WO2018001256A1 - 具有不对称膜结构的可逆交联聚合物囊泡、抗肿瘤药物及其制备方法 - Google Patents

具有不对称膜结构的可逆交联聚合物囊泡、抗肿瘤药物及其制备方法 Download PDF

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WO2018001256A1
WO2018001256A1 PCT/CN2017/090427 CN2017090427W WO2018001256A1 WO 2018001256 A1 WO2018001256 A1 WO 2018001256A1 CN 2017090427 W CN2017090427 W CN 2017090427W WO 2018001256 A1 WO2018001256 A1 WO 2018001256A1
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molecular weight
pei
peg
rccps
drug
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French (fr)
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孟凤华
邹艳
杨炜静
钟志远
方媛
孟浩
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苏州大学
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Priority claimed from CN201610501766.7A external-priority patent/CN106177975B/zh
Priority claimed from CN201610559279.6A external-priority patent/CN105997880B/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
    • C08G81/02—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers at least one of the polymers being obtained by reactions involving only carbon-to-carbon unsaturated bonds

Definitions

  • the invention belongs to the technical field of drugs, in particular to a reversible cross-linked biodegradable polymer vesicle having an asymmetric membrane structure, an antitumor drug and a preparation method thereof.
  • siRNAs are further developed into anti-drugs.
  • Oncology drugs still face enormous challenges; the large size of the protein enters the cell, and it is easily degraded by proteases in the body fluid, which greatly affects its anticancer effect.
  • some water-soluble small molecule anticancer drugs especially those with negative charge in physiological environment, are difficult to enter cells efficiently due to the negative charge of the cells themselves, resulting in low bioavailability of the drug and low anticancer effect.
  • the existing PEI vector has the defects of high gene compounding and transfection efficiency, but the cytotoxicity is small, but the cytotoxicity is small, but its gene complexation and transfection efficiency are poor.
  • the molecular chain of the polymer comprises a hydrophilic segment, a hydrophobic segment and a PEI which are sequentially connected a molecule;
  • the hydrophobic segment comprises a polycarbonate segment and/or a polyester segment;
  • the hydrophilic segment has a molecular weight of 3000-10000 Da; and the hydrophobic segment has a molecular weight of 2.3-8.4 times the molecular weight of the hydrophilic segment
  • the molecular weight of PEI is from 10% to 60% of the molecular weight of the hydrophilic segment.
  • the chemical structural formula of the polymer of the present invention is as follows:
  • R 1 is selected from one of the following groups:
  • R 2 is selected from one of the following groups:
  • the polyethyleneimine (PEI) of the present invention is branched and linear, and the chemical structural formula of the obtained polymer is one of the following structural formulas:
  • the molecular weight of PEG is 3000-10000 Da; the total molecular weight of PTMC or PDLLA is 2-6 times of the molecular weight of PEG; the total molecular weight of PDTC is 15%-40% of the total molecular weight of PTMC or PDLLA; the molecular weight of PEI is 10%-60% of the molecular weight of PEG.
  • the structure and molecular weight of the PEI are defined, and the toxicity is small as a carrier, and the PEG segment and the hydrophobic segment can be combined to form a good drug-encapsulating effect even when the siRNA content is as high as 80 wt.%, and other drug contents. Up to 30wt.%, the vesicle can still completely and tightly wrap the drug; at the same time, the polymer of the invention avoids the instability of the existing PEI combined with the drug by physical entanglement, positively and easily migrates with the cell.
  • the polymer vesicle is a reduction-sensitive reversible cross-linking, intracellular decrosslinkable biodegradable polymer vesicle having an asymmetric membrane structure;
  • the polymer is PEG-P (TMC-DTC)- PEI or PEG-P (DLLA-DTC)-PEI, ie the polymer consists of a hydrophilic segment of PEG, a hydrophobic segment and a PEI molecule, wherein the structure of the hydrophobic segment is:
  • R 2 When it is a PTMC segment; when R 2 is In the case of the PDLLA segment, the hydrophobic segment consists of PTMC-PDTC or PDLLA-PDTC.
  • the PEG molecular weight is 3000-8000 Da; the total molecular weight of PTMC or PDLLA is 2.5-5 times the molecular weight of PEG; the total molecular weight of PDTC is 18%-38% of the total molecular weight of PTMC or PDLLA; and the molecular weight of PEI is 15 of the molecular weight of PEG unit. %-50%.
  • the inner shell of the vesicle membrane is used for compounding drugs such as DNA and siRNA, and can escape the endosomes through the proton sponge effect;
  • vesicle membrane Reversibly cross-linked biodegradable and biocompatible PTMC or PDLLA, the side chain dithiolane is similar to the human body's natural antioxidant lipoic acid, providing reductive sensitive reversible cross-linking, not only supporting biopharmaceuticals in the blood
  • the long cycle in the medium also ensures rapid cross-linking in the cells, releasing the nucleic acid drug into the target cell.
  • the polymer vesicle designed by the invention has the outer surface of the vesicle membrane composed of non-adhesive polyethylene glycol (PEG), and the inner surface of the vesicle membrane is composed of lower molecular weight PEI for efficient loading.
  • PEG polyethylene glycol
  • Proteins include granzyme B, cytochrome C or apoptin, polypeptides and negatively charged small molecule drugs such as pemetrexed disodium, methotrexate disodium, etc.; crosslinked vesicles protect drugs Not degraded, leaking, and long-circulating in the body, the nano-size of the vesicle and the surface-specific tumor-targeting molecules enable the vesicle to deliver drugs into the tumor cells; the protein is easy to escape due to the proton sponge effect of PEI The endosomes prevent protein denaturation, and in the intracellular reducing environment, the vesicles are decrosslinked, and the drug is released into the cytoplasm to exert its function.
  • crosslinked vesicles protect drugs Not degraded, leaking, and long-circulating in the body, the nano-size of the vesicle and the surface-specific tumor-targeting molecules enable the vesicle to deliver drugs into the tumor cells; the protein is easy to escape due to the proton sponge effect
  • the invention also discloses a preparation method of the above reversible cross-linked biodegradable polymer vesicle having an asymmetric membrane structure, comprising the following steps:
  • Reversible cross-linked biodegradable polymer vesicles having an asymmetric membrane structure were prepared by a solvent replacement method using a polymer as a raw material.
  • it may be PEG-P(TMC-DTC)-PEI or PEG-P(DLLA-DTC)-PEI, PEG-P(TMC-DTC)-PEI and PEG-P(TMC-DTC) -PEI as raw material, PEG-P (DLLA-DTC)-PEI and targeted PEG-P (DLLA-DTC)-PEI as raw materials, PEG-P(TMC-DTC)-PEI and targeted PEG-P ( TMC-DTC) is a raw material, which is based on PEG-P (DLLA-DTC)-PEI and targeted PEG-P (TMC-DTC).
  • the outer shell is PEG-based, and the targeting molecule can bind to cancer cells with high specificity. Increase the targeting of the vector.
  • the targeting molecule can be the polypeptides cNGQ, cRGD and CC9 or folic acid or galactose.
  • TMC-DTC tumor active targeting molecule
  • TMC-DTC tumor active targeting molecule
  • cNGQ/RCCPs polymer asymmetric vesicles with asymmetric membrane structure
  • the present invention can be crosslinked at room temperature with or without a reducing agent such as dithiothreitol (DTT) and glutathione (GSH) to obtain a reversibly crosslinked biodegradable polymer vesicle having an asymmetric membrane structure.
  • a reducing agent such as dithiothreitol (DTT) and glutathione (GSH)
  • the invention further discloses an anti-tumor drug obtained by loading the drug with the reversible cross-linked biodegradable polymer vesicle having the asymmetric membrane structure, such as DNA or siRNA nucleic acid drug, protein and polypeptide drug or small molecule anticancer drug. .
  • the medicament of the present invention exhibits excellent efficacy and low toxicity in treating tumors in mice.
  • Anti-tumor drugs are prepared by solvent replacement using polymers and drugs as raw materials, such as PEG-P(TMC-DTC)-PEI and drugs, and PEG-P(DLLA-DTC)-PEI and drugs as raw materials.
  • PEG-P(TMC-DTC)-PEI targeted PEG-P(TMC-DTC)-PEI and drug as raw materials
  • PEG-P(DLLA-DTC)-PEI targeted PEG-P (DLLA-DTC )-PEI and drug as raw materials
  • PEG-P (TMC-DTC)-PEI targeted PEG-P (TMC-DTC) and drugs as raw materials
  • PEG-P (DLLA-DTC)-PEI, targeted PEG -P(TMC-DTC) and drugs are used as raw materials.
  • the invention also discloses the above-mentioned application of the reversible cross-linked biodegradable polymer vesicle having an asymmetric membrane structure as a drug carrier, for example, as a siRNA and a DNA carrier, preferably a polymer, the molecular weight of PEG is 4000-8000Da; PEI The molecular weight is 25%-50% of the molecular weight of PEG; as a small molecule drug carrier for protein drugs, polypeptide drugs and physiological environment, it is preferred that the molecular weight of PEG is 3000-8000Da; the molecular weight of PEI is 10%-50% of the molecular weight of PEG.
  • the invention also discloses the application of the above reversible cross-linked biodegradable polymer vesicle having an asymmetric membrane structure in preparing an antitumor drug.
  • the invention also discloses a polymer, the molecular chain of the polymer comprising a hydrophilic segment, a hydrophobic segment and a PEI molecule which are sequentially connected;
  • the hydrophobic segment comprises a polycarbonate segment and/or a polyester chain a segment;
  • the hydrophilic segment has a molecular weight of 3000-10000 Da;
  • the hydrophobic segment has a molecular weight of 2.3-8.4 times the molecular weight of the hydrophilic segment;
  • the PEI has a molecular weight of 10%-60% of the hydrophilic segment molecular weight;
  • the chemical structural formula of the polymer is as described above.
  • the present invention has the following advantages:
  • the present invention designs a crosslinked polymer vesicle having an asymmetric membrane structure for in vivo drug delivery; the inner shell is PEI for a composite drug; the vesicle membrane is reversibly cross-linked, biodegradable and biocompatible.
  • Good PTMC the side chain of dithiolane is similar to the human body's natural antioxidant lipoic acid, which provides reduction-sensitive reversible cross-linking, which not only supports the long-term circulation of nano-drugs in the blood, but also rapidly cross-links and releases in the cells.
  • the drug is in the target cell; the outer shell has a targeting molecule in the background of PEG, and can bind to the cancer cell with high specificity.
  • the present invention combines various functional advantages by compounding a functional drug with a crosslinked polymer vesicle having an asymmetric membrane structure, including simple manipulation of preparation, excellent biocompatibility, and excellent control of drugs. Release (low physiological leakage / rapid release in tumor cells), superior in vivo circulation stability, superior targeting to cancer cells, significant specific gene silencing, superior ability to inhibit tumor growth and metastasis Therefore, the vesicle system of the present invention is expected to be a nanosystem platform that combines the advantages of convenience, stability, and versatility, and is used for efficient and active targeted delivery of drugs to tumors including in situ tumors.
