WO2017148431A1 - 卵巢癌特异靶向的生物可降解双亲性聚合物、由其制备的聚合物囊泡及应用 - Google Patents

卵巢癌特异靶向的生物可降解双亲性聚合物、由其制备的聚合物囊泡及应用 Download PDF

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WO2017148431A1
WO2017148431A1 PCT/CN2017/075529 CN2017075529W WO2017148431A1 WO 2017148431 A1 WO2017148431 A1 WO 2017148431A1 CN 2017075529 W CN2017075529 W CN 2017075529W WO 2017148431 A1 WO2017148431 A1 WO 2017148431A1
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polymer
ovarian cancer
self
vesicle
clps
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PCT/CN2017/075529
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English (en)
French (fr)
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孟风华
邹艳
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 EP17759282.1A priority Critical patent/EP3421519B1/en
Priority to JP2018565450A priority patent/JP6677914B2/ja
Priority to CA3016655A priority patent/CA3016655C/en
Priority to KR1020187028348A priority patent/KR102190093B1/ko
Priority to AU2017226517A priority patent/AU2017226517B2/en
Publication of WO2017148431A1 publication Critical patent/WO2017148431A1/zh
Priority to US16/121,606 priority patent/US20180360766A1/en

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Definitions

  • the invention relates to a biodegradable polymer material and application thereof, in particular to a biodegradable amphiphilic polymer specifically targeted by ovarian cancer, a polymer vesicle prepared therefrom and a targeted therapy for ovarian cancer Application, belonging 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 functional group, and it is often difficult to provide a circulating stable drug carrier.
  • 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. There are toxic side effects.
  • Micellar nano-drugs prepared by functional biodegradable polymers can be stably circulated in vivo, but can only be loaded with hydrophobic small molecule anticancer drugs, but can not be used for hydrophilic small molecule anticancer drugs with stronger penetrability.
  • the earth limits its use as a drug carrier.
  • Ovarian cancer is a malignant tumor of ovarian tumor. It refers to a malignant tumor that grows on the ovary. 90% to 95% of it is a primary ovarian cancer, and another 5% to 10% of the primary cancer is transferred to other parts. Ovary. Due to the complexity of embryonic development, tissue anatomy and endocrine function of the ovary, the tumor it may have is benign or malignant. Due to the lack of specific symptoms in the early stage of ovarian cancer, the screening effect is limited, and the tissue type and Benign and malignant is quite difficult, so early diagnosis is more difficult.
  • ovarian cancer In ovarian cancer, only 30% of tumors are confined to the ovary during laparotomy. Most of them have spread to the bilateral attachments of the uterus, omentum and pelvic organs; % to 70% are advanced, while advanced cases are not effective.
  • ovarian cancer has been a major problem in diagnosis and treatment. Therefore, although the incidence of ovarian cancer is lower than that of cervical cancer and endometrial cancer in gynecological malignancies, the mortality rate exceeds the sum of cervical cancer and endometrial cancer, which is the highest in gynecological cancer and is a serious threat to women. The biggest health condition.
  • ovarian cancer is a high-grade female malignant tumor. Although the absolute number of cases is not very large, the mortality rate is very high, mainly due to early detection and difficulty in early diagnosis. Most of them are diagnosed as advanced and missed surgery. The best time for resection; and its treatment also has the characteristics of low cure rate, easy transfer and resistance. Nano-drugs can change the distribution of traditional chemotherapeutic drugs, increase the concentration of drugs in tumors, and improve the therapeutic effect. It is a key point and hope for the treatment of ovarian cancer.
  • DOXIL PEGylated liposomal doxorubicin
  • DOXIL is the earliest FDA-approved liposome vesicle nano drug DOXIL (PEGylated liposomal doxorubicin), which is clinically effective in the treatment of ovarian cancer.
  • DOXIL also has problems.
  • DOXIL is a passive targeting effect based on the EPR effect, due to the huge tumors. Individual differences, it is difficult to use a common unified mechanism to transport nanomedicine to all tumor tissues and tumor cells (see: S. Eetezadi, SN. Ekdawi, C.
  • the object of the present invention is to provide a biodegradable amphiphilic polymer specifically targeted by ovarian cancer, a polymer vesicle prepared thereby, and a carrier as an anti-ovarian cancer drug for preparing a ovarian cancer targeted therapeutic drug.
  • a biodegradable amphiphilic polymer specifically targeted by ovarian cancer polymerized by a monomer containing dithiocarbonate
  • the target binding molecule is prepared; the targeting molecule is GE11 (polypeptide), FA (folate), transferrin (transferrin) or Herceptin protein; chemical structure of the polymer containing the dithiocarbonate monomer Is one of the following structural formulas:
  • 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 15 to 45
  • y is from 80 to 300
  • m is from 220 to 280.
  • the hydrophobic block of the dithiocarbonate monomer-containing polymer disclosed in the present invention has a cyclic carbonate unit having a side chain containing a disulfide five-membered ring functional group; and may be a diblock polymer:
  • R1 is selected from one of the following groups:
  • the R 2 is selected from one of the following groups:
  • the k is 113 to 170, x is 20 to 40, and y is 125 to 250.
  • the molecular weight of the polymer disclosed in the present invention is controllable, and the composition and ratio of each structural unit are suitable for forming a self-crosslinking to form a stable polymer vesicle structure.
  • the biodegradable amphiphilic polymer specifically targeted by the ovarian cancer disclosed in the invention has biodegradability, and the molecular weight of the hydrophobic portion is about 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 a film.
  • a polymer vesicle structure is prepared by a method such as hydration.
  • the prepared polymer vesicles are nano-sized, with a particle size of 50-160 nm, and can be used as a carrier for treating ovarian cancer; the hydrophobic membrane of the vesicle is loaded with a hydrophobic small molecule anti-ovarian cancer drug paclitaxel, docetaxel, ar Magnesium, olrapani, gefitinib, etc., can also be loaded with hydrophilic anti-ovarian cancer drugs in the large hydrophilic cavity of vesicles, especially hydrophilic small molecule anticancer drugs such as doxorubicin hydrochloride Star, epirubicin hydrochloride, irinotecan hydrochloride and mitoxantrone hydrochloride.
  • hydrophilic small molecule anticancer drugs such as doxorubicin hydrochloride Star, epirubicin hydrochloride, irinotecan hydrochloride and mitoxantrone hydrochloride.
  • micellar carrier formed by the amphiphilic polymer which can only be loaded with the hydrophobic drug and the defect of the carrier of the hydrophilic small molecule anticancer drug which can be efficiently loaded and stabilized in the prior art.
  • the invention also discloses a polymer vesicle which can be prepared from the above-mentioned dithiocarbonate polymer-containing polymer containing a dithiocarbonate monomer; or a biodegradable parent specifically targeted by the above ovarian cancer Preparing a polymer; or preparing from the above-mentioned dithiocarbonate polymer-containing dithiocarbonate-containing polymer and a biodegradable amphiphilic polymer specifically targeted by ovarian cancer, such as the above-mentioned dithiocarbonate-containing Polymers containing dithiocarbonate monomers and biodegradable amphiphilic polymers specifically targeted by ovarian cancer are mixed in different ratios to prepare polymer vesicles with different targeting densities, ie, ovarian cancer targeting Self-crosslinking vesicles to increase the uptake of vesicle nanodrugs in ovarian cancer cells; outer surface vesicles of vesicles prepared from polymers containing dithiocarbon
  • the polymer capsule of the present invention is prepared from a biodegradable amphiphilic polymer specifically targeted by ovarian cancer and a polymer containing a dithiocarbonate polymer containing a dithiocarbonate monomer;
  • the biodegradable amphiphilic polymer specifically targeted to ovarian cancer is used in an amount of from 1 to 40% by weight.
  • the dithiocarbonate monomer-containing polymer of the present invention and the ovarian cancer-specifically targeted biodegradable amphiphilic polymer can be self-crosslinked without adding any substance to obtain a self-crosslinking polymer vesicle.
  • the polymer When the polymer is applied as a drug carrier, the most basic and most critical property for achieving optimal targeting and therapeutic effects is long-term circulation in the body.
  • the formation of the crosslinked structure is a necessary process for the polymer carrier to circulate in the in vivo environment for a long time.
  • the polymer nanocarrier forms a stable crosslinked structure by adding a crosslinking agent, but the addition of the crosslinking agent is not only It will increase the complexity of the nano drug preparation process, increase the production cost of nano drugs, and reduce the final purity of the drug, which is not conducive to the amplification production of nano drug clinical application, but also affects drug loading efficiency, drug release level, and increases toxic side effects and reduces
  • the biocompatibility of the polymer carrier nano drug; the polymer structure disclosed in the first time of the invention can self-crosslink without forming a cross-linking agent, and form a stable chemical cross-linking structure in the vesicle hydrophobic film.
  • the long circulation can be stabilized in the body, and the side effects of the cross-linking agent are not only avoided, but also after the drug-loaded polymer vesicles reach the tumor and are endocytosed into the cancer cells, the cells can be quickly dissected in the presence of a large amount of reducing substances in the cells. Joint, the maximum amount of drug release, efficient killing of ovarian cancer cells; the stability of self-crosslinking polymers, etc.
  • the present invention avoids the interference of the cross-linking agent on some drugs, and successfully uses self-crosslinking polymer vesicles to load drugs, not only avoiding existing small molecules
  • the side effects of drugs expand the use of anticancer drugs, and can be applied to different individuals with large differences in constitution.
  • the invention also discloses the preparation of the biodegradable amphiphilic polymer specifically targeted by the above ovarian cancer
  • the invention relates to the application of the nano drug for treating ovarian cancer; further, the invention also discloses the application of the above polymer vesicle in preparing a nano drug for treating ovarian cancer; in particular, the self-crosslinking polymer vesicle is protected as a carrier in the preparation of the treatment
  • self-crosslinking polymer vesicles avoid the use of cross-linking agents, further enhancing drug safety and reducing drug assembly steps.
  • the anti-ovarian cancer nano drug prepared based on the polymer of the present invention is a vesicle anti-ovarian cancer nano drug.
