WO2017107934A1 - 生物可降解双亲性聚合物、由其制备的聚合物囊泡及在制备肺癌靶向治疗药物中的应用 - Google Patents

生物可降解双亲性聚合物、由其制备的聚合物囊泡及在制备肺癌靶向治疗药物中的应用 Download PDF

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WO2017107934A1
WO2017107934A1 PCT/CN2016/111385 CN2016111385W WO2017107934A1 WO 2017107934 A1 WO2017107934 A1 WO 2017107934A1 CN 2016111385 W CN2016111385 W CN 2016111385W WO 2017107934 A1 WO2017107934 A1 WO 2017107934A1
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polymer
self
lung cancer
crosslinking
vesicle
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PCT/CN2016/111385
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English (en)
French (fr)
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孟风华
邹艳
方媛
钟志远
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博瑞生物医药（苏州）股份有限公司
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Priority to EP16877744.9A priority Critical patent/EP3392289B1/en
Priority to AU2016374669A priority patent/AU2016374669B2/en
Priority to KR1020187021220A priority patent/KR102144749B1/ko
Priority to US16/064,317 priority patent/US10759905B2/en
Priority to CA3009252A priority patent/CA3009252C/en
Priority to JP2018533090A priority patent/JP6768069B2/ja
Publication of WO2017107934A1 publication Critical patent/WO2017107934A1/zh

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Definitions

  • the invention relates to a biodegradable polymer material and application thereof, in particular to a biodegradable amphiphilic polymer and a polymer vesicle with a side chain containing a disulfide five-membered ring functional group and in targeted therapy of lung cancer
  • the application belongs to the field of medical materials.
  • Biodegradable polymers have very unique properties and are widely used in various fields of biomedicine, such as surgical sutures, bone fixation devices, biological tissue engineering scaffold materials, and drug controlled release carriers.
  • Synthetic biodegradable polymers are mainly aliphatic polyesters (polyglycolide PGA, polylactide PLA, lactide-glycolide copolymer PLGA, polycaprolactone PCL), polycarbonate (polytrimethylene) Base ring carbonate PTMC) is the most commonly used biodegradable polymer and has been approved by the US Food and Drug Administration (FDA).
  • biodegradable polymers such as PTMC, PCL, PLA, and PLGA have relatively simple structures, lacking a modifiable functional group, and it is often difficult to provide a cyclically stable drug carrier or a stable surface-modifying coating.
  • the degradation products of polycarbonate are mainly carbon dioxide and neutral glycols, which do not produce acidic degradation products.
  • the functional cyclic carbonate monomer can be copolymerized with cyclic ester monomers such as GA, LA and ⁇ -CL, and other cyclic carbonate monomers to obtain biodegradable polymers of different properties.
  • the biodegradable nanocarrier obtained by the biodegradable polymer prepared by the prior art has the problems of unstable circulation in the body, low uptake of tumor cells, and low intracellular drug concentration, which leads to low efficacy of the nano drug.
  • Micellar nanoparticles can be prepared from functional biodegradable polymers, which are stable in vivo, but can only be loaded with hydrophobic small molecule anticancer drugs, but Hydrophilic small molecule anticancer drugs with strong penetrability and hydrophilic biomacromolecules such as protein drugs and nucleic acid drugs with little toxic side effects are incapable, which greatly limits their application as drug carriers.
  • R1 is selected from one of the following groups:
  • R2 is selected from one of the following groups:
  • k is from 43 to 170
  • x is from 10 to 30
  • y is from 40 to 200
  • m is from 86 to 340.
  • the hydrophobic block contains a cyclic carbonate unit containing a disulfide five-membered ring functional group; and may be a diblock polymer:
  • R1 is selected from one of the following groups:
  • R2 is selected from one of the following groups:
  • k is from 113 to 170
  • x is from 20 to 26
  • y is from 100 to 190
  • m is from 226 to 340.
  • the above biodegradable amphiphilic polymer side chain contains disulfide, and can be composed of a cyclic carbonate monomer containing a disulfide five-membered ring functional group and other cyclic ester monomers and rings in the presence of an initiator in a solvent.
  • the ring-opening polymerization of a carbonate monomer; the other cyclic carbonate monomer includes trimethylene cyclic carbonate (TMC), a cyclic carbonate containing trimethoxybenzaldehyde in a side chain (PTMBPEC), and a side chain containing Bicyclic thiopyridine cyclic carbonate (PDSC) and acrylate trimethylolethane cyclocarbonate (AEC).
  • the other cyclic ester monomers include lactide (LA), glycolide (GA), and caprolactone (CL).
  • cyclic carbonate monomer can be copolymerized in methylene chloride with monomethoxypolyethylene glycol as initiator, bis(bistrimethylsilyl)amine zinc as catalyst and TMC ring-opening copolymerization.
  • monomethoxypolyethylene glycol as initiator
  • bis(bistrimethylsilyl)amine zinc as catalyst
  • TMC ring-opening copolymerization Forming a randomly arranged diblock polymer of CDC and TMC units; the reaction formula is as follows:
  • the bisulfide-containing amphiphilic polymer disclosed in the present invention has biodegradability, and the molecular weight of the hydrophobic portion is three times or more of the molecular weight of the hydrophilic portion, and can be replaced by a solvent replacement method, a dialysis method, or the like. Or a method such as a film hydration method to prepare a polymer vesicle structure.
  • the prepared polymer vesicles are nanometer-sized, with a particle size of 40-180 nm, and can be used as a carrier for treating lung cancer; the hydrophobic membrane of the vesicle is loaded with a hydrophobic small molecule anti-lung cancer drug paclitaxel, docetaxel, etc.
  • Hydrophilic anti-lung cancer drugs are loaded into the large hydrophilic lumen of vesicles, especially hydrophilic small molecule anticancer drugs such as doxorubicin hydrochloride, epirubicin hydrochloride, irinotecan hydrochloride and mitre hydrochloride Hey.
  • hydrophilic small molecule anticancer drugs such as doxorubicin hydrochloride, epirubicin hydrochloride, irinotecan hydrochloride and mitre hydrochloride Hey.
  • the end of the hydrophilic segment PEG of the above biodegradable amphiphilic polymer can be chemically coupled to a tumor-specific targeting molecule such as cRGD, cNGQ or cc-9 to prepare a tumor-specific targeted biodegradable amphiphilic polymer. .
  • the invention also discloses a polymer vesicle which can be prepared from the above biodegradable amphiphilic polymer; or prepared by the above-mentioned tumor-specific targeted biodegradable amphiphilic polymer; or by the above biodegradable amphiphilic polymerization
  • the biodegradable amphiphilic polymer specifically targeted by the tumor is prepared, for example, the biodegradable amphiphilic polymer and the tumor-specific biodegradable amphiphilic polymer are mixed in different proportions, and different targets can be prepared.
  • Density of polymer vesicles can increase the intake of vesicle nano drugs in lung cancer cells; cross-linked vesicles prepared from biodegradable amphiphilic polymers or The outer surface of self-crosslinking vesicles is coupled with tumor cell-specific targeting molecules to prepare lung cancer-targeted cross-linked vesicles and lung cancer-targeted self-crosslinking vesicles to increase lung cancer cell uptake, such as vesicles in vesicles.
  • the end is bonded to cRGD, cNGQ or cc-9 by Michael addition.
  • the above biodegradable amphiphilic polymer and the tumor-specific targeted biodegradable amphiphilic polymer can be self-crosslinked without adding any substance to obtain self-crosslinking polymer vesicles and lung cancer targeted self-crosslinking polymerization.
  • a vesicle; or a catalyzed amount of a reducing agent such as dithiothreitol (DTT) or glutathione (GSH) to prepare a crosslinked polymer vesicle and a lung cancer targeted cross-linked polymer capsule bubble.
  • Self-crosslinking vesicles, lung cancer targeting self-crosslinking vesicles, cross-linked vesicles, and lung cancer targeting cross-linked vesicles Stable chemical cross-linking is formed in the vesicle hydrophobic membrane, which can stabilize long circulation in vivo; but after endocytosis into cancer cells, the formation of cross-linking will be quickly released (dissolved) in the presence of a large amount of reducing substances in the cells. Link), quickly release drugs, and effectively kill lung cancer cells.
  • the present invention claims the use of the above biodegradable amphiphilic polymer in the preparation of a nano drug for treating lung cancer; further, the present invention also discloses the use of the above polymer vesicle in the preparation of a nano drug for treating lung cancer, including Polymeric vesicles, self-crosslinking polymer vesicles prepared from side-chain disulfide-containing biodegradable amphiphilic polymers, biodegradable amphiphilic polymers specifically targeted by tumors, or biodegradable amphiphilic Polymer-prepared lung cancer-targeted self-crosslinking polymer vesicles, lung cancer-targeted cross-linked polymer vesicles for the preparation of nanomedicines targeted for the treatment of lung cancer.
  • the anti-lung cancer nano drug prepared based on the polymer of the present invention is a vesicle anti-lung cancer nano drug.
  • the present invention has the following advantages compared with the prior art:
  • the present invention utilizes a cyclic carbonate monomer containing a disulfide five-membered ring functional group, a polyethylene glycol as an initiator, and TMC or LA to obtain a molecular weight controllable molecular weight by active controlled ring-opening polymerization.
  • a narrowly distributed side chain containing a disulfide biodegradable amphiphilic polymer since the sulfur-sulfur five-membered ring group does not affect the ring-opening polymerization of the cyclic carbonate monomer, the polymerization process does not require protection and removal in the prior art. The protection process simplifies the operation steps.
  • the side chain bisulfide-containing biodegradable amphiphilic polymer disclosed in the invention has biodegradability, can prepare polymer vesicles and lung cancer targeting vesicles, and can be loaded with drugs of different properties without adding any substances. Self-crosslinking to form a stable self-crosslinking polymer vesicle nanomedicine, thereby overcoming the defects of the prior art nano drug in vivo instability, easy drug release, and toxic side effects.
  • the cross-linking of the self-crosslinking vesicle nano drug disclosed by the invention is reversible, that is, it supports long circulation in the body and can be highly enriched in lung cancer cells; but after entering the lung cancer cell, it can quickly cross-link and release the drug. To achieve efficient and specific killing of lung cancer cells without toxic side effects.
  • the cross-linked nano drug is too stable, and the drug release in the cell is slow, resulting in drug resistance. defect.
  • the biodegradable polymer vesicle and the lung cancer targeting vesicle disclosed by the invention can prepare the self-crosslinking vesicle without adding any substance, and the preparation method is simple, thereby overcoming the preparation of the crosslinked nano drug in the prior art.
  • the presence of substances such as cross-linking agents and the need for complicated handling and purification processes are present.
  • the self-crosslinking polymer vesicle prepared by self-assembly of the amphiphilic polymer disclosed in the invention can be used for the controlled release system of a hydrophilic small molecule anticancer drug, thereby overcoming the existing biodegradable nanomicelle carrier for loading only. Defects of hydrophobic small molecule drugs and defects in the prior art that do not efficiently load and stabilize circulating small molecule anticancer drugs; further, lung cancer-targeted self-crosslinking vesicles can be prepared in lung cancer Efficient targeted therapy has a wider application value.
  • Example 1 is a hydrogen nuclear magnetic spectrum of a polymer PEG5k-P (CDC4.9k-co-TMC19k) in Example 2;
  • Figure 3 is a cross-linked vesicle PEG5k-P (CDC4.9k-co-TMC19k) particle size distribution (A) and an electron projection microscope image (B), the stability of cross-linked vesicles in the fifteenth embodiment (C) And reduction responsiveness test (D) map;
  • Figure 4 is a diagram showing the in vitro release of DOX ⁇ HCl cross-linked vesicle PEG5k-P (CDC4.9k-co-TMC19k) in Example 15;
  • Figure 5 is a diagram showing the in vitro release of DOX ⁇ HCl cross-linked vesicles cRGD20/PEG6k-P (CDC4.6k-co-TMC18.6k) in Example 24;
  • Figure 6 is a graph showing the toxicity of targeted cross-linked vesicle cRGD/PEG6k-P (CDC4.6k-co-TMC18.6k) to A549 lung cancer cells in Example 26;
  • Figure 7 is a DOX ⁇ HCl targeted cross-linked vesicle carrying the twenty-sixth embodiment.
  • Figure 8 is a graph showing the results of blood circulation studies of DOX ⁇ HCl-targeted cross-linked vesicle cRGD/PEG6k-P (CDC4.6k-co-TMC18.6k) in mice in Example 28;
  • Figure 9 is a graph showing the results of blood circulation studies of DOX ⁇ HCl-targeted cross-linked vesicle cNGQ/PEG6k-P (CDC4.6k-co-TMC18.6k) in mice in Example 29;
  • Figure 10 is a graph showing the results of biodistribution of DOX ⁇ HCl-targeted cross-linked vesicle cRGD/PEG6k-P (CDC4.6k-co-TMC18.6k) in subcutaneous lung cancer mice;
  • Figure 11 is a graph showing the results of biodistribution of DOX ⁇ HCl-targeted cross-linked vesicle cNGQ/PEG6k-P (CDC4.6k-co-TMC18.6k) in subcutaneous lung cancer mice;
  • Figure 12 is a treatment diagram of a DOX ⁇ HCl-targeted cross-linked vesicle cRGD/PEG6k-P (CDC4.6k-co-TMC18.6k) in a subcutaneous lung-bearing mouse, in which A is a tumor growth.
  • Curve B is the tumor picture after treatment in mice, C is the change in body weight, and D is the survival curve;
  • Figure 13 is a treatment diagram of DOX ⁇ HCl-targeted cross-linked vesicle cNGQ/PEG6k-P (CDC4.6k-co-TMC18.6k) in subcutaneous lung cancer mice in Example 37, wherein A is tumor growth Curve, B is the weight change curve, and C is the survival curve;
  • Figure 14 is a treatment diagram of DOX ⁇ HCl-targeted cross-linked vesicle cRGD/PEG6k-P (CDC4.6k-co-TMC18.6k) in mice bearing lung cancer in Example 39, wherein A is a tumor Growth curve, B is the weight change curve, and C is the survival curve;
  • Figure 15 is a diagram showing the treatment of DOX ⁇ HCl-targeted cross-linked vesicle cNGQ/PEG6k-P (CDC4.6k-co-TMC18.6k) in mice bearing lung cancer in situ in Example 40, wherein A is tumor growth Curve, B is the weight change curve, and C is the survival curve.
