WO2018010624A1 - 内膜具有正电的可逆交联生物可降解聚合物囊泡及其制备方法与在制备抗肿瘤药物中的应用 - Google Patents

内膜具有正电的可逆交联生物可降解聚合物囊泡及其制备方法与在制备抗肿瘤药物中的应用 Download PDF

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WO2018010624A1
WO2018010624A1 PCT/CN2017/092424 CN2017092424W WO2018010624A1 WO 2018010624 A1 WO2018010624 A1 WO 2018010624A1 CN 2017092424 W CN2017092424 W CN 2017092424W WO 2018010624 A1 WO2018010624 A1 WO 2018010624A1
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peg
dtc
cross
molecular weight
polymer
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French (fr)
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孟凤华
方媛
杨炜静
邹艳
钟志远
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苏州大学
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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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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

Definitions

  • the invention belongs to the technical field of medicines, and particularly relates to a reversible cross-linked biodegradable polymer vesicle with positive inner membrane and a preparation method and application thereof.
  • the existing vesicles have low loading efficiency for biomacromolecules and small-molecule anticancer drugs with negative electrophysiological environment, which greatly limits their application in these pharmaceutical preparations; genetic drugs are easily degraded by nucleases and have poor cell entry ability. Non-specific off-target and high immunogenicity have hindered their clinical application.
  • the virus is a nucleic acid drug carrier, although the transfection efficiency is high, the safety is worrying, and there is high immunogenicity and potential carcinogenicity. Therefore, non-viral gene carriers, especially cationic polymer gene carriers, have become the research hotspots.
  • the results of loading nucleic acids with nanocarriers such as cationic liposomes and polyion complexes are also unsatisfactory. , poor targeting, gene complexing and transfection efficiency, or high cytotoxicity. There is currently no solution to solve these problems at the same time.
  • the molecular chain of the polymer comprises a hydrophilic segment, a hydrophobic segment and a finely connected An amine molecule;
  • the hydrophobic segment comprises a polycarbonate segment and/or a polyester segment;
  • the hydrophilic segment has a molecular weight of 2000-8000 Da; and the hydrophobic segment has a molecular weight of 2.3-8.4 of the hydrophilic segment molecular weight. Times.
  • 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 molecular weight of PEG is 3000-10000 Da; the total molecular weight of PTMC or PLA is 2-6 times the molecular weight of PEG; the total molecular weight of PDTC is 15%-40% of the total molecular weight of PTMC or PLA.
  • spermine as a carrier is less toxic, and a PEG segment and a hydrophobic segment can be combined to form a good drug-encapsulating effect, and even when the siRNA content is as high as 80 wt.%, the vesicle can be completely and tightly.
  • the siRNA is encapsulated, and the protein such as cytochrome C can be efficiently loaded; at the same time, the polymer of the invention avoids the instability of the existing cationic polymer system by physically entanglement, and the positively charged and easily migrated with the cell.
  • the invention combines nucleic acid, protein or negatively charged small molecule drug by electrostatic force, and then the crosslinked vesicle membrane is separated from the outside to avoid loss caused by cell adhesion during transport. And toxic side effects, can be efficiently delivered to the lesion, and in the body of high concentration of salt and reducing agent GSH, rapid release of nucleic acid drugs.
  • the polymer vesicle is a bioredegradable polymer vesicle having a positive charge-reductive reduction-sensitive reversible cross-linking, intracellular cleavable cross-linking;
  • the polymer is PEG-P (TMC-DTC)- SP or PEG-P(LA-DTC)-SP, that is, the polymer consists of a hydrophilic segment of PEG, a hydrophobic segment, and a spermine 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 a PLA segment, the hydrophobic segment consists of P(TMC-co-DTC) or P(LA-co-DTC).
  • the PEG has a molecular weight of 5000-7500 Da; the total molecular weight of PTMC or PLA is 2.5-5 times that of PEG; and the total molecular weight of PDTC is 18% to 38% of the total molecular weight of PTMC or PLA.
  • the reversible cross-linked biodegradable polymer vesicles based on the positively charged spermine in the inner membrane designed by the invention can realize efficient loading of biomacromolecules and negatively charged small molecule anticancer drugs.
  • Spermine contains two amino groups and two imino groups, which are present in bacteria and most animal cells and are important substances that promote cell proliferation.
  • the molecular weight of the amine is 202Da, which is much smaller than the molecular weight of PEG.
  • cross-linked polymer vesicles with positive charge in the inner membrane are obtained.
  • the inner shell of the vesicle membrane is used for complex biomacromolecules such as protein. , DNA and siRNA and negatively charged small molecule drugs in physiological environment; vesicle membrane is reversible cross-linked biodegradable and biocompatible PTMC or PLA, side chain dithiolane is similar to human natural antioxidant sulfur Caprylic acid can provide reductive-sensitive reversible cross-linking, which not only supports the long circulation of biopharmaceuticals in the blood, but also ensures rapid cross-linking in cells and releases drugs into target cell cells.
  • the invention also discloses a preparation method of the positive inner reversible cross-linked biodegradable polymer vesicle of the inner membrane, comprising the following steps:
  • TMC-DTC TMC-DTC
  • LA-DTC PEG-P
  • PEG-P(TMC-DTC)-SP was used as raw material
  • PEG-P(LA-DTC)-SP was used as raw material
  • PEG-P(TMC-DTC)-SP and targeted PEG-P (TMC) -DTC)-SP as raw material
  • PEG-P(LA-DTC)-SP and targeted PEG-P(LA-DTC)-SP as raw materials
  • PEG-P(TMC-DTC)-SP and targeted PEG -P(TMC-DTC) is a raw material or PEG-P(LA-DTC)-SP and targeted PEG-P (TMC-DTC) are used as raw materials to prepare positively reversible cross-linked organisms by solvent replacement method.
  • Degradable polymer vesicles are used as raw materials to prepare positively reversible cross-linked organisms by solvent replacement method.
  • PEG-P(TMC-DTC)-SP and targeted PEG-P (TMC-DTC) are used as raw materials, or PEG-P(LA-DTC)-SP and targeted PEG-P (LA-DTC) are used as Raw material blending, self-assembly, cross-linking, polymerized vesicles with active tumor targeting, positive membrane in the inner membrane, PEG as the background, targeted molecules can bind to cancer cells with high specificity, and increase the targeting of the vector.
  • the targeting molecule can be the polypeptide DP8, cNGQ, cRGD, CC9, folate FA or galactose Gal.
  • the inner membrane has a positive charge of polymer vesicles (DP8-RCCPs).
  • the above preparation method specifically includes the following steps:
  • Step (1) is to dissolve PEG-P (TMC-DTC) or PEG-P (LA-DTC), a hydroxyl activator p-nitrophenyl chloroformate NPC in a dry solvent, then precipitate, filter, and vacuum dry Get Activated PEG-P(TMC-DTC)-NPC or PEG-P(LA-DTC)-NPC; add PEG-P(TMC-DTC)-NPC or PEG-P(LA-DTC)-NPC solution dropwise After the reaction in the spermine solution, dialysis, precipitation, suction filtration, vacuum drying to obtain PEG-P (TMC-DTC)-SP or PEG-P (LA-DTC)-SP; step (2) is to dissolve the polymer In the organic solvent with the targeting molecule such as DMSO or DMF; the step (3) is to add the raw material solution to the non-ion buffer solution such as HEPES, leave it at room temperature for a little, then dialyze the same buffer solution, incubate and
  • 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 positively charged reversible cross-linked biodegradable polymer vesicle having an inner membrane.
