4, and the molecular weight is 2100.
Synthesis of polyethylene glycol monomethyl ether compound
2.5g of polyethylene glycol monomethyl ether (0.5mmol) with the molecular weight of 5000 is dissolved in 50mL of toluene, water is removed under reflux until the evaporated toluene becomes clear, 50mL of dichloromethane is added under the protection of nitrogen, the temperature is reduced in an ice bath, 300mg of dicyclohexylcarbodiimide (1mmol) is added under the condition of the ice bath, and small molecular PETTC with the equivalent of 4 and 20mL of dichloromethane containing 12mg of 4-dimethylaminopyridine (0.1mmol) are slowly and dropwise added. The reaction was carried out overnight at room temperature, washed 3 times with 1N aqueous hydrochloric acid, 3 times with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and recrystallized from ether at-20 ℃ by standing, the precipitate was collected, washed three times with cold anhydrous ether, and dried in a vacuum oven at room temperature to give the product (2.1g, 74% yield). The nuclear magnetic spectrum of the obtained methoxy polyethylene glycol compound is shown in figure 1.
The nuclear magnetic pattern peaks of the polyethylene glycol monomethyl ether compound were examined as follows:1H NMR of mPEG5K-PETTC inCDCl3.(400MHz,CDCl3,298K):δ(ppm)＝1.90(s,3H,-CH3),2.37-2.61(m,4H,-CH2),3.0(t,2H,-CH2),3.38(s,3H,-CH3),3.43-3.83(m,456H),7.23-7.34(m,5H).
synthesis of MMESSN-38
2g (5mmol) of 7-ethyl-10-hydroxycamptothecin, 2.4g (10mmol) of mono [2- [ (2-methyl-acryloyl) oxy ] ethyl ] succinate are dissolved in 150mL of dichloromethane, 18mL of anhydrous pyridine is added, stirring is carried out in an ice bath environment, 1.9g (10mmol) of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) is dissolved in 25mL of dichloromethane solution and slowly dropped through a dropping funnel, and stirring is carried out at room temperature overnight after dropping is completed. Reacting until the solution is clear, filtering, collecting filtrate, washing with 1N dilute hydrochloric acid solution for 3 times, extracting, concentrating by rotary evaporation, recrystallizing with-20 deg.C glacial ethyl ether to obtain crude MMESSN-38 product, and purifying by column chromatography on 200 mesh silica gel column with 95:5 volume ratio dichloromethane/anhydrous methanol eluent to obtain MMESSN-381.4 g (46% yield) nuclear magnetic spectrum as shown in FIG. 2.
The NMR peaks of MMESSN-38 were examined as follows:1H NMR of MMESSN-38in CDCl3.(400MHz,CDCl3,298K)δ＝8.32(d,J＝9.2Hz,1H),7.94-7.70(m,2H),7.58(dd,J＝9.2,2.3Hz,1H),6.13(s,1H),5.74(d,J＝16.4Hz,1H),5.62-5.46(m,1H),4.53-4.18(m,4H),3.18(q,J＝7.6Hz,2H),3.00(t,J＝6.6Hz,2H),2.85(t,J＝6.6Hz,2H),2.10-1.69(m,5H),1.41(t,J＝7.6Hz,3H),1.04(t,J＝7.4Hz,3H).
synthesis of MACHL
9.0g of cholesteryl chloroformate (20mmol) was dissolved in 100mL of dry methylene chloride, and 22mol of anhydrous pyridine (1.75g) was added. 30mL of dry methylene chloride containing 3.9g of hydroxyethyl methacrylate (30mol) was added dropwise under ice-bath conditions, and the mixture was stirred at room temperature overnight. Washing with 1N diluted hydrochloric acid solution for 3 times, drying, concentrating by rotary evaporation, purifying with 200 mesh silica gel column, eluting with dichloromethane/N-hexane (3: 1), collecting filtrate, drying in vacuum oven overnight to obtain cholesterol monomer (5.4g, 47% yield) with nuclear magnetic spectrum as shown in FIG. 3.