  • the nuclear magnetic diagram is shown in Figure 1, 1 H NMR (400 MHz, CDCl 3 ): 2.08 (t, -COCH 2 CH 2 CH 2 O-), 3.08 (s, -CCH 2 ), 3.30 (m, -OCH 3 ), 3.65 (t, -OCH 2 CH 2 O-), 4.28 (t, -COCH 2 CH 2 CH 2 O-), 4.31 (m, -CCH 2 ).
  • 0.1 g (0.52 mmol) of CDC monomer and 0.4 g (3.85 mmol) of TMC were dissolved in 3 mL of dichloromethane, added to a sealed reactor, and then 0.1 g (0.015 mmol) of NHS-PEG 6500 and 0.5 were added.
  • mL of catalyst bis(bistrimethylsilyl)amine zinc in dichloromethane (0.1 mol/L) then seal the reactor, transfer it out of the glove box, react for 2 days in a 40 °C oil bath, and stop the reaction with glacial acetic acid.
  • the synthesis of the cyclic polypeptide CSNIDARAC (cc9) coupled polymer CC9-PEG7.5k-P (CDC3.8k-co-LA13.8k) is divided into two steps.
  • the first step is to prepare NHS-PEG7.5k as in the tenth embodiment.
  • -P (CDC3.8k-co-LA13.8k);
  • the second step is the bonding of CC9 to it by amidation reaction.
  • the synthesis of the cyclic polypeptide c (RGDfC) (cRGD-SH) coupled polymer cRGD-PEG6k-P (CDC3.6k-co-LA18.6k) is divided into two steps.
  • the first step is to prepare Mal as in the eighth embodiment.
  • PEG6k-P (CDC3.6k-co-LA18.6k); the thiol group of the second step cRGD-SH is bonded to it by a Michael addition reaction.
  • the polymer Mal-PEG6k-P (CDC3.6k-co-LA18.6k) was dissolved in 0.5 ml of DMF, 2 ml of boric acid buffer solution (pH 8.0) was added, and 1.5 times the molar amount of cRGD-SH was added. Reaction at 30 ° C for two days, dialysis, freeze-drying to obtain the final product cRGD-PEG6k-P (CDC3.6k-co-LA18.6k), calculated by nuclear magnetic and BCA protein kit test, the grafting rate of cRGD was 94%.
  • the synthesis of the cyclic polypeptide c (RGDfK) (cRGD) coupled polymer cRGD-PEG6.5k-P (CDC4.6k-co-TMC18.6k) is divided into two steps.
  • the first step is to prepare NHS- as in the fourth embodiment.
  • PEG6.5k-P (CDC4.6k-co-TMC18.6k); the amino group of the second step cRGD is bonded to it by an amidation reaction.
  • the above polymer NHS-PEG6.5k-P (CDC4.6k-co-TMC18.6k) was first dissolved in DMF, and twice the molar amount of cRGD was added.
  • cRGD-PEG6.5k-P (CDC4.6k-co-TMC18.6k)
  • the grafting rate of cRGD was 88%.
  • the synthesis of the cyclic polypeptide cNGQGEQc(cNGQ) coupled polymer cNGQ-PEG6.5k-P (CDC4.6k-co-TMC18.6k) is divided into two steps.
  • the first step is to prepare NHS-PEG6.5k as in the fourth embodiment.
  • -P (CDC4.6k-co-TMC18.6k);
  • the second step is the bonding of the amino group of cNGQ to it by amidation reaction.
  • a variety of side chain disulfide-containing biodegradable amphiphilic polymers can be prepared by the similar preparation method described above. The proportions and characterization of the raw materials are shown in Table 1.
  • Polymer vesicles were prepared by solvent displacement. 100 ⁇ L of PEG5k-P (CDC4.9k-co-TMC19k) in DMF solution (10 mg/mL) was added dropwise to 900 ⁇ L of phosphate buffer solution (PB, 10 mM, pH 7.4) and placed in a shaker at 37 ° C (200 rpm). Self-crosslinking was carried out overnight, and then dialyzed overnight in a dialysis bag (MWCO 7000) for five times of water, and the dialysis medium was PB (10 mM, pH 7.4). The size of the obtained self-crosslinking vesicles was 130 nm by the dynamic light scattering particle size analyzer (DLS), and the particle size distribution was very narrow. See FIG.
  • DLS dynamic light scattering particle size analyzer
  • FIG. 3A the TEM measured the nanoparticles as Hollow vesicle structure, self-crosslinking vesicles maintain a constant particle size and particle size distribution in the presence of high-dilution and fetal bovine serum (Fig. 3C), but rapidly release in the simulated tumor cell reduction environment, decrosslinking (Fig. 3D). It can be seen that the obtained vesicles can be self-crosslinked and have the property of reduction-sensitive decrosslinking.
  • Polymer vesicles were prepared by dialysis. 100 ⁇ L of PEG5k-P (CDC4.9k-co-TMC19k) in DMF solution (10 mg/mL) was placed in a dialysis bag (MWCO 7000) in a PB (10 mM, pH 7.4), 37 ° C (200 rpm) shaker Place overnight for self-crosslinking, then dialyze for 24 hours in PB and change for five times. The DLS measures cross-linked vesicles at about 80 nm and a particle size distribution of 0.08.
  • Polymer vesicles were prepared by thin film hydration. 2 mg of PEG5k-P (CDC4.9k-co-TMC19k) is dissolved in 0.5 mL of low boiling organic solvent, such as dichloromethane or acetonitrile, in a 25 ml sharp-bottomed flask, and steamed to form a film at the bottom. Then continue to drain for a further 24 hours under a vacuum of 0.1 mBar.
  • organic solvent such as dichloromethane or acetonitrile
  • PB mM, pH 7.4
  • the size of the self-crosslinking vesicles measured by DLS was about 180 nm, and the particle size distribution was 0.25.
  • the polymer vesicles were prepared as in Example 15. After the addition, DTT (concentration: 0.09 ⁇ M) was added, and the mixture was crosslinked at 37 ° C for 12 hours, and then dialyzed into a dialysis bag (MWCO 7000) overnight for five times.
  • the size of the obtained self-crosslinking vesicle is about 109 nm, and the particle size distribution is 0.13.
  • the target polymer cNGQ-PEG6.5k-P (CDC4.6k-co-TMC18.6k) obtained in Example 14 and the PEG5k-P (CDC4.9k-co-TMC19k) obtained in Example 2 were mixed.
  • cNGQ-conjugated self-crosslinking polymer vesicles were prepared as in Example 15.
  • the PEG molecular weight of the targeting polymer is longer than that of the non-targeted PEG, ensuring that the targeting molecule is better at the surface.
  • the self-crosslinking vesicles having different targeting molecules on the surface can be prepared by mixing the two in different ratios.
  • the former content is 5-30 wt.%.
  • the DLS has a size of about 90-120 nm and a particle size distribution of 0.05-0.15.
  • the cRGD-conjugated targeted self-crosslinking polymer vesicles were prepared by membrane hydration. 1.6 mg of the DM5k-P (CDC4.9k-co-TMC19k) DMF solution (10 mg/mL) obtained in Example 2 and 0.4 mg of the cRGD-PEG6.5k-P obtained in Example 13 (CDC4.6k) -co-TMC18.6k) is dissolved in 0.5 mL of a low boiling organic solvent such as dichloromethane or acetonitrile.
  • the self-crosslinking vesicles prepared as in Example 17 have a particle size distribution of about 0.08.
  • the self-crosslinking vesicles having different targeting molecules on the surface can be prepared by mixing the two in different ratios. Preferably, the former content is 5-30 wt.%.
  • the Mal-PEG6k-P (CDC3.6k-LA18.6k) prepared in Example 8 and P(CDC3.8k-LA18.8k)-PEG4k-P (CDC3.8k-LA18.8k) mixing
  • the vesicles were prepared according to the dialysis method as described in Example 16. Then, 0.5 ml of a 4 M boric acid buffer solution (pH 8.0) was added to adjust the pH of the solution to 7.5-8.0, and then added to CC9 at 1.5 times the molar amount of Mal, and bonded by a Michael addition reaction, and reacted at 30 ° C for two days, followed by dialysis.
  • the DLS was measured to be 110 nm and the particle size distribution was 0.16.
  • the nuclear magnetic and BCA protein kit tests calculate the grafting ratio of the polypeptide to 90%.
  • Self-crosslinking vesicles having different targeting molecules on the surface can be prepared by mixing two polymers in different ratios.
  • the former content is 5-30 wt.%.
  • a variety of self-crosslinking polymer vesicles and targeted self-crosslinking polymer vesicles can be prepared by the similar preparation methods described above. The ratio of the raw materials and the characterization are shown in Table 2.
  • the polymer vesicles were prepared by solvent displacement method.
  • the DOX ⁇ HCl was loaded by pH gradient method, and the hydrophilic drug DOX ⁇ HCl was coated by the difference of pH inside and outside the vesicle.
  • 100 ⁇ L of PEG5k-P (CDC4.9k-co-TMC19k) in DMF solution (10 mg/mL) was added dropwise to 900 ⁇ L sodium citrate/citrate buffer solution (10 mM, pH 4.0) at 37 ° C (200 rpm) shaker Place for 5 hours, then add 0.05 mL of PB (4M, pH 8.1) to establish a pH gradient, then immediately add DOX ⁇ HCl, place in the shaker for 5-10 hours to allow the drug to enter the vesicle while self-crosslinking.
  • the solution was dialyzed overnight in a dialysis bag (MWCO 7000), and the water was changed five times with a dialysis medium of PB (10 mM, pH 7.4).
  • Self-crosslinking vesicles containing different ratios of drugs (10%-30%) have a particle size of 105-124 nm and a particle size distribution of 0.10-0.15.
  • the encapsulation efficiency of DOX ⁇ HCl was determined by fluorescence spectrometry to be 63%-77%.
  • the in vitro release of DOX ⁇ HCl was performed by shaking (200 rpm) in a 37 ° C constant temperature shaker with three replicates in each group.
  • DOX ⁇ HCl-loaded self-crosslinking vesicles were added to 10 mM GSH simulated intracellular reducing environment PB (10 mM, pH 7.4); the second group, DOX ⁇ HCl-loaded self-crosslinking vesicles in PB (10 mM) , pH 7.4); the concentration of drug-loaded self-crosslinking vesicles was 30 mg / L, 0.6 mL was placed in a dialysis bag (MWCO: 12,000), and each tube was added with 25 mL of the corresponding dialysis solvent at predetermined time intervals. The 5.0 mL dialysis bag external medium was taken out for testing, and 5.0 mL of the corresponding medium was added to the test tube.
  • Figure 4 shows the relationship between the cumulative release of DOX ⁇ HCl and time. It can be seen from the figure that the release of GSH in simulated tumor cells is significantly faster than that of samples without GSH, indicating self-crosslinking vesicles. The drug can be effectively released in the presence of 10 mM GSH.
  • Polymer vesicles were prepared by solvent displacement. 10 ⁇ L of Paclitaxel PTX in DMF solution (10 mg/mL) and 90 ⁇ L of Ally-PEG6k-P (CDC2.9k-CL14.2k) in DMF solution (10 Mg/mL) was mixed, then added dropwise to 900 ⁇ L of phosphate buffer solution (10 mM, pH 7.4, PB), placed in a 37 ° C (200 rpm) shaker overnight for self-crosslinking, and then loaded into a dialysis bag (MWCO 7000) The dialysis was carried out overnight, and the water was changed five times. The dialysis medium was PB (10 mM, pH 7.4).
  • the content of PTX is 0-20 wt.%, and the obtained self-crosslinking vesicle has a size of 130-170 nm and a particle size distribution of 0.1-0.2.
  • the TEM was measured as a vesicle structure with a reduction-sensitive decrosslinking property.
  • the package efficiency of PTX is 50%-70%).
  • the in vitro release experiment design was the same as in Example 22. After the addition of GSH, the release of hydrophobic drugs was significantly faster than the absence of GSH samples.
  • Polymer vesicles were prepared by membrane hydration and DOX ⁇ HCl was loaded by pH gradient method.
  • 1.6 mg of PEG5k-P (CDC4.9k-co-TMC19k) and 0.4 mg of cRGD-PEG6.5k-P (CDC4.6k-co-TMC18.6k) are dissolved in 0.5 mL of low boiling organic solvent, such as In a methyl chloride or acetonitrile, in a 25 ml sharp-bottomed flask, a film was formed by rotary evaporation at the bottom, and then dried under a vacuum of 0.1 mBar for 24 hours.
  • low boiling organic solvent such as In a methyl chloride or acetonitrile
  • the particle size is 112-121 nm
  • the particle size distribution is 0.10-0.15
  • the encapsulation efficiency of DOX ⁇ HCl is 61%-77%.
  • the in vitro release experiment design was the same as in Example 22, and Figure 5 shows that after the addition of 10 mM GSH, the drug was effectively released at a faster rate than the sample without GSH.
  • the vesicles were prepared by dialysis method, and the epirubicin hydrochloride (Epi ⁇ HCl) was loaded by a pH gradient method.
  • 80 ⁇ L A solution of PEG5k-P (CDC4.9k-co-TMC19k) in DMF (10 mg/mL) and 20 ⁇ L of cNGQ-PEG6.5k-P (CDC4.6k-co-TMC18.6k) in DMF (10 mg/mL)
  • MWCO 7000 dialysis bag
  • a sodium citrate / citrate buffer solution (10 mM, pH 4.0
  • the medium was dialyzed for 12 hours and replaced with five times.
  • the DLS measured cross-linked vesicles were 96 nm and the particle size distribution was 0.18.
  • 0.05 mL of PB (4 M, pH 8.5) was added to the above vesicle solution to establish a pH gradient, followed immediately by the addition of Epi ⁇ HCl, and placed in a shaker for 5-10 hours. It was then placed in a dialysis bag (MWCO 7000) and dialyzed against PB overnight for five times. Loaded in different proportions (10%-30%), particle size 98-118nm, particle size distribution 0.10-0.15, Epi ⁇ HCl package efficiency of 64%-79%.