  • a reducing agent such as dithiothreitol (DTT) and glutathione (GSH)
  • PEG-P (TMC-DTC) and NPC were dissolved in dry dichloromethane (DCM) in an ice water bath for 12-24 hours, then precipitated in ice diethyl ether, filtered and dried in vacuo to give PEG-P (TMC-DTC) -NPC; then PEG-P(TMC-DTC)-NPC was dissolved in dry DCM, added dropwise to DCM of spermine at 30-40 ° C for 12-24 hours, in DCM and methanol (volume ratio 1: 1) Dialysis for 24-48 hours, followed by precipitation, suction filtration, and vacuum drying to obtain the product PEG-P(TMC-DTC)-SP; PEG-P(TMC-DTC)-SP and targeted PEG-P (TMC-DTC) The DMSO solution was mixed and added to HEPES buffer, left at room temperature overnight, dialyzed, and incubated for 4 h with or without a reducing agent such as dithiothreitol (DTT
  • the invention further discloses an anti-tumor drug obtained by loading a drug with a positively charged reversible cross-linked biodegradable polymer vesicle in the inner membrane; the drug is a small molecule drug negatively charged in a protein, a nucleic acid or a physiological environment, Such as pemetrexed, methotrexate.
  • the medicament of the invention adopts active targeting, reducing sensitive reversible cross-linking nano vesicles as a carrier, loading anti-tumor drugs, and exhibiting excellent curative effect and low toxicity in treating tumors in mice.
  • the anti-tumor drug is prepared by mixing a vesicle material solution with a drug or a non-ion buffer solution, placing it at room temperature, then dialysis, incubating and cross-linking to obtain an anti-tumor drug;
  • the vesicle material is the following group: PEG-P ( TMC-DTC)-SP, PEG-P(LA-DTC)-SP, PEG-P(TMC-DTC)-SP and targeted PEG-P(TMC-DTC)-SP, PEG-P(LA-DTC) -SP and targeted PEG-P (LA-DTC)-SP, PEG-P (TMC-DTC)-SP and targeted PEG-P (TMC-DTC), PEG-P (LA-DTC)-SP and target To PEG-P (TMC-DTC).
  • the polymer solution, the antitumor drug solution and the nonionic buffer solution are mixed together to obtain a drug-loaded inner membrane having positively charged reversibly crosslinked biodegradable polymer vesicles, preferably under polymerization conditions.
  • the solution is added to a HEPES buffer solution containing a protein, DNA, and a negatively charged small molecule drug in a physiological environment, or the polymer solution is mixed with the siRNA solution and then added to the HEPES.
  • the invention also discloses the application of the positive inner reversible cross-linked biodegradable polymer vesicle as an anti-tumor nano drug carrier, for example, as a protein, siRNA, DNA and pemetrexed, methotrexate and the like.
  • an anti-tumor nano drug carrier for example, as a protein, siRNA, DNA and pemetrexed, methotrexate and the like.
  • the invention also discloses the use of the positive inner membrane reversible cross-linked biodegradable polymer vesicles for preparing bio-antitumor drugs.
  • R 1 is selected from one of the following groups:
  • R 2 is selected from one of the following groups:
  • the molecular weight of PEG is 2000-8000 Da; the total molecular weight of PTMC or PLA is 2-6 times of PEG; the total molecular weight of PDTC is 15%-40% of the total molecular weight of PTMC or PLA.
  • the invention designs a crosslinked polymer vesicle with a positive charge of the inner membrane for in vivo delivery of the antitumor drug; firstly, the block polymer PEG-P(TMC-DTC)-SP is synthesized, and the inner vesicle membrane is refined.
  • Amines are used to efficiently load small molecules such as pemetrexed and methotrexate, which are negatively charged in proteins, DNA, siRNA and physiological environment; vesicle membranes are reversibly cross-linked biodegradable and biocompatible PTMC.
  • the side chain of dithiolane is similar to the human body's natural antioxidant, lipoic acid, which provides reduction-sensitive reversible cross-linking.
  • the outer shell has a targeting molecule in the background of PEG, which can bind to cancer cells with high specificity; the nano-size of the vesicle and the specific targeting of the tumor enable the vesicle to transport the nucleic acid efficiently into the tumor cells.
  • Cross-linked polymer vesicles with positively charged spermine can be used to load complex functional siRNA and DNA drugs with significant gene silencing effects in vitro and in vivo.
  • Spermine is a natural organism
  • the formed complex is unstable, positively charged, easily combined with cells, and has poor migration force and poor release efficiency.
  • the inner membrane has a positive charge, a reduction-sensitive reversible cross-linking, and an intracellular decrosslinkable vesicle carrier avoids the low bioavailability caused by the low loading efficiency, protein variability, or slow protein release of the existing protein nanocarriers.
  • the invention has the advantages of low efficacy; the polymer carrier of the invention avoids the lack of a suitable carrier for the negatively charged small molecule drug in the physiological environment, low loading efficiency, slow release, low bioavailability of the drug, low efficacy, etc. defect.
  • the vesicle system has a number of unique advantages, including easy handling, excellent biocompatibility, excellent controlled release of the drug, superior systemic circulation stability, superior targeting to cancer cells, and significant Specific gene silencing ability, excellent ability to inhibit tumor growth and metastasis. Therefore, the vesicle of the present invention is expected to be a nano-drug platform that is convenient, stable, and multifunctional, and is used for efficient and active targeted delivery of drugs to tumors.
  • Example 2 is a graph showing the stability, TEM, and reduction response of the targeted cross-linked vesicles in Example 8;
  • Figure 3 is a diagram showing the release of FITC-CC cross-linked vesicle FITC-CC-DP 8 /RCCPs in Example 11 and the toxicity of DP 8 /RCCPs on MCF-7 breast cancer cells in Example 17;
  • Figure 4 is a graph showing the cytotoxicity of GrB-DP 8 /RCCPs against MCF-7 and HepG2 in Example 18;
  • Figure 5 is a graph showing the pharmacokinetics of FITC-CC-DP 8 /RCCPs against MCF-7 and HepG2 in Example 19 and CC-Cy5-DP 8 /RCCPs in Example 20 in mice;
  • Example 6 is a biodistribution map of CC-Cy5-DP 8 /RCCPs to MCF-7 model in Example 20;
  • Example 7 is an image diagram of the MCF-7 model of CC-Cy5-DP 8 /RCCPs in Example 20;
  • Figure 8 is a graph showing the treatment of PMW subcutaneous lung cancer with PEM-CC9-RCCPs in Example 21.
  • the synthesis is divided into two steps.
  • the PEG5k-P (DTC4.6k-TMC13.5k) diblock copolymer is prepared by ring-opening polymerization.
  • PEG5k-P (DTC4.6k-TMC13.5k) (0.2g, hydroxyl group 0.0087mmol) and pyridine (3.5 ⁇ L) were dissolved in dry dichloromethane (DCM), and NPC (9.2mg, 0.046) was slowly added dropwise in an ice bath. A solution of mmol of DCM was reacted at room temperature for 12 hours, then twice in ice diethyl ether, filtered and dried in vacuo to give PEG5k-P (DTC 4.6k-TMC13.5k)-NPC.
  • DCM dry dichloromethane
  • 1A is a nuclear magnetic spectrum of PEG5k-P (DTC4.6k-TMC13.5k)-SP, and its 1 H NMR (400 MHz, DTCl 3 ) characterization shows that in addition to PEG and P (DTC-TMC) peaks (PEG: ⁇ 3. 38, 3.65; TMC: ⁇ 4.24, 2.05; DTC: ⁇ 4.32, 3.02), and the characteristic peaks of spermine are ⁇ 2.6-2.8 and 3.23.
  • Example 2 The synthesis of Mal-PEG6k-P(DTC4.8k-TMC15.2k)-SP was similar to that of Example 1.
  • the initiator MeO-PEG-OH in the first step was changed to Mal-PEG6k-OH to obtain Mal-PEG6k- P (DTC4.8k-TMC15.2k), then its terminal hydroxyl group was activated by NPC, and then reacted with primary amine of spermine; yield: 90.2%.
  • 1H NMR 400MHz, DTCl3): PEG: ⁇ 3.38, 3.65; TMC: ⁇ 4.24, 2.05; DTC: ⁇ 4.32, 3.02, and characteristic peaks of Mal and spermine.
  • the number average molecular weight of the polymer was calculated to be 6.0-(4.8-15.2)-0.2 kg/mol by the characteristic peak area integral ratio.
  • the synthesis was similar to that of Example 1.