  • the inner surface of the biodegradable polymer vesicle of the invention consists of low molecular weight PEI for efficient loading of drugs, and the crosslinked vesicle membrane protects the drug from degradation and can circulate in the body for long periods of time, vesicles Nano-size and tumor-specific targeting enable vesicles to transport drugs into tumor cells efficiently. In the reducing environment of cells, vesicles are decrosslinked, and drug dissociation is released into the cytoplasm; the low molecular weight PEI defined here is toxic as a carrier.
  • the polymer of the invention avoids the instability caused by the complex formed by the existing PEI combined with the drug by electrostatic interaction.
  • the positive electricity is easy to combine with cells, and the migration is poor and the release efficiency is poor.
  • the polymer vesicle having an asymmetric membrane structure of the antitumor drug disclosed in the present invention is a crosslinked vesicle, and the PEI is combined with a hydrophilic segment and a hydrophobic segment, thereby having a stable structure and circulating well in the body, even if
  • the drug content is as high as 35 wt.% (the siRNA content is as high as 80 wt.%)
  • the vesicle can still completely and tightly wrap the drug, which proves that the antitumor drug of the present invention has excellent stability when it is incubated for 20 hours in the presence of 10 mM GSH. Because of the cross-linking and swelling of cross-linked vesicles, most of the drugs are released; it is a good drug controlled release carrier for tumor treatment.
  • Figure 3 is a gel electrophoresis pattern of DNA cross-linked vesicle DNA-RCCPs carrying in Example 7.
  • Example 5 is a flow cytometry in Example 8 for measuring the entry of siCy5-cNGQ/RCCPs into A549 cells;
  • Figure 6 is a confocal microscope image of siCy5-cNGQ/RCCPs entering A549 cells in Example 8;
  • Figure 7 is the result of luciferin gene silencing of A549-Luc by siGL3-cNGQ/RCCPs in Example IX (A) and the PLK1 gene silencing result of A549-Luc by siPLK1-cNGQ/RCCPs in Example IX (B);
  • Figure 8 is the toxicity of siGL3-cNGQ/RCCPs to A549-Luc cells in Example IX (A) and gene silencing of mice bearing A549-Luc orthotopic lung cancer (B);
  • Figure 9 is a quantitative gene silencing effect of siGL3-cNGQ/RCCPs on A549-Luc orthotopic lung cancer in Example 10 (A) and the drug of siCy5-cNGQ/RCCPs in A549-Luc orthotopic lung cancer mice in Example 10. Generational dynamics results (B);
  • Figure 10 is a near-infrared fluorescence imaging of siCy5-cNGQ/RCCPs in a sample of A549-Luc orthotopic lung cancer mice in Example 10;
  • Figure 11 is a diagram showing the ex vivo fluorescence imaging (A) of the main organs of siCy5-cNGQ/RCCPs in mice bearing A549-Luc orthotopic lung cancer in Example 11 and the quantitative biodistribution in vivo (B);
  • Figure 12 is a diagram showing the bioluminescence (A) and the main organ ex vivo fluorescence imaging (B) of mice treated with siPLK1-cNGQ/RCCPs against A549-Luc orthotopic lung cancer mice in Example 12;
  • Figure 13 is a treatment of mice bearing A549-Luc orthotopic lung cancer with siPLK1-cNGQ/RCCPs in Example 12.
  • A is fluorescence quantitative
  • B is a picture of lung in mice
  • C is a change in body weight
  • D is a survival curve
  • Figure 14 is a histological analysis of siPLK1-cNGQ/RCCPs in the treatment of mice bearing A549-Luc orthotopic lung cancer after treatment in Example 12;
  • Figure 15 is an in vitro release of MTX-CPP33-RCCPs in Example 13;
  • Figure 16 is the toxicity of CPP33-RCCPs to A549 cells in Example 14 (A) and the toxicity of CPP-MTX-RCCPs to A549 cells (B);
  • Figure 17 is the toxicity of CC 9 -PEM-RCCPs to H460 cells in Example 14 (A) and the toxicity of CPP-GrB-RCCPs to A549 cells (B);
  • Figure 18 is a diagram showing the biodistribution of CC 9 -RCCPs-Cy5 in mouse blood (A) and the CPP-MTX-RCCPs in Example 17 in subcutaneous A549 lung cancer mice (B);
  • Figure 19 is a view showing the biodistribution of CC 9 -PEM-RCCPs in subcutaneous H460 lung cancer mice in Example 18;
  • Figure 20 is a multi-dose treatment of MTX-CPP-RCCPs in subcutaneous A549 lung cancer mice in Example 19.
  • Figure 21 is a multi-dose treatment of PEM-CC 9 -RCCPs in subcutaneous H460 lung cancer mice in Example 20.
  • DCM methyl chloride
  • a ring-opening polymerization catalyst bis(bistrimethylsilyl)amine zinc (29 mg, 75 ⁇ mol) was quickly added.
  • the closed reactor was sealed and placed in a 40 ° C oil bath for 2 days under magnetic stirring. After the reaction was terminated by glacial acetic acid, the mixture was precipitated twice in iced diethyl ether, filtered, and dried under vacuum at room temperature to afford PEG5k-P (DTC 4.4k-TMC19.8k).
  • PEG5k-P (DTC4.4k-TMC19.8k) (0.4g, hydroxyl 0.013mmol) and NPC (40mg, 0.07mmol) were dissolved in dry DCM and reacted at 0 ° C for 24 hours, then precipitated in ice diethyl ether and filtered Vacuum drying gave PEG5k-P (DTC4.4k-TMC19.8k)-NPC.
  • Figure 1 shows the nuclear magnetic spectrum of PEG5k-P (DTC4.4k-TMC19.8k)-bPEI1.8k. Its 1 H NMR characterization shows that in addition to the PEG and P (DTC-TMC) peaks, the characteristic peak of PEI is ⁇ 2. 59-2.79, by integration, the molecular weight of the polymer is 5.0-(4.4-19.8)-1.8 kg/mol.
  • the number average molecular weight of the polymer was calculated to be 6.0-(4.8-19.2)-1.2 kg/mol by the integral ratio of the characteristic peak areas.
  • Mal-PEG6k-P (DTC3.2k-TMC15.4k) was reacted with primary amine of bPEI1.8k to prepare Mal-PEG6k-P (DTC3.2k-TMC15.4k)-bPEI1.8k; Yield: 90.2 %, 1 H NMR (400 MHz, CDCl 3 ): PEG: ⁇ 3.38, 3.65; TMC: ⁇ 4.24, 2.05; DTC: ⁇ 4.32, 3.02, PEI: ⁇ 2.56-2.98, and characteristic peaks of Mal; The first step of MeO-PEG-OH was changed to azide-functionalized Azide-PEG6.5k-OH, and ring-opening polymerization of LA and DTC gave Azide-PEG6.5k-P (DTC4.0k-DLLA15.3), and then The terminal hydroxyl group was activated
  • cNGQ-PEG7k-P (DTC2.8k-TMC14.2k) is similar to that of Example 1, and is also divided into two steps, replacing the initiator MeO-PEG-OH in the first step with N-hydroxysuccinimide.
  • Functionalized NHS-PEG-OH, ring-opening polymerization of TMC and DTC gave NHS-PEG7k-P (DTC4.8k-TMC19.2k); the latter (0.5 g, 0.017 mmol) was reacted with cNGQ (20 mg, 0.033 mmol).
  • the product cNGQ-PEG7k-P (DTC4.8k-TMC19.2k). Yield: 81.2%.
  • the target polymer cNGQ-PEG6k-P (DTC4.8k-TMC19.2k)-bPEI1.8k is obtained by reacting with a targeting molecule containing a free thiol group such as polypeptide cNGQ-SH or folic acid FA-SH at room temperature; Alkynyl-PEG5k -P(DTC5.8k-LA23k)-lPEI1.2k and the azide-functionalized polypeptide cNGQ-N3 or galactose Gal-N3, the target polymer Gal-PEG5k-P is obtained by azide-alkynyl click chemistry ( DTC5.8k-LA23k)-lPEI1.2k; alkynyl-functional
  • siRNA-RCCPs were prepared by solvent exchange and compounded with non-specific, control siRNA (siScramble).
  • 100 ⁇ L of PEG5k-P (DTC4.4k-TMC19.8k)-bPEI1.8k in DMSO (5.0mg/mL) was mixed with a predetermined amount of siRNA buffer solution (1mg/mL, 10mM HEPES, pH 7.4), and then slowly entered 900 ⁇ L of HEPES (10 mM, pH 6.8), left overnight at room temperature, dialyzed in HEPES, incubated at 25 ° C for 4 h, self-crosslinking to obtain siRNA-RCCPs.
  • Fig. 2 is a vesicle particle size distribution diagram and a TEM image obtained.
  • DTT can also be omitted, and self-crosslinking vesicles can be formed in the vesicle membrane to avoid interference of the crosslinking agent.
  • the vesicles are called Gal-RCCPs; the same method, the vesicles prepared by mixing CC9-PEG3k-P(DTC4k-TMC12k)-bPEI0.6k and PEG3k-P(DTC4k-TMC12k)-bPEI0.6k are called CC9-RCCPs; The manner in which targeted vesicles (mixed with untargeted triblock polymers) were prepared from Alkynyl-PEG5k-P (DTC 5.8k-LA23k) and Azide-PEG3k-P (DTC4k-TMC12k) was similar to the above examples.
  • Mal-PEG6k-P (DTC4.8k-TMC19.2k)-bPEI1.8k or AA-PEG6.5k-P (DTC4.6k-TMC18.6k)-bPEI1.8k and the corresponding inactive polymer PEG5k-P (DTC4.6k-TMC18.6k)-bPEI1.8k is dissolved in DMSO and then injected into HEPES solution to prepare cross-linked vesicles.
  • a target molecule containing free thiol group such as peptide cNGQ-SH or folic acid FA- SH or CPP33-SH, targeted by Michael addition reaction and vesicle bonding with active Mal or AA on the surface Polymer vesicles CPP33-RCCPs, FA-RCCP, etc.
  • Preparation of vesicles, peptides such as cNGQ by mixing Mal-PEG6k-P (DTC3.2k-TMC15.4k) and AA-PEG5k-P (DTC4.5k-TMC19.3k) and triblock polymers containing PEI
  • the manner in which the targeted vesicles are prepared is similar to the above examples.
  • 80% by weight of PEG5k-P (DTC4.4k-TMC19.8k)-bPEI1.8k and 20% of cNGQ-PEG6.5k-P (DTC4.6k-TMC18.6k)-bPEI1.8 were dissolved in DMSO ( In 5.0 mg/mL), it was first mixed with a predetermined amount of siRNA (siScramble) buffer solution (1 mg/mL, 10 mM HEPES, pH 7.4), and then slowly added to 900 ⁇ L of HEPES (10 mM, pH 6.8), and allowed to stand at room temperature overnight. Dialysis and incubation for 4 h in HEPES gave tumor-targeted, siRNA-crosslinked siRNA-cNGQ/RCCPs.
  • siRNA siRNA
  • siRNA was prepared by adding PEG5k-P (DTC4.4k-TMC19.8k)-bPEI1.8k and 20% cNGQ-PEG7k-P (DTC4.8k-TMC19.2k) in a weight ratio of 80% to siRNA prepared by solvent exchange. -cNGQ/RCCPs.