  • the experimental design of Epi ⁇ HCl in vitro release was the same as in Example 22.
  • a variety of self-crosslinking polymer vesicles and targeted self-crosslinking polymer vesicles can be studied by a similar preparation method as described above for various hydrophilic anticancer small molecule drugs such as doxorubicin hydrochloride (DOX ⁇ HCl), hydrochloric acid.
  • hydrophilic anticancer small molecule drugs such as doxorubicin hydrochloride (DOX ⁇ HCl), hydrochloric acid.
  • vesicle cRGD/PEG6.5k-P (CDC4.6k-co-TMC18.6k), another cell blank control well and medium blank well (complex 4 well).
  • FIG. 6 is a cytotoxicity result of self-crosslinking vesicles. It can be seen that when the concentration of cross-linked vesicles is increased from 0.75 to 1.5 mg/mL, the survival rate of A549 is still higher than 90%, indicating that the cross-linked capsule The foam has good biocompatibility.
  • Example 27 MTT assay for the toxicity of drug-loaded self-crosslinking vesicles and drug-loaded self-crosslinking vesicles to A549 lung cancer cells.
  • the toxicity of vesicles to A549 cells was tested by MTT assay.
  • the culture of the cells was the same as that of the twenty-sixth embodiment, except that the drug-loaded cross-linked vesicles and the drug-loaded drug were targeted to the self-crosslinking vesicles, and the DOX ⁇ HCl-loaded self-crosslinking vesicles of Example 22 were applied.
  • Figure 7 is the toxicity of drug-loaded self-crosslinking vesicle cRGD/PEG6.5k-P (CDC4.6k-co-TMC18.6k) to A549 cells; it can be seen that 30% cRGD containing DOX ⁇ HCl is targeted from The semi-lethal concentration (IC 50 ) of cross-linked vesicles to A549 cells was 2.13 ⁇ g/mL, which was much lower than that of non-targeted control vesicles and lower than that of free drugs (4.89 ⁇ g/mL), indicating drug loading of the present invention.
  • Targeted self-crosslinking vesicles can effectively target lung cancer cells, release drugs in cells, and ultimately kill cancer cells.
  • Example 28 TMT method for testing drug-loaded self-crosslinking vesicles and drug-loaded self-crosslinking vesicle pairs Toxicity of H460 cells.
  • the toxicity of vesicles to H460 human lung cancer cells was tested by MTT assay.
  • the culture of the cells was the same as that of the twenty-sixth embodiment. Only when the wells of the experimental group were loaded, the drug-loaded vesicles with different cc-9 contents and different doses were targeted to self-crosslinking with CPT ⁇ HCl.
  • vesicle CC9/P (CDC3.8k-LA18.8k)-PEG4k-P (CDC3.8k-LA18.8k) was added to each corresponding well, and the concentration of CPT ⁇ HCl was 0.01, 0.1, 0.5, 1, 5, 10, 20 and 40 ⁇ g/mL; target molecular content from 10%, 20% to 30%; no drug-loaded cross-linked vesicles, and free CPT ⁇ HCl group as a control group.
  • the samples were aspirated and replaced with fresh medium for a further 44 h.
  • the MTT was then added, processed and measured for absorbance as in Example 26.
  • the toxicity of various drug-loaded self-crosslinking polymer vesicles and self-crosslinking polymer vesicles on lung cancer cells was studied by a similar method as described above.
  • the drug is a hydrophilic anticancer small molecule drug which is doxorubicin hydrochloride ( DOX ⁇ HCl), epirubicin hydrochloride (Epi ⁇ HCl), irinotecan hydrochloride (CPT ⁇ HCl) and mitoxantrone hydrochloride (MTO ⁇ HCl) and hydrophobic anticancer drugs paclitaxel and docetaxel.
  • DOX ⁇ HCl hydrophilic anticancer small molecule drug
  • Epi ⁇ HCl epirubicin hydrochloride
  • CPT ⁇ HCl irinotecan hydrochloride
  • MTO ⁇ HCl mitoxantrone hydrochloride
  • mice All animal experiments were conducted in accordance with the regulations of the Animal Experimental Center of Suzhou University. The experiment used Balb/C nude mice weighing about 18-20 grams and 4-6 weeks old.
  • the vesicles were composed of PEG5k-P (CDC4.9k-co-TMC19k) and cRGD-PEG6.5k-P (CDC4.6k-co-TMC18.6k) and PEG5k-P (CDC4.9k-co- mixed in different ratios).
  • TMC19k composition when the cRGD ratio is 20%, the particle size is 100 nm, and the particle size distribution is 0.10, named For cRGD20/CLPs, the drug is DOX ⁇ HCl.
  • DOX ⁇ HCl-free targeting vesicle CLPs, targeted vesicle cRGD20/CLPs, non-crosslinking targeting vesicles cRGD20/PEG-PTMC and DOX ⁇ HCl were injected into mice via tail vein (DOX dose was 10 mg) /kg), take about 10 ⁇ L at 0, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours, calculate the blood weight accurately by differential method, plus add 100 ⁇ L of 1% Triton After extraction with 500 ⁇ L of DMF (containing 20 mM DTT, 1 M HCl); then centrifugation (20,000 rpm, 20 minutes), the supernatant was taken and the amount of DOX ⁇ HCl at each time point was measured by fluorescence.
  • DMF containing 20 mM DTT, 1 M HCl
  • centrifugation 20,000 rpm, 20 minutes
  • the abscissa is time, and the ordinate is the total DOX injection amount (ID%/g) of DOX ⁇ HCl per gram of blood.
  • the cycle time of DOX ⁇ HCl is very short, DOX is difficult to detect in 2 hours, and the crosslinked vesicles still have 8 ID%/g after 24 hours.
  • Example 30 Drug-loaded self-crosslinking vesicle CLPs and blood circulation targeting self-crosslinking vesicles cNGQ20/CLPs
  • the fluorescent substance cy-7-labeled cRGD20/CLPs and untargeted CLPs were injected into the mice through the tail vein, and then lived with small animals at different time points 1, 2, 4, 6, 8, 12, 24, 48 hours.
  • the imager tracks the whereabouts of the vesicles.
  • the experimental results show that cRGD20/CLPs accumulate rapidly at the tumor site, and the fluorescence is still strong after 48 hours. This indicates that cRGD20/CLPs can actively target and enrich tumor sites.
  • the operation and calculation methods of other in vivo imaging experiments targeting self-crosslinking vesicles and self-crosslinking vesicles were the same, and the results are shown in Table 4.
  • Example 32 In vivo imaging experiment of drug-loaded self-crosslinking vesicle CLPs and targeted self-crosslinking vesicle cNGQ20/CLPs in mice bearing A549 lung cancer
  • Tumor inoculation and tail vein administration in the in vivo imaging experiment were the same as in Example 31.
  • Example 25 Epi ⁇ HCl-loaded, cy-7-labeled CLPs and cNGQ20/CLPs were found to accumulate rapidly at the tumor site, CLPs disappeared in 4-6 hours, and cNGQ20/CLPs were The fluorescence of the tumor site remained strong after 48 hours, indicating that cNGQ20/CLPs can actively target and enrich the tumor site.
  • Table 4 The results are in Table 4.
  • Example 33 In vivo imaging experiment of drug-loaded self-crosslinking vesicle CLPs and drug-loaded self-crosslinking vesicles CC9/CLPs in mice bearing H460 lung cancer
  • In vivo imaging experiments were performed on Balb/C nude mice weighing about 18-20 g and 4-6 weeks old, and subcutaneously injecting 5 ⁇ 10 6 H460 human lung cancer cells. After about 3 to 4 weeks, the tumor size was 100-200 mm. The experiment started at 3 o'clock.
  • Targeted self-crosslinking vesicles CC9/CLPs and drug-loaded preparations prepared from CC9-PEG6.5k-P (CDC3.8k-co-LA13.8k) and PEG5k-P (CDC3.7k-co-LA14.6k)
  • the cross-linked vesicle CLPs were labeled with cy-5 and loaded with the hydrophobic drug docetaxel DTX, and the 32nd operation of the example was used to study in vivo imaging.
  • the experimental results show that CC9/CLPs carrying DTX can accumulate rapidly in the tumor site, and the fluorescence of the tumor site is still strong after 48 hours. It is indicated that CC9/CLPs can actively target and enrich the tumor site, while the drug-free self-crosslinking vesicles are metabolized quickly after entering the tumor in 2 hours, and the intensity is low.
  • Table 4 The results are shown in Table 4.
  • the amount of DOX ⁇ HCl accumulated in tumors after cRGD20/CLPs, CLPs and DOX ⁇ HCl injection for 12 hours were 6.54, 2.53 and 1.02 ID%/g, respectively, and cRGD20/CLPs were 3 and 6 times higher than CLPs and DOX ⁇ HCl, indicating drug loading.
  • cRGD20/CLPs accumulated more at the tumor site by active targeting, and the results are shown in Table 4.
  • DOX ⁇ HCl-containing cNGQ20/CLPs, non-targeted CLPs, and liposomal doxorubicin-rich DOX-LPs were injected into mice (DOX ⁇ HCl: 10 mg/kg). After 6 hours, the amount of DOX ⁇ HCl accumulated in tumors of cNGQ20/CLPs, CLPs and DOX-LP were 8.63, 3.52 and 1.82 ID%/g, respectively, and cNGQ20/CLPs were 2 and 5 times of the latter two, indicating that drug-loaded cNGQ20 /CLPs through active targeting More accumulation in the tumor site. The result is shown in Figure 11.
  • Modeling of H460-loaded lung cancer mice was the same as in Example 33, tail vein administration and animal operation as in Example 34.
  • DTX-loaded CC9/CLPs, untargeted CLPs, and DOX-LPs were administered intravenously. After 6 hours, the amount of DTX in CC9/CLPs, CLPs and DOX-LPs accumulated in tumors was 9.02, 2.42 and 1.82 ID%/g, respectively.
  • CC9/CLPs were 4 and 5 times higher than CLPs and DOX-LPs, indicating drug-loaded CC9/ CLPs accumulate at the tumor site by active self-targeting, as shown in Table 4.
  • Example 37 Anti-tumor effect, body weight change and survival rate of drug-targeted self-crosslinking vesicle cRGD20/CLPs and self-crosslinking vesicle CLPs in mice bearing A549 subcutaneous lung cancer
  • the body weight of the mice was weighed every two days, and the tumor volume was measured by a vernier caliper.
  • the survival of the mice was continuously observed for 45 days. As can be seen from Fig. 12, tumors were significantly inhibited at 18 days in the cRGD20/CLPs treatment group, while tumors in the drug-loaded CLPs group had a certain increase. Although DOX ⁇ HCl also inhibited tumor growth, the body weight of the mice decreased by 21% at 12 days, indicating that the toxic side effects on mice were large.
  • mice in the cRGD20/CLPs and CLPs groups showed little change in body weight, indicating that the drug-loaded self-crosslinking vesicles had no toxic side effects on mice.
  • the cRGD20/CLPs treatment group survived after 60 days, the DOX ⁇ HCl group had all died at 42 days, and the PBS group also died at 43 days. Therefore, the targeted self-crosslinking vesicles of the present invention can effectively inhibit the growth of tumors, have no toxic side effects on mice, and can prolong the survival time of tumor-bearing mice.
  • Example 38 Anti-tumor effect, body weight change and survival rate of drug-targeted self-crosslinking vesicle cNGQ/CLPs and self-crosslinking vesicle CLPs in mice bearing A549 subcutaneous lung cancer
  • DOX ⁇ HCl-loaded self-crosslinking capsule prepared by mixing 1:5 with cNGQ-PEG6.5k-P (CDC4.6k-co-TMC18.6k) and PEG5k-P (CDC4.9k-co-TMC19k) Bubble cNGQ20/CLPs, non-targeted CLPs, DOX-LPs, and PBS tail vein injection.
  • the tumors were significantly inhibited at 18 days in the cNGQ20/CLPs treatment group, while the tumor-bearing CLPs group had tumor growth and the mice had almost no change in body weight.
  • DOX-LPs Although DOX-LPs also inhibited tumor growth, the body weight of mice in the DOX-LPs group decreased by 18% at 12 days, indicating that the toxic side effects on mice were large.
  • Example 39 Anti-tumor effect, body weight change and survival rate of drug-targeted self-crosslinking vesicle CC9/CLPs and self-crosslinking vesicle CLPs in mice bearing H460 subcutaneous lung cancer
  • the subcutaneous H460 tumor model was established as in the thirty-third example, and the tail vein administration method and data collection were the same as in the thirty-seventh embodiment.
  • the experiment was started when the tumor size was 30-50 mm 3 , and mixed by CC5-PEG6.5k-P (CDC3.8k-co-LA13.8k) and PEG5k-P (CDC3.7k-co-LA14.6k) at 1:5.
  • CC5-PEG6.5k-P CDC3.8k-co-LA13.8k
  • PEG5k-P CDC3.7k-co-LA14.6k
  • Example 40 drug-loaded self-crosslinking vesicle cRGD20/CLPs and self-crosslinking vesicle CLPs Antitumor effect, body weight change and survival rate in mice bearing A549 orthotopic lung cancer
  • the experiment used Balb/C nude mice weighing about 18-20 g and 4-6 weeks old, and directly injected 5 ⁇ 10 6 A549 human lung cancer cells (A549-Luc) with bioluminescence in the lung, about 10 days later.
  • A549-Luc human lung cancer cells
  • DOX ⁇ prepared by mixing 1:5 with cRGD-PEG6.5k-P (CDC4.6k-co-TMC18.6k) and PEG5k-P (CDC4.9k-co-TMC19k) HCl-targeted self-crosslinking vesicles cRGD20/CLPs, CLPs, DOX ⁇ HCl, and PBS were injected into mice by tail vein at 0, 4, 8 and 12 days (DOX ⁇ HCl: 10 mg/kg). From 0 to 16 days, the body weight of the mice was weighed every four days, and the bioluminescence of the lung tumors of the mice was monitored by a small animal live imager, and the survival of the mice was observed for 45 days.