  • the present invention has the following advantages compared with the prior art:
  • the side chain disulfide-containing biodegradable amphiphilic polymer disclosed in the invention has biodegradability, excellent ovarian cancer targeting property, can prepare polymer vesicles, and can load drugs of different properties without adding any The cross-linking agent self-crosslinks to form a stable self-crosslinking polymer vesicle nano-drug, thereby overcoming the defects of the prior art nano drug in vivo instability, easy release of the drug, 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 ovarian cancer cells; however, it can be quickly cross-linked and released after entering the ovarian cancer cells.
  • the drug is used to achieve high-efficiency and specific killing of ovarian cancer cells without toxic and side effects, and overcomes the defects in the prior art that the cross-linked nano drug is too stable, and the drug release in the cell is slow, resulting in drug resistance.
  • the ovarian cancer-specific biodegradable amphiphilic polymer disclosed in the present invention can prepare self-crosslinking vesicles without adding any cross-linking agent, and the preparation method is simple, thereby overcoming the preparation of cross-linked nano-drugs in the prior art. At the time, it is necessary to add substances such as cross-linking agents and defects such as complicated operation and purification processes, which is beneficial to the clinical application of nano-drugs.
  • the self-crosslinking polymer vesicle prepared by self-assembly of the biodegradable amphiphilic polymer specifically targeted by the ovarian cancer disclosed in the invention can be used for the controlled release system of a hydrophilic small molecule anticancer drug, thereby overcoming the existing organism
  • the degradable nanomicelle carrier is only suitable for the defect of loading a small hydrophobic molecule drug and the defect of the hydrophilic small molecule anticancer drug which can be efficiently loaded and stabilized in the prior art in the prior art; further, the ovarian cancer target can be prepared Self-crosslinking vesicles have wider application value in the efficient targeted therapy of ovarian cancer.
  • Figure 1 is a cross-linked vesicle PEG5k-P (CDC5.8k-co-TMC23k) 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;
  • Example 2 is a diagram showing the in vitro release of DOX ⁇ HCl cross-linked vesicle PEG5k-P (CDC5.8k-co-TMC23k) in Example XX;
  • Figure 3 is a diagram showing the in vitro release of DOX ⁇ HCl cross-linked vesicles GE11-CLPs in Example XX;
  • Figure 4 is a graph showing the results of toxicity of targeted cross-linked vesicles GE11-CLPs against SKOV3 ovarian cancer cells in Example 21;
  • Figure 5 is a graph showing the toxicity of DOX ⁇ HCl-targeted cross-linked vesicles GE11-CLPs on SKOV3 ovarian cancer cells in Example 22;
  • Figure 6 is a graph showing the results of semi-lethal toxicity of DOX ⁇ HCl-targeted cross-linked vesicles GE11-CLPs on SKOV3 ovarian cancer cells in Example 22;
  • Figure 7 is a graph showing the results of endocytosis of SKOV3 ovarian cancer cells by DOX ⁇ HCl-targeted cross-linked vesicles GE11-CLPs in Example 24;
  • Figure 8 is a graph showing the blood circulation results of DOX ⁇ HCl-targeted cross-linked vesicles GE11-CLPs in mice in Example 29;
  • Figure 9 is a graph showing the results of biodistribution of DOX ⁇ HCl-targeted cross-linked vesicles GE11-CLPs in subcutaneous ovarian cancer mice in Example 30;
  • Figure 10 is a graph showing the maximum tolerated dose of DOX ⁇ HCl-targeted cross-linked vesicle GE11-CLPs in mice in Example 31;
  • Figure 11 is a multi-dose treatment diagram of DOX ⁇ HCl-targeted cross-linked vesicles GE11-CLPs in subcutaneous ovarian cancer mice, in which the A is a tumor growth curve and B is a tumor image after treatment in mice. , C is the change in body weight, and D is the survival curve;
  • Figure 12 is a single-dose treatment of DOX ⁇ HCl-targeted cross-linked vesicles GE11-CLPs in subcutaneous ovarian cancer mice, in which the A is a tumor growth curve and B is a tumor image after treatment in mice. , C is the weight change curve, and D is the survival curve.
  • the 1 H NMR characteristic peak was the same as in Example 2.
  • the synthesis of the transferrin-transferrin-conjugated polymer is divided into two steps.
  • the first step is to prepare Mal-PEG7.5k-P (CDC3.8k-co-LA13.8k) as in the eighth embodiment; the second step is to transfer the ferferrin Michael reacts to bond.
  • the above polymer Mal-PEG7.5k-P (CDC3.8k-co-LA13.8k) was first dissolved in DMF, twice the molar amount of transferrin was added, reacted at 30 ° C for two days, dialyzed, and lyophilized to obtain transferrin- PEG6.5k-P (CDC3.8k-co-LA13.8k), calculated by the nuclear magnetic and BCA protein kit test, the transferrin grafting rate was 95%.
  • the synthesis of the human epidermal growth factor antibody Herceptin-conjugated polymer is divided into three steps.
  • the first step is to prepare Mal-PEG6k-P (CDC3.6k-co-LA18.6k) as in the eighth embodiment;
  • the second step is Mal-PEG6k- P (CDC3.6k-co-LA18.6k) reacts with cysteamine Michael addition to convert the terminal group to amino group;
  • the carboxyl group of FA in the third step is bonded to it by amidation reaction:
  • the polymer obtained in the previous step is firstly Dissolve in 0.5 ml of DMF, add 2 ml of boric acid buffer solution (pH 8.0), add 2 times molar amount of Herceptin, react at 30 ° C for two days, dialysis, freeze-drying to obtain the final product Herceptin-PEG6k-P (CDC3.6k -co-LA18.6k), the nuclear magnetic and BCA protein tests calculated the FA grafting rate to be 96%.
  • the synthesis of folic acid (FA) coupled polymer is divided into two steps.
  • the first step is to prepare NHS-PEG6.5k-P (CDC6k-co-TMC22.6k) as in the fourth step; the amino group of FA in the second step is passed through the amide.
  • Chemical reaction bonding first dissolving the above polymer in DMF, adding twice the molar amount of FA, reacting at 30 ° C for two days, dialysis to remove free FA, and freeze-drying to obtain FA-PEG6.5k-P (CDC6k-co-TMC22 .6k), the FA grafting ratio was calculated to be 88% by the nuclear magnetic test.
  • the synthesis of the cyclic polypeptide YHWYGYTPQNVI (GE11) coupled polymer is divided into two steps.
  • the first step is to prepare NHS-PEG6.5k-P (CDC6k-co-TMC22.6k) as in the fourth embodiment; the second step is GE11.
  • the amino group is bonded to it by amidation reaction: firstly dissolving the above polymer in DMF, adding twice the molar amount of GE11, reacting at 30 ° C for two days, dialysis to remove free GE11, and freeze-drying to obtain GE11-PEG6.5k-P (CDC6k) -co-TMC22.6k), the GE11 graft ratio was calculated to be 96% by the NMR and BCA protein kits.
  • the bismuth carbonate-containing polymer-bonded targeting molecules of Examples 2 to 10 and Table 1 can be used to prepare ovarian cancer-specific targeting organisms.
  • Polymer vesicles were prepared by solvent displacement. 100 ⁇ L of PEG5k-P (CDC5.8k-co-TMC23k) 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 performed overnight, and then dialyzed overnight in a dialysis bag (MWCO 7000) for five times of media PB.
  • Figure 1 is a graph showing the particle size distribution (A) and electron transmission microscopy of the self-crosslinking vesicle PEG5k-P (CDC5.8k-co-TMC23k).
  • the size of the self-crosslinking vesicles obtained is determined by a dynamic light scattering particle size analyzer (DLS).
  • the bubble is 130 nm, and the particle size distribution is very narrow, as shown in Fig. 1A.
  • the TEM detects that the nanoparticles are hollow vesicle structures, and the self-crosslinking vesicles remain unchanged in the presence of high dilution and fetal bovine serum.
  • Particle size and particle size distribution (Fig. 1C), but rapid release in the simulated tumor cell reduction environment, decrosslinking (Fig. 1D).
  • the obtained vesicles can be self-crosslinked and have the property of reduction-sensitive decrosslinking.
  • PEG5k-P CDC4.9k-co-TMC19k
  • PEG5k-P CDC4.9k-co-TMC19k
  • Self-crosslinking polymer vesicles were prepared by dialysis. 100 ⁇ L of PEG5k-P (CDC5.8k-co-TMC23k) in DMF solution (10 mg/mL) was placed in a dialysis bag (MWCO 7000), and placed in PB, 37 ° C (200 rpm) shaker for overnight self-crosslinking. Then dialyze for 24 hours in PB and change for five times. The DLS measured cross-linked vesicles were about 60 nm, and the particle size distribution was 0.08.
  • Self-crosslinking polymer vesicles were prepared by thin film hydration. 2 mg of PEG5k-P (CDC5.8k-co-TMC23k) 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 160 nm, and the particle size distribution was 0.24.
  • the target polymer GE11-PEG6.5k-P (CDC6k-co-TMC22.6k) obtained in Example 14 and the PEG5k-P (CDC5.8k-co-TMC23k) obtained in Example 2 were dissolved and dissolved.
  • a self-crosslinking polymer vesicle was prepared by a solvent displacement method 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 polymer vesicles GE11-CLPs having different target molecular content on the surface can be prepared by mixing the two in different ratios.
  • the target polymer GE11-PEG6.5k-P (CDC6k-co-TMC22.6k) is used in an amount of 10 to 30 wt.%.
  • the self-crosslinking polymer vesicles prepared by the DLS assay have a size of about 85-130 nm and a particle size distribution of 0.01-0.20.
  • DM5k-P (CDC5.8k-co-TMC23k) DMF solution (10 mg/mL) obtained in Example 2 and 0.4 mg of FA-PEG6.5k-P obtained in Example 13 (CDC6k-co) -TMC22.6k)
  • a low boiling organic solvent such as dichloromethane or acetonitrile
  • the self-crosslinking polymer vesicle prepared by the film hydration method as in Example 17 is about 88 nm.
  • the diameter distribution is 0.08.
  • the self-crosslinking vesicle FA-CLPs having different target molecular content on the surface can be prepared by mixing the two in different ratios.