  • the DLS results of siRNA-cNGQ/RCCPs showed that the average particle size of cNGQ/RCCPs entrained with 10 wt.% siRNA was 109 nm and the particle size distribution was 0.13; TEM showed it to be a clear spherical hollow structure.
  • Table 3 shows the relationship between the particle size of siRNA-cNGQ/RCCPs and the siRNA content; as the siRNA content increased from 0 wt.% to 50 wt.%, the particle size of siRNA-cNGQ/RCCPs also increased from 109 to 175 nm.
  • Example 4 describes a manner in which a plurality of functional polymers are bonded to a polypeptide.
  • the target polypeptide is modified, for example, as follows, by ring-opening polymerization and The PEI reaction gave the polymer Mal-PEG6k-P (DTC4.8k-TMC19.2k)-bPEI1.8k, and the siRNA was combined with the above solvent exchange method to obtain Mal-siRNA-RCCPs, followed by CNGQ-SH Michael Plus at room temperature. Targeted cross-linked vesicles siScramble-cNGQ/RCCPs were obtained.
  • DNA-RCCPs can be prepared and complex encapsulated by solvent exchange to obtain DNA-RCCPs.
  • the DNA is pcDEF3-CD8IL-36 ⁇ (pIL-36 ⁇ ), pcDEF3-CD8IL-12 (pIL-12), calf thymus DNA, and the like.
  • the DLS results showed that the DNA-RCCPs particle size increased with the increase of the percentage of DNA. See Table 4, and the TEM confirmed the hollow structure. Gel electrophoresis showed that the cross-linked vesicles could effectively complex the DNA (pIL-12). image 3.
  • DNA-cNGQ-RCCPs 80 ⁇ L of PEG5k-P (DTC4.4k-TMC19.8k)-bPEI1.8k and 20 ⁇ L of cNGQ-PEG7k-P (DTC4.8k-TMC19.2k) in DMSO (5.0mg/mL) slowly into the predetermined amount of DNA buffer The solution was cross-linked to obtain DNA-cNGQ-RCCPs.
  • the agarose gel retention method showed that even when the DNA content was as high as 50 wt.%, DNA-cNGQ-RCCPs could completely and tightly encapsulate the DNA, proving DNA-cNGQ- RCCPs are excellent in stability.
  • Cy5-siRNA Cy5 is introduced at the 5' inverted strand end. See CN201610501766.7 for experiments: Figure 5 shows the results of flow analysis. Flow cytometry reveals that cNGQ functionalization significantly increases RCCPs endocytosis by A549 cells; Figure 6 shows CLSM maps, showing that Cy5-siRNA-cNGQ/RCCPs can effectively escape connotation/ Lysosomal. In addition, the fluorescence intensity of Cy5-siRNA-cNGQ/RCCPs incubated cells was significantly stronger than that of the non-targeted group Cy5-siRNA-RCCPs and free siRNA groups.
  • FIG. 7A is a graph showing the expression of luciferase gene. The results show that luciferase expression is significantly down-regulated by siGL3-cNGQ/RCCPs;
  • Figure 7B is a graph of targeting and sequence-specific gene silencing ability, siPLK1- When cNGQ/RCCPs were incubated for 48 h in A549 cells, the amount of PLK1 mRNA in siPLK1-cNGQ/RCCPs group was significantly lower than that of siPLK1-cNGQ/RCCPs and siScramble-cNGQ/RCCPs, demonstrating its targeting and sequence-specific gene silencing ability.
  • the cNGQ/RCCPs of the present invention contain siGL3 or siPLK1, and the vesicles can effectively encapsulate the siRNA in the cell culture environment, and the internal cells are effectively internalized by the receptor-mediated endocytosis, and the cytoplasmic reducing environment is escaped due to the PEI proton sponge effect. Rapid release of siRNA under such a high level of gene silencing ability.
  • Figure 8A is a graph showing the cytotoxicity results of the CCK-8 kit, indicating that both siGL3-cNGQ/RCCPs and siGL3-RCCPs are non-toxic, demonstrating the excellent biocompatibility of the vesicles of the present invention.
  • FIG. 8B is a picture showing the changes of lung fluorescence in nude mice bearing A549-Luc orthotopic lung cancer xenografts before and after administration of siGL3-cNGQ/RCCPs;
  • Fig. 9A is a bioluminescence map of lungs, after siGL3-cNGQ/RCCPs injection for 24 and 48 hours, The bioluminescence intensity of lungs decreased by 76% and 53%, respectively, which proved that siGL3-cNGQ/RCCPs induced the expression of luciferase gene in lung tissue.
  • changes in lung fluorescence intensity in the siScramble-cNGQ/RCCPs control group were not observed, confirming that only specific sequences could cause bioluminescence gene silencing.
  • PEG5k-P (DTC3k-TMC15k)-bPEI1.8k and cNGQ-PEG7k-P (DTC3k-TMC15k) polymers were obtained as siE6-cNGQ/RCCPs by 4:1 complex siE6 gene.
  • the results showed that siE6-cNGQ/RCCPs induced the E6 gene of Hela cells to be effectively down-regulated, while the siScramble-cNGQ/RCCPs as control group showed no significant change, confirming that only the specific sequence was confirmed.
  • the column can cause the bioluminescent gene to silence.
  • Figure 9B is a Cy5-siRNA map at different time points in plasma.
  • the results show that Cy5-siRNA-cNGQ/RCCPs and Cy5-siRNA-RCCPs demonstrate relatively long blood relative to free Cy5-siRNA. Cycle time, and longer than other cationic complex siRNA vectors reported in the literature, (the elimination half-lives of Cy5-siRNA-cNGQ/RCCPs and Cy5-siRNA-RCCPs were 1.35 and 1.21h, respectively, while the free Cy5-siRNA was 0.17h)
  • Figure 10 shows the Cy5-siRNA fluorescence map of the tumor site.
  • Figure 11A shows the fluorescence intensity of Cy5-siRNA in different parts. The results showed that the fluorescence intensity of Cy5-siRNA in the lungs of Cy5-siRNA-cNGQ/RCCPs group was significantly stronger than that of other major organs. In the targeted control group, Cy5-siRNA-RCCPs showed weak Cy5-siRNA fluorescence in the lungs and strong fluorescence in the liver and kidney.
  • Figure 11B is a graph of Cy5-siRNA enrichment rate at different sites. Fluorescence quantification showed that Cy5-siRNA-cNGQ/RCCPs were 4.02% in the lung and 3.5 times in the Cy5-siRNA-RCCPs (1.13% ID/g) group.
  • Figure 12A shows that siPLK1-cNGQ/RCCPs significantly inhibited tumor growth, and some tumor growth was inhibited by siPLK1-RCCPs;
  • Figure 12B is an isolated image of mouse organs, after 10 days of treatment with siPLK1-cNGQ/RCCPs, This group showed that the lung tissue biofluorescence was significantly lower than the other control groups.
  • images of siScramble-cNGQ/RCCPs and PBS mice showed extensive metastasis of liver tumors to the heart and liver, while other organs in the siPLK1-cNGQ/RCCPs.
  • FIG. 13 from left to The right is the A549-Luc content map of the lung, the mouse body weight and the survival rate map, showing the A549-Luc content in the lungs of each treatment group.
  • siPLK1-cNGQ/RCCPs demonstrated high tumor suppressive capacity, significantly stronger than the untargeted control group siPLK1-RCCPs;
  • Figure 14 is a histological analysis of H&E staining, showing lung tissue of siPLK1-cNGQ/RCCPs treated mice Similar to the appearance of the lungs of normal mice, there were still a large number of tumor cells in the lungs of other groups.
  • siPLK1-cNGQ/RCCPs induced apoptosis of large-area lung tumor cells compared with other groups, but had less damage to heart, liver and kidney. The results indicated that siPLK1-cNGQ/RCCPs mediate safe, efficient, targeted delivery of siRNA to the Dutch Lung cancer mice.
  • PEG5k-P (DTC4.4k-TMC19.8k)-bPEI1.8k and Mal-PEG6k-P (DTC4.8k-TMC19.2)-bPEI1.8k are packaged with different amounts of MTX.2Na (10-30wt%) to form Vesicles, and reacted with cRGD-SH to obtain drug-loaded targeted cross-linked vesicles MTX-cRGD-RCCPs, with a particle size of 80-120 nm, particle size distribution of 0.08-0.17, drug encapsulation efficiency of 70%-85%; by PEG7k- P(DTC4k-LA18)-lPEI3.5k loaded with different amounts of MTX.2Na (10%-30wt%), the particle size of the vesicles was 90-150nm, the particle size distribution was 0.12-0.19, and the wrapping efficiency of MTX.2Na was 70%. -85%.
  • the in vitro release test of MTX.2Na is shown in CN201610559279.6. The results are shown in Fig. 15. It can be seen that after the addition of simulated intracellular GSH, the drug release is significantly faster than the sample without GSH; the different ratios of PEM.Na (10%- 30% by weight of the crosslinked PEM-CC 9 -RCCPs vesicles have a particle size of 55-120 nm and a particle size distribution of 0.12-0.18.
  • the UV-spectrometer measures the encapsulation efficiency of PEM . Na from 65% to 80%.
  • the in vitro release test is as above. The relationship between cumulative release and time shows that after adding GSH in simulated cells, the drug release is significantly faster than that without GSH; indicating that the drug-loaded vesicles can be present in the presence of 10 mM GSH. Effective release of the drug.
  • PEG3k-P(DTC4k-TMC12k)-bPEI0.6k and CC9-PEG3k-P(DTC4k-TMC12k)-bPEI0.6k were packaged with different PEM.Na (10%-30wt%) and cross-linked to obtain drug-loaded target
  • the vesicles PEM-CC9-RCCPs have a particle size of 50-100 nm, a particle size distribution of 0.14-0.18, and a drug encapsulation efficiency of 55%-75%.
  • PEG5k-P (DTC4.4k-TMC19.8k)-bPEI1.8k and cRGD-PEG6k-P (DTC4.8k-TMC19.2k)-bPEI1.8k were mixed at a mass ratio of 4:1 to contain different concentrations of protein FITC- CC, preparation of FITC-CC-cRGD-RCCPs.
  • the crosslinked vesicles carrying different proportions of CC (1%-5wt%) have a particle size of 90-120 nm and a particle size distribution of 0.13-0.19.
  • the encapsulation efficiency of FITC-CC was determined by fluorescence spectrometry to be 95%-100%.
  • the drug-loaded targeted cross-linked vesicles have a particle size of 90-130 nm, a particle size distribution of 0.14-0.17, and a protein encapsulation efficiency close to 100%.
  • the drug-loaded vesicles obtained from PEG8k-P(DTC8k-LA30)-bPEI1.2k and Gal-PEG8k-P(DTC8k-LA30)-bPEI1.2k with different amounts of Caspase3 (1-5wt%) have a particle size of 110-150nm.
  • the particle size distribution is 0.14-0.17, and the wrapping efficiency is close to 100%.
  • PEG4k-P (DTC5.7k-LA18.8k)-PEI2.0k and cNGQ-PEG5k-P (DTC5.7k-LA18.8k) are loaded with different amounts of insulin (1-5wt%) to obtain drug-loaded cross-linked vesicles.
  • the particle size is 100-120 nm, the particle size distribution is 0.15-0.18, and the wrapping efficiency is close to 100%.