  • the initiator MeO-PEG-OH in the first step of Example 1 was changed to NHS-PEG6.5k-OH, and ring-opening polymerization TMC and DTC were carried out to obtain NHS-PEG6.5k-P (DTC6k). -TMC15k);
  • the polypeptide DMAPPVLP (DP8) was added in a molar ratio of 3:1 to the amino group and NHS-PEG6.5k-P (DTC6k-TMC15k), and the amidation reaction was carried out at 30 degrees for 24-72 hours.
  • DP8 freeze-dried to obtain DP8-PEG6.5k-bP (DTC6k-TMC15k), and the grafting ratio of DP8 was calculated to be close to 100% by nuclear magnetic (Fig. 1B) and TNBSA.
  • the synthesis was similar to that of Example 1, replacing the initiator MeO-PEG-OH in the first step with N-hydroxysuccinimide-functionalized NHS-PEG-OH, ring-opening polymerization of TMC and DTC to obtain NHS-PEG7k-P. (DTC4.8k-TMC19.2k); Secondly, polypeptide C (NGQGEQ) (cNGQ) having a free primary amine and NHS-PEG7k-P (DTC4.8k-TMC19.2k) were bonded by an amidation reaction. Yield: 81.2%.
  • the synthesis was similar to that of Example 1.
  • the initiator of the first step, MeO-PEG-OH was changed to azide-functionalized Azide-PEG6.5k-OH, and the ring-opening polymerization of LA and DTC gave Azide-PEG6.5k-P ( DTC4.0k-LA15.3), then its terminal hydroxyl group was activated by NPC and then reacted with primary amine of spermine; yield: 90.2%.
  • 1 H NMR 400 MHz, DTCl 3 ): PEG: ⁇ 3.38, 3.65; TMC: ⁇ 4.24, 2.05; DTC: ⁇ 4.32, 3.02, and characterized.
  • the number average molecular weight of the polymer was calculated from the characteristic peak area integral ratio to be 6.5-(4.0-15.3)-0.2 kg/mol.
  • Polymers of different molecular weights can be obtained by adjusting the ratio of raw materials used, as shown in Table 1.
  • Polymers 1-15 were: PEG5k-P (DTC4.4k-TMC19.8k)-SP, Mal-PEG6k-P (DTC4.8k-TMC19.2)-SP, AA-PEG6.5k-P (DTC4. 6k-TMC18.6k)-SP, Alkynyl-PEG5k-P(DTC5.8k-TMC23k)-SP, Azide-PEG3k-P(DTC4k-TMC12k)-SP, NHS-PEG7k-P(DTC3k-TMC15k)-SP, PEG5k-P(DTC3k-TMC15k)-SP, Azide-PEG6.5k-P(DTC4.0k-LA15.3)-SP, PEG7k-P(DTC4k-LA18k)-SP, PEG3k-P(DTC0.9k-TMC6k )-SP, PEG8k-P(DTC8k-LA30k)-SP, NHS-PEG7.5
  • RCCPs reversible nuclear cross-linked vesicles
  • Alkynyl-PEG5k-OH functionalized by alkyne initiates ring-opening polymerization of DTC with LA, activation of terminal hydroxyl groups, and reaction with spermine to obtain Alkynyl-PEG5k-P (DTC5.8k-LA23k)-SP with a terminally active alkynyl group;
  • Azido-functionalized targeting molecules such as the polypeptide cNGQ-N3 or galactose Gal-N3, are targeted by the azide-alkynyl click chemistry to give the targeting polymer Gal-PEG5k-P(DTC5.8k-LA23k)-SP .
  • the target vesicles are then prepared in Gal-PEG5k-P (DTC5.8k-LA23k)-SP and PEG5k-P(DTC5.8k-LA23k)-SP was mixed into DMSO and then injected into HEPES solution to prepare Gal-RCCPs as in Example 6.
  • Azide-functionalized Azide-PEG3k-OH initiates ring-opening polymerization of DTC with TMC, activation of terminal hydroxyl groups, and reaction with spermine to obtain Azide-PEG3k-P (DTC4k-TMC12k)-SP with terminally active azide group.
  • the alkynyl-functionalized targeting molecule such as alk-CC9 or cRGD-alk, is targeted to the polymer CC9-PEG3k-P(DTC4k-TMC12k)-SP by azide-alkynyl click chemistry.
  • the target vesicles were prepared by mixing CC9-PEG3k-P(DTC4k-TMC12k)-SP and PEG3k-P(DTC4k-TMC12k)-SP in DMSO and then into HEPES solution, which was prepared in the same manner as in the sixth embodiment.
  • CC9-RCCPs were prepared by mixing CC9-PEG3k-P(DTC4k-TMC12k)-SP and PEG3k-P(DTC4k-TMC12k)-SP in DMSO and then into HEPES solution, which was prepared in the same manner as in the sixth embodiment. CC9-RCCPs.
  • the azide or alkynyl functionalized polymer is a diblock polymer without terminal spermine, namely Alkynyl-PEG5k-P (DTC5.8k-LA23k) and Azide-PEG3k-P (DTC4k-TMC12k), which are bonded
  • Alkynyl-PEG5k-P DTC5.8k-LA23k
  • Azide-PEG3k-P DTC4k-TMC12k
  • a target molecule containing a free thiol group such as the polypeptide cNGQ-SH or folic acid FA-SH or CPP33-SH is added to the vesicle solution, and the target is obtained by a Michael addition reaction and a vesicle bond with a surface active Mal or AA.
  • a target molecule containing a free thiol group such as the polypeptide cNGQ-SH or folic acid FA-SH or CPP33-SH
  • Maleimide Mal and acrylate functionalized block polymers are diblock polymers without terminal spermine, namely Mal-PEG6k-P (DTC3.2k-TMC15.4k) and AA-PEG5k-P ( DTC4.5k-TMC19.3k), which is prepared by mixing a triblock polymer having a spermine terminal at the end to prepare a vesicle, a polypeptide such as cNGQ, and a method of preparing a targeted vesicle is similar to the above example.
  • Targeted polymersomes are prepared in several ways. The first is based on triblock polymers and targeted diblock polymers. For example, DP 8 -PEG6.5k-P (DTC6k-TMC15k) and PEG5k-P (DTC4.6k-TMC13.5k)-SP prepared in Example 3 are mixed in a specific ratio, dissolved in DMSO, and added to HEPES (5 mM In the buffer of pH 6.8), the same example 6 was allowed to stand, cross-link, and dialyze to obtain targeted cross-linked vesicles, which were labeled as DP8-RCCPs.
  • DTC6k-TMC15k PEG5k-P (DTC4.6k-TMC13.5k)-SP prepared in Example 3 are mixed in a specific ratio, dissolved in DMSO, and added to HEPES (5 mM In the buffer of pH 6.8), the same example 6 was allowed to stand, cross-link, and dialyze to obtain targeted cross-linked vesicles, which
  • the content of the targeting polymer DP 8 -PEG6.5k-bP (DTC6k-TMC15k) is 5 to 40 wt.%.
  • the self-crosslinking polymer vesicles prepared by DLS assay have a size of about 80-125 nm and a particle size distribution of 0.11-0.16.
  • Figure 2 is a test (A) and electron transmission microscopy (B) of the self-crosslinking vesicle particle size distribution and stability, cross-linked vesicles and reduction responsiveness test (C); obtained self-crosslinking vesicles
  • the size of the nanoparticle vesicles measured by the dynamic light scattering particle size analyzer (DLS) is 80-125 nm, the particle size distribution is very narrow, and the particle size and particle size distribution remain unchanged after high-fold dilution (Fig. 2A);
  • the nanoparticles measured by TEM are hollow vesicle structures; however, they are rapidly released and decrosslinked in a simulated tumor cell reducing environment (Fig. 2C). It can be seen that the obtained vesicles can be self-crosslinked and have the property of reduction-sensitive decrosslinking.
  • Targeted polymer vesicles were prepared based on two triblock polymers. For example, two PEG5k-P (DTC4.6k-TMC13.5k)-SP and cRGD-PEG6.5k-P (DTC6k-TMC15k)-SP mixed in a specific ratio are dissolved in DMSO and added to HEPES (5 mM, pH). 6.8) In the buffer solution, the same as in Example 6, the DTT solution (final 0.1 m M) corresponding to a DTC molar amount of 10% to 30% was added, and dialyzed to obtain a targeted cross-linked vesicle, which was labeled as cRGD-RCCPs.