  • the bioluminescence intensity of lung tumors continued to decrease within 16 days of the cRGD20/CLPs treatment group, while the bioluminescence intensity of the lungs in the drug-loaded CLPs group increased to some extent, but the body weight of the two groups was almost unchanged.
  • DOX ⁇ HCl also inhibited tumor growth
  • the body weight of mice in the DOX ⁇ HCl group decreased by 21% at 4 days, indicating that the toxic side effects on mice were large.
  • the cRGD20/CLPs treatment group survived after 45 days, the DOX ⁇ HCl group had all died at 30 days, and the PBS group also died at 20 days. Therefore, drug-loaded self-crosslinking vesicles cRGD20/CLPs can effectively inhibit the growth of orthotopic lung cancer tumors, have no toxic side effects on mice, and effectively prolong the survival time of tumor-bearing mice.
  • Example 41 Anti-tumor effect, body weight change and survival rate of drug-targeted self-crosslinking vesicle cNGQ20/CLPs and self-crosslinking vesicle CLPs in mice bearing A549 orthotopic lung cancer
  • the mouse model of A549 orthotopic lung cancer was established, administered, and tested in the same manner as in Example 40.
  • DOX ⁇ HCl-loaded self-crosslinking capsule prepared by mixing 1:5 with cNGQ-PEG6.5k-P (CDC4.6k-co-TMC18.6k) and PEG5k-P (CDC4.9k-co-TMC19k) Bubble cNGQ20/CLPs, non-targeted CLPs, DOX-LPs, and PBS tail vein injection. The results are shown in Figure 15.
  • the bioluminescence intensity of the tumor continued to decrease within 16 days, while the tumor bioluminescence intensity of the drug-loaded CLP group increased to some extent, and the body weight of the mice hardly changed. change.
  • DOX-LPs also inhibited tumor growth
  • the body weight of DOX-LPs mice decreased by 21% at 4 days.
  • the cNGQ20/CLPs treatment group survived after 45 days, the DOX-LPs group had all died at 32 o'clock, and the PBS group also died at 23 days. Therefore, drug-loaded self-crosslinking vesicle cNGQ20/CLPs can also effectively inhibit the growth of lung cancer in situ, have no toxic side effects on mice, and can prolong the survival time of tumor-bearing mice.
  • Example 42 Anti-tumor effect, body weight change and survival rate of drug-targeted self-crosslinking vesicle CC9/CLPs and self-crosslinking vesicle CLPs in mice bearing A549 orthotopic lung cancer
  • mice The mouse model of A549 orthotopic lung cancer was established, administered, and tested in the same manner as in Example 40.
  • Preparation of CPT ⁇ HCl-loaded self-crosslinking by mixing cc9-PEG6.5k-P (CDC3.8k-co-LA13.8k) and PEG5k-P (CDC3.7k-co-LA14.6k) at 1:5 Vesicles CC9/CLP, untargeted CLPs, CPT ⁇ HCl, and PBS were injected into mice.
  • CC9/CLPs treatment group the tumor bioluminescence intensity decreased, while the bioluminescence intensity of the drug-loaded CLPs group increased, and the body weight of the mice hardly changed.
  • CPT ⁇ HCl also inhibited tumor growth
  • the body weight of mice in the CPT ⁇ HCl group decreased by 21% at 3 days, indicating that the toxic side effects on mice were large.
  • the CC9/CLPs treatment group survived after 40 days, all of them died in the CPT ⁇ HCl group at 34, and all died in the PBS group at 21 days. Therefore, drug-loaded self-crosslinking vesicles CC9/CLPs can effectively inhibit the growth of lung cancer in situ, have no toxic side effects, and can prolong the survival time of tumor-bearing mice.
  • Example 43 Anti-tumor effect, body weight change and survival rate of drug-targeted self-crosslinking vesicle cRGD/CLPs and self-crosslinking vesicle CLPs in mice bearing A549 orthotopic lung cancer
  • PTX-loaded self-crosslinking vesicles were prepared by mixing 1:5 with AA-PEG3k-P (CDC3.9k-PDSC4.8k) and PEG1.9k-P (CDC3.6k-PDSC4.6k).
  • the PTX-loaded self-crosslinking vesicle cRGD/CLP was then prepared by Michael addition reaction of the acrylate (AA) and cRGDfC thiol groups on the surface of the vesicles as in Example 21.
  • the DLS was measured to be 85 nm and the particle size distribution was 0.10.
  • the graft ratio of the calculated molecular weight of the nuclear magnetic and BCA protein kits was 92%.
  • mice The establishment, administration and detection of a mouse model of A549 orthotopic lung cancer are the same as in the fourth embodiment. ten.
  • PGT-loaded cRGD/CLPs, non-targeted self-crosslinking vesicle CLPs, Taxol, and PBS were injected into mice, respectively.
  • the tumor bioluminescence intensity continued to decrease, while the untargeted CLPs group showed an increase in tumor bioluminescence intensity, and the body weight of the two groups of mice hardly changed.
  • PTX also inhibited tumor growth, the weight of mice in the PTX group decreased by 10% at 12 days, indicating that the toxic side effects on mice were large.
  • the pRGD/CLPs-treated group with PTX survived for 41 days, the mice in the PTX group all died at 29 o'clock, and the PBS group died at 32 days. Therefore, the PRGD/CLPs loaded with PTX can effectively inhibit the growth of lung cancer in situ, have no toxic side effects, and prolong the survival time of tumor-bearing mice.

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Abstract

本发明公开了一种侧链含双硫的生物可降解双亲性聚合物及其自交联聚合物囊泡以及在肺癌靶向治疗中的应用。所述聚合物基于含有双硫五元环功能基团的环碳酸酯单体通过活性可控开环聚合得到，其分子量可控、分子量分布较窄，无需保护和脱保护过程；利用本发明所述的环碳酸酯单体开环聚合得到的聚合物具有生物可降解性，可用于控制药物释放体系，制备的肺癌靶向的还原敏感可逆交联的囊泡纳米药物载体支持体内稳定长循环，但在肺癌组织高富集并高效进入细胞，在细胞内快速解交联、释放出药物，高效特异性地杀死癌细胞，有效抑制肿瘤的生长而不造成毒副作用。

Description

生物可降解双亲性聚合物、由其制备的聚合物囊泡及在制备肺癌靶向治疗药物中的应用 技术领域
本发明涉及一种生物可降解聚合物材料及其应用，具体涉及一种侧链含双硫五元环功能基团的生物可降解双亲性聚合物及聚合物囊泡以及在肺癌靶向治疗中的应用，属于医药材料领域。
背景技术
生物可降解聚合物具有非常独特的性能而被广泛应用于生物医学的各个领域，如手术缝合线、骨固定器械、生物组织工程支架材料、和药物控制释放载体等。合成的生物可降解聚合物主要有脂肪族聚酯(聚乙交酯PGA、聚丙交酯PLA、丙交酯-乙交酯共聚物PLGA、聚己内酯PCL)、聚碳酸酯(聚三亚甲基环碳酸酯PTMC)等是最常用的生物可降解聚合物，已获得美国食品药物管理部门(FDA)的许可。
但是，现有的生物可降解聚合物如PTMC、PCL、PLA和PLGA等结构比较单一，缺乏可修饰官能团，往往难以提供循环稳定的药物载体或是稳定的表面修饰涂层。聚碳酸酯的降解产物主要是二氧化碳和中性的二元醇，不产生酸性降解产物。其中功能性环状碳酸酯单体可和环酯类单体如GA、LA和ε-CL等，以及其它环状碳酸酯单体共聚，得到不同性能的生物可降解聚合物。
另外，由现有技术制备的生物可降解聚合物得到的生物可降解纳米载体存在体内循环不稳定、肿瘤细胞摄取低、细胞内药物浓度低的问题，这导致纳米药物的药效不高，还存在毒副作用。由功能性生物可降解聚合物可制备胶束纳米，其在体内循环稳定，但只能装载疏水性小分子抗癌药物，而对 穿透能力强的亲水性小分子抗癌药物以及毒副作用小的亲水性生物大分子药物如蛋白药物和核酸类药物无能为力，极大地限制了其作为药物载体的应用。