  • the dialysis bag MWCO of the FITC-CC in vitro release test was 300 kDa, and the concentration of the drug in the solution was measured using a fluorometer. The results showed that the cumulative release of FITC-CC was significantly faster than that of the sample without DTT after adding 10 mM DTT, indicating that the drug-loaded cross-linked vesicles could effectively release the drug in the presence of 10 mM DTT.
  • Example 14 MTT assay for cytotoxicity of polymer vesicles
  • the concentration was increased to 0.5 mg/mL, the survival rate of H460 was still higher than 90%; the MTX-CPP33-RCCPs were tested, and the results are shown in Fig. 16B.
  • the 20% CPP33-targeted cross-linked polymer vesicles containing MTX.2Na were applied to A549.
  • the semi-lethal concentration (IC 50 ) of the cells was 2.8 ⁇ g/mL, and the semi-lethal concentration of the non-targeted vesicles was about 9.8 ⁇ g/mL, which was 20 and 5 times smaller than that of the free drug.
  • the PEM-CC 9 -RCCPs were tested.
  • the semi-lethal concentration (IC 50 ) of the targeted cross-linked vesicles containing 20% CC9 carrying PEM to A549 cells was 3.6 ⁇ g/mL, and the semi-lethal concentration of non-targeted vesicles was 9.2 ⁇ g/mL.
  • free drug was 4.5 ⁇ g / mL
  • the test GrB-CPP-RCCPs the results can be seen from FIG. 17B, contained 20% of GrB-containing crosslinked polymer vesicle targeting CPP LC50 A549 cells (IC 50) 0.32 Gg/mL, far lower than the free drug, is 3 times smaller than the semi-lethal concentration of the non-targeted vesicles.
  • FITC-MTX-CPP-RCCPs see CN201610559279.6 for experiments; the results indicate that FITC-MTX-CPP-RCCPs can be more efficiently endocytosed into A549 cells and FITC- by mediating relative to untargeted FITC-MTX-RCCPs. MTX is rapidly released in cells, causing effective apoptosis.
  • CC9-RCCPs-Cy5 can be more efficiently endocytosed into H460 cells by mediating relative to RCCPs-Cy5.
  • the endocytosis test of HepG2 cells from FITC-CC-Gal-RCCPs showed that FITC-CC-Gal-RCCPs can be more efficiently endocytosed into HepG2 cells by FITC-CC-RCCPs, and FITC -CC is rapidly released in cells, causing effective apoptosis.
  • Cy5-labeled polymer PEG5.0k-P (DTC3.0k-co-TMC15.0k) was prepared by amidation reaction using Cy5-NHS and PEG5.0k-P (DTC3.0k-co-TMC15.0k)-PEI1.8k. )-PEI1.8k-Cy5 (1 Cy5/molecular chain).
  • the experiment is referred to CN201610559279.6, and the result is FIG. 18A.
  • vesicles formed from PEG8.0k-P (DTC9.0k-LA32.0k)-PEI3.2k-Cy5 and Gal-PEG8.5k-P (DTC9.2k-LA32.0k) have longer vesicles.
  • Cycle time targeted cross-linked polymer vesicles, non-targeted cross-linked polymer vesicles in mice have an elimination half-life of 8.16 and 8.5 hours, respectively; by PEG4.0k-P (DTC2.4k-TMC8.0k) -PEI0.6k-Cy5 and CC9-PEG5.0k-P (DTC2.6k-TMC8.2k) form vesicles with relatively long cycle times, targeting cross-linked polymer vesicles, non-targeted cross-linked polymers The elimination half-lives of vesicles in mice were 6.16 and 6.5 hours, respectively.
  • MTX.2Na amount in the tumor accumulation of MTX-CPP33-RCCPs, MTX-RCCPs and MTX.2Na for 8 hours is 5.4, 1.6 and 0.7 ID%/g, respectively
  • MTX- CPP33-RCCPs are 3.4 and 7.7 times higher than MTX-RCCPs and MTX.2Na, indicating that MTX-CPP33-RCCPs accumulate more in tumors through active targeting.
  • Example 19 Therapeutic effect and survival rate of MTX-CPP33-RCCPs in subcutaneous A549 lung cancer mice
  • the PEM-CC 9 -RCCPs treatment group survived after 60 days, the PEM-RCCPs group had all died at 42 days, and the Alimta group also died at 38 days.
  • the PBS group also died at 30 days. Therefore, the targeted cross-linked vesicle of the present invention can effectively inhibit the growth of the tumor after being loaded with the drug, has no toxic side effects on the mouse, and can prolong the survival time of the tumor-bearing mouse.
  • the vesicle of the invention can efficiently load nucleic acid drugs and other drugs, and can transport the cells into living tumor cells to induce apoptosis, and the preparation thereof has the advantages of simple manipulation, excellent biocompatibility and excellent control of drugs. Release, superior in vivo circulation stability, superior cancer cell targeting, significant specific gene silencing ability, etc. Therefore, it is expected to be a nanosystem platform that combines the advantages of simplicity, stability, and versatility for efficient and active targeted delivery of nucleic acids to tumors in situ.

Abstract

一种可逆交联生物可降解聚合物囊泡及其制备方法，所述囊泡由聚合物自组装后交联得到；所述聚合物的分子链包括依次连接的亲水链段、疏水链段以及聚乙烯亚胺分子；所述疏水链段包括聚碳酸酯链段和/或聚酯链段；所述亲水链段的分子量为3000-10000Da；疏水链段的分子量为亲水链段分子量的2.3-8.4倍；PEI的分子量为亲水链段分子量的10%-60%。

Description

具有不对称膜结构的可逆交联聚合物囊泡、抗肿瘤药物及其制备方法 技术领域
本发明属于药物载体技术，具体涉及一种具有不对称膜结构的可逆交联生物可降解聚合物囊泡、抗肿瘤药物及其制备方法。
背景技术
基因药物易被血清中的核酸酶降解，且进入细胞能力差因此很难细胞核进行基因转染，由于非特异性脱靶、免疫源性高、siRNA易于降解和细胞摄取低等问题，siRNAs进一步发展成为抗肿瘤药物仍然面临巨大挑战；蛋白质尺寸大进入细胞能力差，同时易被体液中的蛋白酶降解，大大影响了其发挥抗癌作用。另外，一些水溶性小分子抗癌药物尤其是在生理环境下带负电荷的药物由于细胞本身的负电荷难以高效进入细胞，导致药物的生物利用率低下，抗癌效果不高。因此设计能够保护药物不被降解且安全输送进入靶细胞核的载体十分重要。研究发现现有PEI载体存在基因复合和转染效率高但细胞毒性很大、细胞毒性虽小但其基因复合和转染效率差的缺陷。