  • the obtained vesicles have an average particle diameter of about 50 to 125 nm and a particle size distribution of 0.04 to 0.17. It has the characteristics of vesicles, is stable in vitro, and has reducing sensitivity.
  • the resulting vesicles have an average particle size of 58-130 nm and a particle size distribution of 0.06-0.17.
  • the vesicles are stable in vitro and have a reducing sensitivity.
  • cross-linked vesicles carrying proteins such as cytochrome C, granzyme B and DNA was the same as in Example 7, except that different concentrations of protein or DNA were previously dissolved in the HEPES solution.
  • cross-linked polymer vesicles containing different ratios (2-30 wt.%) of FITC-labeled cytochrome C have a particle size of 90-108 nm and a particle size distribution of 0.13-0.19.
  • FITC-CC-DP8-RCCPs Its fluorescence spectrophotometer measures the encapsulation efficiency of FITC-CC from 50% to 100%.
  • In vitro release is performed by dialysis. Taking the in vitro release of FITC-CC as an example, it was carried out by shaking (200 rpm) in a 37 ° C constant temperature shaker, and each group had three parallel samples. In the first group, the cross-linked polymer vesicles carrying FITC-CC were added to the reducing environment PB (10 Mm, pH 7.4) of 10 mM DTT; the second group, the polymer vesicles carrying FITC-CC were in PB (10 Mm, Ph7.
  • the concentration of drug-loaded cross-linked vesicles is 50mg / L, take 0.5ml into a dialysis bag (MWCO: 350kDa), add 25ml of the corresponding dialysis medium to each test tube, take 5.0ml dialysis at the scheduled time
  • the outer bag medium was used as a test and 5.0 ml of fresh medium was added.
  • the concentration of the drug in the solution was measured by a fluorescence spectrometer.
  • Figure 3A shows the relationship between the cumulative release of FITC-CC and time. It can be seen that after the addition of simulated intracellular DTT, the release is significantly faster than the sample without DTT, indicating that the drug-loaded cross-linked vesicles are at 10 mM DTT. In the presence of it, it can effectively release the drug.
  • DNA-encapsulated DNA-RCCPs can be prepared by solvent exchange.
  • the DNA is pcDEF3-CD8IL-36 ⁇ (pIL-36 ⁇ ), pcDEF3-CD8IL-12 (pIL-12) or calf thymus DNA.
  • PEG5k-P (DTC3.0k-TMC15k)-SP to prepare DNA-RCCPs 100 ⁇ L PEG5k-P (DTC3.0k-TMC15k)-SP in DMSO solution (5.0mg/mL) slowly into 900 ⁇ L dissolved
  • a predetermined amount of DNA (1 mg/mL) in a mixed solution of HEPES (10 mM, pH 6.8) was allowed to stand overnight at room temperature, dialyzed, and a catalytic amount of reducing agent dithiothreitol (DTT) was added to crosslink to obtain DNA-RCCPs.
  • DTT reducing agent dithiothreitol
  • DNA/Polymer (wt.%) 0 10 20 30 40 50 Size a (nm) 96 118 127 141 167 173 PDI b 0.1 0.1 0.1 0.2 0.2 0.3
  • DNA-cNGQ/RCCPs were prepared as in Example 12.
  • PEG5k-P (DTC4.4k-TMC19.8k)-SP and cNGQ-PEG7k-P (DTC4.8k-TMC19.2k) were mixed at a weight ratio of 4:1.
  • the DNA used was pcDEF3-CD8IL-36 ⁇ (pIL-36 ⁇ ) and pcDEF3-CD8IL-12 (pIL-12).
  • 0.7 g of agarose was added to 70 mL of tetrabromoethane (TBE) buffer solution, and the agarose powder was dissolved by heating. After cooling, 1 ⁇ L of ethidium bromide was added to obtain agarose gel.
  • TBE tetrabromoethane
  • DNA-cNGQ-RCCPs or The weight percentage (wt.%) of DNA-RCCPs in DNA and polymer was set to 10%, 20%, 30%, 40%, and 50%, respectively.
  • 20 ⁇ L of DNA-cNGQ-RCCPs, DNA-RCCPs, and free DNA were added to the gel, and the gel was run in TBE running buffer (100 V, 30 min). Thereafter, gel images were photographed by Molecular Imager FX (Bio-Rad, Hercules, Ex/Em: 532/605 nm) and analyzed by Quantity One software (Bio-Rad).
  • siRNA-RCCPs crosslinked vesicles loaded with siRNA were prepared by solvent exchange.
  • a method of loading a non-specific control siRNA (siScramble) to obtain siRNA-RCCPs A predetermined number of siRNAs were mixed with HEPES buffer solution (1 mg/mL, 10 mM, pH 7.4) and 100 ⁇ L of PEG5k-P (DTC4.4k-TMC19.8k)-SP in DMSO (5.0 mg/mL), and then injected into 900 ⁇ L of HEPES. (10 mM, pH 6.8), left overnight at room temperature, dialyzed in HEPES, and incubated for 4 h to obtain siRNA-RCCPs.
  • HEPES buffer solution 1 mg/mL, 10 mM, pH 7.4
  • PEG5k-P (DTC4.4k-TMC19.8k)-SP in DMSO 5.0 mg/mL
  • the DLS results showed that when the 10 wt% siRNA was coated, the particle size was about 100 nm, and the TEM confirmed the 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 to 50 wt.%, the particle size of siRNA-cNGQ/RCCPs also increased from 109 to 175 nm. Subsequent cell experiments and in vivo tests were performed on samples with 10% by weight of siRNA, unless otherwise specified.
  • the vesicles loaded with fluorescently labeled Cy5-siRNA or luciferase gene marker siGL3 or therapeutic siPLK1 are similar.
  • the particle size is 90-180 nm.
  • siScramble-cNGQ/RCCPs were prepared as in Example 14.
  • the gel was prepared and run as in Example 13.
  • the weight percentage (wt.%) of siRNA and cNGQ/RCCPs or siRNA-RCCPs in siRNA and polymer was set to 10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%.
  • 20 ⁇ L of siRNA-cNGQ/RCCPs, siRNA-RCCPs, free siRNA, and siRNA-cNGQ/RCCPs after treatment with 10 mM GSH for 20 h were added to the gel.
  • the cross-linked vesicles loaded with a negatively charged small molecule drug such as pemetrexed in a physiological environment were prepared by a solvent exchange method, similar to the entrapment of the protein in Example 11.
  • 100 ⁇ L of polymer PEG5k ⁇ P (DTC4.4k ⁇ TMC19.8k)-SP in DMSO (5.0 mg/mL) was injected into 900 ⁇ L of HEPES buffer solution containing a predetermined amount of pemetrexed (1 mg/mL, 10 mM). , pH 6.6), placed at room temperature overnight, dialysis in HEPES, incubation for 4 h cross-linking to obtain PEM-RCCPs.
  • the crosslinked polymer vesicles carrying 20% by weight of pemetrexed had a particle size of 90 nm and a particle size distribution of 0.14.
  • the obtained vesicles were PEM-RCCPs. Its UV spectrophotometer measures the encapsulation efficiency of PEM to be 89%.
  • Example 17 MTT assay for toxicity of vesicles to breast cancer cell MCF-7
  • the MTT method used human breast cancer cells (MCF-7) to calculate cell viability.
  • MCF-7 human breast cancer cells
  • cross-linked vesicles having different targeting molecules on the surface were prepared.
  • the effect of FA system on ovarian cancer SKOV-3 cells and human oral epidermoid carcinoma cells KB cells was studied by the MTT method, and the cNGQ system was applied to lung cancer A549 cells.
  • the Gal system has cytotoxicity against hepatoma cells HepG2, and cRGD system on melanoma B16 cells, and the results also indicate that these empty cross-linking targeting vesicles have good biocompatibility.