  • the amount of FA-PEG6.5k-P (CDC6k-co-TMC22.6k) is 5-30 wt.%, and the prepared self-crosslinking polymer vesicles have a size of about 85-130 nm and a particle size distribution of 0.01-0.20.
  • the Mal-PEG6k-P (CDC3.6k-LA18.6k) prepared in Example 8 was mixed with P(CDC3.8k-LA18.8k)-PEG4k-P (CDC3.8k-LA18.8k), and then 0.5 ml was added.
  • 4M boric acid buffer solution (pH 8.0) adjust the pH of the solution to 7.5-8.0, add ferferrin according to 1.5 times the molar amount of Mal, bind by Michael addition reaction, react at 30 ° C for two days, and dialyze, according to Example 16
  • the dialysis method was used to prepare vesicular transferin-CLPs.
  • the DLS was measured to be 115 nm and the particle size distribution was 0.12.
  • the graft ratio of the calculated peptides in the nuclear magnetic and BCA protein kits was 94%.
  • the targeting polymer and the non-targeting polymer are dosed according to the mass ratio in the preparation of the vesicle, and the self-crosslinking vesicle transferin-CLPs of different targeting molecules can be obtained, the size is about 85-130 nm, and the particle size distribution is 0.01- 0.20.
  • a variety of self-crosslinking polymer vesicles can be prepared by the similar preparation methods described above, and the proportions and characterization of the raw materials are shown in Table 2.
  • PEG5k-P (CDC5.8k-co-TMC23k) self-crosslinking polymer vesicles were prepared by solvent displacement method.
  • DOX ⁇ HCl was loaded by pH gradient method, and the hydrophilic drug DOX ⁇ HCl was encapsulated by the difference of pH inside and outside the vesicle. .
  • the self-crosslinking vesicles carrying different proportions of the drug (10%-30%) have a particle size of 108-128 nm and a particle size distribution of 0.10-0.14.
  • the encapsulation efficiency of DOX ⁇ HCl was determined by fluorescence spectrometry to be 68%-85%.
  • 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.
  • FIG. 2 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.
  • the self-crosslinking polymer vesicle PEG5k-P (CDC4.9k-co-TMC19k) loaded with DOX ⁇ HCl was prepared in the same manner as above.
  • Self-crosslinking vesicles containing different ratios of drugs (10% to 30%) have a particle size of 100-125 nm and a particle size distribution of 0.10-0.14.
  • the encapsulation efficiency of DOX ⁇ HCl was determined by fluorescence spectrometry to be 60% to 80%.
  • Ally-PEG6k-P (CDC2.9k-CL14.2k) self-crosslinking 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 and then added dropwise to 900 ⁇ L of phosphate buffer solution (10 mM, pH 7.4, PB) and placed in a shaker at 37 ° C (200 rpm) overnight.
  • phosphate buffer solution (10 mM, pH 7.4, PB
  • Self-crosslinking was carried out, followed by dialysis overnight in a dialysis bag (MWCO 7000), five times of water, and 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 assay was designed as above, and the release of hydrophobic drugs after GSH was significantly faster than the absence of GSH samples.
  • the drug-loaded PEG6.5k-P (CDC6k-co-TMC22.6k)-based self-crosslinking polymer vesicle FA-CLPs were prepared by membrane hydration method, and DOX ⁇ HCl was loaded by pH gradient method.
  • 1.6 mg of PEG5k-P (CDC5.8k-co-TMC23k) and 0.4 mg of FA-PEG6.5k-P (CDC6k-co-TMC22.6k) are dissolved in 0.5 mL of low boiling organic solvent such as dichloromethane Or in 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.
  • the particle size is 112-121 nm, the particle size distribution is 0.10-0.15, and the encapsulation efficiency of DOX ⁇ HCl is 61%-77%.
  • the in vitro release assay was designed as above. After the addition of 10 mM GSH, the drug was effectively released at a faster rate than the sample without GSH.
  • the drug-loaded self-crosslinking polymer vesicle GE11-CLPs based on PEG6.5k-P was prepared by dialysis method, and doxorubicin hydrochloride (Epi ⁇ HCl) was loaded by pH gradient method.
  • a variety of self-crosslinking polymer vesicles and targeted self-crosslinking polymer vesicles can be studied for a variety of hydrophilic anti-cancer small molecule drugs and genes such as doxorubicin hydrochloride (DOX ⁇ HCl) using similar preparation methods described above.
  • hydrophilic anti-cancer small molecule drugs and genes such as doxorubicin hydrochloride (DOX ⁇ HCl) using similar preparation methods described above.
  • epirubicin hydrochloride Epi ⁇ HCl
  • CPT ⁇ HCl irinotecan hydrochloride
  • MTO ⁇ HCl mitoxantrone hydrochloride
  • the amount and encapsulation rate are shown in Table 3.
  • cytotoxicity of empty vesicles was tested by MTT assay using SKOV3 human ovarian cancer cells.
  • SKOV3 cells were seeded at a concentration of 5 ⁇ 10 4 /mL in a 96-well plate at 100 ⁇ L per well, and after 24 hours, the cells were adhered to about 70% of the cells.
  • vesicle samples containing different concentrations 0.5, 1.0 mg/mL were added to each well of the experimental group (taking the empty self-crosslinking polymer vesicles of Example 15 and Example 19 as an example), Cell blank control wells and medium blank wells (replicate 4 wells) were set. After 24 hours of culture, 10 ⁇ L of MTT (5.0 mg/mL) was added to each well.
  • FIG. 4 is a graph showing the cytotoxicity of self-crosslinking polymer vesicles. It can be seen that when the concentration of self-crosslinking polymer vesicles is increased from 0.5 to 1.0 mg/mL, the survival rate of SKOV3 is still higher than 92%. It is indicated that the self-crosslinking polymer vesicle of the present invention has good biocompatibility.
  • the culture of the cells was the same as in Example 21 except that the PEG5k-P (CDC5.8k-co-TMC23k) self-crosslinking polymer vesicles carrying DOX ⁇ HCl were loaded with DOX ⁇ HCl in each well of the experimental group.
  • PEG5k-P CDC5.8k-co-TMC23k
  • Self-crosslinking polymer vesicle GE11-CLPs composed of PEG5k-P (CDC5.8k-co-TMC23k) and GE11-PEG6.5k-P (CDC6k-co-TMC22.6k) (where GE11 content is 10%, respectively) , 20%, 30%) added to each corresponding well, DOX ⁇ HCl concentration range is 0.01, 0.1, 0.5, 1, 5, 10, 20, 40 and 80 ⁇ g / mL; target molecular content from 10%, 20% to 30%; doxorubicin liposome in multiple groups as a control group. After 4 hours of co-cultivation, the samples were aspirated and replaced with fresh medium for a further 44 h.
  • FIG. 5 and Figure 6 are the toxicity of drug-loaded self-crosslinking polymer vesicles GE11-CLPsGE11/ to SKOV3 cells; it can be seen that 20% GE11-CLPsGE11 self-crosslinking polymer vesicles carrying DOX ⁇ HCl to SKOV3 cells
  • the semi-lethal concentration (IC 50 ) is 2.01 ⁇ g/mL, which is much lower than that of PEG5k-P (CDC5.8k-co-TMC23k) self-crosslinking polymer vesicles, and lower than that of doxorubicin liposomes. (14.23 ⁇ g/mL), indicating that the drug-loaded self-crosslinking vesicles of the present invention can effectively target ovarian cancer cells, release drugs in cells, and finally kill cancer cells.
  • the culture of the cells was the same as in the twenty-first example, except that when the wells of the experimental group were loaded, the self-crosslinking polymer vesicles for different transferrin contents and different doses were loaded with CPT ⁇ HCl, and Mal-PEG6k- Self-crosslinking polymer vesicles of ferritin-CLPs prepared from P(CDC3.6k-LA18.6k) and P(CDC3.8k-LA18.8k)-PEG4k-P (CDC3.8k-LA18.8k) (Example 10 IX)
  • the corresponding concentration of CPT ⁇ HCl is 0.01, 0.1, 0.5, 1, 5, 10, 20 and 40 ⁇ g/mL
  • the target molecular content is from 10%, 20% to 30%
  • (CDC3.8k-LA18.8k)-PEG4k-P (CDC3.8k-LA18.8k) drug-loaded crosslinked polymer vesicles and free CPT ⁇ HCl group were used as a control group.
  • the drug-loaded self-crosslinking polymer vesicle of the invention can effectively target ovarian cancer cells, release drugs in cells, and finally kill cancer cells, especially after binding target molecules, greatly enhancing the ovary
  • the specificity of cancer cells significantly increases the lethality of drugs against ovarian cancer cells.
  • the toxicity of various drug-loaded self-crosslinking polymer vesicles on ovarian cancer cells was studied by the similar method described above.
  • the drug is a hydrophilic anticancer small molecule drug and the gene drug is doxorubicin hydrochloride (DOX ⁇ HCl), hydrochloric acid.
  • DOE doxorubicin hydrochloride
  • Epirubicin Epi ⁇ HCl
  • irinotecan hydrochloride CPT ⁇ HCl
  • MTO ⁇ HCl mitoxantrone hydrochloride
  • hydrophobic anticancer drugs paclitaxel, docetaxel and olaparib the results are shown in Table 4. .
  • SKOV3 cells were seeded at 6 ⁇ 10 5 / mL in a 6-well plate at 900 ⁇ L per well, and after 24 hours, the cells were adhered to about 70% of the cells. Then, the drug-loaded vesicle samples CLPs and GE11-CLPs were added to each well of the experimental group, and a cell blank control well and a saline control group (two wells) were additionally set.
  • FIG. 7 shows the results of cell uptake of drug-loaded cross-linked vesicles. It can be seen that the uptake of self-crosslinking vesicle cells targeting drug-loaded cells is higher than that of non-targeted cross-linked vesicles and doxorubicin liposomes. , indicating that targeted self-crosslinking vesicles can actively take up endocytosis by ovarian cancer cells.