技术问题
用含阳离子的脂质体和聚离子复合物等纳米载体来装载核酸的研究结果也并不令人满意，存在着体内不稳定、靶向性差、基因复合和转染效率不高、仍有细胞毒性的问题。目前尚无能同时解决这些问题的方案。
问题的解决方案
技术解决方案
本发明的目的是公开一种具有不对称膜结构的可逆交联的生物可降解聚合物囊泡及其制备方法。
为达到上述发明目的，本发明采用如下技术方案：
一种具有不对称膜结构的可逆交联生物可降解聚合物囊泡，由聚合物自组装后交联得到；所述聚合物的分子链包括依次连接的亲水链段、疏水链段以及PEI分子；所述疏水链段包括聚碳酸酯链段和/或聚酯链段；所述亲水链段的分子量为3000-10000Da；疏水链段的分子量为亲水链段分子量的2.3-8.4倍；PEI的分子量为亲水链段分子量的10％-60％。
优选的，本发明的聚合物化学结构式如下：
Figure PCTCN2017090427-appb-000001
其中，R1选自以下基团中的一种：
Figure PCTCN2017090427-appb-000002
R2选自以下基团中的一种：
Figure PCTCN2017090427-appb-000003
PEI的化学结构式如下：
Figure PCTCN2017090427-appb-000004
本发明的聚乙烯亚胺(PEI)为支化和线性两种，得到聚合物的化学结构式为以下结构式中的一种：
Figure PCTCN2017090427-appb-000005
所述聚合物中，PEG的分子量为3000-10000Da；PTMC或PDLLA的总分子量为PEG分子量的2-6倍；PDTC的总分子量为PTMC或PDLLA总分子量的15％-40％；PEI的分子量为PEG分子量的10％-60％。
本发明的聚合物中，限定PEI的结构与分子量，作为载体时毒性小，结合PEG链段与疏水链段，可以形成良好的药物包载效果，即使当siRNA含量高达80wt.％、其他药物含量高达30wt.％，该囊泡仍可以完全、紧实包裹药物；同时本发明的聚合物避免了现有PEI通过物理缠绕的方式结合药物带来的不稳定、带正电易与细胞结合而迁移力差、释放效率差的缺陷；通过静电作用力结合药物，再被交联的囊泡膜和外界分隔，避免在输送过程被细胞黏附而造成损失和毒副作用，能够高效迁移至病灶处，并在体内高浓度盐和还原剂GSH的作用下，快速释放药物，解决疾病问题。
本发明中，聚合物囊泡为具有不对称膜结构的还原敏感可逆交联、细胞内可解交联的生物可降解聚合物囊泡；所述聚合物为PEG-P(TMC-DTC)-PEI或者PEG-P(DLLA-DTC)-PEI，即聚合物由PEG亲水链段、疏水链段以及PEI分子组成，其中疏水链段的结构为：
Figure PCTCN2017090427-appb-000006
当R2为
Figure PCTCN2017090427-appb-000007
时，为PTMC链段；当R2为
Figure PCTCN2017090427-appb-000008
时，为PDLLA链段，即疏水链段由PTMC-PDTC或者PDLLA-PDTC组成。
优选方案为：PEG分子量为3000-8000Da；PTMC或PDLLA总分子量为PEG分子量的2.5-5倍；PDTC总分子量为PTMC或PDLLA总分子量的18％-38％；PEI的分子量为PEG单元分子量的15％-50％。
上述嵌段聚合物PEG-P(TMC-DTC)-PEI或PEG-P(DLLA-DTC)-PEI，其中中间嵌段的TMC或DLLA与DTC呈无规排列；PEI分子量小于PEG分子量，在自组装、交联后得到具有不对称膜结构的交联的聚合物囊泡，囊泡膜的内壳为PEI用于复合药物如DNA和siRNA，并能通过质子海绵效应逃逸内涵体；囊泡膜为可逆交联的生物可降解且生物相容性好的PTMC或者PDLLA，侧链的二硫戊环类似人体天然的抗氧化剂硫辛酸，可提供还原敏感的可逆交联，不但支持生物药物在血液中的长循环，还可保证在细胞内快速解交联，释放核酸药物到靶细胞细胞内。本发明设计的聚合物囊泡，其囊泡膜的外表面由具有不粘附性的聚乙二醇(PEG)组成，囊泡膜的内表面由较低分子量的PEI组成，用于高效装载蛋白质包括颗粒酶B、细胞色素C或者凋亡素、多肽和生理环境带负电荷的小分子药物如培美曲塞二钠、甲氨蝶呤二钠等；交联的囊泡膜可保护药物不被降解、不泄漏，并可在体内长循环，囊泡的纳米尺寸以及表面的肿瘤特异性靶向分子使得囊泡可定向输送药物进入肿瘤细胞；由于PEI的质子海绵效应而使蛋白质易于逃离内涵体防止了蛋白质变性，在细胞内还原环境下，囊泡解交联，药物被释放进入细胞质发挥其作用。
本发明还公开了上述具有不对称膜结构的可逆交联生物可降解聚合物囊泡的制备方法，包括以下步骤：
(1)将PEG-P(TMC-DTC)或PEG-P(DLLA-DTC)的末端用羟基活化剂比如氯甲酸对硝基苯酯NPC活化，再与PEI反应制得PEG-P(TMC-DTC)-PEI或PEG-P(DLLA-DTC)-PEI；
(2)在PEG-P(TMC-DTC)-PEI或PEG-P(DLLA-DTC)–PEI的PEG端偶联肿瘤特异性靶向分子，得到靶向PEG-P(TMC-DTC)-PEI或靶向PEG-P(DLLA-DTC)-PEI；
(3)以聚合物为原料，通过溶剂置换法制备具有不对称膜结构的可逆交联生物可降解聚合物囊泡。
具体可以为以PEG-P(TMC-DTC)-PEI或PEG-P(DLLA-DTC)-PEI为原料，以PEG-P(TMC-DTC)-PEI和靶向PEG-P(TMC-DTC)-PEI为原料，以PEG-P(DLLA-DTC)-PEI和靶向PEG-P(DLLA-DTC)-PEI为原料，以PEG-P(TMC-DTC)-PEI和靶向PEG-P(TMC-DTC)为原料，以PEG-P(DLLA-DTC)-PEI和靶向PEG-P(TMC-DTC)为原料，外壳为以PEG为背景、靶向分子对癌细胞可高特异性结合，增加载体的靶向性。靶向分子可为多肽cNGQ、cRGD及CC9或是叶酸、半乳糖。比如通过PEG-P(TMC-DTC)-PEI或PEG-P(DLLA-DTC)-PEI和偶联了肿瘤主动靶向分子的二嵌段聚合物如cNGQ-PEG-P(TMC-DTC)混合，共自组装、装载药物、交联后得到肿瘤主动靶向、具有不对称膜结构的聚合物囊泡(cNGQ/RCCPs)；所述cNGQ-PEG-P(TMC-DTC)的化学结构式为：
Figure PCTCN2017090427-appb-000009
本发明可在加或不加还原剂如二硫代苏糖醇(DTT)和谷胱甘肽(GSH)下室温交联得到具有不对称膜结构的可逆交联生物可降解聚合物囊泡。
本发明进一步公开了一种抗肿瘤药物，由上述具有不对称膜结构的可逆交联生物可降解聚合物囊泡装载药物得到，如DNA或siRNA核酸药物、蛋白质和多肽药物或者小分子抗癌药物。本发明的药物在小鼠体内治疗肿瘤表现了卓越的疗效和低毒性。以聚合物与药物为原料，通过溶剂置换法制备抗肿瘤药物，比如以PEG-P(TMC-DTC)-PEI与药物为原料，以PEG-P(DLLA-DTC)-PEI与药物为原料，以PEG-P(TMC-DTC)-PEI、靶向PEG-P(TMC-DTC)-PEI与药物为原料，以PEG-P(DLLA-DTC)-PEI、靶向PEG-P(DLLA-DTC)-PEI与药物为原料，以PEG-P(TMC-DTC)-PEI、靶向PEG-P(TMC-DTC)与药物为原料，以PEG-P(DLLA-DTC)-PEI、靶向PEG-P(TMC-DTC)与药物为原料。
本发明还公开了上述具有不对称膜结构的可逆交联生物可降解聚合物囊泡作为药物载体的应用，比如作为siRNA和DNA载体，优选聚合物中，PEG的分子量为4000-8000Da；PEI的分子量为PEG分子量的25％-50％；作为蛋白质药物、多肽药物和生理环境带负电的小分子药物载体，优选聚合物中，PEG的分子量为 3000-8000Da；PEI的分子量为PEG分子量的10％-50％。本发明还公开了上述具有不对称膜结构的可逆交联生物可降解聚合物囊泡在制备抗肿瘤药物中的应用。
本发明还公开了一种聚合物，所述聚合物的分子链包括依次连接的亲水链段、疏水链段以及PEI分子；所述疏水链段包括聚碳酸酯链段和/或聚酯链段；所述亲水链段的分子量为3000-10000Da；疏水链段的分子量为亲水链段分子量的2.3-8.4倍；PEI的分子量为亲水链段分子量的10％-60％；或者所述聚合物的化学结构式如上所述。
与现有技术相比，本发明具有如下优点：
1.本发明设计了具有不对称膜结构的交联聚合物囊泡用于药物的体内传递；其内壳为PEI用于复合药物；囊泡膜为可逆交联的生物可降解且生物相容性好的PTMC，侧链的二硫戊环类似人体天然抗氧化剂硫辛酸，可提供还原敏感的可逆交联，既支持纳米药物在血液中长循环，还可在细胞内快速解交联，释放药物到靶细胞细胞内；外壳以PEG为背景同时具有靶向分子，对癌细胞可高特异性结合。
2.本发明通过对具有不对称膜结构的交联聚合物囊泡来复合功能性药物，拥有多种独特优点，包括制备的简易操控性、杰出的生物相容性、对药物极好的控制释放性(生理条件泄漏量低/肿瘤细胞内快速释放)、超强的体内循环稳定性、对癌细胞的优越靶向性、显著的特异性基因沉默能、卓越的抑制肿瘤生长和转移的能力；因此，本发明的囊泡体系有望成为集便捷、稳定、多功能等优点于一身的纳米系统平台，用于高效、主动靶向输送药物至肿瘤包括原位肿瘤。
3.本发明的生物可降解聚合物囊泡内表面由低分子量的PEI组成，用于高效装载药物，交联的囊泡膜可保护药物不被降解，并可在体内长循环，囊泡的纳米尺寸以及肿瘤特异性靶向使得囊泡可输送药物高效进入肿瘤细胞，在细胞内的还原环境下，囊泡解交联，药物解离释放进入细胞质；这里限定的低分子量PEI作为载体时毒性小，在结合PEG链段与疏水链段后却可以形成良好的药物包载效果；同时本发明的聚合物避免了现有PEI通过静电相互作用结合药物形成的复合物带来的不稳定、带正电易与细胞结合而迁移力差、释放效率差的缺陷。
4.本发明公开的抗肿瘤药物的具有不对称膜结构的聚合物囊泡为交联囊泡，PEI配合亲水链段以及疏水链段，从而具有稳定的结构，在体内循环良好，能够即使当药物含量高达35wt.％(siRNA含量高达80wt.％)，该囊泡仍可完全、紧实包裹药物，证明本发明的抗肿瘤药物稳定性优异，当它在10mM GSH存在下孵育20h后发现，由于交联囊泡的解交联及溶胀大部分药物释放出来；是一种良好的药物控释载体，用于肿瘤治疗。
对附图的简要说明
图1为实施例一中PEG5k-P(DTC4.4k-TMC19.8k)-bPEI1.8k核磁谱图；
图2为实施例五中siScramble-cNGQ/RCCPs的粒径分布及TEM图；
图3为实施例七中载DNA交联囊泡DNA-RCCPs的凝胶电泳图
图4为实施例七中siScramble-cNGQ/RCCPs的凝胶电泳图；
图5为实施例八中流式细胞仪测定siCy5-cNGQ/RCCPs进入A549细胞；
图6为实施例八中siCy5-cNGQ/RCCPs进入A549细胞的共聚焦显微镜图；
图7为实施例九中siGL3-cNGQ/RCCPs对A549-Luc的荧光素基因沉默结果(A)以及实施例九中siPLK1-cNGQ/RCCPs对A549-Luc的PLK1基因沉默结果(B)；
图8为实施例九中siGL3-cNGQ/RCCPs对A549-Luc细胞的毒性(A)以及对荷A549-Luc原位肺癌小鼠的基因沉默(B)；
图9为实施例十中siGL3-cNGQ/RCCPs对A549-Luc原位肺癌的定量基因沉默效果(A)以及实施例十中siCy5-cNGQ/RCCPs在荷A549-Luc原位肺癌小鼠体内的药代动力学结果(B)；