  • Example 18 Toxicity of targeted cross-linked vesicles carrying GrB to breast cancer cell MCF-7 by MTT assay
  • the test object is the eleventh GrB-DP8-RCCPs.
  • the semi-lethal concentration of MCF-7 cells containing 30% DP 8 targeting crosslinked polymer vesicles carrying GrB (IC 50 ) ) is 0.188 ⁇ g / mL, which is much lower than the semi-lethal concentration of non-targeted vesicles, indicating that the vesicles of the present invention can deliver the drug well into the cells, and effectively release, eventually killing the cancer cells, and the target
  • the effect on the nanoparticles is better, and the cell survival rate of the capped cells is above 70%.
  • Cross-linked vesicles with different targeting molecules on the surface, pemetrexed, methotrexate, cytochrome C, or apoptin were prepared, and the FA system was used to study ovarian cancer SKOV-3 cells by MTT assay.
  • the cytotoxicity of hepatoma cells HepG2 and cRGD system on melanoma B16 cells all showed the specificity of targeting, and the drug-targeted polymer vesicles could enter cells faster and exert their effects.
  • FITC-CC-DP8-RCCPs the drug-loaded vesicle FITC-CC-DP8-RCCPs as an example
  • CLSM laser confocal microscope
  • the drug-loaded cross-linked vesicles with different targeting molecules were prepared, and the effect of FA system on ovarian cancer SKOV-3 cells and human oral epidermoid carcinoma cells KB cells was studied by CLSM.
  • the endocytosis and intracellular release behavior of hepatoma cells HepG2, and cRGD system on melanoma B16 cells showed that the targeted polymer vesicles could be swallowed faster and more efficiently by target cells and rapidly release the drug.
  • Example 20 Blood circulation of vesicles, distribution in vivo of MCF-7 mice, and in vivo imaging
  • Vesicle Cy5-CC-DP8-RCCPs (130 nm, particle size distribution 0.17) and non-targeted Cy5-CC-RCCPs were injected into mice (Cy5 concentration 4 ⁇ M) at 0, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours to take blood about 10 ⁇ L, accurately calculate the blood weight by differential method, add 100 ⁇ L, 1% concentration of Triton and 500 ⁇ L dimethyl sulfoxide extraction (which contains 20mM DTT) Then, the supernatant was taken by centrifugation (20,000 rpm, 20 minutes), and the amount of Cy5 at each time point was measured by a fluorescence spectrometer.
  • the cycle time of several representative samples in mice was studied.
  • the results showed that the drug-loaded PEG8k-P (DTC8k-LA30k)-SP cross-linked vesicles, PEG7k-P (DTC4k-LA18k)-SP cross-linking
  • the blood circulation time of the vesicles and PEG3k-P (DTC0.9k-TMC6k)-SP cross-linked vesicles in mice was 7.56 hours, 6.51 hours and 2.18 hours, respectively.
  • 1 ⁇ 10 7 MCF-7 cells were injected subcutaneously, and the experiment was started about 3 to 4 weeks after the tumor size was 100 to 200 mm 3 .
  • Cy5-CC-DP8-RCCPs free protein Cy5-CC and non-targeted Cy5-CC-RCCPs were injected into mice (Cy5-CC: 0.25 mg equiv./kg), and the mice were sacrificed 8 hours later.
  • Heart, liver, spleen, lung and kidney tissues were taken out, washed and added, and 500 ⁇ L of 1% Triton was added to a homogenizer for grinding, and then 900 ⁇ L of dimethyl sulfoxide (containing 20 mM of DTT) was added thereto. The supernatant was taken by centrifugation (20,000 rpm, 20 minutes), and the amount of Cy5-CC at each time point was measured by a fluorescence spectrometer.
  • Cy5-CC-RCCPs and Cy5-CC-DP8-RCCPs were injected into the tail vein mice, and the small animal live imager was used to track the vesicles at 4, 8, 12, and 24 hours.
  • Fig. 7 Cy5-CC-DP8-RCCPs accumulate rapidly at the tumor site, and the fluorescence is still strong after 24 hours.
  • Cy5-CC-DP8-RCCPs can actively target the breast cancer site and have strong specificity to breast cancer cells.
  • the cross-linked vesicles of Cy5-CC-RCCPs are metabolized quickly after entering the tumor. Low strength.
  • the animals were selected as in the twelfth embodiment, and 1 ⁇ 10 7 H460 human lung cancer cells were injected subcutaneously, and the experiment was started about 3 to 4 weeks after the tumor size was 100 to 200 mm 3 .
  • PEM-CC9-RCCPs, PEM-RCCPs, clinical injection Alimta, and PBS were injected into mice by tail vein at 0, 4, 8 and 12 days (PEM: 12.5 mg/kg; Alimta: 25 mg/kg).
  • PEM 12.5 mg/kg
  • Alimta 25 mg/kg
  • the body weight of the mice was weighed every two days, and the tumor volume was measured by a vernier caliper.
  • the width, H is the thickness of the tumor).
  • the survival of the mice was continuously observed to 60 days. From Fig. 8 (in which the tumor growth curve of each group during treatment, B is the tumor map of each group after treatment, C is the body weight change curve), it can be seen that the tumor was significantly inhibited in the PEM-CC 9 -RCCPs treatment group for 20 days. There was a certain increase in tumors in the PEM-RCCPs group. In contrast, mice in the PEM-CPP-RCCPs and PEM-RCCPs groups showed little change in body weight, indicating that the drug-loaded cross-linked vesicles had no toxic side effects on mice.
  • the PEM-CC 9 -RCCPs treatment group survived after 60 days, and all of the PEM-RCCPs, Alimta, and PBS groups died at 42 days, 38 days, and 30 days, respectively. 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.
  • a similar experimental method was used to study the cross-linked vesicles and targeted cross-linked vesicles of different drug-loaded drugs (apoptotic protein, plant toxin protein, methotrexate, therapeutic DNA and siRNA) in tumor-bearing mice. The results showed that all of them can effectively inhibit tumor growth, have no toxic side effects on mice, and can prolong the survival time of tumor-bearing mice.