癌症是威胁人类健康的主要杀手，其发病率和死亡率呈逐年上升的趋势。肺癌在世界上尤其是在我国的发病率居高不下。手术只能对早期的肺癌患者有利，而对于中晚期无效。肺癌的治疗存在难以早期诊断、愈后差、易转移、易耐药的特点。纳米药物是治疗肺癌的一个关键点和希望所在。但是现有技术中，尚缺乏在体内循环稳定、特异性靶向肺癌、细胞内快速释放药物、毒副作用小的高效纳米药物，尤其是缺少能够运输亲水性小分子抗癌药物的纳米载体。
发明内容
本发明的目的是提供一种生物可降解双亲性聚合物、由其制备的聚合物囊泡、及其作为抗肺癌药物的载体在制备肺癌靶向治疗药物中的应用。
为达到上述目的，本发明具体的技术方案为：
一种生物可降解双亲性聚合物，其化学结构式为：
Figure PCTCN2016111385-appb-000001
其中，R1选自以下基团中的一种：
Figure PCTCN2016111385-appb-000002
R2选自以下基团中的一种：
Figure PCTCN2016111385-appb-000003
其中，k为43～170，x为10～30，y为40～200；m为86～340。
本发明公开的生物可降解双亲性聚合物，疏水嵌段含有含双硫五元环功能基团的环碳酸酯单元；可以为二嵌段聚合物：
Figure PCTCN2016111385-appb-000004
也可以为三嵌段聚合物：
Figure PCTCN2016111385-appb-000005
优选的技术方案中，R1选自以下基团中的一种：
Figure PCTCN2016111385-appb-000006
R2选自以下基团中的一种：
Figure PCTCN2016111385-appb-000007
优选的，上述生物可降解双亲性聚合物化学结构式中，k为113～170，x为20～26，y为100～190，m为226～340。
上述生物可降解双亲性聚合物侧链含有双硫，可以在引发剂存在下，在溶剂中，由含双硫五元环功能基团的环状碳酸酯单体和其他环酯单体、环碳酸酯单体开环聚合得到；所述其他环碳酸酯单体包括三亚甲基环碳酸酯(TMC)、侧链含有三甲氧基苯甲缩醛的环状碳酸酯(PTMBPEC)、侧链含有双硫吡啶的环状碳酸酯(PDSC)和丙烯酸酯三羟甲基乙烷环碳酸酯(AEC)。所述其他环酯单体包括丙交酯(LA)、乙交酯(GA)和己内酯(CL)。
含双硫五元环功能基团的环状碳酸酯单体(CDC)的化学结构式如下：
Figure PCTCN2016111385-appb-000008
比如，上述环碳酸酯单体(CDC)可以在二氯甲烷中、以单甲氧基聚乙二醇为引发剂、双(双三甲基硅基)胺锌为催化剂和TMC开环共聚合，形成CDC与TMC单元无规排列的二嵌段聚合物；其反应式如下：
Figure PCTCN2016111385-appb-000009
本发明公开的侧链含双硫的双亲性聚合物具有生物可降解性，其疏水部分的分子量是亲水部分分子量的三倍及以上，可通过溶剂置换法、透析法、 或薄膜水化法等方法来制备得到聚合物囊泡结构。制备的聚合物囊泡为纳米尺寸，粒径40-180纳米，可以作为治疗肺癌的药物的载体；在囊泡的疏水膜中装载疏水性小分子抗肺癌药物紫杉醇、多西紫杉醇等，也可以在囊泡的大的亲水内腔中装载亲水性抗肺癌药物，尤其是亲水性小分子抗癌药物如盐酸多柔比星、盐酸表阿霉素、盐酸伊利替康和盐酸米托蒽醌。这样克服了现有的由双亲性聚合物形成的胶束载体只能装载疏水药物的缺陷和现有技术中没有能高效装载、并稳定体内循环的亲水性小分子抗癌药物的载体的缺陷；上述生物可降解双亲性聚合物的亲水段PEG的末端可以化学偶联肿瘤特异性靶向分子如cRGD、cNGQ或者cc-9等多肽制备得到肿瘤特异靶向的生物可降解双亲性聚合物。
本发明还公开了聚合物囊泡，可以由上述生物可降解双亲性聚合物制备得到；或者由上述肿瘤特异靶向的生物可降解双亲性聚合物制备得到；或者由上述生物可降解双亲性聚合物与肿瘤特异靶向的生物可降解双亲性聚合物制备得到，比如上述生物可降解双亲性聚合物和肿瘤特异靶向的生物可降解双亲性聚合物按照不同比例混合，可制备具有不同靶向密度的聚合物囊泡，即得到肺癌靶向自交联囊泡，来可以增加囊泡纳米药物在肺癌细胞中的摄取量；也可以由生物可降解双亲性聚合物制备的交联囊泡或自交联囊泡的外表面偶联肿瘤细胞特异性靶向分子来制备肺癌靶向交联囊泡和肺癌靶向自交联囊泡，以增加肺癌细胞的摄取量，比如在囊泡的PEG端通过迈克尔加成键合cRGD、cNGQ或者cc-9。
上述生物可降解双亲性聚合物和肿瘤特异靶向的生物可降解双亲性聚合物可在不加入任何物质的情况下自行交联，得到自交联聚合物囊泡和肺癌靶向自交联聚合物囊泡；或是在催化量的还原剂如二硫代苏糖醇(DTT)或谷胱甘肽(GSH)催化下，制备得到交联聚合物囊泡和肺癌靶向交联聚合物囊泡。自交联囊泡、肺癌靶向自交联囊泡、交联囊泡和肺癌靶向交联囊泡在 囊泡疏水膜内形成了稳定的化学交联，从而可在体内稳定长循环；但内吞进入癌细胞后，在细胞内大量还原性物质存在环境下，形成的交联会快速解除(解交联)，快速释放出药物，高效杀死肺癌细胞。所以本发明请求保护上述生物可降解双亲性聚合物在制备治疗肺癌的纳米药物中的应用；进一步的地，本发明还公开了上述聚合物囊泡在制备治疗肺癌的纳米药物中的应用，包括由侧链含双硫的生物可降解双亲性聚合物制备的聚合物囊泡、自交联聚合物囊泡，由肿瘤特异靶向的生物可降解双亲性聚合物独自或者和生物可降解双亲性聚合物制备的肺癌靶向自交联聚合物囊泡、肺癌靶向交联聚合物囊泡在制备靶向治疗肺癌的纳米药物中的应用。基于本发明聚合物制备的抗肺癌纳米药物为囊泡抗肺癌纳米药物。
由于上述方案的实施，本发明与现有技术相比，具有以下优点：
1.本发明利用含双硫五元环功能基团的环状碳酸酯单体、以聚乙二醇为引发剂、和TMC或LA通过活性可控开环聚合共聚合得到分子量可控、分子量分布较窄的侧链含双硫的生物可降解双亲性聚合物；由于硫硫五元环基团不影响环碳酸酯单体的开环聚合，因此聚合过程无需现有技术中的保护和脱保护过程，简化了操作步骤。
2.本发明公开的侧链含双硫的生物可降解双亲性聚合物具有生物可降解性，可以制备聚合物囊泡和肺癌靶向囊泡，装载不同性质的药物，可以不加入任何物质而自行交联，形成稳定的自交联聚合物囊泡纳米药物，从而克服了现有技术中纳米药物体内循环不稳定、药物易早释、造成毒副作用的缺陷。
3.本发明公开的自交联囊泡纳米药物的交联具有可逆性，即支持体内长循环，可在肺癌细胞高富集；但是进入肺癌细胞内后却可以快速解交联，释放出药物，实现高效特异性地杀死肺癌细胞而不具有毒副作用。克服了现有技术中交联纳米药物过于稳定、而在细胞内药物释放缓慢、造成耐药性的 缺陷。
4.本发明公开的生物可降解聚合物囊泡和肺癌靶向囊泡，可以不加入任何物质而制备自交联囊泡，制备方法简便，从而克服了现有技术中制备交联纳米药物时候存在的必须加入交联剂等物质以及需要复杂的操作和提纯过程等缺陷。
5.本发明公开的双亲性聚合物自组装制备的自交联聚合物囊泡可用于亲水小分子抗癌药物的控制释放体系，从而克服了现有生物可降解纳米胶束载体仅适用装载疏水小分子药物的缺陷和现有技术中没有能高效装载、并稳定体内循环的亲水性小分子抗癌药物的缺陷；进一步地，可制备肺癌靶向的自交联囊泡，在肺癌的高效靶向治疗方面具有更广泛的应用价值。
附图说明
图1为实施例二中聚合物PEG5k-P(CDC4.9k-co-TMC19k)的氢核磁谱图；
图2为实施例六中聚合物PEG5k-P(CDC3.7k-co-LA14.6k)的核磁谱图；
图3是实施例十五中交联囊泡PEG5k-P(CDC4.9k-co-TMC19k)粒径分布(A)及电子投射显微镜图片(B)，交联囊泡稳定性的测试(C)及还原响应性测试(D)图；
图4为实施例十五中载DOX·HCl交联囊泡PEG5k-P(CDC4.9k-co-TMC19k)的体外释放图；
图5是实施例二十四中载DOX·HCl交联囊泡cRGD20/PEG6k-P(CDC4.6k-co-TMC18.6k)的体外释放图；
图6是实施例二十六中靶向交联囊泡cRGD/PEG6k-P(CDC4.6k-co-TMC18.6k)对A549肺癌细胞的毒性结果图；
图7是实施例二十六中载DOX·HCl靶向交联囊泡 cRGD/PEG6k-P(CDC4.6k-co-TMC18.6k)对A549肺癌细胞的毒性结果图；
图8是实施例二十八中载DOX·HCl靶向交联囊泡cRGD/PEG6k-P(CDC4.6k-co-TMC18.6k)在小鼠体内的血液循环研究结果图；
图9为实施例二十九中载DOX·HCl靶向交联囊泡cNGQ/PEG6k-P(CDC4.6k-co-TMC18.6k)在小鼠体内的血液循环研究结果图；
图10为实施例三十三中载DOX·HCl靶向交联囊泡cRGD/PEG6k-P(CDC4.6k-co-TMC18.6k)对荷皮下肺癌小鼠的生物分布结果图；
图11为实施例三十四中载DOX·HCl靶向交联囊泡cNGQ/PEG6k-P(CDC4.6k-co-TMC18.6k)对荷皮下肺癌小鼠的生物分布结果图；
图12为实施例三十六中载DOX·HCl靶向交联囊泡cRGD/PEG6k-P(CDC4.6k-co-TMC18.6k)在皮下荷肺癌小鼠的治疗图，其中A为肿瘤生长曲线，B为小鼠治疗后肿瘤图片，C为体重变化，D为生存曲线；
图13为实施例三十七中载DOX·HCl靶向交联囊泡cNGQ/PEG6k-P(CDC4.6k-co-TMC18.6k)在皮下荷肺癌小鼠的治疗图，其中A为肿瘤生长曲线，B为体重变化曲线，C为生存曲线；
图14为实施例三十九中载DOX·HCl靶向交联囊泡cRGD/PEG6k-P(CDC4.6k-co-TMC18.6k)在原位荷肺癌小鼠的治疗图，其中A为肿瘤生长曲线，B为体重变化曲线，C为生存曲线；
图15为实施例四十中载DOX·HCl靶向交联囊泡cNGQ/PEG6k-P(CDC4.6k-co-TMC18.6k)在原位荷肺癌小鼠的治疗图，其中A为肿瘤生长曲线，B为体重变化曲线，C为生存曲线。
具体实施方式
下面结合实施例和附图对本发明作进一步描述：
实施例一含双硫五元环功能基团的环状碳酸酯单体(CDC)的合成
一水合硫氢化钠(28.25g，381.7mmol)溶在400mL N，N-二甲基甲酰胺(DMF)中，50℃加热至完全溶解，逐滴加入二溴新戊二醇(20g，76.4mmol)，反应48小时。反应物减压蒸馏除去溶剂DMF，然后用200mL蒸馏水稀释，用250mL乙酸乙酯萃取四次，最后有机相旋蒸得到黄色粘稠状化合物A，产率：70％；溶解在400mL的四氢呋喃(THF)中的化合物A在空气中放置24小时，分子间巯基氧化成硫硫键，得到化合物B，产率；>98％；在氮气保护下，化合物B(11.7g，70.5mmol)溶于干燥过的THF(150mL)中，搅拌至完全溶解。接着冷却到0℃，加入氯甲酸乙酯(15.65mL，119.8mmol)，然后逐滴加入Et3N(22.83mL，120.0mmol)。待滴加完毕后，该体系在冰水浴条件下继续反应4h。反应结束后，过滤掉产生的Et3N·HCl，滤液经旋转浓缩，最后用乙醚进行多次重结晶，得到黄色晶体，即含双硫五元环功能基团的环状碳酸酯单体(CDC)，产率：64％。
实施例二两嵌段侧链含双硫五元环聚合物PEG5k-P(CDC4.9k-co-TMC19k)的合成
在氮气环境下，0.1g(0.52mmol)CDC单体和0.4g(3.85mmol)的三亚甲基碳酸酯(TMC)溶在3mL二氯甲烷中，加入密封反应器里，然后加入0.1g(0.02mmol)CH3O-PEG5000和0.5mL的催化剂双(双三甲基硅基)胺锌的二氯甲烷溶液(0.1mol/L)，接着把反应器密封好，转移出手套箱，40℃油浴中反应2天后，冰乙酸终止反应，冰乙醚中沉淀，最终经过过滤、真空干燥得到PEG5k-P(CDC4.9k-co-TMC19.0k)。核磁图见附图1，1H NMR(400MHz,CDCl3):2.08(t,-COCH2CH2CH2O-),3.08(s,-CCH2),3.30(m，-OCH3)，3.65(t，-OCH2CH2O-)，4.28(t,-COCH2CH2CH2O-),4.31(m,-CCH2)。核磁计算出下式中k＝114，x＝26，y＝186。GPC测分子量：34.5kDa， 分子量分布：1.48。
Figure PCTCN2016111385-appb-000010
实施例三两嵌段侧链含双硫五元环聚合物Mal-PEG6k-P(CDC4.8k-co-TMC19.2k)的合成
在氮气环境下，0.1g(0.52mmol)CDC单体和0.4g(3.85mmol)的TMC溶在3mL二氯甲烷中，加入密封反应器里，然后加入0.12g(0.02mmol)Mal-PEG6000和0.1mol/L的催化剂双(双三甲基硅基)胺锌的二氯甲烷溶液(0.1mol/L)，接着把反应器密封好，转移出手套箱，40℃油浴反应2天后，冰乙酸终止反应，冰乙醚中沉淀，最终经过过滤、真空干燥得到Mal-PEG6k-P(CDC4.8k-co-TMC19.2k)。1H NMR(400MHz,CDCl3):2.08(t,-COCH2CH2CH2O-),3.08(s,-CCH2),3.30(m，-OCH3)，3.65(t，-OCH2CH2O-)，4.28(t,-COCH2CH2CH2O-),4.31(m,-CCH2)，和6.70(s，Mal)。核磁计算出下式中，k＝136，x＝25，y＝188。GPC测的分子量：38.6kDa，分子量分布：1.42。
Figure PCTCN2016111385-appb-000011
实施例四两嵌段侧链含双硫聚合物NHS-PEG6.5k-P(CDC4.6k-co-TMC18.6k)的合成
在氮气环境下，0.1g(0.52mmol)CDC单体和0.4g(3.85mmol)的TMC溶在3mL二氯甲烷中，加入密封反应器里，然后加入0.1g(0.015mmol)NHS-PEG6500和0.5mL的催化剂双(双三甲基硅基)胺锌的二氯甲烷溶液 (0.1mol/L)，接着把反应器密封好，转移出手套箱，40℃油浴反应2天后，冰乙酸终止反应，冰乙醚中沉淀，最终经过过滤、真空干燥得到NHS-PEG6.5k-P(CDC4.6k-co-TMC18.6k)。1H NMR(400MHz,CDCl3):2.08(t,-COCH2CH2CH2O-),3.08(s,-CCH2),3.30(m，-OCH3)，3.65(t，-OCH2CH2O-)，4.28(t,-COCH2CH2CH2O-),4.31(m,-CCH2)，和2.3(s，NHS)。核磁计算出下式中k＝145，x＝24.0，y＝182。GPC测分子量：37.6kDa，分子量分布：1.38。