图10为是实施例十中siCy5-cNGQ/RCCPs在荷A549-Luc原位肺癌小鼠体内的近红外荧光成像；
图11为是实施例十一中siCy5-cNGQ/RCCPs在荷A549-Luc原位肺癌小鼠的主要器官的离体荧光成像图(A)以及体内的定量生物分布(B)；
图12为实施例十二中siPLK1-cNGQ/RCCPs对荷A549-Luc原位肺癌小鼠的治疗期间小鼠的生物发光(A)以及主要器官离体荧光成像(B)；
图13为实施例十二中siPLK1-cNGQ/RCCPs对荷A549-Luc原位肺癌小鼠的治疗，A为荧光定量，B为小鼠肺部图片，C为体重变化，D为生存曲线；
图14为实施例十二中siPLK1-cNGQ/RCCPs对荷A549-Luc原位肺癌小鼠治疗后各主要器官的组织学分析；
图15为实施例十三中MTX-CPP33-RCCPs体外释放；
图16为实施例十四中CPP33-RCCPs对A549细胞的毒性(A)以及CPP-MTX-RCCPs对A549细胞的毒性(B)；
图17为实施例十四中CC9-PEM-RCCPs对H460细胞的毒性(A)以及CPP-GrB-RCCPs对A549细胞的毒性(B)；
图18为实施例十六中CC9-RCCPs-Cy5在小鼠血液中循环图(A)以及实施例十七中CPP-MTX-RCCPs在荷皮下A549肺癌小鼠体内的生物分布(B)；
图19为实施例十八中CC9-PEM-RCCPs对荷皮下H460肺癌小鼠体内的生物分布；
图20为实施例十九中MTX-CPP-RCCPs在荷皮下A549肺癌小鼠多剂量治疗；
图21为实施例二十中PEM-CC9-RCCPs在荷皮下H460肺癌小鼠多剂量治疗。
发明实施例
本发明的实施方式
实施例一 PEG5k-P(DTC4.4k-TMC19.8k)-bPEI1.8k嵌段共聚物的合成
在氮气手套箱内，依次称取MeO-PEG-OH(Mn＝5.0kg/mol,0.50g,100μmol), TMC(2.0g,19.2mmol)和DTC(0.50g,2.60mmol)并溶解在二氯甲烷(DCM，7.0mL)中，快速加入开环聚合催化剂双(双三甲基硅基)胺锌(29mg,75μmol)。密闭反应器密封好放置40℃油浴中磁力搅拌下反应2天。冰醋酸终止反应后在冰乙醚中沉淀两次、抽滤、常温真空干燥后得到PEG5k-P(DTC4.4k-TMC19.8k)。
PEG5k-P(DTC4.4k-TMC19.8k)(0.4g,羟基0.013mmol)和NPC(40mg,0.07mmol)溶于干燥的DCM中在0℃下反应24小时，然后在冰乙醚中沉淀、过滤、真空干燥得到PEG5k-P(DTC4.4k-TMC19.8k)-NPC。然后将产物溶于3mL DCM后滴加到3mL溶有bPEI(Mn＝1.8kg/mol)(180mg,0.10mmol)的DCM中，30℃下反应24小时后，在DCM和甲醇(体积比为1:1)中透析(MWCO 7000)48小时，接着在冰乙醚中沉淀两次、抽滤并室温真空干燥得到产物PEG5k-P(DTC4.4k-TMC19.8k)-bPEI1.8k。产率：91.6％。1H NMR(400MHz,CDCl3)：PEG:δ3.38,3.65；TMC:δ4.24,2.05；DTC:δ4.32,3.02,PEI:δ2.56-2.98。图1为PEG5k-P(DTC4.4k-TMC19.8k)-bPEI1.8k的核磁图谱，其1H NMR表征显示除了PEG及P(DTC-TMC)峰外，还有PEI的特征峰在δ2.59-2.79，通过积分可知，聚合物的分子量为5.0-(4.4-19.8)-1.8kg/mol。
Figure PCTCN2017090427-appb-000010
实施例二 Mal-PEG6k-P(DTC4.8k-TMC19.2k)-lPEI1.2k嵌段共聚物的合成
与实施例一类似，只是将其第一步中的MeO-PEG-OH换为马来酰亚胺官能化的Mal-PEG6k-OH，开环聚合TMC和DTC得到Mal-PEG6k-P(DTC4.8k-TMC19.2k)，其末端羟基用NPC活化，再与线性PEI(lPEI)的伯胺(Mn＝1.2kg/mol)反应制得，产率：93.2％。1H NMR(400MHz,CDCl3)：PEG:δ3.38,3.65；TMC:δ4.24,2.05；DTC:δ4.32,3.02,PEI:δ2.56-2.98。聚合物的数均分子量通过特征峰面积的积分比值，计算为6.0-(4.8-19.2)-1.2kg/mol。类似的，Mal-PEG6k-P(DTC3.2k-TMC15.4k)与bPEI1.8k的伯胺反应制得Mal-PEG6k-P(DTC3.2k-TMC15.4k)-bPEI1.8k；产率：90.2％，1H NMR(400MHz,CDCl3)：PEG:δ3.38,3.65；TMC:δ4.24,2.05；DTC:δ4.32,3.02，PEI:δ2.56-2.98,以及Mal的特征峰；将第一步的MeO-PEG-OH换为叠氮官能化的Azide-PEG6.5k-OH，开环聚合LA和DTC得到Azide-PEG6.5k-P(DTC4.0k-DLLA15.3)，然后其末端羟基用NPC活化，再与线性PEI(lPEI0.7k)的伯胺反应制得Azide-PEG6.5k-P(DTC4.0k-LA15.3)-lPEI0.7k；产率：90.2％。1H NMR(400MHz,CDCl3)：PEG:δ3.38,3.65； TMC:δ4.24,2.05；DTC:δ4.32,3.02，以及PEI的特征峰(δ2.56-2.98)。
实施例三 cNGQ-PEG7k-P(DTC4.8k-TMC19.2k)二嵌段共聚物的合成
cNGQ-PEG7k-P(DTC2.8k-TMC14.2k)的合成与实施例一类似，也是分为两步，将第一步中的引发剂MeO-PEG-OH换为N-羟基琥珀酰亚胺官能化的NHS-PEG-OH，开环聚合TMC和DTC得到NHS-PEG7k-P(DTC4.8k-TMC19.2k)；后者(0.5g,0.017mmol)与cNGQ(20mg,0.033mmol)反应得到产物cNGQ-PEG7k-P(DTC4.8k-TMC19.2k)。产率：81.2％。1H NMR(400MHz,DMSO-d6):PEG:δ3.51；TMC:δ4.23,1.94；DTC:δ4.13,2.99；cNGQ:δ6.84–7.61。BCA蛋白试剂盒测得cNGQ的接枝率为89.7％。通过调整原料比可得到不同分子量的聚合物(表1)。
表1 各个聚合物制备条件和产物的核磁表征确定的分子量结果
Figure PCTCN2017090427-appb-000011
实施例四 靶向聚合物的合成
聚合物的合成参考前文，Mal-PEG6k-P(DTC4.8k-TMC19.2k)-bPEI1.8k或者AA-PEG6.5k-P(DTC4.6k-TMC18.6k)-bPEI1.8k通过迈克尔加成与含有自由巯基的靶向分子如多肽cNGQ-SH或叶酸FA-SH，室温下反应后得到靶向聚合物cNGQ-PEG6k-P(DTC4.8k-TMC19.2k)-bPEI1.8k；Alkynyl-PEG5k-P(DTC5.8k-LA23k)-lPEI1.2k与叠氮功能化的多肽cNGQ-N3或半乳糖Gal-N3，通过叠氮-炔基的点击化学得到靶向聚合物Gal-PEG5k-P(DTC5.8k-LA23k)-lPEI1.2k；炔基功能化的alk-CC9或cRGD-alk和Azide-PEG3k-P(DTC4k-TMC12k)-bPEI0.6k通过叠氮-炔基的点击得到靶向聚合物CC9-PEG3k-P(DTC4k-TMC12k)-bPEI0.6k；当功能化聚合物为二嵌段聚合物、没有PEI的情况，其键合cNGQ等多肽的方式与上类似；多肽CPP33-SH与AA-PEG6.5k-P(DTC3.0k-co-TMC15.0k)通过迈克尔反应而得CPP-PEG6.5k-P(DTC3.0k-co-TMC15.0k)，产率：85.2％。BCA蛋白试剂盒测得 CPP33的接枝率为91.7％。通过调整原料比可得到不同分子量的聚合物，见表2。
表2 各个聚合物制备条件和产物的核磁表征结果
Figure PCTCN2017090427-appb-000012
实施例五 制备交联聚合物囊泡
siRNA-RCCPs通过溶剂交换法制备并复合包裹无特异性的、对照siRNA(siScramble)。100μL PEG5k-P(DTC4.4k-TMC19.8k)-bPEI1.8k的DMSO溶液(5.0mg/mL)与预定数量的siRNA缓冲溶液(1mg/mL,10mM HEPES,pH 7.4)混合，再缓慢打入900μL的HEPES(10mM,pH 6.8)，室温下放置过夜、在HEPES中透析、25℃孵育4h，自交联得到siRNA-RCCPs。DLS结果显示包裹10wt％siRNA时，粒径为100纳米左右，且TEM证实了其中空结构。不加药物由此得到可逆核交联的囊泡，简称为RCCPs。图2为得到的囊泡粒径分布图及TEM图。在制备该囊泡时也可不加DTT，囊泡膜内可形成自交联囊泡，避免交联剂的干扰。
混合两种聚合物Gal-PEG5k-P(DTC5.8k-LA23k)-lPEI1.2k和PEG5k-P(DTC5.8k-LA23k)-lPEI1.2k的DMSO溶液后，打入HEPES缓冲溶液中，得到的囊泡称为Gal-RCCPs；同法，由CC9-PEG3k-P(DTC4k-TMC12k)-bPEI0.6k和PEG3k-P(DTC4k-TMC12k)-bPEI0.6k混合制备的囊泡称为CC9-RCCPs；由Alkynyl-PEG5k-P(DTC5.8k-LA23k)和Azide-PEG3k-P(DTC4k-TMC12k)制备靶向囊泡(和无靶向的三嵌段聚合物混合)的方式与上述例子类似。
Mal-PEG6k-P(DTC4.8k-TMC19.2k)-bPEI1.8k或AA-PEG6.5k-P(DTC4.6k-TMC18.6k)-bPEI1.8k和相应的无活性端的聚合物PEG5k-P(DTC4.6k-TMC18.6k)-bPEI1.8k混合溶于DMSO中后，打入HEPES溶液中，制备得到交联囊泡，加入含有自由巯基的靶向分子如多肽cNGQ-SH或叶酸FA-SH或CPP33-SH，通过迈克尔加成反应和表面有活性Mal或AA的囊泡键合，得到靶向 聚合物囊泡CPP33-RCCPs、FA-RCCP等。Mal-PEG6k-P(DTC3.2k-TMC15.4k)和AA-PEG5k-P(DTC4.5k-TMC19.3k)和含有PEI的三嵌段聚合物混合制备囊泡、键合cNGQ等多肽的方式和制备靶向囊泡的方式与上述例子类似。
实施例六 载药交联囊泡的制备
重量含量为80％的PEG5k-P(DTC4.4k-TMC19.8k)-bPEI1.8k及20％的cNGQ-PEG6.5k-P(DTC4.6k-TMC18.6k)-bPEI1.8溶解于DMSO(5.0mg/mL)中，先与预定数量的siRNA(siScramble)缓冲溶液(1mg/mL,10mM HEPES,pH 7.4)混合，再缓慢加入到900μL的HEPES(10mM,pH 6.8)，室温放置过夜，在HEPES中透析、孵育4h，得到肿瘤靶向、装siRNA的交联siRNA-cNGQ/RCCPs。
加入重量比为80％的PEG5k-P(DTC4.4k-TMC19.8k)-bPEI1.8k及20％cNGQ-PEG7k-P(DTC4.8k-TMC19.2k)由溶剂交换法包载siRNA制备得到siRNA-cNGQ/RCCPs。siRNA-cNGQ/RCCPs的DLS结果显示包载10wt.％siRNA的cNGQ/RCCPs平均粒径为109nm，粒径分布为0.13；TEM显示它为清晰的球状中空结构。表3为siRNA-cNGQ/RCCPs的粒径与siRNA含量的关系；随着siRNA含量从0wt.％增到50wt.％，siRNA-cNGQ/RCCPs的粒径也由109增长到175nm。