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Abstract

内膜具有正电的可逆交联生物可降解聚合物囊泡及其制备方法与在制备抗肿瘤药物中的应用；基于嵌段聚合物PEG-P(TMC-DTC)-SP或者PEG-P(LA-DTC)-SP的肿瘤靶向、具有带正电荷内膜的、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，能高效装载保护生物大分子如蛋白质、DNA和siRNA和生理环境带负电的小分子药物，并能输送其到活体的肿瘤细胞内，诱导其凋亡。该体系拥有多种独特优点，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、超强的体内循环稳定性、优越的癌细胞靶向性、显著的癌细胞凋亡能力等。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。

Description

内膜具有正电的可逆交联生物可降解聚合物囊泡及其制备方法与在制备抗肿瘤药物中的应用 技术领域
本发明属于药物技术领域，具体涉及一种内膜具有正电的可逆交联生物可降解聚合物囊泡及其制备方法与应用。
背景技术
现有囊泡对生物大分子药物以及生理环境带负电的小分子抗癌药物的装载效率低，极大地限制了其在这些药物制剂上的应用；基因药物易被核酸酶降解，进入细胞能力差、非特异性脱靶、免疫源性高等都阻碍了其临床应用。以病毒为核酸药物的载体虽然转染效率很高，但安全性堪忧，存在高免疫原性和潜在的致癌性。因此非病毒基因载体尤其是阳离子聚合物基因载体成为研究的热点，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、或细胞毒性高的问题。目前尚无能同时解决这些问题的方案。
发明内容
为达到上述发明目的，本发明采用如下技术方案：
一种内膜具有正电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、疏水链段以及精胺分子；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2000-8000Da；疏水链段的分子量为亲水链段分子量的2.3-8.4倍。
优选的，本发明的聚合物化学结构式如下：
Figure PCTCN2017092424-appb-000001
其中，R1选自以下基团中的一种：
Figure PCTCN2017092424-appb-000002
R2选自以下基团中的一种：
Figure PCTCN2017092424-appb-000003
所述聚合物中，PEG的分子量为3000-10000Da；PTMC或PLA的总分子量为PEG分子量的2-6倍；PDTC的总分子量为PTMC或PLA总分子量的15％-40％。
本发明的聚合物中，精胺作为载体时毒性小，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.％，该囊泡仍可以完全、紧实包裹siRNA，并且能高效装载蛋白质如细胞色素C；同时本发明的聚合物避免了现有阳离子聚合物体系通过物理缠绕的方式结合核酸带来的不稳定、带正电易与细胞结合而迁移力差、释放效率差的缺陷；本发明通过静电作用力复合核酸、蛋白质或是带负电的小分子药物，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，能够高效送至病灶处，并在体内高浓度盐和还原剂GSH的作用下，快速释放核酸药物。
本发明中，聚合物囊泡为内膜具有正电荷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)-SP或者PEG-P(LA-DTC)-SP，即聚合物由PEG亲水链段、疏水链段以及精胺分子组成，其中疏水链段的结构为：
Figure PCTCN2017092424-appb-000004
当R2为
Figure PCTCN2017092424-appb-000005
时，为PTMC链段；当R2为
Figure PCTCN2017092424-appb-000006
时，为PLA链段，即疏水链段由P(TMC-co-DTC)或者P(LA-co-DTC)组成。
优选的，PEG分子量为5000-7500Da；PTMC或PLA的总分子量为PEG的2.5-5倍；PDTC的总分子量为PTMC或PLA总分子量的18％-38％。
本发明设计的基于内膜具有正电荷的精胺的、可逆交联的生物可降解聚合物囊泡，能够实现对生物大分子药物和带负电的小分子抗癌药物的高效装载。精胺含有两个氨基和两个亚氨基，存在于细菌和大多数动物细胞中，是促进细胞增殖的重要物质。上述三嵌段聚合物PEG-P(TMC-DTC)-SP或者PEG-P(LA-DTC)-SP，其中中间嵌段的TMC或者LA与DTC呈无规排列；精 胺的分子量为202Da，远小于PEG分子量，在自组装、交联后得到内膜具有正电荷的交联的聚合物囊泡，囊泡膜的内壳为精胺用于复合生物大分子如蛋白质、DNA和siRNA和生理环境带负电的小分子药物；囊泡膜为可逆交联的生物可降解且生物相容性好的PTMC或者PLA，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，释放药物到靶细胞细胞内。
本发明还公开了上述内膜具有正电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)或者PEG-P(LA-DTC)的末端用羟基活化剂比如氯甲酸对硝基苯酯NPC活化，再与精胺反应制得PEG-P(TMC-DTC)-SP或者PEG-P(LA-DTC)-SP；
(2)在PEG-P(TMC-DTC)-SP或者PEG-P(LA-DTC)–SP的PEG端偶联肿瘤特异性靶向分子，得到靶向PEG-P(TMC-DTC)-SP或者靶向PEG-P(LA-DTC)-SP；
(3)以PEG-P(TMC-DTC)-SP为原料、以PEG-P(LA-DTC)-SP为原料、以PEG-P(TMC-DTC)-SP和靶向PEG-P(TMC-DTC)-SP为原料、以PEG-P(LA-DTC)-SP和靶向PEG-P(LA-DTC)-SP为原料、以PEG-P(TMC-DTC)-SP和靶向PEG-P(TMC-DTC)为原料或者以PEG-P(LA-DTC)-SP和靶向PEG-P(TMC-DTC)为原料，通过溶剂置换法制备内膜具有正电的可逆交联生物可降解聚合物囊泡。
优选以PEG-P(TMC-DTC)-SP和靶向PEG-P(TMC-DTC)为原料，或者以PEG-P(LA-DTC)-SP和靶向PEG-P(LA-DTC)为原料共混，自组装、交联得到肿瘤主动靶向、内膜具有正电荷的聚合物囊泡，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。靶向分子可以为多肽DP8、cNGQ、cRGD、CC9、叶酸FA或半乳糖Gal。比如通过PEG-P(TMC-DTC)-SP或者PEG-P(LA-DTC)-SP和偶联了肿瘤主动靶向分子的二嵌段聚合物如DP8-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、内膜具有正电荷的聚合物囊泡(DP8-RCCPs)。
上述制备方法，具体包括以下步骤：
步骤(1)为将PEG-P(TMC-DTC)或者PEG-P(LA-DTC)、羟基活化剂氯甲酸对硝基苯酯NPC溶于干燥的溶剂中反应，然后沉淀、过滤、真空干燥得到 活化的PEG-P(TMC-DTC)-NPC或者PEG-P(LA-DTC)-NPC；将PEG-P(TMC-DTC)-NPC或者PEG-P(LA-DTC)-NPC溶液滴加到精胺溶液中反应后，透析、沉淀、抽滤、真空干燥得到PEG-P(TMC-DTC)-SP或者PEG-P(LA-DTC)-SP；步骤(2)为将得到聚合物溶于带有靶向分子的有机溶剂如DMSO或DMF中；步骤(3)为将原料溶液中加入非离子缓冲溶液中如HEPES，室温放置少许后对相同缓冲溶液透析，孵育交联，得到内膜具有正电的可逆交联生物可降解聚合物囊泡。本发明可以在加或不加还原剂如二硫代苏糖醇(DTT)和谷胱甘肽(GSH)下室温交联得到内膜具有正电的可逆交联生物可降解聚合物囊泡。
比如：
PEG-P(TMC-DTC)和NPC溶于干燥的二氯甲烷(DCM)中冰水浴下反应12-24小时，然后在冰乙醚中沉淀、过滤、真空干燥得到PEG-P(TMC-DTC)-NPC；然后将PEG-P(TMC-DTC)-NPC溶于干燥DCM，滴加到精胺的DCM中30-40℃下反应12-24小时后，在DCM和甲醇(体积比为1:1)中透析24-48小时，接着沉淀、抽滤、真空干燥得到产物PEG-P(TMC-DTC)-SP；PEG-P(TMC-DTC)-SP和靶向PEG-P(TMC-DTC)的DMSO溶液混合后加入HEPES缓冲液中，室温下放置过夜、透析、在加或不加还原剂如二硫代苏糖醇(DTT)或谷胱甘肽(GSH)时孵育4h囊泡交联，得到内膜具有正电荷的交联聚合物囊泡。