Figure PCTCN2016111385-appb-000012
实施例五两嵌段侧链含双硫五元环聚合物PEG1.9k-P(CDC1.9k-co-TMC4.1k)的合成
在氮气环境下，0.1g(0.52mmol)CDC单体和0.2g(1.93mmol)的TMC溶在1mL二氯甲烷中，加入密封反应器里，然后加入0.1g(0.05mmol)CH3O-PEG1900和0.5mL的催化剂双(双三甲基硅基)胺锌的二氯甲烷溶液(0.1mol/L)，40℃油浴反应2天，后处理同实施例二，得到PEG1.9k-P(CDC1.9k-co-TMC3.9k)。反应式和1H NMR特征峰同实施例二。核磁计算出式中k＝46，x＝10，y＝40。GPC测分子量：14.5kDa，分子量分布：1.36。
实施例六两嵌段侧链含双硫聚合物PEG5k-P(CDC3.7k-co-LA14.6k)的合成
在氮气环境下，0.08g(0.42mmol)CDC和0.3g(2.1mmol)的丙交酯(LA)溶在2mL二氯甲烷中，加入密封反应器里，然后加入0.1g(0.02mmol)CH3O-PEG5000和0.1mol/L的催化剂双(双三甲基硅基)胺锌的二氯甲烷 溶液(0.1mL)，40℃油浴反应2天，后处理同实施例二，得到PEG5k-P(CDC3.7k-co-LA14.6k)。核磁见附图2，1H NMR(400MHz,CDCl3):1.,59(s,-COCH(CH3)O-),3.08(s,-CCH2),3.30(m，-OCH3)，3.65(t，-OCH2CH2O-)，4.31(m,-CCH2)，5.07(s,-COCH(CH3)O-)。核磁计算出下式中，k＝114，x＝19，y＝101。GPC测的分子量：24.3kDa，分子量分布：1.32。
Figure PCTCN2016111385-appb-000013
实施例七两嵌段侧链含双硫聚合物PEG6.5k-P(CDC5.8k-co-LA28.3k)的合成
在氮气环境下，0.1g(0.57mmol)CDC和0.5g(3.5mmol)的LA溶在3mL二氯甲烷中，加入密封反应器，然后加入0.11g(0.015mmol)CH3O-PEG6500和0.5mL的催化剂双(双三甲基硅基)胺锌的二氯甲烷溶液(0.1mol/L)，40℃油浴反应2天，后处理同实施例二，得到PEG6.5k-P(CDC5.8k-co-LA28.3k)。反应式和1H NMR特征峰同实施例六。核磁计算出式中k＝148，x＝30，y＝200。GPC测分子量：42.4kDa，分子量分布：1.43。
实施例八两嵌段侧链含双硫聚合物Mal-PEG6k-P(CDC3.6k-co-LA18.6k)的合成
在氮气环境下，0.1g(0.52mmol)CDC和0.5g(5.56mmol)的LA溶在4mL二氯甲烷中，加入密封反应器里，然后加入0.15g(0.025mmol)Mal-PEG6000和0.1mol/L的催化剂双(双三甲基硅基)胺锌的二氯甲烷溶液(0.1mL)40℃油浴反应2天，后处理同实施例二，得到Mal-PEG6k-P(CDC3.6k-co-LA18.6k)。1H NMR(400MHz,CDCl3):1.59(s,-COCH(CH3)O-),3.08(s,-CCH2),3.30(m，-OCH3)，3.65(t，-OCH2CH2O-)， 4.31(m,-CCH2)，5.07(s,-COCH(CH3)O-)，和6.70(s，Mal)。核磁计算出下式中k＝136，x＝19，y＝129。GPC测分子量：32.5kDa，分子量分布：1.44。
Figure PCTCN2016111385-appb-000014
实施例九三嵌段聚合物P(CDC3.8k-TMC18.8k)-PEG5k-P(CDC3.8k-TMC18.8k)的合成
在氮气环境下0.8g(7.84mmol)的TMC和0.16g(0.83mmol)CDC溶在8mL二氯甲烷中，加入密封反应器里，后加入0.1g(0.02mmol)的HO-PEG-OH5000和1mL的催化剂双(双三甲基硅基)胺锌的二氯甲烷溶液(0.2mol/L)，40℃油浴反应2天，后处理同实施例二，得到三嵌段聚合物P(CDC3.8k-TMC18.8k)-PEG5k-P(CDC3.8k-TMC18.8k)。1H NMR特征峰同实施例二。核磁计算出下式中，m＝114，x＝20，y＝184。GPC测的分子量：78.9kDa，分子量分布：1.54。
Figure PCTCN2016111385-appb-000015
实施例十两嵌段侧链含双硫聚合物NHS-PEG7.5k-P(CDC3.8k-co-LA13.8k)的合成
在氮气环境下，0.1g(0.52mmol)CDC和0.4g(2.8mmol)的LA溶在3mL二氯甲烷中，加入密封反应器里，然后加入0.013mmol的NHS-PEG7500和1mL的催化剂双(双三甲基硅基)胺锌的二氯甲烷溶液(0.1mol/L)，密封反应器，转移出手套箱，40℃油浴反应2天，后处理 同实施例二，得到NHS-PEG7.5k-P(CDC4.8k-co-LA19.0k)。1H NMR(400MHz,CDCl3):1.,59(s,-COCH(CH3)O-),3.08(s,-CCH2),3.30(m，-OCH3)，3.65(t，-OCH2CH2O-)，4.31(m,-CCH2)，5.07(s,-COCH(CH3)O-)和2.3(s，NHS)。核磁计算出下式中，k＝170，x＝20，y＝96。GPC测分子量：42.3kDa，分子量分布：1.45。
Figure PCTCN2016111385-appb-000016
实施例十一靶向两嵌段聚合物CC9-PEG7.5k-P(CDC3.8k-co-LA13.8k)的合成
环状多肽CSNIDARAC(cc9)偶联的聚合物CC9-PEG7.5k-P(CDC3.8k-co-LA13.8k)的合成分为两步，第一步如实施例十制备NHS-PEG7.5k-P(CDC3.8k-co-LA13.8k)；第二步为CC9与其通过酰胺化反应键合。先将上述聚合物NHS-PEG7.5k-P(CDC3.8k-co-LA13.8k)溶解在DMF中，加入两倍摩尔量的CC9，30℃反应两天、透析、冻干，得到CC9-PEG6.5k-P(CDC3.8k-co-LA13.8k)，通过核磁和BCA蛋白试剂盒测试计算CC9接枝率为91％。
实施例十二靶向两嵌段聚合物cRGD-PEG6k-P(CDC3.6k-co-LA18.6k)的合成
环状多肽c(RGDfC)(cRGD-SH)偶联的聚合物cRGD-PEG6k-P(CDC3.6k-co-LA18.6k)的合成分为两步，第一步如实施例八制备Mal-PEG6k-P(CDC3.6k-co-LA18.6k)；第二步cRGD-SH的巯基与其通过迈克尔加成反应键合。先将聚合物Mal-PEG6k-P(CDC3.6k-co-LA18.6k)溶在0.5毫升DMF中，加入2毫升的硼酸缓冲溶液(pH 8.0)，再加入1.5倍摩尔量的cRGD-SH，30℃反应两天，透析、冷冻干燥得到最终产物 cRGD-PEG6k-P(CDC3.6k-co-LA18.6k)，通过核磁和BCA蛋白试剂盒测试计算cRGD接枝率为94％。
实施例十三靶向两嵌段聚合物cRGD-PEG6.5k-P(CDC4.6k-co-TMC18.6k)的合成
环状多肽c(RGDfK)(cRGD)偶联的聚合物cRGD-PEG6.5k-P(CDC4.6k-co-TMC18.6k)的合成分为两步，第一步如实施例四制备NHS-PEG6.5k-P(CDC4.6k-co-TMC18.6k)；第二步cRGD的氨基与其通过酰胺化反应键合。先将上述聚合物NHS-PEG6.5k-P(CDC4.6k-co-TMC18.6k)溶解在DMF中，加入两倍摩尔量的cRGD，30℃反应两天后，透析除去游离cRGD，冷冻干燥得到cRGD-PEG6.5k-P(CDC4.6k-co-TMC18.6k)，通过核磁和BCA蛋白试剂盒测试计算cRGD接枝率为88％。
实施例十四靶向两嵌段聚合物cNGQ-PEG6.5k-P(CDC4.6k-co-TMC18.6k)的合成
环状多肽cNGQGEQc(cNGQ)偶联的聚合物cNGQ-PEG6.5k-P(CDC4.6k-co-TMC18.6k)的合成分为两步，第一步如实施例四制备NHS-PEG6.5k-P(CDC4.6k-co-TMC18.6k)；第二步为cNGQ的氨基与其通过酰胺化反应键合。先将上述聚合物NHS-PEG6.5k-P(CDC4.6k-co-TMC18.6k)溶在DMF中，加入两倍摩尔量的cNGQ，30℃反应两天后，透析除去游离cNGQ，冷冻干燥得到cNGQ-PEG6.5k-P(CDC4.6k-co-TMC18.6k)，通过核磁和BCA蛋白试剂盒计算cNGQ接枝率为92％。
采用上述类似的制备方法可以制备多种侧链含双硫的生物可降解双亲性聚合物，原料比例以及表征见表1。
表1各个聚合物制备条件、产物的核磁及GPC表征结果
Figure PCTCN2016111385-appb-000017
实施例十五溶剂置换法制备自交联聚合物囊泡PEG5k-P(CDC4.9k-co-TMC19k)
采用溶剂置换法制备聚合物囊泡。100μL的PEG5k-P(CDC4.9k-co-TMC19k)的DMF溶液(10mg/mL)滴加到900μL磷酸盐缓冲溶液(PB，10mM，pH 7.4)中，在37℃(200rmp)摇床中放置过夜进行自交联，然后装入透析袋(MWCO 7000)中透析过夜，换五次水，透析介质为PB(10mM，pH 7.4)。得到的自交联囊泡的尺寸由动态光散射粒度分析仪(DLS)测的形成的纳米囊泡为130nm，粒径分布很窄，见图3A，由图3B可知，TEM测得纳米粒子为中空的囊泡结构，自交联囊泡在高倍稀释和胎牛血清存在下仍然保持不变的粒径和粒径分布(图3C)，但在模拟肿瘤细胞还原环境下快速释放，解交联(图3D)。由此可知，得到的囊泡可自交联，并具有还原敏感的解交联的性质。
实施例十六透析法制备自交联聚合物囊泡PEG5k-P(CDC4.9k-co-TMC19k)
采用透析法制备聚合物囊泡。100μL的PEG5k-P(CDC4.9k-co-TMC19k)的DMF溶液(10mg/mL)装入透析袋(MWCO 7000)中，在PB(10mM，pH 7.4)中、37℃(200rmp)摇床中放置过夜自行交联，然后PB中透析24小时，换五次液。DLS测交联囊泡为80nm左右，粒径分布0.08。
实施例十七薄膜水化法制备自交联聚合物囊泡PEG5k-P(CDC4.9k-co-TMC19k)
采用薄膜水化法制备聚合物囊泡。2mg的PEG5k-P(CDC4.9k-co-TMC19k)的溶于0.5mL的低沸点有机溶剂中，如二氯甲烷或乙腈中，在25毫升的尖底烧瓶中，旋蒸在底部形成薄膜，然后再0.1mBar的真空度下继续抽干24小时。加入2mL的PB(10mM，pH 7.4)在37℃下搅拌把薄膜剥离表面，并搅碎，超声20分钟(200rmp)，不断搅拌24小时，得到的囊泡自行交联。DLS测定自交联囊泡的尺寸为180nm左右，粒径分布0.25。
实施例十八溶剂置换法制备交联聚合物囊泡PEG5k-P(CDC4.9k-co-TMC19k)
如实施例十五制备聚合物囊泡，滴加完毕后加入DTT(浓度为0.09μM)，在37℃交联12小时，然后装入透析袋(MWCO 7000)中透析过夜，换五次液。得到的自交联囊泡的尺寸为109nm左右，粒径分布0.13.
实施例十九cNGQ偶联的靶向自交联聚合物囊泡cNGQ/PEG5k-P(CDC4.9k-co-TMC19k)制备
实施例十四中得到的靶向聚合物cNGQ-PEG6.5k-P(CDC4.6k-co-TMC18.6k)和实施例二得到的PEG5k-P(CDC4.9k-co-TMC19k)二者混合溶于DMF中，如实施例十五制备cNGQ偶联的靶向自交联聚合物囊泡。靶向聚合物的PEG分子量比非靶向的PEG要长，保证靶向分子更好的支出表面。两者按不同比例混合可制备表面具有不同靶向分子的自交联囊泡。优选方案为前者含量为5-30wt.％。DLS测定其尺寸为90-120nm左右，粒径分布0.05-0.15。
实施例二十cRGD偶联的靶向自交联囊泡cRGD/PEG6.5k-P(CDC4.6k-co-TMC18.6k)的制备
采用薄膜水化法制备cRGD偶联的靶向自交联聚合物囊泡。1.6mg的实施例二得到的PEG5k-P(CDC4.9k-co-TMC19k)的DMF溶液(10mg/mL)及0.4mg的实施例十三中得到的cRGD-PEG6.5k-P(CDC4.6k-co-TMC18.6k)溶于0.5mL的低沸点有机溶剂中，如二氯甲烷或乙腈中，如实施例十七制备得到的自交联囊泡为88nm左右，粒径分布0.08。两者按不同比例混合可制备表面具有不同靶向分子的自交联囊泡。优选方案为前者含量为5-30wt.％。
实施例二十一CC9偶联的靶向自交联囊泡CC9/P(CDC3.8k-LA18.8k)-PEG4k-P(CDC3.8k-LA18.8k)的制备
实施例八制备的Mal-PEG6k-P(CDC3.6k-LA18.6k)和 P(CDC3.8k-LA18.8k)-PEG4k-P(CDC3.8k-LA18.8k)混合按照如实施例十六所述透析方法制备囊泡。然后加入0.5毫升的4M硼酸缓冲溶液(pH 8.0)调节溶液pH至7.5-8.0，再按照Mal摩尔量的1.5倍加入CC9，通过迈克尔加成反应键合，30℃反应两天后，透析。DLS测定为110nm，粒径分布0.16。核磁和BCA蛋白试剂盒测试计算多肽的接枝率为90％。两种聚合物者按不同比例混合可制备表面具有不同靶向分子的自交联囊泡。优选方案为前者含量为5-30wt.％。
采用上述类似的制备方法可以制备多种自交联聚合物囊泡和靶向自交联聚合物囊泡，原料比例以及表征见表2。
表2自交联聚合物囊泡和靶向自交联聚合物囊泡的制备和表征
Figure PCTCN2016111385-appb-000018
实施例二十二自交联聚合物囊泡PEG5k-P(CDC4.9k-co-TMC19k)的药物装载及体外释放