表3 siRNA-cNGQ/RCCPs的粒径与siScramble含量的关系
Figure PCTCN2017090427-appb-000013
实施例四介绍了多种功能性聚合物键合多肽的方式，除此以外，还可以通过溶剂交换法制备纳米粒之后，再后修饰靶向多肽，具体举例如下，通过开环聚合并与支化PEI反应得到聚合物Mal-PEG6k-P(DTC4.8k-TMC19.2k)-bPEI1.8k，与上述的溶剂交换法复合siRNA得到Mal-siRNA-RCCPs，接着在室温下与cNGQ-SH迈克尔加成得到靶向交联囊泡siScramble-cNGQ/RCCPs。
实施例七 PEG5k-P(DTC3.0k-TMC15k)-bPEI1.8k制备DNA-RCCPs
参见CN201610501766.7d的实验：通过溶剂交换法可制备并复合包裹核酸DNA得到DNA-RCCPs。DNA为pcDEF3-CD8IL-36γ(pIL-36γ)，pcDEF3-CD8IL-12(pIL-12)、小牛胸腺DNA等。DLS结果显示，DNA-RCCPs粒径随着DNA百分比的增加而增大，见表4，且TEM证实了其中空结构，凝胶电泳显示交联囊泡可有效复合DNA(pIL-12)，见图3。
表4 DNA-cNGQ/RCCPs的粒径与DNA含量的关系
Figure PCTCN2017090427-appb-000014
80μL PEG5k-P(DTC4.4k-TMC19.8k)-bPEI1.8k与20μL cNGQ-PEG7k-P(DTC4.8k-TMC19.2k)的DMSO溶液(5.0mg/mL)缓慢打入预定数量的DNA缓冲溶液，按步骤交联得到DNA-cNGQ-RCCPs；琼脂糖凝胶阻留法表明，即使当DNA含量高达50wt.％，DNA-cNGQ-RCCPs仍可以完全、紧实包裹DNA，证明DNA-cNGQ-RCCPs稳定性优异。然而，当它在10mM GSH存在下孵育20h后发现，由于交联囊泡的解交联及溶胀大部分DNA释放出来。图4的琼脂糖凝胶阻留法表明，即使当siRNA含量高达80wt.％，cNGQ/RCCPs仍可以完全、紧实包裹siRNA，证明siRNA-cNGQ/RCCPs稳定性优异。然而，当它在10mM GSH存在下孵育20h后发现，由于交联囊泡的解交联及溶胀大部分siRNA释放出来。实施例八流式细胞仪及共聚焦显微镜(CLSM)实验
基因使用Cy5-siRNA(Cy5在5′反转链末端引入)。实验参见CN201610501766.7：图5为流式分析结果图，流式分析显露cNGQ功能化显著提高RCCPs被A549细胞内吞；图6为CLSM图，显示Cy5-siRNA-cNGQ/RCCPs能有效逃离内涵/溶酶体。此外，Cy5-siRNA-cNGQ/RCCPs孵育的细胞Cy5荧光强度明显强于无靶向组Cy5-siRNA-RCCPs及游离siRNA组。
实施例九 siGL3-cNGQ/RCCPs体外荧光素及细胞毒性实验
siRNA使用萤火虫荧光素酶报告基因siRNA(siGL3)。实验参见CN201610501766.7：图7A为荧光素酶基因表达结果图，结果显示荧光素酶表达被siGL3-cNGQ/RCCPs显著下调；图7B为靶向性及序列特异性基因沉默能力结果图，siPLK1-cNGQ/RCCPs在A549细胞中孵育48h，发现siPLK1-cNGQ/RCCPs组PLK1 mRNA量与siPLK1-cNGQ/RCCPs和siScramble-cNGQ/RCCPs相比显著降低，证明其靶向性及序列特异性基因沉默能力。本发明的cNGQ/RCCPs包载siGL3或siPLK1，囊泡在细胞培养基环境下能有效包裹siRNA，通过受体介导内吞方式有效细胞内在化，由于PEI质子海绵效应逃离内涵体，细胞质还原环境下快速释放siRNA，从而有如此高的基因沉默能力。另外，图8A为CCK-8试剂盒检测的细胞毒性结果图，表明siGL3-cNGQ/RCCPs和siGL3-RCCPs均无毒性，佐证了本发明的囊泡优异的生物相容性。图8B为荷A549-Luc原位肺癌异种移植裸鼠肺部荧光在siGL3-cNGQ/RCCPs给药前后的变化图片；图9A为肺部生物荧光图，注射siGL3-cNGQ/RCCPs 24及48h后，肺部生物荧光强度分别降低76％及53％，证明siGL3-cNGQ/RCCPs引诱肺组织荧光素酶基因有效表达。相反，没有观察到siScramble-cNGQ/RCCPs对照组小鼠肺部荧光强度的变化，证实了只有特异序列才能致使生物荧光基因沉默。PEG5k-P(DTC3k-TMC15k)-bPEI1.8k和cNGQ-PEG7k-P(DTC3k-TMC15k)聚合物按4:1复合siE6基因得到siE6-cNGQ/RCCPs。结果证明siE6-cNGQ/RCCPs诱导Hela细胞的E6基因有效下调，而作为对照组的siScramble-cNGQ/RCCPs则无明显变化，证实了只有特异序 列才能致使生物荧光基因沉默。
实施例十 Cy5-siRNA-cNGQ/RCCPs药代动力学及体内活体成像
实验参见CN201610501766.7：图9B为血浆中不同时间点的Cy5-siRNA图，结果表明，Cy5-siRNA-cNGQ/RCCPs和Cy5-siRNA-RCCPs证明相对于游离Cy5-siRNA，它们有相当长的血液循环时间，并且长于文献中报道的其他阳离子复合物siRNA载体，(Cy5-siRNA-cNGQ/RCCPs和Cy5-siRNA-RCCPs的消除半衰期分别为1.35和1.21h，而自由Cy5-siRNA则为0.17h)；图10为肿瘤部位Cy5-siRNA荧光图，图片显示Cy5-siRNA-cNGQ/RCCPs组小鼠在注射2h后，观察到肿瘤部位Cy5-siRNA荧光很强。相比之下，无靶向组Cy5-siRNA-RCCPs在肿瘤部位积累量显著减少，尽管它们含有相近的循环时间。这些结果表明主动靶向在肿瘤高富集及久持续上发挥着重要作用。
实施例十一 siPLK1-cNGQ/RCCPs离体成像及生物分布
实验参见CN201610501766.7：图11A为不同部位Cy5-siRNA荧光强度图，结果表明Cy5-siRNA-cNGQ/RCCPs组小鼠肺部Cy5-siRNA荧光强度明显强于其他主要器官，相比之下，无靶向对照组Cy5-siRNA-RCCPs肺部呈现出较弱的Cy5-siRNA荧光，而在肝及肾荧光很强。图11B为不同部位Cy5-siRNA富集率图，荧光定量表明Cy5-siRNA-cNGQ/RCCPs在肺部富集量为4.02％，为Cy5-siRNA-RCCPs(1.13％ID/g)组3.5倍。
实施例十二 荷A549-Luc原位肺癌裸鼠的治疗实验
实验参见CN201610501766.7：图12A表明siPLK1-cNGQ/RCCPs显著抑制肿瘤增长，siPLK1-RCCPs造成部分肿瘤增长被抑制；图12B为小鼠器官离体成像图，经siPLK1-cNGQ/RCCPs治疗10天后，此组别显示其肺组织生物荧光显著低于其他对照组。此外，siScramble-cNGQ/RCCPs和PBS组小鼠图像显示肝肿瘤大面积转移至心脏及肝脏，而siPLK1-cNGQ/RCCPs.组小鼠其他器官几乎没有荧光，证明肺癌没有转移；图13从左至右分别为肺部A549-Luc含量图、小鼠体重以及生存率图，显示各治疗组肺部A549-Luc含量。siPLK1-cNGQ/RCCPs展示出高效的肿瘤抑制能力，显著强于无靶向对照组siPLK1-RCCPs；图14为H&E染色的组织学分析图，表明，siPLK1-cNGQ/RCCPs治疗的小鼠的肺组织和外观和正常小鼠的肺部类似，其他组老鼠肺部仍有大量肿瘤细胞存在。siPLK1-cNGQ/RCCPs比其他组引发了大面积肺肿瘤细胞的凋亡，但对心、肝及肾伤害较小，结果指明siPLK1-cNGQ/RCCPs介导安全、高效、靶向传递siRNA至荷原位肺癌小鼠。
实施例十三 交联囊泡MTX-CPP33-RCCPs及体外释放
PEG5.0k-P(DTC3.0k-co-TMC15.0k)-PEI1.8k和CPP-PEG6.5k-P(DTC3.0k-co-TMC15.0k)(质量比4:1)包载甲氨蝶呤钠盐(MTX.2Na)，实验参见CN201610559279.6，结果显示载不同比例的MTX.2Na(10％-30wt％)的交联囊泡 的粒径在60-120nm，粒径分布0.12-0.19。紫外光谱仪测定MTX.2Na的包裹效率为60％-85％。得到的载药可逆核交联囊泡称为MTX-CPP33-RCCPs，表示载的药物为MTX.2Na，靶向分子为CPP33，其他命名以此类推。
PEG5k-P(DTC4.4k-TMC19.8k)-bPEI1.8k和Mal-PEG6k-P(DTC4.8k-TMC19.2)-bPEI1.8k包载不同量MTX.2Na(10-30wt％)后形成囊泡，并和cRGD-SH反应得到载药靶向交联囊泡MTX-cRGD-RCCPs，粒径80-120nm，粒径分布0.08-0.17，药物包裹效率为70％-85％；由PEG7k-P(DTC4k-LA18)-lPEI3.5k装载不同量MTX.2Na(10％-30wt％)后形成囊泡的粒径90-150nm，粒径分布0.12-0.19，MTX.2Na的包裹效率为70％-85％。
MTX.2Na的体外释放实验参见CN201610559279.6，结果为图15，可看出，加入模拟细胞内GSH后，药释明显快于没加GSH的样本；载不同比例的PEM.Na(10％-30wt％)的交联PEM-CC9-RCCPs囊泡的粒径在55-120nm，粒径分布0.12-0.18。紫外光谱仪测定PEM.Na的包裹效率为65％-80％。其体外释放实验如上，累积释放量与时间的关系可看出，加入模拟细胞内GSH后，药释明显快于没加GSH的样本；说明载药交联囊泡在10mM的GSH的存在下能有效释放药物。
由PEG3k-P(DTC4k-TMC12k)-bPEI0.6k和CC9-PEG3k-P(DTC4k-TMC12k)-bPEI0.6k包载不同PEM.Na(10％-30wt％)后交联得到载药靶向交联囊泡PEM-CC9-RCCPs，粒径50-100nm，粒径分布0.14-0.18，药物包裹效率为55％-75％。
按质量比4:1把PEG5k-P(DTC4.4k-TMC19.8k)-bPEI1.8k和cRGD-PEG6k-P(DTC4.8k-TMC19.2k)-bPEI1.8k混合包载不同浓度蛋白质FITC-CC，制备FITC-CC-cRGD-RCCPs。载不同比例CC(1％-5wt％)的交联囊泡粒径在90-120nm，粒径分布0.13-0.19。荧光光谱仪测定FITC-CC的包裹效率为95％-100％。
由Gal-PEG5k-P(DTC5.8k-LA23k)-lPEI1.2k和PEG5k-P(DTC5.8k-LA23k)-lPEI1.2制备的Gal-RCCPs在包载不同量凋亡蛋白apoptin(1-5wt％)得到载药靶向交联囊泡的粒径90-130nm，粒径分布0.14-0.17，蛋白质包裹效率接近100％。由PEG8k-P(DTC8k-LA30)-bPEI1.2k和Gal-PEG8k-P(DTC8k-LA30)-bPEI1.2k包载不同量Caspase3(1-5wt％)得到的载药囊泡粒径110-150nm，粒径分布0.14-0.17，包裹效率接近100％。PEG4k-P(DTC5.7k-LA18.8k)-PEI2.0k和cNGQ-PEG5k-P(DTC5.7k-LA18.8k)包载不同量胰岛素(1-5wt％)，得到载药交联囊泡粒径100-120nm，粒径分布0.15-0.18，包裹效率接近100％。