本发明进一步公开了一种抗肿瘤药物，由上述内膜具有正电的可逆交联生物可降解聚合物囊泡装载药物得到；所述药物为蛋白质、核酸或生理环境带负电的小分子药物，如培美曲塞、甲氨蝶呤。本发明的药物以主动靶向、还原敏感可逆交联的纳米囊泡为载体、装载抗肿瘤药物，在小鼠体内治疗肿瘤表现了卓越的疗效和低毒性。
上述抗肿瘤药物的制备方法为将囊泡原料溶液与药物、非离子缓冲液混合，室温放置，然后透析、孵育交联得到抗肿瘤药物；所述囊泡原料为以下一组：PEG-P(TMC-DTC)-SP、PEG-P(LA-DTC)-SP、PEG-P(TMC-DTC)-SP和靶向PEG-P(TMC-DTC)-SP、PEG-P(LA-DTC)-SP和靶向PEG-P(LA-DTC)-SP、PEG-P(TMC-DTC)-SP和靶向PEG-P(TMC-DTC)、PEG-P(LA-DTC)-SP和靶向PEG-P(TMC-DTC)。
其中，聚合物溶液、抗肿瘤药物溶液和非离子缓冲溶液三者共同混合得到包载药物的内膜具有正电的可逆交联生物可降解聚合物囊泡，优选条件为聚合 物溶液加入至含蛋白质、DNA和生理环境带负电的小分子药物的HEPES缓冲溶液中，或聚合物溶液与siRNA溶液混合后再加入HEPES中。
本发明还公开了上述内膜具有正电的可逆交联生物可降解聚合物囊泡作为抗肿瘤纳米药物载体的应用，比如作为蛋白质、siRNA、DNA和培美曲塞、甲氨蝶呤等生理环境带负电的小分子药物的载体的应用。
本发明还公开了上述内膜具有正电的可逆交联生物可降解聚合物囊泡在制备生物抗肿瘤药物中的应用。
本发明还公开了一种聚合物，其特征在于，所述聚合物的化学结构式如下：
Figure PCTCN2017092424-appb-000007
其中，R1选自以下基团中的一种：
Figure PCTCN2017092424-appb-000008
R2选自以下基团中的一种：
Figure PCTCN2017092424-appb-000009
聚合物中，PEG的分子量为2000-8000Da；PTMC或PLA的总分子量为PEG的2-6倍；PDTC的总分子量为PTMC或PLA总分子量的15％-40％。
本发明设计了内膜具有正电荷的交联聚合物囊泡用于抗肿瘤药物的体内传递；首先合成了嵌段聚合物PEG-P(TMC-DTC)-SP，囊泡膜内壳的精胺用于高效装载蛋白质、DNA、siRNA和生理环境带负电的小分子药物如培美曲塞、甲氨蝶呤；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，不但支持纳米药物在血液中长循环，还可保证在细胞内快速解交联，释放核酸到靶细胞细胞内；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；囊泡的纳米尺寸以及肿瘤特异性靶向使得囊泡可输送核酸高效进入肿瘤细胞。通过对内部具有带正电的精胺的交联聚合物囊泡能用来装载复合功能性siRNA和DNA药物、其体内外具有显著的基因沉默效果。精胺是生物体天然 存在的多胺，无毒，在结合PEG链段与疏水链段后可形成囊泡结构，具有良好的药物包载效果；聚合物载体避免了现有非病毒阳离子聚合物通过静电相互作用结合核酸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。内膜具有正电荷的、还原敏感可逆交联、细胞内可解交联囊泡载体避免了现有蛋白质纳米载体装载效率低、蛋白质易变性、或是蛋白质释放慢等导致的生物利用率低、药效低的缺陷；本发明的聚合物载体避免了现有在生理环境下带负电荷的小分子药物缺乏合适的载体、装载效率低、释放慢、药物的生物利用率低、药效低等缺陷。该囊泡体系拥有多种独特的优点，包括制备易操控、杰出的生物相容性、对药物极好的控制释放性、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，本发明的囊泡有望成为集便捷、稳定、多功能等于一身的纳米药物平台，用于高效、主动靶向输送药物至肿瘤。
附图说明
图1为实施例一中PEG5k-P(DTC4.6k-TMC13.5k)-SP、实施例三中DP8-PEG6.5k-P(DTC6k-TMC15k)核磁谱图；
图2为实施例八中靶向交联囊泡的稳定性、TEM、还原响应图；
图3为实施例十一中载FITC-CC交联囊泡FITC-CC-DP8/RCCPs的释放图、实施例十七中DP8/RCCPs对MCF-7乳腺癌癌细胞的毒性图；
图4为实施例十八中GrB-DP8/RCCPs对MCF-7、HepG2的细胞毒性图；
图5为实施例十九中FITC-CC-DP8/RCCPs对MCF-7、HepG2的CLSM图、实施例二十中CC-Cy5-DP8/RCCPs在小鼠体内的药力动力学曲线图；
图6为实施例二十中CC-Cy5-DP8/RCCPs对MCF-7模型的生物分布图；
图7为实施例二十中CC-Cy5-DP8/RCCPs对MCF-7模型的成像图；
图8为实施例二十一中PEM-CC9-RCCPs对荷H460皮下肺癌治疗实验图。
具体实施方式
实施例一 合成聚合物PEG5k-P(DTC4.6k-TMC13.5k)-SP
合成分为两步，首先是开环聚合制备PEG5k-P(DTC4.6k-TMC13.5k)二嵌段共聚物，具体操作如下，在氮气环境下，依次称取MeO-PEG-OH(Mn＝5.0kg/mol,0.20g,40μmol),TMC(0.6g,5.9mmol)和DTC(0.192g,1.0mmol)并溶解在二氯甲烷(DCM，6.8mL)中，快速加入开环聚合催化剂，如双(双三甲基硅基)胺锌(7.7mg,20μmol)。密闭反应器密封好放置40℃油浴中磁力搅拌下反应24小时。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产物。产率：90.3％。1H NMR(400MHz,DTCl3)：PEG:δ3.38,3.65；TMC:δ4.24,2.05；DTC:δ4.32,3.02，经核磁计算可知聚合物各段的分子量为5.0-(4.6-13.5)kg/mol。GPC测得分子量分布：1.39。
Figure PCTCN2017092424-appb-000010
PEG5k-P(DTC4.6k-TMC13.5k)(0.2g,羟基0.0087mmol)和吡啶(3.5μL)溶于干燥的二氯甲烷(DCM)中，冰浴下缓慢滴加NPC(9.2mg,0.046mmol)的DCM溶液，后室温反应12小时，然后在冰乙醚中沉淀两次、过滤、真空干燥得到PEG5k-P(DTC4.6k-TMC13.5k)-NPC。接下来，将产物溶于3mL DCM后滴加到2mL溶有精胺(SP，34.7mg,0.172mmol，Mn＝202.34kg/mol)的DCM中，25℃下反应24小时后在DCM和甲醇(体积比为1:1)中透析(MWCO 3500)48小时，浓缩、冰乙醚中沉淀两次、抽滤并室温真空干燥得到产物PEG5k-P(DTC4.6k-TMC13.5k)-SP。产率：87.3％。附图1A为PEG5k-P(DTC4.6k-TMC13.5k)-SP的核磁图谱，其1H NMR(400MHz,DTCl3)表征显示除了PEG及P(DTC-TMC)峰外(PEG:δ3.38,3.65；TMC:δ4.24,2.05；DTC:δ4.32,3.02)，还有精胺的特征峰在δ2.6-2.8和3.23。
Figure PCTCN2017092424-appb-000011
实施例二 合成嵌段共聚物Mal-PEG6k-P(DTC4.8k-TMC15.2k)-SP
Mal-PEG6k-P(DTC4.8k-TMC15.2k)-SP的合成与实施例一类似，将第一步中的引发剂MeO-PEG-OH换为Mal-PEG6k-OH，得到Mal-PEG6k-P(DTC4.8k-TMC15.2k)，然后其末端羟基用NPC活化，再与精胺的伯胺反应制得；产率：90.2％。1H NMR(400MHz,DTCl3)：PEG:δ3.38,3.65；TMC:δ4.24,2.05；DTC:δ4.32,3.02，以及Mal和精胺的特征峰。聚合物数均分子量通过特征峰面积积分比值计算为6.0-(4.8-15.2)-0.2kg/mol。
实施例三 合成靶向二嵌段聚合物DP8-PEG6.5k-P(DTC6k-TMC15k)
合成与实施例一类似，将实施例一的第一步中的引发剂MeO-PEG-OH换为NHS-PEG6.5k-OH，开环聚合TMC和DTC得到NHS-PEG6.5k-P(DTC6k-TMC15k)；第二步，多肽DMAPTVLP(DP8)按照其氨基和NHS-PEG6.5k-P(DTC6k-TMC15k)摩尔比3:1加入，30度酰胺化反应24-72小时，透析除去游离的DP8，冷冻干燥得到DP8-PEG6.5k-b-P(DTC6k-TMC15k)，通过核磁(附图1B)和TNBSA计算DP8接枝率接近100％。