  • Example 25 Blood circulation of drug-loaded self-crosslinking polymer vesicles (CLPs and FA-CLPs)
  • the experiment used Balb/C nude mice weighing 18-20 g and 4-6 weeks old.
  • the vesicles were prepared by mixing FA-PEG6.5k-P (CDC6k-co-TMC22.6k) and PEG5k-P (CDC5.8k-co-TMC23k) in different proportions, named FA-CLPs, when FA was in the polymer capsule.
  • the targeting ratio in the bubble is 20%
  • the particle size is 100 nm
  • the particle size distribution is 0.10
  • the name is FA20-CLPs
  • the drug is DOX ⁇ HCl.
  • DOX ⁇ HCl-loaded CLPs vesicles, FA-CLPs vesicles, and DOX ⁇ HCl were injected into mice via the 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, and add 100 ⁇ L of 1% Triton And 500 ⁇ L of the extract (DMF containing 20 mM DTT and 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 Triton And 500 ⁇ L of the extract
  • centrifugation 20,000 rpm, 20 minutes
  • a self-crosslinking vesicle GE11-CLPs was prepared by mixing GE11-PEG6.5k-P (CDC6k-co-TMC22.6k) and PEG5k-P (CDC5.8k-co-TMC23k) to GE11-CLPs self-crosslinking vesicles and self-crosslinking vesicle CLPs were loaded with DOX ⁇ HCl, and then injected into Balb/C nude mice to study their blood circulation. DOX ⁇ HCl and Lido multi-DOX-LPs were used for comparison. group. As a result, as shown in Fig.
  • the fluorescent substance cy-7-labeled FA20-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 FA20-CLPs accumulate rapidly at the tumor site, and the fluorescence is still strong after 48 hours. These results indicate that FA20-CLPs can actively target and enrich ovarian cancer tumor sites, and have strong specificity for ovarian cancer cells.
  • the operation and calculation methods of the in vivo imaging experiments of other self-crosslinking polymer vesicles were the same, and the results are shown in Table 4.
  • Epi ⁇ HCl-loaded, cy-7-labeled CLPs and GE11-CLPs were prepared.
  • the tumor inoculation and tail vein administration were the same in the in vivo imaging experiment. Both of them were found to accumulate rapidly in the ovarian tumor site.
  • CLPs were 4-6. The hour disappeared, and the fluorescence of the tumor site remained strong after 48 hours of GE11-CLPs.
  • GE11-CLPs can actively target and enrich ovarian tumor sites.
  • Example 28 In vivo imaging experiment of drug-loaded self-crosslinking polymer vesicle CLPs and transferrin-CLPs in A2780 ovarian cancer mice
  • 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 A2780 human ovarian cancer cells. After about 3 to 4 weeks, the tumor size was 100 ⁇ . The experiment was started at 200 mm 3 .
  • Self-crosslinking vesicle CLPs were labeled with cy-5 and loaded with the hydrophobic drug docetaxel DTX, and the same procedure was used to study in vivo imaging.
  • the experimental results show that the DTX-containing transferrin-CLPs can accumulate rapidly in the tumor site, and the fluorescence of the tumor site is still strong after 48 hours. It indicated that transferrin-CLPs can actively target and enrich tumor sites, while drug-loaded CLPs self-crosslinking vesicles metabolize quickly after entering the tumor for 2 hours, and the intensity is low.
  • FA20-CLPs and CLPs were injected into mice (DOX ⁇ HCl: 10 mg/kg). After 12 hours, the mice were sacrificed, and the tumor and heart, liver, spleen, lung and kidney tissues were taken out, washed and added to 500 ⁇ L 1%. The Triton was ground by a homogenizer and further extracted with 900 ⁇ L of DMF (containing 20 mM DTT, 1 M HCl).
  • the abscissa is the tissue organ, and the ordinate is the total DOX ⁇ HCl injection amount (ID%/g) per gram of tumor or tissue.
  • the amount of DOX ⁇ HCl accumulated in tumors after injection of FA-CLPs, CLPs and DOX ⁇ HCl for 12 hours were 6.54, 2.53 and 1.02 ID%/g, respectively, and FA-CLPs were 3 and 6 times higher than CLPs and DOX ⁇ HCl, indicating drug loading.
  • FA-CLPs accumulate more in the tumor site by active targeting, and have obvious specificity to ovarian cancer cells, which is conducive to killing ovarian cancer cells. The results are shown in Table 4.
  • DO11 ⁇ HCl-loaded GE11-CLPs, CLPs, and liposomal doxorubicin-rich DOX-LPs were injected into mice (DOX ⁇ HCl: 10 mg/kg). After 6 hours, GE11-CLPs, CLPs and DOX-LP were in tumor mass The amount of accumulated DOX ⁇ HCl was 8.63, 3.52 and 1.82 ID%/g, respectively, and GE11-CLPs were 2 and 5 times of the latter two, indicating that the drug-loaded GE11CLPs accumulated more at the tumor site by active targeting (Fig. 9).
  • Example 31 The maximum tolerated dose (MTD) of drug-loaded self-crosslinking polymer vesicles GE11-CLPs to Balb/C mice
  • mice Balb/C nude mice weighing about 18-20 grams and 4-6 weeks old were used.
  • Single-dose injection of self-crosslinking polymer vesicles GE11-CLPs and doxorubicin liposomes including doxorubicin concentrations of 120mg/kg, 140mg/kg, 160mg/kg, 180mg/kg and At 200 mg/kg, the concentration of sputum in the doxorubicin liposome was 20 mg/kg, and five mice in each group. On the last 10 days, the mental state of the mice was observed and the body weight was measured every day. The maximum tolerated dose was based on non-accidental death in mice and less than 15% in mice.
  • the maximum uncomfortable dose of drug-loaded self-crosslinking vesicles was 160 mg/kg, while the maximum tolerated dose of sputum in doxorubicin liposomes. It is 20 mg/kg, and it can be seen that the targeted drug-loaded self-crosslinking vesicles have high tolerance to mice and greatly improve the therapeutic window.
  • Example 32 Anti-tumor effect, body weight change and survival rate of drug-loaded self-crosslinking polymer vesicles GE11-CLPs and CLPs in mice bearing SKOV3 subcutaneous ovarian cancer
  • tumors were significantly inhibited in the GE11-CLPs treatment group at 18 days, while tumors in the drug-loaded CLPs group increased, and the mice had almost no change in body weight.
  • 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.
  • the GE11-CLPs treatment group survived after 62 days, the DOX-LPs group had all died at 42 days, and the PBS group also died at 42 days. Therefore, drug-loaded self-crosslinking vesicles can effectively inhibit tumors, have no toxic side effects on mice, and prolong the survival time of tumor-bearing mice.
  • Example 33 Single-dose anti-tumor effect, body weight change and survival rate of drug-loaded self-crosslinking polymer vesicle GE11-CLPs in mice bearing SKOV3 subcutaneous ovarian cancer
  • DO11 ⁇ CLPs containing DOX ⁇ HCl, DOX-LPs in doxorubicin liposomes, and PBS A single-dose injection of DOX ⁇ HCl in GE11-CLPs self-crosslinking vesicles with doxorubicin doses of 20 mg/kg, 40 mg/kg, and 60 mg/kg, while DOX-LPs have a doxorubicin concentration of 10 mg/kg. Kg and 15mg/kg. As can be seen from Fig.
  • Example 34 Anti-tumor effect, body weight change and survival rate of drug-loaded self-crosslinking polymer vesicles of passenger-CLPs and CLPs in subcutaneous ovarian cancer mice bearing A2780
  • the establishment of the subcutaneous A2780 tumor model, the tail vein administration method and data collection were the same as those in Example 32.
  • the experiment was started when the tumor size was 30-50 mm 3 and mixed by transferrin-PEG6.5k-P (CDC3.8k-co-LA13.8k) and PEG5k-P (CDC3.7k-co-LA14.6k) at 1:5.
  • transferrin-PEG6.5k-P CDC3.8k-co-LA13.8k
  • PEG5k-P CDC3.7k-co-LA14.6k
  • DOX ⁇ HCl-loaded self-crosslinking vesicle cRGD-CLPs prepared by mixing 1:5 with cRGD-PEG6k-P (CDC4.6k-co-TMC18.6k) and PEG5k-P (CDC4.9k-co-TMC19k) As a control group.
  • the results showed that tumors were significantly inhibited at 18 days of treatment with transferrin-CLPs, while tumors in the drug-loaded CLPs group showed a small increase in tumor weight and almost no change in body weight.
  • the body weight of the cRGD-CLPs group did not change, but the tumor inhibition was significantly weaker than the former, and the tumor size was 3 times that of the former, indicating that cRGD had no obvious targeting to ovarian cancer.
  • CPT ⁇ HCl also inhibited tumor growth, the weight of mice in the CPT ⁇ HCl group decreased by 18% at 10 days.
  • the transferrin-CLPs treatment group survived after 72 days, the CPT ⁇ HCl group had all died at 28 days, and the PBS group also died at 37 days.
  • Example 35 Anti-tumor effect, body weight change and survival rate of drug-loaded self-crosslinking polymer vesicles GE11-CLPs and CLPs in mice bearing SKOV3 orthotopic ovarian cancer
  • DOX ⁇ HCl-loaded self-crosslinking vesicles GE11-CLPs, non-targeting CLPs, DOX-LPs, and PBS were injected intravenously into mice bearing SKOV3 orthotopic ovarian cancer.
  • 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.
  • DOX-LPs also inhibited tumor growth, the body weight of DOX-LPs mice decreased by 21% at 4 days.
  • the GE11-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, the drug-loaded self-crosslinking vesicles GE11-CLPs of the binding targeting molecule can effectively inhibit the growth of ovarian cancer in situ, without toxicity. It can also prolong the survival time of tumor-bearing mice.