FITC-CC的体外释放实验的透析袋MWCO为300kDa，使用荧光仪测定溶液中药物浓度。结果表明，加入10mM DTT后，FITC-CC累积释放量明显快于没加DTT的样本，说明载药交联囊泡在10mM的DTT的存在下，能有效释放药物。
PEG5.0k-P(DTC3.0k-co-TMC15.0k)-PEI1.8k和CPP-PEG6.5k-P(DTC3.0k-co-TMC15.0k)按质量比4：1混合于DMSO溶液中，同上制备载GrB交联靶向囊泡 GrB-CPP-RCCPs。载不同比例GrB(1％-5wt％)的交联囊泡粒径在90-120nm，粒径分布在0.12-0.17，囊泡包裹效率接近100％。更换聚合物以及药物，可得到不同的载药聚合物囊泡的载药量、包封率，结果见表5。
表5 载药聚合物囊泡的载药量、包封率
Figure PCTCN2017090427-appb-000015
实施例十四 MTT法测试聚合物囊泡的细胞毒性
实验参见CN201610559279.6：MTT法测试CC9-RCCPs和RCCPs囊泡样品(实施例五)，结果显示，当交联囊泡的浓度从0.1增到0.5mg/mL时，H460的存活率仍高于90％，说明本发明的交联囊泡具有良好的生物相容性；测试CPP33-RCCPs和RCCPs囊泡样品(实施例五)，结果由图16A显示，当交联囊泡的浓度从0.1增到0.5mg/mL时，H460的存活率仍高于90％；测试MTX-CPP33-RCCPs，结果由图16B可知，载MTX.2Na的含20％CPP33靶向交联聚合物囊泡对A549细胞的半致死浓度(IC50)为2.8μg/mL，无靶向囊泡的半致死浓度约为9.8μg/mL，比自由药小20和5倍，测试PEM-CC9-RCCPs，结果由图17A可知，载PEM的含20％CC9的靶向交联囊泡对A549细胞的半致死浓度(IC50)为3.6μg/mL，无靶向囊泡的半致死浓度为9.2μg/mL，自由药为4.5μg/mL，测试GrB-CPP-RCCPs，由图17B结果可知，载GrB的含20％CPP靶向交联聚合物囊泡对A549细胞的半致死浓度(IC50)为0.32μg/mL，远低于自由药，比无靶向囊泡的半致死浓度小3倍，以上结合表6说明本发明的囊泡能很好的将药物传送到细胞内，并有效的释放，最终杀死癌细胞，而靶向纳米粒的效果要更好；其他聚合物囊泡的细胞毒性的测定与此类似，毒性均很小，具有良好的生物相容性。实施例十五靶向载药囊泡FITC-MTX-CPP-RCCPs的内吞和细胞内释放实验
靶向载药囊泡的内吞和细胞内释放实验以载FITC标记的MTX的囊泡 FITC-MTX-CPP-RCCPs为例，实验参见CN201610559279.6；结果表明FITC-MTX-CPP-RCCPs相对于无靶向FITC-MTX-RCCPs可通过介导作用更有效内吞进入A549细胞且FITC-MTX在细胞内可快速释放，引起有效细胞凋亡。同样地，CLSM跟踪CC9-RCCPs-Cy5在H460细胞的内吞实验表明，CC9-RCCPs-Cy5相对于RCCPs-Cy5可通过介导作用更有效内吞进入H460细胞。再如，FITC-CC-Gal-RCCPs的肝癌细胞HepG2的内吞实验结果表明，FITC-CC-Gal-RCCPs相对于FITC-CC-RCCPs可通过介导作用更有效内吞进入HepG2细胞，且FITC-CC在细胞内可快速释放，引起有效细胞凋亡。
实施例十六 RCCPs-Cy5和CC9-RCCPs-Cy5交联囊泡的血液循环
用Cy5-NHS和PEG5.0k-P(DTC3.0k-co-TMC15.0k)-PEI1.8k通过酰胺化反应制备Cy5标记的聚合物PEG5.0k-P(DTC3.0k-co-TMC15.0k)-PEI1.8k-Cy5(1个Cy5/分子链)。Cy5标记的囊泡CC9-RCCPs-Cy5由PEG5.0k-P(DTC3.0k-co-TMC15.0k)-PEI1.8k-Cy5、PEG5.0k-P(DTC3.0k-co-TMC15.0k)-PEI1.8k和CC9-PEG6.5k-P(DTC3.0k-co-TMC15.0k)按1:3:1混合而成，形成的聚合物囊泡粒径为100纳米，粒径分布为0.14。实验参见CN201610559279.6，结果为图18A，可知靶向交联聚合物囊泡、非靶向交联聚合物囊泡在小鼠体内的消除半衰期分别为7.46、7.5小时，所以本发明的交联聚合物囊泡在小鼠体内稳定，有较长循环时间。
按照上述方法，由PEG8.0k-P(DTC9.0k-LA32.0k)-PEI3.2k-Cy5和Gal-PEG8.5k-P(DTC9.2k-LA32.0k)形成的囊泡有较长的循环时间，靶向交联聚合物囊泡、非靶向交联聚合物囊泡在小鼠体内的消除半衰期分别为8.16和8.5小时；由PEG4.0k-P(DTC2.4k-TMC8.0k)-PEI0.6k-Cy5和CC9-PEG5.0k-P(DTC2.6k-TMC8.2k)形成的囊泡有相对长的循环时间，靶向交联聚合物囊泡、非靶向交联聚合物囊泡在小鼠体内的消除半衰期分别为6.16和6.5小时。
实施例十七 MTX-CPP33-RCCPs在荷A549肺癌小鼠的体内生物分布
实验参见CN201610559279.6，结果为图18B，可知MTX-CPP33-RCCPs、MTX-RCCPs和MTX.2Na注射8小时在肿瘤积累的MTX.2Na量分别为5.4、1.6和0.7ID％/g，MTX-CPP33-RCCPs是MTX-RCCPs和MTX.2Na的3.4和7.7倍，说明MTX-CPP33-RCCPs通过主动靶向在肿瘤积累较多。
实施例十八 PEM-CC9-RCCPs在荷H460肺癌小鼠的体内生物分布
实验参见CN201610559279.6，结果为图19，PEM-CC9-RCCPs、PEM-RCCPs和PEM注射8小时在肿瘤积累的PEM量分别为6.8、2.1和0.8ID％/g，PEM-CC9-RCCPs是PEM-RCCPs和PEM的3.2和8.5倍，说明PEM-CC9-RCCPs通过主动靶向在肿瘤积累较多，结果见表6。
实施例十九 MTX-CPP33-RCCPs在荷皮下A549肺癌小鼠中的治疗效果和存活率
实验参见CN201610559279.6，结果为图20，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，MTX-CPP33-RCCPs组治疗18天内，肿瘤得到明显抑制，而MTX-RCCPs组肿瘤有一定的增长。两组的小鼠体重几乎没有改变，说明载药交联囊泡对小鼠没有毒副作用。MTX-CPP33-RCCPs治疗组在70天后全部存活，Trexall组在48天时全死亡，PBS组在40天时也全部死亡。因此，本发明的靶向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。
实施例二十 PEM-CC9-RCCPs在荷皮下H460肺癌小鼠中的治疗效果和存活率
实验参见CN201610559279.6，结果为图21，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，PEM-CC9-RCCPs治疗组20天时，肿瘤得到明显抑制，而载药PEM-RCCPs组肿瘤有一定的增长。相比之下，PEM-CPP-RCCPs和PEM-RCCPs组的小鼠体重几乎没有改变，说明载药交联囊泡对小鼠没有毒副作用。PEM-CC9-RCCPs治疗组在60天后全部存活，PEM-RCCPs组在42天时已全部死亡，Alimta组在38天时也全部死亡PBS组在30天时也全部死亡。因此，本发明的靶向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。
表6 载药交联囊泡对肺癌的体内外抗肿瘤结果
Figure PCTCN2017090427-appb-000016
本发明的囊泡能高效装载保护核酸药物及其他药物，并能输送其到活体的肿瘤细胞内，诱导其凋亡，其制备简易操控性强、生物相容性杰出、对药物极好的控制释放性、超强的体内循环稳定性、优越的癌细胞靶向性、显著的特异性基因沉默能力等。因此，其有望成为集简易、稳定、多功能等优点于一身的纳米系统平台，用于高效、主动靶向输送核酸至原位肿瘤。

Claims (10)

  1. 一种具有不对称膜结构的可逆交联聚合物囊泡，由聚合物自组装后交联得到；所述聚合物的分子链包括依次连接的亲水链段、疏水链段以及PEI分子；所述疏水链段包括聚碳酸酯链段和/或聚酯链段；所述亲水链段的分子量为3000-10000Da；疏水链段的分子量为亲水链段分子量的2.3-8.4倍；PEI的分子量为亲水链段分子量的10％-60％。
  2. 根据权利要求1所述具有不对称膜结构的可逆交联聚合物囊泡，其特征在于，所述聚合物的化学结构式如下：
    Figure PCTCN2017090427-appb-100001
    其中，R1选自以下基团中的一种：
    Figure PCTCN2017090427-appb-100002
    R2选自以下基团中的一种：
    Figure PCTCN2017090427-appb-100003
    PEI的化学结构式如下结构式中的一种：
    Figure PCTCN2017090427-appb-100004
  3. 根据权利要求2所述具有不对称膜结构的可逆交联聚合物囊泡，其特征在于，PEG的分子量为4000-8000Da；PTMC或PDLLA的总分子量为PEG分子量的2.5-5倍；PDTC总分子量为PTMC或PDLLA总分子量的18％-38％；PEI的分子量为PEG分子量的15％-50％。
  4. 权利要求1-3所述任意一项具有不对称膜结构的可逆交联聚合物囊泡的制备方法，其特征在于，包括以下步骤：
    (1)将PEG-P(TMC-DTC)或者PEG-P(DLLA-DTC)的末端通过羟基活化剂活化，再与PEI反应制得PEG-P(TMC-DTC)-PEI或者PEG-P(DLLA-DTC)-PEI；
    (2)在PEG-P(TMC-DTC)-PEI或者PEG-P(DLLA-DTC)-PEI的PEG端偶联肿瘤特异性靶向分子，得到靶向PEG-P(TMC-DTC)-PEI或者靶向PEG-P(DLLA-DTC)-PEI；
    (3)以聚合物为原料，通过溶剂置换法制备具有不对称膜结构的可逆交联 生物可降解聚合物囊泡。
  5. 根据权利要求4所述具有不对称膜结构的可逆交联聚合物囊泡的制备方法，其特征在于，所述肿瘤特异性靶向分子为叶酸、半乳糖或者多肽。
  6. 一种抗肿瘤药物，由权利要求1-3所述任意一项具有不对称膜结构的可逆交联聚合物囊泡装载药物得到；所述药物为核酸药物、蛋白质药物、多肽药物或者小分子抗癌药物。
  7. 权利要求6所述抗肿瘤药物的制备方法，其特征在于，包括以下步骤，以聚合物与药物为原料，通过溶剂置换法制备抗肿瘤药物。
  8. 权利要求6所述抗肿瘤药物的制备方法，其特征在于，所述药物占原料的质量比为1％-35％。
  9. 权利要求1-3所述任意一项具有不对称膜结构的可逆交联聚合物囊泡在制备生物抗肿瘤药物中的应用或者作为核酸药物的载体的应用。
  10. 一种聚合物，其特征在于，所述聚合物的分子链包括依次连接的亲水链段、疏水链段以及PEI分子；所述疏水链段包括聚碳酸酯链段和/或聚酯链段；所述亲水链段的分子量为3000-10000Da；疏水链段的分子量为亲水链段分子量的2.3-8.4倍；PEI的分子量为亲水链段分子量的10％-60％。
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