The NMR peaks for MACHL were examined as follows:1H NMR of MMESSN-38in CDCl3(400MHz,CDCl3,298K):δ(ppm)＝0.55-2.04(m,41H,-CH3,-CH(CH3)-,-CH-,-CH2-),2.32-2.51(m,2H,-CH2),4.32(s,4H,-CO-O-CH2-CH2-O-CO-),4.48-4.50(m,1H,-CHO-),5.39-5.40(m,1H,-C(CH2-)＝CH-),5.55-5.60(m,1H,CH3-C＝CH2),6.10-6.20(m,1H,CH3-C＝CH2).
synthesis of tetra, PEG-PSN38-PCHL
The block polymer PEG-PSN-38 is synthesized by RAFT free radical polymerization, as shown in the figure, taking macromolecular chain transfer agents PEG-PETTC 213mg (0.04mmol), SN-38 monomer MMESSN-38250 mg (0.42mmol), cholesterol monomer MACHL100mg (0.17mmol) and AIBN (3.3mg, 0.02mmol), dissolving in 1mL calcium hydride-dehydrated DMSO and 1mL dry dioxane together, and fully dissolving by ultrasound. Bubbling nitrogen for 30min, removing water and oxygen by a freeze-thaw method for 3-5 times, then sealing the polymerization bottle, and placing the polymerization bottle in an oil bath kettle at 70 ℃ for polymerization reaction for 16 h. After the reaction is finished, the polymer bottle is placed in liquid nitrogen for freezing, the bottle stopper is opened, the temperature is slowly raised in an ethanol solution, the solution is placed in a semipermeable membrane dialysis bag with the molecular weight cutoff of 3500Da, 250mL of DMSO solution is used for dialysis 3 times at the temperature of 40 ℃ for 12 hours each time, and then 250mL of DMF solution is used for dialysis 3 times at the temperature of 40 ℃ for 12 hours each time. DMF was replaced with water and frozen to give the product (280mg, 50% yield) which had a NMR spectrum as shown in FIG. 4.
The nuclear magnetic pattern peaks of PEG-PSN38-PCHL were examined as follows:1H NMR of PEG-PSN38-PCHL in CDCl3.(400MHz,CDCl3,298K)δ(ppm)＝7.02-8.52(dd,48H),6.47(s,14H),5.82-5.73(d,48H),3.78-4.58(d,64H),3.39-3.83(m,452H),2.96(d,J＝28.0Hz,12H),2.83-2.63(m,12H),2.63-2.27(m,32H),2.14(s,48H),1.84(s,48H),1.55-0.05(m,50H).
the gel exclusion chromatogram of the resulting product is shown in FIG. 5, and the retention time of the product PEG-PSN38-PCHL is significantly lower than that of the macroinitiator PEG-PETTC. PEG-PSN38-PCHL spontaneously assembles into nanoparticles in water, and the critical micelle concentration required for forming the nanoparticles is measured by a Nile red method, as shown in FIG. 6, and is 46.64 mu g/mL.
The synthetic route is as follows:
Figure BDA0002128588210000131
preparation of calcipotriol-loaded nanoparticles assembled by five, 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug
Weighing 5mg of PEG-PSN-38-PCHL polymer powder, dissolving in 1mL of DMSO, dissolving 10mg of calcipotriol in 1mL of DMSO to prepare a calcipotriol stock solution, adding 75 mu L of the calcipotriol stock solution into 1mL of DMSO solution of the polymer, supplementing to 1.5mL of DMSO, fully mixing and ultrasonically treating for 5 minutes, slowly dropwise adding 5mL of deionized water into the mixture, fully stirring for fifteen minutes, and dialyzing with a cellulose acetate semipermeable membrane (with molecular weight cutoff of 3500Da) in 500mL of deionized water for three times, two hours each time. After completion of dialysis, the mixture was centrifuged at 3300rpm for 5 minutes, and the supernatant was subjected to distillation at 35 ℃ under reduced pressure to concentrate the polymer to an appropriate concentration. The concentration of calcipotriol and 7-ethyl-10-hydroxycamptothecin in the solution is measured by high performance liquid chromatography, and the encapsulation efficiency is calculated.