Abstract

提供一种卵巢癌特异靶向的生物可降解双亲性聚合物、由其制备的聚合物囊泡及其应用。所述生物可降解双亲性聚合物由含双硫碳酸酯单体的聚合物键合靶向分子制得。由所述生物可降解双亲性聚合物制备的聚合物囊泡可自行交联而无需外加交联剂，且该交联具有原敏感性，因而其可用于药物控释体系，有利于纳米药物临床应用生产。

Description

卵巢癌特异靶向的生物可降解双亲性聚合物、由其制备的聚合物囊泡及应用 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，具体涉及一种卵巢癌特异靶向的生物可降解双亲性聚合物、由其制备的聚合物囊泡以及在卵巢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。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药物在体内循环稳定，但只能装载疏水性小分子抗癌药物，而对穿透能力更强的亲水性小分子抗癌药物无能为力，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。卵巢癌是卵巢肿瘤的一种恶性肿瘤，是指生长在卵巢上的恶性肿瘤，其中90％～95％为卵巢原发性的癌，另外5％～10％为其他部位原发的癌转移到卵巢。由于卵巢的胚胎发育、组织解剖及内分泌功能较复杂，它所患的肿瘤可能为良性或恶性。由于卵巢癌早期缺少特异性症状，筛查作用又有限，鉴别其组织类型及 良恶性相当困难，因此早期诊断比较困难，卵巢癌行剖腹探查术中发现肿瘤局限于卵巢的仅占30％，大多数已扩散到子宫双侧附件，大网膜及盆腔各器官；就诊时60％～70％已为晚期，而晚期病例疗效不佳。直到目前为止，卵巢癌无论在诊断和治疗上确是一大难题。因此，虽然卵巢癌的发病例低于宫颈癌和子宫内膜癌居妇科恶性肿瘤的第三位，但死亡率却超过宫颈癌和子宫内膜癌之和，高居妇科癌症首位，是严重威胁妇女健康的最大疾患。
简而言之，卵巢癌是高发性的女性恶性肿瘤，虽然发病的绝对人数不是很多，但是死亡率很高，主要是由于早期难以觉察，难以早期诊断，多数一经诊断即为晚期，错过了手术切除的最佳时间；而且其治疗还存在着治愈率低、易转移、易耐药的特点。而纳米药物能够改变传统化疗药物的体内分布，增强肿瘤内药物的浓度，提高治疗效果，是治疗卵巢癌的一个关键点和希望所在。DOXIL(PEG化的脂质体阿霉素)是FDA批准使用最早的一个脂质体囊泡纳米药物DOXIL(PEG化的脂质体阿霉素)，临床上对卵巢癌的治疗有效。但是，DOXIL也存在问题。，其一是其最大耐受剂量(MTD)较小，故治疗窗口相对较窄、还有易出现毒副作用的问题；其二，DOXIL是基于EPR效应的被动靶向作用，由于不同肿瘤的巨大的个体差异性，很难利用一个通用的统一机理来把纳米药物运输到所有的肿瘤组织和肿瘤细胞中(参见：S.Eetezadi,SN.Ekdawi,C.Allen,Adv.Drug Deliv Rev,2015,91,7-22)；对于不同肿瘤，由于不同肿瘤表面性质差别很大，相同肿瘤在不同病人间的差别也很大；即便是同一个肿瘤中不同的肿瘤细胞也不尽相同，因此个性化的治疗显得尤为重要。因此我们必须个性化地设计适合目标肿瘤的靶向体系，而不能把适用于一个适应症的药物随便用到其他病症上，因此个性化的治疗显得尤为重要。所以，需要研发针对特定肿瘤的主动靶向纳米药物，具有肿瘤特异性，来实现纳米药物的其他的优点如提高肿瘤细胞的有效药物浓度、提高体内外的疗效。
发明内容
本发明的目的是提供一种卵巢癌特异靶向的生物可降解双亲性聚合物及其制备的聚合物囊泡以及作为抗卵巢癌药物的载体在制备卵巢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：
一种卵巢癌特异靶向的生物可降解双亲性聚合物，由含双硫碳酸酯单体的聚合 物键合靶向分子制备得到；所述靶向分子为GE11(多肽)、FA(叶酸)、transferrin(转铁蛋白)或者Herceptin蛋白；所述含双硫碳酸酯单体的聚合物的化学结构式为以下结构式中的一种：
Figure PCTCN2017075529-appb-000001
式Ⅰ
Figure PCTCN2017075529-appb-000002
式Ⅱ
其中，R1选自以下基团中的一种：
Figure PCTCN2017075529-appb-000003
R2选自以下基团中的一种：
Figure PCTCN2017075529-appb-000004
其中，k为113～170，x为15～45，y为80～300，m为220～280。
本发明公开的含双硫碳酸酯单体的聚合物的疏水嵌段具有侧链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 PCTCN2017075529-appb-000005
也可以为三嵌段聚合物：
Figure PCTCN2017075529-appb-000006
优选的技术方案中，R1选自以下基团中的一种：
Figure PCTCN2017075529-appb-000007
所述R2选自以下基团中的一种：
Figure PCTCN2017075529-appb-000008
所述k为113～170，x为20～40，y为125～250。
优选的，当含双硫碳酸酯单体的聚合物的化学结构式为式Ⅰ时，分子量为30～55kDa；当含双硫碳酸酯单体的聚合物的化学结构式为式Ⅱ时，分子量为60～95kDa；本发明公开的聚合物的分子量可控，其各结构单元的组成和比例适合于形成自交联形成稳定的聚合物囊泡结构。
本发明公开的卵巢癌特异靶向的生物可降解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左右及以上，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50～160纳米，可以作为治疗卵巢癌的药物的载体；在囊泡的疏水膜中装载疏水性小分子抗卵巢癌药物紫杉醇、多西紫杉醇、阿霉素、奥拉帕尼、吉非替尼等，也可以在囊泡的大亲水内腔中装载亲水性抗卵巢癌药物，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。
本发明还公开了一种聚合物囊泡，可以由上述含双硫碳酸酯聚合物含双硫碳酸酯单体的聚合物制备得到；或者由上述卵巢癌特异靶向的生物可降解双亲 性聚合物制备得到；或者由上述含双硫碳酸酯聚合物含双硫碳酸酯单体的聚合物与卵巢癌特异靶向的生物可降解双亲性聚合物制备得到，比如上述含双硫碳酸酯聚合物含双硫碳酸酯单体的聚合物和卵巢癌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，即得到卵巢癌靶向自交联囊泡，来可以增加囊泡纳米药物在卵巢癌细胞中的摄取量；也可以由含双硫碳酸酯聚合物含双硫碳酸酯单体的聚合物制备的囊泡的外表面偶联肿瘤细胞特异性靶向分子来制备卵巢癌靶向囊泡，以增加卵巢癌细胞的摄取量，比如在囊泡的PEG端通过迈克尔加成或酰胺化反应键合GE11、FA、transferrin或者Herceptin等。优选的，本发明的聚合物囊由卵巢癌特异靶向的生物可降解双亲性聚合物与含双硫碳酸酯聚合物含双硫碳酸酯单体的聚合物制备得到；按质量百分数，所述卵巢癌特异靶向的生物可降解双亲性聚合物的用量为1～40wt.％。
本发明的含双硫碳酸酯单体的聚合物和卵巢癌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。聚合物作为药物载体应用时，为达到最佳靶向和治疗效果，最基本也是最关键的性能为就是在体内长时间循环。而形成交联结构是聚合物载体能够在体内环境长时间循环的必要过程，现有技术都是通过添加交联剂使得聚合物纳米载体形成稳定的交联结构，但是交联剂的加入，不仅会增加纳米药物制备流程的复杂程度、增加纳米药物的生产成本、降低药物的最终纯度，不利于纳米药物临床应用的放大生产，还会影响药物装载效率、药物释放水平，并增加毒副作用、降低聚合物载体纳米药物的生物相容性；本发明首次公开的聚合物结构，可以在无需外加交联剂的条件下，自行交联，而在囊泡疏水膜内形成稳定的化学交联结构，从而可在体内稳定长循环，不仅避免了交联剂的副作用，而且在载药聚合物囊泡到达肿瘤并内吞进入癌细胞后，在细胞内大量还原性物质存在环境下，可以快速解交联，最大量释放药物，高效杀死卵巢癌细胞；同时自交联聚合物的稳定性等同甚至优于交联剂交联聚合物囊泡；更为重要的，本发明避免了交联剂对一些药物的干扰，成功利用自交联聚合物囊泡装载药物，不仅避免了现有小分子药物的副作用，拓展抗癌药物的利用空间，而且可以适用体质差异大的不同个体。
本发明还公开了上述卵巢癌特异靶向的生物可降解双亲性聚合物在制备治 疗卵巢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卵巢癌的纳米药物中的应用；尤其保护自交联聚合物囊泡作为载体在制备治疗卵巢癌的药物中的应用，自交联聚合物囊泡避免交联剂的使用，进一步增强了药物安全性，减少药物组装步骤。基于本发明聚合物制备的抗卵巢癌纳米药物为囊泡抗卵巢癌纳米药物。
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、优异的卵巢癌靶向性，可以制备聚合物囊泡，装载不同性质的药物，可以不加入任何交联剂而自行交联，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。
2.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，可在卵巢癌细胞高富集；但是进入卵巢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卵巢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。
3.本发明公开的卵巢癌特异靶向的生物可降解双亲性聚合物可以不加入任何交联剂而制备自交联囊泡，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，有利于纳米药物的临床应用。
4.本发明公开的卵巢癌特异靶向的生物可降解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，可制备卵巢癌靶向的自交联囊泡，在卵巢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是实施例十五中交联囊泡PEG5k-P(CDC5.8k-co-TMC23k)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2为实施例二十中载DOX·HCl交联囊泡PEG5k-P(CDC5.8k-co-TMC23k)的体外释放图；
图3是实施例二十中载DOX·HCl交联囊泡GE11-CLPs的体外释放图；
图4是实施例二十一中靶向交联囊泡GE11-CLPs对SKOV3卵巢癌细胞的毒性结果图；
图5是实施例二十二中载DOX·HCl靶向交联囊泡GE11-CLPs对SKOV3卵巢癌细胞的毒性结果图；
图6是实施例二十二中载DOX·HCl靶向交联囊泡GE11-CLPs对SKOV3卵巢癌细胞的半致死量毒性结果图；
图7为实施例二十四中载DOX·HCl靶向交联囊泡GE11-CLPs对SKOV3卵巢癌细胞的细胞内吞结果图；
图8为实施例二十九中载DOX·HCl靶向交联囊泡GE11-CLPs对小鼠的血液循环结果图；
图9为实施例三十中载DOX·HCl靶向交联囊泡GE11-CLPs对荷皮下卵巢癌小鼠的生物分布结果图；
图10为实施例三十一中载DOX·HCl靶向交联囊泡GE11-CLPs对小鼠的最大耐受量结果图；
图11为实施例三十二中载DOX·HCl靶向交联囊泡GE11-CLPs在皮下荷卵巢癌小鼠的多剂量治疗图，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12为实施例三十三中载DOX·HCl靶向交联囊泡GE11-CLPs在皮下荷卵巢癌小鼠的单剂量治疗图，其中A为肿瘤生长曲线，B为小鼠治疗后肿瘤图片，C为体重变化曲线，D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Et3N·HCl，滤液经旋蒸浓缩，乙醚进行多次重结晶，得到黄色晶体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 合成两嵌段聚合物PEG5k-P(CDC5.8k-co-TMC23k)的