采用溶剂置换法制备聚合物囊泡，DOX·HCl的装载采用pH梯度法，利用囊泡内外pH的不同来包裹亲水药物DOX·HCl。100μL的PEG5k-P(CDC4.9k-co-TMC19k)的DMF溶液(10mg/mL)滴加到900μL柠檬酸钠/柠檬酸缓冲溶液(10mM，pH 4.0)中，在37℃(200rmp)摇床中放置5小时，然后加入0.05mL的PB(4M，pH 8.1)建立pH梯度，随后立即加入DOX·HCl，摇床中放置5-10小时允许药物进入囊泡中，同时自交联。最后装入透析袋(MWCO 7000)中透析过夜，换五次水，透析介质为PB(10mM，pH 7.4)。载不同比例的药(10％-30％)的自交联囊泡的粒径在105-124nm，粒径分布在0.10-0.15。荧光光谱仪测定DOX·HCl的包裹效率为63％-77％。DOX·HCl的体外释放实验是在37℃恒温摇床中震荡(200rpm)进行，每组各有三个平行样。第一组，载DOX·HCl的自交联囊泡在加入10mM GSH模拟细胞内还原环境PB(10mM,pH 7.4)中；第二组，载DOX·HCl的自交联囊泡在PB(10mM,pH 7.4)；载药自交联囊泡的浓度为30mg/L，取0.6mL放入透析袋(MWCO:12,000)中，每个试管中加入相应的透析溶剂25mL，在预定的时间间隔，取出5.0mL透析袋外部介质用作测试，同时向试管中补加5.0mL相应介质。使用荧光仪测定溶液中药物浓度。附图4为DOX·HCl累积释放量与时间的关系，从图中可以看出，加入模拟肿瘤细胞内GSH后，其释放明显要快于没加GSH的样本，说明载药自交联囊泡在10mM的GSH的存在下，能有效释放药物。
实施例二十三靶向自交联囊泡Ally-PEG6k-P(CDC2.9k-CL14.2k)的载疏水药物PTX及释放
用溶剂置换法制备聚合物囊泡。10μL的紫杉醇PTX的DMF溶液(10mg/mL)和90μL的Ally-PEG6k-P(CDC2.9k-CL14.2k)的DMF溶液(10 mg/mL)混合，然后滴加到900μL磷酸盐缓冲溶液(10mM，pH 7.4，PB)中，在37℃(200rmp)摇床中放置过夜进行自交联，然后装入透析袋(MWCO 7000)中透析过夜，换五次水，透析介质为PB(10mM，pH 7.4)。PTX的含量在0-20wt.％，得到的自交联囊泡尺寸为130-170nm，粒径分布0.1-0.2。TEM测得为囊泡结构，具有还原敏感的解交联性质。PTX的包裹效率为50％-70％)。体外释放实验设计同实施例二十二，加GSH后疏水药物释放明显要快于没加GSH样本。
实施例二十四靶向自交联囊泡cRGD20/PEG6.5k-P(CDC4.6k-co-TMC18.6k)的载药及释放
用薄膜水化法制备聚合物囊泡,pH梯度法装载DOX·HCl。1.6mg的PEG5k-P(CDC4.9k-co-TMC19k)及0.4mg的cRGD-PEG6.5k-P(CDC4.6k-co-TMC18.6k)溶于0.5mL的低沸点有机溶剂中，如二氯甲烷或乙腈中，在25毫升的尖底烧瓶中，旋蒸在底部形成薄膜，然后再0.1mBar的真空度下继续抽干24小时。加入2mL柠檬酸钠/柠檬酸缓冲溶液(10mM，pH4.0)中，在37℃下搅拌把薄膜剥离表面，并搅碎，超声20分钟(200rmp)，不断搅拌24小时，自交联。DLS测定交联囊泡的尺寸为90nm左右，粒径分布0.10。在上述囊泡溶液中加入0.05mL的PB(4M，pH8.1)建立pH梯度，随后立即加入DOX·HCl，摇床中放置5-10小时。然后装入透析袋(MWCO 7000)中对PB透析过夜，换五次液。载不同比例药量(10％-30％)之后，粒径112-121nm，粒径分布0.10-0.15，DOX·HCl的包裹效率61％-77％。体外释放实验设计同实施例二十二，附图5可看出，加入10mM GSH后，药物有效释放，速度明显要快于没加GSH的样本。
实施例二十五靶向自交联囊泡cNGQ20/PEG6.5k-P(CDC4.6k-co-TMC18.6k)的载药及释放
采用透析法制备囊泡，pH梯度法装载盐酸表阿霉素(Epi·HCl)。80μL 的PEG5k-P(CDC4.9k-co-TMC19k)的DMF溶液(10mg/mL)及20μL的cNGQ-PEG6.5k-P(CDC4.6k-co-TMC18.6k)的DMF溶液(10mg/mL)均匀混合之后，直接装入透析袋(MWCO 7000)中，在柠檬酸钠/柠檬酸缓冲溶液(10mM，pH4.0)中，在37℃摇床中放置4小时自交联，后对相同的介质透析12小时，换五次液。DLS测交联囊泡为96nm，粒径分布0.18。在上述囊泡溶液中加入0.05mL的PB(4M，pH 8.5)建立pH梯度，随后立即加入Epi·HCl，摇床中放置5-10小时。然后装入透析袋(MWCO 7000)中对PB透析过夜，换五次液。载不同比例药(10％-30％)，粒径98-118nm，粒径分布0.10-0.15，Epi·HCl的包裹效率为64％-79％。Epi·HCl体外释放实验设计同实施例二十二。
采用上述类似的制备方法可以研究多种自交联聚合物囊泡和靶向自交联聚合物囊泡对多种亲水抗癌小分子药物如盐酸多柔比星(DOX·HCl)、盐酸表阿霉素(Epi·HCl)、盐酸伊利替康(CPT·HCl)和盐酸米托蒽醌(MTO·HCl)以及疏水抗癌药物紫杉醇、多烯紫杉醇的载药量和包封率，见表3。
表3自交联聚合物囊泡和靶向自交联聚合物囊泡载亲水药物的载药量、包封率
Figure PCTCN2016111385-appb-000019
实施例二十六MTT法测试空自交联囊泡和空靶向自交联囊泡对A549细胞的毒性
采用MTT法测试空的囊泡的细胞毒性，使用A549人肺癌细胞。以5×104个/mL将A549细胞种于96孔板，每孔100μL，24小时后养至细胞贴壁70％左右。然后，实验组各孔中分别加入含有不同浓度(0.0001-1.5mg/mL)的囊 泡样品(以实施例十五的空的自交联囊泡和实施例十九的空靶向自交联囊泡cRGD/PEG6.5k-P(CDC4.6k-co-TMC18.6k)为例)，另设细胞空白对照孔和培养基空白孔(复4孔)。培养24小时后，每孔加入MTT(5.0mg/mL)10μL，继续培养4小时后每孔加入150μL DMSO溶解生成的结晶子，用酶标仪于492nm处测吸光度值(A)，以培养基空白孔调零，计算细胞存活率。附图6为自交联囊泡的细胞毒性结果，可看出，当交联囊泡的浓度从0.75增到1.5mg/mL时，A549的存活率仍高于90％，说明该交联囊泡具有良好的生物相容性。
实施例二十七MTT法测载药自交联囊泡和载药靶向自交联囊泡对A549肺癌细胞的毒性。
用MTT法测试囊泡对A549细胞的毒性。细胞的培养和实施例二十六相同，只是实验组各孔加样时，把载药交联囊泡和载药靶向自交联囊泡、实施例二十二的载DOX·HCl的自交联囊泡、实施例二十四的载DOX·HCl靶向自交联囊泡cRGD/PEG6.5k-P(CDC4.6k-co-TMC18.6k)和实施例二十五的载DOX·HCl靶向自交联囊泡cNGQ/PEG6.5k-P(CDC4.6k-co-TMC18.6k)加入各对应孔中，DOX·HCl浓度范围为0.01、0.1、0.5、1、5、10、20和40μg/mL；靶向分子含量从10％、20％到30％；无靶向载药自交联囊泡及游离DOX·HCl组作为对照组。共同培养4小时后，吸出样品换上新鲜培养基继续孵育44h后。而后的MTT加入、处理和测定吸光度同实施例二十六。附图7是载药自交联囊泡cRGD/PEG6.5k-P(CDC4.6k-co-TMC18.6k)对A549细胞的毒性；可看出，载DOX·HCl的30％cRGD靶向自交联囊泡对A549细胞的半致死浓度(IC50)为2.13μg/mL，远远低于无靶向对照囊泡，也低于自由药物(4.89μg/mL)，说明本发明的载药靶向自交联囊泡能有效靶向到肺癌细胞，在细胞内释放药物，最终杀死癌细胞。
实施例二十八MTT法测试载药自交联囊泡和载药靶向自交联囊泡对 H460细胞的毒性。
采用MTT法测试囊泡对H460人肺癌细胞的毒性。细胞的培养和实施例二十六相同，只是实验组各孔加样时，把不同cc-9含量、不同药量的载药靶向交联囊泡，以载CPT·HCl靶向自交联囊泡CC9/P(CDC3.8k-LA18.8k)-PEG4k-P(CDC3.8k-LA18.8k)为例，加入各对应孔中，CPT·HCl浓度范围为0.01、0.1、0.5、1、5、10、20和40μg/mL；靶向分子含量从10％、20％到30％；无靶向载药交联囊泡、和游离CPT·HCl组作为对照组。共同培养4小时后，吸出样品换上新鲜培养基继续孵育44h后。而后的MTT加入、处理和测定吸光度同实施例二十六。结果表明，无靶向载药自交联囊泡对H460细胞的IC50为4.85μg/mL，特别是载DOX·HCl的30％CC9靶向交联囊泡对H460细胞的IC50为2.17μg/mL，远低于DOX脂质体注射液里葆多DOX-LPs(35.2μg/mL)，低于自由药物(3.09μg/mL。说明本发明的载药靶向交联囊泡能有效靶向到肺癌细胞，并在细胞内释放药物，最终杀死癌细胞。
采用上述类似的方法研究了多种载药自交联聚合物囊泡和靶向自交联聚合物囊泡对肺癌细胞的毒性，药物为亲水抗癌小分子药物为盐酸多柔比星(DOX·HCl)、盐酸表阿霉素(Epi·HCl)、盐酸伊利替康(CPT·HCl)和盐酸米托蒽醌(MTO·HCl)以及疏水抗癌药物紫杉醇、多烯紫杉醇，结果见表4。
实施例二十九载药的自交联囊泡CLPs和靶向自交联囊泡cRGD20/CLPs的血液循环
所有动物实验操作符合苏州大学动物实验中心规定。实验选用体重为18～20克左右，4～6周龄的Balb/C裸鼠。囊泡由PEG5k-P(CDC4.9k-co-TMC19k)以及按不同比例混合的cRGD-PEG6.5k-P(CDC4.6k-co-TMC18.6k)和PEG5k-P(CDC4.9k-co-TMC19k)组成，当cRGD比例为20％时，粒径为100纳米，粒径分布为0.10，命名 为cRGD20/CLPs，药物为DOX·HCl。将载DOX·HCl的无靶向囊泡CLPs、靶向囊泡cRGD20/CLPs、无交联靶向囊泡cRGD20/PEG-PTMC和DOX·HCl通过尾静脉注射小鼠体内(DOX药量为10mg/kg)，在0、0.25、0.5、1、2、4、8、12和24小时定点取血约10μL，通过差量法准确计算血液重量，再加如100μL浓度为1％的曲拉通和500μL DMF萃取(其中含有20mM的DTT，1M的HCl)；然后离心(20000转/分钟，20分钟)后，取上层清液，通过荧光测得每个时间点DOX·HCl的量。图8中横坐标为时间，纵坐标为每克血液中的DOX·HCl占总的DOX注射量(ID％/g)。由图可知，DOX·HCl的循环时间很短，2小时已很难检测到DOX，而交联囊泡在24小时后仍有8ID％/g。由计算可知，靶向载药自交联囊泡、载药自交联囊泡和无交联靶向囊泡在小鼠体内的消除半衰期分别为4.49、4.26和1.45小时，而DOX·HCl的仅为0.27小时，所以靶向载药自交联囊泡在小鼠体内稳定，有较长循环时间。其他载药靶向自交联囊泡、载药自交联囊泡的血液循环实验的操作和计算方法相同，结果在表4。
实施例三十载药的自交联囊泡CLPs和靶向自交联囊泡cNGQ20/CLPs的血液循环
如实施例二十五，由cNGQ-PEG6.5k-P(CDC4.6k-co-TMC18.6k)和PEG5k-P(CDC4.9k-co-TMC19k)组成的靶向自交联囊泡cNGQ20/CLPs，以及无靶向的自交联囊泡CLP装载DOX·HCl后，尾静脉注射入Balb/C裸鼠中，同实施例二十九研究其血液循环，DOX·HCl和脂质体阿霉素里葆多DOX-LPs用于对照组。结果如图9所示，cNGQ20/CLPs和CLPs在48小时后仍有5.0ID％/g。由计算可知，靶向自交联囊泡和自交联囊泡在小鼠体内的消除半衰期分别为4.99和4.79小时，所以其在小鼠体内稳定，有较长的循环时间。结果在表4。
实施例三十一自交联囊泡和靶向自交联囊泡在荷A549肺癌小鼠的活 体成像
活体成像实验选用体重为18～20克左右，4～6周龄的Balb/C裸鼠，在皮下注射5×106个A549人肺癌细胞，大约3～4周后，肿瘤大小为100～200mm3时开始实验。以由cRGD-PEG6.5k-P(CDC4.6k-co-TMC18.6k)和PEG5k-P(CDC4.9k-co-TMC19k)制备的自交联囊泡cRGD20/CLPs和无靶向自交联囊泡CLPs为例。将荧光物质cy-7标记的cRGD20/CLPs和无靶向的CLPs通过尾静脉注射小鼠体内，然后在不同时间点1、2、4、6、8、12、24、48小时用小动物活体成像仪来追踪囊泡的去向。实验结果可知，cRGD20/CLPs在肿瘤部位很快积累，且在48小时后荧光仍然很强。说明cRGD20/CLPs能主动靶向及富集到肿瘤部位。其他靶向自交联囊泡、自交联囊泡的活体成像实验的操作和计算方法相同，结果在表4。
实施例三十二载药自交联囊泡CLPs和靶向自交联囊泡cNGQ20/CLPs在荷A549肺癌小鼠的活体成像实验
活体成像实验中肿瘤的接种以及尾静脉给药同实施例三十一。如实施例二十五制备的载Epi·HCl的、cy-7标记的CLPs和cNGQ20/CLPs，发现二者都可在肿瘤部位很快积累，CLPs在4-6小时消失，而cNGQ20/CLPs在48小时后肿瘤部位荧光仍然很强，说明cNGQ20/CLPs能主动靶向及富集到肿瘤部位。结果在表4。
实施例三十三载药自交联囊泡CLPs和载药靶向自交联囊泡CC9/CLPs在荷H460肺癌小鼠的活体成像实验
活体成像实验选用体重为18～20克左右，4～6周龄的Balb/C裸鼠，在皮下注射5×106个H460人肺癌细胞，大约3～4周后，肿瘤大小为100～200mm3时开始实验。由CC9-PEG6.5k-P(CDC3.8k-co-LA13.8k)和PEG5k-P(CDC3.7k-co-LA14.6k)制备的靶向自交联囊泡CC9/CLPs和载药自交联囊泡CLPs用cy-5标记，装载了疏水药物多烯紫杉醇DTX，同实施例 三十二操作来研究活体成像。实验结果可知，载DTX的CC9/CLPs可在肿瘤部位很快积累，并在48小时后肿瘤部位荧光仍然很强。说明CC9/CLPs能主动靶向及富集到肿瘤部位，而载药无靶向自交联囊泡在2小时进入肿瘤后很快代谢，且强度低，结果在表4。