As shown in fig. 7, the particle size of the nanoparticles encapsulated with calcipotriol was measured by a dynamic light scattering instrument, the obtained particle size was 110 nm, and the morphological characteristics of the obtained nanoparticles were characterized by a transmission electron microscope; as shown in fig. 8A, it can be seen that the nanoparticles formed after the calcipotriol is entrapped are spherical nanoparticles with a uniform size. Further enlarging the electron microscope picture, as can be seen from fig. 8B, the particle size of the spherical nanoparticle is about 100 nm, the release rate of calcipotriol and 7-ethyl-10-hydroxycamptothecin in the nanoparticle is determined by dialysis, that is, the obtained nanoparticle is placed in a semipermeable membrane dialysis bag with the molecular weight cutoff of 3500 dalton, and then the dialysis bag is mixed in a buffer solution to determine the concentration of the drug outside the dialysis bag; as shown in figure 9, both calcipotriol and 7-ethyl-10-hydroxycamptothecin are gradually released, and the release speed of calcipotriol is faster than that of 7-ethyl-10-hydroxycamptothecin, so that the controlled slow release effect of the two drugs is achieved.

Claims (6)

1. An amphipathic copolymeric prodrug, comprising: polyethylene glycol monomethyl ether is used as a hydrophilic segment, poly (7-ethyl-10-hydroxycamptothecin) and cholesterol are used as hydrophobic segments, and the poly (7-ethyl-10-hydroxycamptothecin) and the cholesterol are connected with a polyethylene glycol monomethyl ether skeleton by forming degradable chemical bonds; the number average molecular weight of the polyethylene glycol monomethyl ether is 550-20000;
the number average molecular weight of the poly (7-ethyl-10-hydroxycamptothecin) is 600-60000;
the number average molecular weight of the cholesterol is 600-;
the amphiphilic copolymer prodrug is a compound with a structure shown in the following formula I:
Figure FDA0002453113590000011
wherein x is 10-500, y is 1-100, and z is 1-100.
2. The method of preparing an amphipathic copolymer prodrug of claim 1, comprising:
(1) dissolving polyethylene glycol monomethyl ether and a micromolecular chain transfer agent PETTC into an organic solvent, adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine for catalytic reaction, washing, drying, and precipitating with anhydrous ether to obtain a polyethylene glycol monomethyl ether compound with a structure shown in a formula a;
Figure FDA0002453113590000012
whereinXIs 10 to 500;
(2) adding 7-ethyl-10-hydroxycamptothecin and succinic acid mono [2- [ (2-methyl-acryloyl) oxy ] ethyl ] ester into an organic solvent, reacting at room temperature under the action of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, washing, drying, concentrating, and passing through a column to obtain a 7-ethyl-10-hydroxycamptothecin derivative with a structure shown in a formula b;
Figure FDA0002453113590000021
(3) dissolving cholesteryl chloroformate and hydroxyethyl methacrylate in an organic solvent, reacting overnight under the catalysis of anhydrous pyridine, washing, drying, concentrating, and passing through a column to obtain the cholesterol derivative with the structure shown in the formula c;
Figure FDA0002453113590000022
(4) dissolving a polyethylene glycol monomethyl ether compound with a structure shown in a formula a, a 7-ethyl-10-hydroxycamptothecin derivative with a structure shown in a formula b and a cholesterol derivative with a structure shown in a formula c in an organic solvent, carrying out heating reaction under the catalysis of azodiisobutyronitrile and the anhydrous and anaerobic conditions, and dialyzing to obtain the 7-ethyl-10-hydroxycamptothecin amphiphilic copolymer prodrug shown in a formula I.
3. The method of claim 2, wherein the organic solvent comprises one of dichloromethane, anisole, chloroform, tetrahydrofuran, pyridine, dimethylsulfoxide, and N, N' -dimethylformamide.
4. A calcipotriol-entrapped nanoparticle formed by assembly of an amphiphilic copolymer prodrug according to claim 1 with calcipotriol.
5. The calcipotriol-entrapped nanoparticle according to claim 4, wherein said nanoparticle has a size ranging from 10nm to 300 nm.
6. The method of claim 5, wherein the calcipotriol-entrapped nanoparticle is prepared by one of dialysis, thin film method, and solvent evaporation method.
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WO2008114274A1 (en) * 2007-03-19 2008-09-25 Fresenius Kabi Onclology Ltd. Proliposomal and liposomal compositions

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