在氮气环境下，0.1g(0.52mmol)CDC单体和0.4g(4.90mmol)的三亚甲基碳酸酯(TMC)溶在5mL二氯甲烷中，加入密封反应器里，然后加入0.12g(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(CDC5.8k-co-TMC23k)。1H NMR(400MHz,CDCl3):2.08(t,-COCH2CH2CH2O-),3.08(s,-CCH2),3.30(m，-OCH3)，3.65(-OCH2CH2O-)，4.28(t,-COCH2CH2CH2O-),4.31(m,-CCH2)。核磁计算出下式中k＝114，x＝30.2，y＝225.5。GPC测分子量：45.6kDa，分子量分布：1.53。
Figure PCTCN2017075529-appb-000009
实施例三 合成两嵌段聚合物Mal-PEG6k-P(CDC4.8k-co-TMC19.2k)
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 PCTCN2017075529-appb-000010
实施例四 合成两嵌段聚合物NHS-PEG6.5k-P(CDC6k-co-TMC22.6k)
在氮气环境下，0.1g(0.52mmol)CDC单体和0.42g(4.12mmol)的TMC溶在5mL二氯甲烷中，加入密封反应器里，然后加入0.11g(0.017mmol)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(CDC6k-co-TMC22.6k)。1H NMR(400MHz,CDCl3):2.08(t,-COCH2CH2CH2O-),3.08(s,-CCH2),3.30(m，-OCH3)，3.65(-OCH2CH2O-)，4.28(t,-COCH2CH2CH2O-),4.31(m,-CCH2)，和2.3(s，NHS)。核磁计算出下式中k＝148，x＝31.3，y＝221.6。GPC测分子量：51.3kDa，分子量分布：1.43。
Figure PCTCN2017075529-appb-000011
实施例五 合成两嵌段聚合物PEG7.5k-P(CDC5.8k-co-TMC20.0k)
在氮气环境下，60mg(0.31mmol)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75mg(0.01mmol)CH3O-PEG7500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PEG7.5k-P(CDC5.8k-co-TMC20.0k)。反应式和1H NMR特征峰同实施例二。核磁计算出式中k＝170，x＝30，y＝196。GPC测分子量：54.5kDa，分子量分布：1.36。
实施例六 合成两嵌段聚合物PEG5k-P(CDC3.9k-co-LA18.0k)
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(CDC3.9k-co-LA14.6k)。1H NMR(400MHz,CDCl3):1.,59(s,-COCH(CH3)O-),3.08(s,-CCH2),3.30(m，-OCH3)，3.65(-OCH2CH2O-)，4.31(m,-CCH2)，5.07(s,-COCH(CH3)O-)。核磁计算下式中，k＝114，x＝20，y＝125。GPC测分子量：34.3kDa，分子量分布：1.32。
Figure PCTCN2017075529-appb-000012
实施例七 合成两嵌段聚合物PEG6.5k-P(CDC5.8k-co-LA28.3k)
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。核磁计算式中k＝148，x＝30，y＝190。GPC分子量：42.4kDa，分子量分布：1.43。
实施例八 合成两嵌段聚合物Mal-PEG6k-P(CDC3.6k-co-LA18.6k)
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＝20，y＝129。GPC测分子量：32.5kDa，分子量分布：1.44。
Figure PCTCN2017075529-appb-000013
实施例九 合成三嵌段聚合物P(CDC3.8k-co-TMC18.8k)-PEG10k-P(CDC3.8k-co-TMC18.8k)
在氮气环境下0.8g(7.84mmol)的TMC和0.16g(0.83mmol)CDC溶在8 mL二氯甲烷中，加入密封反应器里，后加入0.2g(0.04mmol)的HO-PEG-OH10000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-co-TMC18.8k)-PEG10k-P(CDC3.8k-co-TMC18.8k)。1H NMR特征峰同实施例二。核磁计算出下式中，m＝227，x＝20，y＝184。GPC测的分子量：92.3kDa，分子量分布：1.46。
Figure PCTCN2017075529-appb-000014
实施例十 合成两嵌段聚合物NHS-PEG7.5k-P(CDC3.8k-co-LA13.8k)
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 PCTCN2017075529-appb-000015
采用上述类似的制备方法可以制备多种含双硫碳酸酯单体的聚合物，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 PCTCN2017075529-appb-000016
Figure PCTCN2017075529-appb-000017
实施例十一 合成靶向聚合物transferrin-PEG7.5k-P(CDC3.8k-co-LA13.8k)
转铁蛋白transferrin偶联的聚合物的合成分为两步，第一步如实施例八制备Mal-PEG7.5k-P(CDC3.8k-co-LA13.8k)；第二步为transferrin与其通过迈克尔反应键合。先将上述聚合物Mal-PEG7.5k-P(CDC3.8k-co-LA13.8k)溶解在DMF中，加入两倍摩尔量的transferrin，30℃反应两天、透析、冻干，得到transferrin-PEG6.5k-P(CDC3.8k-co-LA13.8k)，通过核磁和BCA蛋白试剂盒测试计算transferrin接枝率为95％。
实施例十二 合成靶向聚合物Herceptin-PEG6k-P(CDC3.6k-co-LA18.6k)
人类表皮生长因子抗体Herceptin偶联的聚合物的合成分为三步，第一步如实施例八制备Mal-PEG6k-P(CDC3.6k-co-LA18.6k)；第二步Mal-PEG6k-P(CDC3.6k-co-LA18.6k)与半胱胺迈克尔加成反应，使得端基转变为氨基；第三步FA的羧基与其通过酰胺化反应键合：先将前步得到的聚合物溶在0.5毫升DMF中，加入2毫升的硼酸缓冲溶液(pH 8.0)，再加入2倍摩尔量的Herceptin，30℃反应两天，透析、冷冻干燥得到最终产物Herceptin-PEG6k-P(CDC3.6k-co-LA18.6k)，核磁和BCA蛋白测试计算FA接枝率为96％。
实施例十三 合成靶向聚合物FA-PEG6.5k-P(CDC6k-co-TMC22.6k)
叶酸(FA)偶联的聚合物的合成分为两步，第一步如实施例四制备NHS-PEG6.5k-P(CDC6k-co-TMC22.6k)；第二步FA的氨基与其通过酰胺化反应键合：先将上述聚合物溶解在DMF中，加入两倍摩尔量的FA，30℃反应两天后，透析除去游离FA，冷冻干燥得到FA-PEG6.5k-P(CDC6k-co-TMC22.6k)，通过核磁测试计算FA接枝率为88％。
实施例十四 合成靶向聚合物GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)
环状多肽YHWYGYTPQNVI(GE11)偶联的聚合物的合成分为两步，第一步如实施例四制备NHS-PEG6.5k-P(CDC6k-co-TMC22.6k)；第二步为GE11的氨基与其通过酰胺化反应键合：先将上述聚合物溶在DMF中，加入两倍摩尔量的GE11，30℃反应两天后，透析除去游离GE11，冷冻干燥得到GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)，通过核磁和BCA蛋白试剂盒计算GE11接枝率为96％。
根据实施例十一至实施例十三的方法，可以在实施例二至实施例十以及表1的含双硫碳酸酯单体的聚合物键合靶向分子制备得到卵巢癌特异靶向的生物可降解双亲性聚合物。
实施例十五 制备自交联聚合物囊泡
采用溶剂置换法制备聚合物囊泡。100μL的PEG5k-P(CDC5.8k-co-TMC23k)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，换五次介质PB。图1是上述自交联囊泡PEG5k-P(CDC5.8k-co-TMC23k)粒径分布(A)及电子透射显微镜图片 (B)，交联囊泡稳定性的测试(C)及还原响应性测试(D)图；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1A；由图1B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1C)，但在模拟肿瘤细胞还原环境下快速释放，解交联(图1D)。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。采用相同的制备方法，PEG5k-P(CDC4.9k-co-TMC19k)可形成自交联纳米囊泡，粒径为100nm，粒径分布为0.1。
实施例十六 采用透析法和薄膜水化法制备自交联聚合物囊泡
采用透析法制备自交联聚合物囊泡。100μL的PEG5k-P(CDC5.8k-co-TMC23k)的DMF溶液(10mg/mL)装入透析袋(MWCO 7000)中，在PB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60nm左右，粒径分布0.08。
采用薄膜水化法制备自交联聚合物囊泡。2mg的PEG5k-P(CDC5.8k-co-TMC23k)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160nm左右，粒径分布0.24。
实施例十七 制备基于PEG5k-P(CDC5.8k-co-TMC23k)和GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)的自交联聚合物囊泡GE11-CLPs
实施例十四中得到的靶向聚合物GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)和实施例二得到的PEG5k-P(CDC5.8k-co-TMC23k)二者混合溶于DMF中，采用如实施例十五的溶剂置换法制备自交联聚合物囊泡。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含量的自交联聚合物囊泡GE11-CLPs。本实施例靶向聚合物GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)的用量为10～30wt.％。DLS测定制备的自交联聚合物囊泡尺寸为85-130nm左右，粒径分布0.01-0.20。
实施例十八 制备基于FA-PEG6.5k-P(CDC6k-co-TMC22.6k)和PEG5k-P(CDC5.8k-co-TMC23k)的自交联聚合物囊泡FA-CLPs