实施例三十四载药自交联囊泡CLPs和靶向自交联囊泡cRGD20/CLPs在荷A549肺癌小鼠的体内生物分布
活体成像实验中肿瘤的接种以及尾静脉给药同实施例三十一。由cRGD-PEG6.5k-P(CDC4.6k-co-TMC18.6k)和PEG5k-P(CDC4.9k-co-TMC19k)制备的载DOX·HCl的靶向自交联囊泡cRGD20/CLPs和无靶向自交联囊泡CLPs尾静脉注射小鼠体内(DOX·HCl：10mg/kg)，12小时后处死老鼠，将肿瘤及心，肝，脾，肺和肾组织取出，清洗称重后加入500μL 1％的曲拉通通过匀浆机磨碎，再加入900μL DMF萃取(其中含有20mM的DTT，1M的HCl)。离心(20000转/分钟，20分钟)后，取上层清液，通过荧光测得每个时间点DOX·HCl的量。图10中横坐标为组织器官，纵坐标为每克肿瘤或组织中的DOX·HCl占总DOX·HCl注射量(ID％/g)。cRGD20/CLPs、CLPs和DOX·HCl注射12小时在肿瘤积累的DOX·HCl量分别为6.54、2.53和1.02ID％/g，cRGD20/CLPs是CLPs和DOX·HCl的3和6倍，说明载药cRGD20/CLPs通过主动靶向在肿瘤部位积累较多，结果在表4。
实施例三十五载药自交联囊泡CLPs和靶向自交联囊泡cNGQ/CLPs在荷A549肺癌小鼠的体内生物分布
肿瘤的接种、尾静脉给药以及动物的操作同实施例三十四。载DOX·HCl的cNGQ20/CLPs、无靶向CLPs和脂质体阿霉素里葆多DOX-LPs尾静脉注射小鼠体内(DOX·HCl：10mg/kg)。6小时后，cNGQ20/CLPs、CLPs和DOX-LP在肿瘤积累的DOX·HCl量分别为8.63、3.52和1.82ID％/g，cNGQ20/CLPs是后两者的2和5倍，说明载药cNGQ20/CLPs通过主动靶向 在肿瘤部位积累较多。结果如图11。
实施例三十六载药自交联囊泡CLPs和靶向自交联囊泡CC9/CLPs在荷H460肺癌小鼠的体内生物分布
荷H460肺癌小鼠的建模同实施例三十三，尾静脉给药以及动物的操作同实施例三十四。载DTX的CC9/CLPs、无靶向CLPs和DOX-LPs尾静脉给药。6小时后CC9/CLPs、CLPs和DOX-LPs在肿瘤积累的DTX量分别为9.02、2.42和1.82ID％/g，CC9/CLPs是CLPs和DOX-LPs的4和5倍，说明载药CC9/CLPs通过主动自靶向在肿瘤部位积累，如表4。
实施例三十七载药靶向自交联囊泡cRGD20/CLPs和自交联囊泡CLPs在荷A549皮下肺癌的小鼠中的抑瘤效果、体重变化和存活率
实验选用体重为18～20克左右，4～6周龄的Balb/C裸鼠，在皮下注射5×106个A549人肺癌细胞，大约两周后，肿瘤大小为30～50mm3时开始实验。由cRGD-PEG6.5k-P(CDC4.6k-co-TMC18.6k)和PEG5k-P(CDC4.9k-co-TMC19k)按1∶5混合制备的载DOX·HCl的靶向自交联囊泡cRGD20/CLPs、CLPs、自由DOX·HCl以及PBS分别在0、4、8和12天通过尾静脉注射小鼠体内(DOX药量为10mg/kg)。在0～18天，每两天称量小鼠的体重，游标卡尺测量肿瘤体积，肿瘤体积计算方法为：V＝(L×W×H)/2，(其中L为肿瘤的长度，W为肿瘤的宽度，H为肿瘤的厚度)。持续观察小鼠的生存到45天。由图12中可知，cRGD20/CLPs治疗组18天时，肿瘤得到明显抑制，而载药CLPs组肿瘤有一定的增长。DOX·HCl虽然也能抑制肿瘤的增长，但其小鼠体重在12天时降低了21％，说明对小鼠的毒副作用很大。相比之下cRGD20/CLPs和CLPs组的小鼠体重几乎没有改变，说明载药自交联囊泡对小鼠没有毒副作用。cRGD20/CLPs治疗组在60天后全部存活，DOX·HCl组在42天时已全部死亡，PBS组在43天时也全部死亡。因此，本发明的靶向自交联囊泡载药后可有效抑制肿瘤的增长，对小鼠 没有毒副作用，还可以延长荷瘤老鼠的生存时间。
实施例三十八载药靶向自交联囊泡cNGQ/CLPs和自交联囊泡CLPs在荷A549皮下肺癌的小鼠中的抑瘤效果、体重变化和存活率
皮下A549肿瘤模型的建立、尾静脉给药方式和数据采集同实施例三十七。由cNGQ-PEG6.5k-P(CDC4.6k-co-TMC18.6k)和PEG5k-P(CDC4.9k-co-TMC19k)按1：5混合制备的载DOX·HCl的靶向自交联囊泡cNGQ20/CLPs、无靶向CLPs、DOX-LPs以及PBS尾静脉注射。由图13中可知，cNGQ20/CLPs治疗组18天时，肿瘤得到明显抑制，而载药CLPs组肿瘤有增长，小鼠体重几乎没有改变。DOX-LPs虽然也能抑制肿瘤的增长，但DOX-LPs组的小鼠体重在12天时降低了18％，说明其对小鼠的毒副作用很大。cNGQ20/CLPs治疗组在68天后全部存活，DOX·HCl组在32天时已全部死亡，PBS组42天时也全部死亡。因此，载药靶向自交联囊泡可有效抑制肿瘤，对小鼠没有毒副作用，可延长荷瘤老鼠的生存时间。
实施例三十九载药靶向自交联囊泡CC9/CLPs和自交联囊泡CLPs在荷H460皮下肺癌的小鼠中的抑瘤效果、体重变化和存活率
皮下H460肿瘤模型的建立同实施例三十三，尾静脉给药方式和数据采集同实施例三十七。肿瘤大小为30～50mm3时开始实验，由CC9-PEG6.5k-P(CDC3.8k-co-LA13.8k)和PEG5k-P(CDC3.7k-co-LA14.6k)按1：5混合制备装载CPT·HCl的靶向自交联囊泡CC9/CLPs、无靶向CLPs、自由CPT·HCl以及尾静脉注射。结果发现在CC9/CLPs治疗18天时，肿瘤得到明显抑制，而载药CLPs组肿瘤有少量增长，小鼠体重几乎没有改变。CPT·HCl虽然也能抑制肿瘤的增长，但CPT·HCl组小鼠体重在10天时降低了18％。CC9/CLPs治疗组在72天后全部存活，CPT·HCl组在28天时已全部死亡，PBS组在37天时也全部死亡。
实施例四十载药靶向自交联囊泡cRGD20/CLPs和自交联囊泡CLPs在 荷A549原位肺癌的小鼠中的抑瘤效果、体重变化和存活率
实验选用体重为18～20克左右，4～6周龄的Balb/C裸鼠，在肺部直接注射5×106个带有生物发光的A549人肺癌细胞(A549-Luc)，大约10天后，通过小动物活体成像系统观察，老鼠肺部出现荧光，成功建立A549原位肺癌模型。接着，如实施例二十，由cRGD-PEG6.5k-P(CDC4.6k-co-TMC18.6k)和PEG5k-P(CDC4.9k-co-TMC19k)按1：5混合制备的载DOX·HCl的靶向自交联囊泡cRGD20/CLPs、CLPs、DOX·HCl以及PBS分别在0、4、8和12天通过尾静脉注射小鼠体内(DOX·HCl：10mg/kg)。从0～16天，每四天称量小鼠的体重，用小动物活体成像仪监测小鼠肺部肿瘤生物发光强弱，观察小鼠的生存到45天。见图14，cRGD20/CLPs治疗组16天内，肺部肿瘤生物发光强度持续减弱，而载药CLPs组肺部的肿瘤生物发光强度有一定增长，但两组体重几乎没有改变。DOX·HCl虽然也能抑制肿瘤的增长，但DOX·HCl组的小鼠体重在4天时降低了21％，说明其对小鼠的毒副作用很大。cRGD20/CLPs治疗组在45天后全部存活，DOX·HCl组在30天时已全部死亡，PBS组在20天时也全部死亡。因此，载药靶向自交联囊泡cRGD20/CLPs可有效抑制原位肺癌肿瘤的增长，对小鼠没有毒副作用，并有效延长荷瘤老鼠的生存时间。
实施例四十一载药靶向自交联囊泡cNGQ20/CLPs和自交联囊泡CLPs在荷A549原位肺癌的小鼠中的抑瘤效果、体重变化和存活率
A549原位肺癌的小鼠模型的建立、给药方式以及检测方式同实施例四十。由cNGQ-PEG6.5k-P(CDC4.6k-co-TMC18.6k)和PEG5k-P(CDC4.9k-co-TMC19k)按1：5混合制备的载DOX·HCl的靶向自交联囊泡cNGQ20/CLPs、无靶向CLPs、DOX-LPs以及PBS尾静脉注射。结果如图15所示，cNGQ20/CLPs治疗组16天内，肿瘤生物发光强度持续减弱，而载药CLP组肿瘤生物发光强度有一定增长，小鼠的体重几乎没有改 变。DOX-LPs虽然也能抑制肿瘤增长，但DOX-LPs的小鼠体重在4天时降低了21％。cNGQ20/CLPs治疗组在45天后全部存活，DOX-LPs组在32时已全部死亡，PBS组在23天时也全部死亡。因此，载药靶向自交联囊泡cNGQ20/CLPs同样可有效抑制原位肺癌肿瘤的增长，对小鼠没有毒副作用，还可以延长荷瘤老鼠的生存时间。
实施例四十二载药靶向自交联囊泡CC9/CLPs和自交联囊泡CLPs在荷A549原位肺癌的小鼠中的抑瘤效果、体重变化和存活率
A549原位肺癌的小鼠模型的建立、给药方式以及检测方式同实施例四十。由cc9-PEG6.5k-P(CDC3.8k-co-LA13.8k)和PEG5k-P(CDC3.7k-co-LA14.6k)按1：5混合制备装载CPT·HCl的靶向自交联囊泡CC9/CLP、无靶向CLPs、CPT·HCl以及PBS注射入小鼠。CC9/CLPs治疗组16天时，肿瘤生物发光强度减弱，而载药CLPs组的肿瘤生物发光强度有一定增长，小鼠体重几乎没有发生改变。CPT·HCl虽然也能抑制肿瘤的增长，但CPT·HCl组的小鼠体重在3天时降低了21％，说明其对小鼠的毒副作用很大。CC9/CLPs治疗组在40天后全部存活，CPT·HCl组34时已全部死亡，PBS组21天时全部死亡。因此，载药靶向自交联囊泡CC9/CLPs可有效抑制原位肺癌肿瘤的增长，无毒副作用，并能延长荷瘤老鼠的生存时间。
实施例四十三载药靶向自交联囊泡cRGD/CLPs和自交联囊泡CLPs荷A549原位肺癌的小鼠中的抑瘤效果、体重变化和存活率
由AA-PEG3k-P(CDC3.9k-PDSC4.8k)和PEG1.9k-P(CDC3.6k-PDSC4.6k)按1：5混合制备装载PTX的自交联囊泡。然后如实施例二十一通过囊泡表面的的丙烯酸酯(AA)和cRGDfC的巯基的迈克尔加成反应制备载PTX的靶向自交联囊泡cRGD/CLP。DLS测定为85nm，粒径分布0.10。核磁和BCA蛋白试剂盒测试计算多肽的接枝率为92％。
A549原位肺癌的小鼠模型的建立、给药方式以及检测方式同实施例四 十。载PTX的cRGD/CLPs、无靶向自交联囊泡CLPs、Taxol以及PBS分别注射入小鼠。载PTX的cRGD/CLPs治疗组16天内，肿瘤生物发光强度持续减弱，而无靶向的CLPs组的肿瘤生物发光强度有增长，两组小鼠体重几乎没有发生改变。PTX虽然也能抑制肿瘤的增长，但PTX组的小鼠体重在12天时降低了10％，说明其对小鼠的毒副作用很大。载PTX的cRGD/CLPs治疗组在41天仍然存活，PTX组老鼠在29时已全部死亡，PBS组在32天时也全部死亡。因此，载PTX的cRGD/CLPs可有效抑制原位肺癌肿瘤的增长，无毒副作用，并可延长荷瘤老鼠的生存时间。
采用上述类似的实验方法研究了多种载不同药物的自交联聚合物囊泡和靶向自交联聚合物囊泡对荷肺癌的小鼠的影响，结果见表4。
表4载药自交联聚合物囊泡和靶向自交联聚合物囊泡对肺癌的体内外抗肿瘤结果
Figure PCTCN2016111385-appb-000020

Claims (10)

  1. 一种生物可降解双亲性聚合物，其特征在于：所述生物可降解双亲性聚合物的化学结构式为以下结构式中的一种：
    Figure PCTCN2016111385-appb-100001
    其中，R1选自以下基团中的一种：
    Figure PCTCN2016111385-appb-100002
    R2选自以下基团中的一种：
    Figure PCTCN2016111385-appb-100003
    其中，k为43～170，x为10～30，y为40～200，m为86～340。
  2. 根据权利要求1所述生物可降解双亲性聚合物，其特征在于：所述R1 选自以下基团中的一种：
    Figure PCTCN2016111385-appb-100004
    所述R2选自以下基团中的一种：
    Figure PCTCN2016111385-appb-100005
    所述k为113～170，x为20～26，y为100～190，m为226～340。
  3. 一种肿瘤特异靶向的生物可降解双亲性聚合物，其特征在于：所述肿瘤特异靶向的生物可降解双亲性聚合物由权利要求1或者2所述的生物可降解双亲性聚合物键合靶向分子制备得到。
  4. 根据权利要求3所述肺癌靶向的生物可降解双亲性聚合物，其特征在于：所述靶向分子为cRGD、cNGQ或者cc-9。
  5. 一种聚合物囊泡，其特征在于，所述聚合物囊泡的制备方法为以下制备方法中的一种：
    (1)由权利要求1或者2所述生物可降解双亲性聚合物制备得到；
    (2)由权利要求3所述肿瘤特异靶向的生物可降解双亲性聚合物制备得到；
    (3)由权利要求1或者2所述生物可降解双亲性聚合物与权利要求3所述肿瘤特异靶向的生物可降解双亲性聚合物制备得到；
    (4)在权利要求1或者2所述生物可降解双亲性聚合物制备的囊泡表面偶联靶向分子后得到。
  6. 根据权利要求5所述聚合物囊泡，其特征在于：所述聚合物囊泡为自 交联聚合物囊泡；所述自交联聚合物囊泡的粒径为40～180纳米。
  7. 权利要求5所述聚合物囊泡作为治疗肺癌的药物载体的应用。
  8. 根据权利要求7所述的应用，其特征在于：所述治疗肺癌的药物为亲水性抗癌药物或者疏水抗癌药物。
  9. 权利要求1或者2所述的生物可降解双亲性聚合物在制备治疗肺癌的纳米药物中的应用。
  10. 权利要求5所述聚合物囊泡在制备治疗肺癌的纳米药物中的应用。
PCT/CN2016/111385 2015-12-22 2016-12-21 生物可降解双亲性聚合物、由其制备的聚合物囊泡及在制备肺癌靶向治疗药物中的应用 WO2017107934A1 (zh)

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US20200062897A1 (en) 2020-02-27
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