1.6mg的实施例二得到的PEG5k-P(CDC5.8k-co-TMC23k)的DMF溶液(10mg/mL)及0.4mg的实施例十三中得到的FA-PEG6.5k-P(CDC6k-co-TMC22.6k)溶于0.5mL的低沸点有机溶剂中，如二氯甲烷或乙腈中，采用如实施例十七的薄膜水化法制备得到的自交联聚合物囊泡为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含量的自交联囊泡FA-CLPs。FA-PEG6.5k-P(CDC6k-co-TMC22.6k)的用量为5～30wt.％，制备的自交联聚合物囊泡尺寸为85～130nm左右，粒径分布0.01～0.20。
实施例十九 制备表面偶联transferrin的自交联囊泡transferrin-CLPs
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，然后加入0.5毫升的4M硼酸缓冲溶液(pH8.0)调节溶液pH至7.5-8.0，再按照Mal摩尔量的1.5倍加入transferrin，通过迈克尔加成反应键合，30℃反应两天后，透析，按照实施例十六所述透析方法制备囊泡transferrin-CLPs。DLS测定为115nm，粒径分布0.12。核磁和BCA蛋白试剂盒测试计算多肽的接枝率为94％。靶向聚合物与非靶向聚合物在制备囊泡过程中按照质量比百分数投料，可获得不同靶向分子的自交联囊泡transferrin-CLPs，尺寸为85-130nm左右，粒径分布0.01-0.20。
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，原料比例以及表征见表2。
表2自交联聚合物囊泡的制备和表征
Figure PCTCN2017075529-appb-000018
实施例二十 自交联聚合物囊泡的药物装载及体外释放
采用溶剂置换法制备PEG5k-P(CDC5.8k-co-TMC23k)自交联聚合物囊泡，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(CDC5.8k-co-TMC23k)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，pH8.1)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108-128nm，粒径分布在0.10-0.14。荧光光谱仪测定DOX·HCl的包裹效率为68％-85％。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2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。
采用和上述相同的方法制备了装载DOX·HCl的自交联聚合物囊泡PEG5k-P(CDC4.9k-co-TMC19k)。载不同比例的药(10％～30％)的自交联囊泡的粒径在100-125nm，粒径分布在0.10-0.14。荧光光谱仪测定DOX·HCl的包裹效率为60％～80％。
用溶剂置换法制备Ally-PEG6k-P(CDC2.9k-CL14.2k)自交联聚合物囊泡。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(10mg/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上，加GSH后疏水药物释放明显要快于没加GSH样本。
用薄膜水化法制备载药的基于PEG6.5k-P(CDC6k-co-TMC22.6k)的自交联聚合物囊泡FA-CLPs，pH梯度法装载DOX·HCl。1.6mg的PEG5k-P(CDC5.8k-co-TMC23k)及0.4mg的FA-PEG6.5k-P(CDC6k-co-TMC22.6k)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上，加入10mM GSH后，药物有效释放，速度明显要快于没加GSH的样本。
采用透析法制备载药的基于PEG6.5k-P(CDC6k-co-TMC22.6k)的自交联聚合物囊泡GE11-CLPs，pH梯度法装载盐酸阿霉素(Epi·HCl)。80μL的PEG5k-P(CDC5.8k-co-TMC23k)的DMF溶液(10mg/mL)及20μL的GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)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， DOX·HCl的包裹效率为64％-79％。Epi·HCl体外释放实验设计同上，见图3，加入10mM GSH后，药物有效释放，速度明显要快于没加GSH的样本。
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及基因如盐酸多柔比星(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、多烯紫杉醇和奥拉帕尼的载药量和包封率，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 PCTCN2017075529-appb-000020
实施例二十一 MTT法测试空自交联聚合物囊泡对SKOV3细胞的毒性
采用MTT法测试空囊泡的细胞毒性，使用SKOV3人卵巢癌细胞。以 5×104个/mL将SKOV3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(0.5，1.0mg/mL)的囊泡样品(以实施例十五和实施例十九的空的自交联聚合物囊泡为例)，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4为自交联聚合物囊泡的细胞毒性结果，可看出，当自交联聚合物囊泡的浓度从0.5增到1.0mg/mL时，SKOV3的存活率仍高于92％，说明本发明的自交联聚合物囊泡具有良好的生物相容性。
实施例二十二 MTT法测载药自交联囊泡对SKOV3卵巢癌细胞的毒性
细胞的培养和实施例二十一相同，只是实验组各孔加样时，载DOX·HCl的PEG5k-P(CDC5.8k-co-TMC23k)自交联聚合物囊泡、载DOX·HCl的由PEG5k-P(CDC5.8k-co-TMC23k)和GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)构成的自交联聚合物囊泡GE11-CLPs(其中GE11含量分别为10％、20％、30％)加入各对应孔中，DOX·HCl浓度范围为0.01、0.1、0.5、1、5、10、20、40和80μg/mL；靶向分子含量从10％、20％到30％；阿霉素脂质体里葆多组作为对照组。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二十一。附图5和图6是载药自交联聚合物囊泡GE11-CLPsGE11/对SKOV3细胞的毒性；可看出，载DOX·HCl的20％GE11-CLPsGE11自交联聚合物囊泡对SKOV3细胞的半致死浓度(IC50)为2.01μg/mL，远远低于PEG5k-P(CDC5.8k-co-TMC23k)自交联聚合物囊泡，也低于阿霉素脂质体里葆多(14.23μg/mL)，说明本发明的载药靶向自交联囊泡能有效靶向到卵巢癌细胞，在细胞内释放药物，最终杀死癌细胞。
实施例二十三 MTT法测试载药自交联聚合物囊泡对A2780细胞的毒性
细胞的培养和实施例二十一相同，只是实验组各孔加样时，针对不同transferrin含量、不同药量的载药自交联聚合物囊泡，以载CPT·HCl、由Mal-PEG6k-P(CDC3.6k-LA18.6k)和P(CDC3.8k-LA18.8k)-PEG4k-P(CDC3.8k-LA18.8k)制备的自交联聚合物囊泡transferrin-CLPs(实施例十九)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％；P(CDC3.8k-LA18.8k)-PEG4k- P(CDC3.8k-LA18.8k)载药交联聚合物囊泡和游离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二十一。结果表明，P(CDC3.8k-LA18.8k)-PEG4k-P(CDC3.8k-LA18.8k)载药自交联聚合物囊泡对A2780细胞的IC50为4.15μg/mL，特别是载CPT·HCl的30％transferrin-CLPs对A2780细胞的IC50为2.07μg/mL，低于自由药物CPT·HCl(4.11μg/mL)。
说明本发明的载药自交联聚合物囊泡能有效靶向到卵巢癌细胞，并在细胞内释放药物，最终杀死癌细胞，尤其是键合靶向分子后，极大增强了对卵巢癌细胞的特异性，显著提高了药物对卵巢癌细胞的杀伤力。
采用上述类似的方法研究了多种载药自交联聚合物囊泡对卵巢癌细胞的毒性，药物为亲水抗癌小分子药物及基因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、多烯紫杉醇和奥拉帕尼，结果见表4。
实施例二十四 载药的自交联囊泡(CLPs及GE11-CLPs)的细胞内吞
采用流式法测试载药囊泡的细胞内吞，使用SKOV3人卵巢癌细胞。以2×105个/mL将SKOV3细胞种于6孔板，每孔900μL，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载药囊泡样品CLPs及GE11-CLPs，另设细胞空白对照孔和生理盐水对照组(复2孔)。培养4小时后，每孔加入胰酶消化五分钟，并用生理盐水洗涤三次，用流式细胞仪仪于488nm处测阿霉素荧光吸收强度，以生理盐水组为对照，计算细胞内吞量。附图7为载药交联囊泡的细胞摄取结果，可看出，靶向载药自交联囊泡细胞摄取量高于无靶向交联囊泡及阿霉素脂质体里葆多，说明靶向自交联囊泡能主动被卵巢癌细胞摄取内吞。
实施例二十五 载药的自交联聚合物囊泡(CLPs和FA-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，4～6周龄的Balb/C裸鼠。囊泡由FA-PEG6.5k-P(CDC6k-co-TMC22.6k)和PEG5k-P(CDC5.8k-co-TMC23k)按不同比例混合制备，命名为FA-CLPs，当FA在聚合物囊泡中的靶向比例为20％时，粒径为100纳米，粒径分布为0.10，命名为FA20-CLPs，药物为DOX·HCl。将载DOX·HCl的CLPs囊泡、FA-CLPs囊泡和DOX·HCl通过尾静脉注射小鼠体内(DOX药量为10 mg/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，再加入100μL浓度为1％的曲拉通和500μL萃取液(DMF含20mM的DTT和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。由计算可知，载药FA-CLPs自交联聚合物囊泡-和载药CLPs自交联聚合物囊泡在小鼠体内的消除半衰期分别为4.23和4.16小时，而DOX·HCl的仅为0.27小时，所以本发明公开的自交联聚合物囊泡在小鼠体内稳定，有较长循环时间。其他载药自交联聚合物囊泡的血液循环实验的操作和计算方法相同，结果在表4。
实施例二十六 载药的自交联聚合物囊泡CLPs和GE11-CLPs的血液循环
如实施例二十五，由GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)和PEG5k-P(CDC5.8k-co-TMC23k)混合制备得自交联囊泡GE11-CLPs，以GE11-CLPs自交联囊泡以及自交联囊泡CLPs装载DOX·HCl后，尾静脉注射入Balb/C裸鼠中，研究其血液循环，DOX·HCl和里葆多DOX-LPs用于对照组。结果如图10所示，GE11-CLPs和CLPs囊泡在48小时后仍有5.0ID％/g。由计算可知，GE11-CLPs自交联囊泡和CLPs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。
实施例二十七 自交联囊泡FA-CLPs在荷SKOV3卵巢癌小鼠的活体成像
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SKOV3人卵巢癌细胞，大约3～4周后，肿瘤大小为100～200mm3时开始实验。以由FA-PEG6.5k-P(CDC6k-co-TMC22.6k)和PEG5k-P(CDC5.8k-co-TMC23k)按1:5混合制备的自交联聚合物囊泡FA20-CLPs和自交联囊泡CLPs为例。将荧光物质cy-7标记的FA20-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，FA20-CLPs在肿瘤部位很快积累，且在48小时后荧光仍然很强。说明FA20-CLPs能主动靶向及富集到卵巢癌肿瘤部位，对卵巢癌细胞具有极强的特异性。其他自交联聚合物囊泡的活体成像实验的操作和计算方法相同，结果在表4。
制备载Epi·HCl的、cy-7标记的CLPs和GE11-CLPs，活体成像实验中肿瘤的接种以及尾静脉给药同上，发现二者都可在卵巢肿瘤部位很快积累，CLPs在4-6小时消失，而GE11-CLPs在48小时后肿瘤部位荧光仍然很强，说明 GE11-CLPs能主动靶向及富集到卵巢肿瘤部位。
实施例二十八 载药自交联聚合物囊泡CLPs和transferrin-CLPs在荷A2780卵巢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A2780人卵巢癌细胞，大约3～4周后，肿瘤大小为100～200mm3时开始实验。由transferrin-PEG6.5k-P(CDC3.8k-co-LA13.8k)和PEG5k-P(CDC3.7k-co-LA14.6k)混合制备的靶向自交联囊泡transferrin-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，同实施例二十七操作来研究活体成像。实验结果可知，载DTX的transferrin-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transferrin-CLPs能主动靶向及富集到肿瘤部位，而载药CLPs自交联囊泡在2小时进入肿瘤后很快代谢，且强度低。
实施例二十九 载药自交联聚合物囊泡CLPs和FA-CLPs在荷SKOV3卵巢癌小鼠的体内生物分布
样本FA20-CLPs的制备、生物分布实验中肿瘤的接种以及尾静脉给药同实施例二十七。FA20-CLPs和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8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)。FA-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，FA-CLPs是CLPs和DOX·HCl的3和6倍，说明载药FA-CLPs通过主动靶向在肿瘤部位积累较多，对卵巢癌细胞具有明显的特异性，有利于杀伤卵巢癌细胞，结果在表4。
实施例三十 载药自交联聚合物囊泡CLPs和GE11-CLPs在荷SKOV3卵巢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二十七。载DOX·HCl的GE11-CLPs、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，GE11-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，GE11-CLPs是后两者的2和5倍，说明载药GE11CLPs通过主动靶向在肿瘤部位积累较多(图9)。
实施例三十一 载药自交联聚合物囊泡GE11-CLPs对Balb/C小鼠的最大耐受剂量(MTD)
选用体重为18～20克左右，4～6周龄的Balb/C裸鼠。单剂量注射载药自交联聚合物囊泡GE11-CLPs及阿霉素脂质体里葆多，其中阿霉素的浓度分别为120mg/kg、140mg/kg、160mg/kg、180mg/kg及200mg/kg，阿霉素脂质体里葆多的浓度为20mg/kg，每组小鼠五只，最后的10天，每天观察小鼠的精神状态及测量体重。最大耐受剂量的标准为小鼠非意外性死亡及小鼠体重低于15％。从图10A及10B各组分小鼠体重变化和生存率可知，载药靶向自交联囊泡的最大难受剂量为160mg/kg，而阿霉素脂质体里葆多的最大耐受剂量为20mg/kg，由此可知，靶向性载药自交联囊泡对小鼠有很高的耐受能力，大大的提高了治疗窗口。
实施例三十二 载药自交联聚合物囊泡GE11-CLPs和CLPs在荷SKOV3皮下卵巢癌的小鼠中的抑瘤效果、体重变化和存活率
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SKOV3人卵巢癌细胞，大约两周后，肿瘤大小为30～50mm3时开始实验。由GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)和PEG5k-P(CDC5.8k-co-TMC23k)按1：5混合制备的载DOX·HCl的靶向性自交联囊泡GE11-CLPs、无靶向CLPs、DOX-LPs以及PBS尾静脉注射。由图11中可知，GE11-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。GE11-CLPs治疗组在62天后全部存活，DOX-LPs组在42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。
实施例三十三 载药靶向自交联聚合物囊泡GE11-CLPs在荷SKOV3皮下卵巢癌的小鼠中的单剂量抑瘤效果、体重变化和存活率
皮下SKOV3肿瘤模型的建立、尾静脉给药方式和数据采集同实施例三十二。载DOX·HCl的GE11-CLPs、阿霉素脂质体里葆多DOX-LPs以及PBS尾静 脉单剂量注射，其中载DOX·HCl的GE11-CLPs自交联囊泡的阿霉素药量为20mg/kg、40mg/kg及60mg/kg，而DOX-LPs的阿霉素浓度为10mg/kg及15mg/kg。由图12中可知，GE11-CLPs阿霉素浓度为60mg/kg治疗组18天时，肿瘤得到明显抑制，而阿霉素浓度为20mg/kg及40mg/kg肿瘤有增长，所有组别小鼠体重几乎没有改变。DOX-LPs在阿霉素浓度为10mg/kg及15mg/kg单剂量都不能抑制肿瘤的增长。GE11-CLPs治疗组在49天后全部存活，DOX-LPs组在42及43天时已全部死亡，PBS组34天时也全部死亡。
实施例三十四 载药靶向自交联聚合物囊泡transferrin-CLPs和CLPs在荷A2780皮下卵巢癌的小鼠中的抑瘤效果、体重变化和存活率
皮下A2780肿瘤模型的建立、尾静脉给药方式和数据采集同实施例三十二。肿瘤大小为30～50mm3时开始实验，由transferrin-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transferrin-CLPs、无靶向CLPs、自由CPT·HCl以及尾静脉注射。由cRGD-PEG6k-P(CDC4.6k-co-TMC18.6k)和PEG5k-P(CDC4.9k-co-TMC19k)按1：5混合制备的载DOX·HCl的自交联囊泡cRGD-CLPs作为对照组。结果发现，在transferrin-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。cRGD-CLPs组小鼠体重没有改变，但是肿瘤抑制明显弱于前者，肿瘤尺寸为前者的3倍，表明cRGD对卵巢癌没有明显的靶向性。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％。transferrin-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。
实施例三十五 载药靶向自交联聚合物囊泡GE11-CLPs和CLPs在荷SKOV3原位卵巢癌的小鼠中的抑瘤效果、体重变化和存活率
载DOX·HCl的自交联囊泡GE11-CLPs、无靶向CLPs、DOX-LPs以及PBS尾静脉注射到荷SKOV3原位卵巢癌的小鼠中。GE11-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％。GE11-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，键合靶向分子的载药自交联囊泡GE11-CLPs可有效抑制原位卵巢癌肿瘤的增长，没有毒副作 用，还可以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对荷卵巢癌的小鼠的影响，结果见表4。
表4载药自交联聚合物囊泡对卵巢癌的体内外抗肿瘤结果
Figure PCTCN2017075529-appb-000021

Claims (10)

  1. 一种卵巢癌特异靶向的生物可降解双亲性聚合物，其特征在于：所述卵巢癌特异靶向的生物可降解双亲性聚合物由含双硫碳酸酯单体的聚合物键合靶向分子制备得到；所述靶向分子为GE11多肽、叶酸FA、转铁蛋白transferrin或者Herceptin蛋白；所述含双硫碳酸酯单体的聚合物的化学结构式为式Ⅰ或者式Ⅱ中的一种：
    Figure PCTCN2017075529-appb-100001
    其中，R1选自以下基团中的一种：
    Figure PCTCN2017075529-appb-100002
    R2选自以下基团中的一种：
    Figure PCTCN2017075529-appb-100003
    其中，k为113～170，x为15～45，y为80～300，m为220～280。
  2. 根据权利要求1所述卵巢癌特异靶向的生物可降解双亲性聚合物，其特征在于：当含双硫碳酸酯单体的聚合物的化学结构式为式Ⅰ时，分子量为30～55kDa；当含双硫碳酸酯单体的聚合物的化学结构式为式Ⅱ时，分子量为60～95kDa。
  3. 一种聚合物囊泡，其特征在于，所述聚合物囊泡由以下聚合物制备得到：
    (1)由权利要求1所述卵巢癌特异靶向的生物可降解双亲性聚合物制备得到；
    (2)由权利要求1所述含双硫碳酸酯单体的聚合物制备得到；
    (3)由权利要求1所述卵巢癌特异靶向的生物可降解双亲性聚合物与含双硫碳酸酯单体的聚合物制备得到；
    (4)在权利要求1所述含双硫碳酸酯单体的聚合物制备的囊泡表面偶联靶向分子后得到，所述靶向分子为GE11多肽、叶酸、转铁蛋白或者Herceptin蛋白。
  4. 根据权利要求3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50～160纳米。
  5. 根据权利要求3所述聚合物囊泡，其特征在于：所述聚合物囊泡由权利要求1所述卵巢癌特异靶向的生物可降解双亲性聚合物与含双硫碳酸酯单体的聚合物制备得到；按质量百分数，所述卵巢癌特异靶向的生物可降解双亲性聚合物的用量为1～40wt.％。
  6. 权利要求3～5所述任意一种聚合物囊泡作为治疗卵巢癌药物的载体的应用。
  7. 根据权利要求6所述的应用，其特征在于：所述治疗卵巢癌药物为小分子抗癌药物。
  8. 根据权利要求7所述的应用，其特征在于：所述小分子抗癌药物为紫杉醇、多西紫杉醇、阿霉素、奥拉帕尼、吉非替尼、盐酸多柔比星、盐酸表阿霉素或者盐酸伊利替康。
  9. 权利要求1或者2所述的卵巢癌特异靶向的生物可降解双亲性聚合物在制备治疗卵巢癌的纳米药物中的应用。
  10. 权利要求3所述聚合物囊泡在制备治疗卵巢癌的纳米药物中的应用。
PCT/CN2017/075529 2016-03-04 2017-03-03 卵巢癌特异靶向的生物可降解双亲性聚合物、由其制备的聚合物囊泡及应用 WO2017148431A1 (zh)

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