实施例四 合成靶向二嵌段聚合物cNGQ-PEG7k-P(DTC4.8k-TMC19.2k)
合成与实施例一类似，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)；其次，具有自由伯胺的多肽C(NGQGEQ)(cNGQ)与NHS-PEG7k-P(DTC4.8k-TMC19.2k)通过酰胺化反应而键合。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(Thermo scientific)测得cNGQ的接枝率为89.7％。通过不同的活性PEG可得到不同靶向分子的聚合物。
实施例五 合成聚合物Azide-PEG6.5k-P(DTC4.0k-LA15.3)-SP
合成与实施例一类似，将其第一步的引发剂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-LA15.3)，然后其末端羟基用NPC活化，再与精胺的伯胺反应制得；产率：90.2％。1H NMR(400MHz,DTCl3)：PEG:δ3.38,3.65；TMC:δ4.24,2.05；DTC:δ4.32,3.02，以及精胺的特征峰。聚合物数均分子量通过特征峰面积积分比值计算为6.5-(4.0-15.3)-0.2kg/mol。
Figure PCTCN2017092424-appb-000012
通过调整使用的原料比例可得到不同分子量的聚合物，见表1。
表1 各个聚合物制备条件和产物的核磁表征结果
Figure PCTCN2017092424-appb-000013
Figure PCTCN2017092424-appb-000014
聚合物1-15分别为：PEG5k-P(DTC4.4k-TMC19.8k)-SP、Mal-PEG6k-P(DTC4.8k-TMC19.2)-SP、AA-PEG6.5k-P(DTC4.6k-TMC18.6k)-SP、Alkynyl-PEG5k-P(DTC5.8k-TMC23k)-SP、Azide-PEG3k-P(DTC4k-TMC12k)-SP、NHS-PEG7k-P(DTC3k-TMC15k)-SP、PEG5k-P(DTC3k-TMC15k)-SP、Azide-PEG6.5k-P(DTC4.0k-LA15.3)-SP、PEG7k-P(DTC4k-LA18k)-SP、PEG3k-P(DTC0.9k-TMC6k)-SP、PEG8k-P(DTC8k-LA30k)-SP、NHS-PEG7.5k-P(DTC4k-LA18k)、AA-PEG5k-P(DTC4.5k-TMC19.3k)、Azide-PEG6.5k-P(DTC5.8k-TMC18.7k)、Mal-PEG5k-P(DTC5.7k-LA18.8k)。
实施例六 制备交联聚合物囊泡PEG5k-P(DTC4.6k-TMC13.5k)-SP
溶剂置换法制备。室温下向950μL的HEPES(5mM，pH 6.8)缓冲液中加入50μL浓度为5mg/ml的PEG5k-P(DTC4.6k-TMC13.5k)-SP的DMSO溶液，室温静置1h,缓慢转动混合液，使之分散均匀后，(不)加入相当于DTC摩尔量10％‐30％的DTT溶液(最终0.1mM)后在37℃摇床中震荡12h充分(自)交联。然后透析(MWCO:3500)12h除去有机溶剂和游离蛋白，期间换5次介质，由此得到可逆核交联囊泡，简称为RCCPs。
实施例七 靶向聚合物的合成和靶向聚合物囊泡的制备
炔功能化的Alkynyl-PEG5k-OH引发DTC与LA开环聚合、端羟基活化、与精胺反应得到末端为活性炔基的Alkynyl-PEG5k-P(DTC5.8k-LA23k)-SP；最后，与叠氮功能化的靶向分子，如多肽cNGQ-N3或半乳糖Gal-N3，通过叠氮-炔基的点击化学反应得到靶向聚合物Gal-PEG5k-P(DTC5.8k-LA23k)-SP。然后靶向囊泡的制备即是在Gal-PEG5k-P(DTC5.8k-LA23k)-SP和 PEG5k-P(DTC5.8k-LA23k)-SP混合在DMSO中后打入HEPES溶液中，同实施例六制备得到Gal-RCCPs。
叠氮功能化的Azide-PEG3k-OH引发DTC与TMC开环聚合、端羟基活化、与精胺反应得到末端为活性叠氮基的Azide-PEG3k-P(DTC4k-TMC12k)-SP，最后，与炔基功能化的靶向分子，如alk-CC9或是cRGD-alk，通过叠氮-炔基的点击化学得到靶向聚合物CC9-PEG3k-P(DTC4k-TMC12k)-SP。然后靶向囊泡的制备即是把CC9-PEG3k-P(DTC4k-TMC12k)-SP和PEG3k-P(DTC4k-TMC12k)-SP混合在DMSO中后打入HEPES溶液中，同实施例六制备得到CC9-RCCPs。
叠氮或炔基功能化的聚合物为无末端精胺的二嵌段聚合物，即Alkynyl-PEG5k-P(DTC5.8k-LA23k)和Azide-PEG3k-P(DTC4k-TMC12k)，其键合cNGQ等多肽的方式和制备靶向囊泡(和无靶向的三嵌段聚合物混合)的方式与上述例子类似。
马来酰亚胺Mal功能化的Mal-PEG6k-OH或者丙烯酸酯功能化的AA-PEG6.5k-OH引发DTC与TMC开环聚合、端羟基活化、与精胺反应得到聚合物Mal-PEG6k-P(DTC4.8k-TMC19.2k)-SP或AA-PEG6.5k-P(DTC4.6k-TMC18.6k)-SP。然后，他们和相应的无活性端的聚合物PEG5k-P(DTC4.6k-TMC18.6k)-SP混合溶于DMSO中后，打入HEPES溶液中，同实施例六制备得到交联囊泡。最后，在囊泡溶液中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。
马来酰亚胺Mal和丙烯酸酯功能化的嵌段聚合物为无末端精胺的二嵌段聚合物，即Mal-PEG6k-P(DTC3.2k-TMC15.4k)和AA-PEG5k-P(DTC4.5k-TMC19.3k)，其和末端有精胺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。
实施例八 制备靶向自交联聚合物囊泡DP8-RCCPs
溶剂置换法制备。靶向聚合物囊泡通过几种方式制备。第一种基于三嵌段聚合物和靶向二嵌段聚合物。例如，实施例三制备的DP8-PEG6.5k-P(DTC6k-TMC15k)和PEG5k-P(DTC4.6k-TMC13.5k)-SP按特定比例混合，溶于DMSO中，加入到HEPES(5mM，pH 6.8)缓冲液中，同实施例六静 置、交联、透析，得到靶向交联囊泡，标记为DP8-RCCPs。靶向聚合物DP8-PEG6.5k-b-P(DTC6k-TMC15k)的含量为5～40wt.％。DLS测定制备的自交联聚合物囊泡尺寸为80-125nm左右，粒径分布为0.11-0.16。图2是上述自交联囊泡粒径分布及稳定性的测试(A)及电子透射显微镜图片(B)，交联囊泡及还原响应性测试(C)；得到的自交联囊泡的尺寸由动态光散射粒度分析仪(DLS)测的形成的纳米粒囊泡为80～125nm,粒径分布很窄，且高倍稀释后仍然保持不变的粒径和粒径分布(图2A)；由图2B可知，TEM测得的纳米粒子为中空的囊泡结构；但在模拟肿瘤细胞还原环境下快速释放，解交联(图2C)。由此可知，得到的囊泡可自交联，并具有还原敏感的解交联的性质。
实施例九 制备靶向自交联聚合物囊泡cRGD-RCCPs
基于两种三嵌段聚合物制备靶向聚合物囊泡。例如，按特定比例混合的两种PEG5k-P(DTC4.6k-TMC13.5k)-SP和cRGD-PEG6.5k-P(DTC6k-TMC15k)-SP溶于DMSO中，加入到HEPES(5mM，pH 6.8)缓冲液中，同实施例六静置、加入相当于DTC摩尔量10％‐30％的DTT溶液(最终0.1m M)，透析，得到靶向交联囊泡，标记为cRGD-RCCPs。取决于不同靶向聚合物的比例和聚合物的溶解时间，得到的囊泡平均粒径为50-125nm左右，粒径分布为0.04-0.17。具有囊泡的特征，体外稳定、具有还原敏感性。
实施例十 制备靶向自交联聚合物囊泡FA-RCCPs
基于三嵌段聚合物和有活性基团的二或三嵌段聚合物，例如，实施例二的Mal-PEG6k-P(DTC4.8k-TMC15.2k)-SP和PEG5k-P(DTC4.6k-TMC13.5k)-SP按特定比例混合溶于DMSO，加入到HEPES(5mM，pH 6.8)缓冲液中，同实施例六静置、交联、透析，得到交联囊泡。然后，通过迈克尔加成与含有自由巯基的靶向分子如叶酸FA-SH在室温反应后得到靶向交联囊泡，标记为FA-RCCPs。取决于不同靶向聚合物的比例和聚合物的溶解时间，得到的囊泡平均粒径为58-130nm，粒径分布为0.06-0.17。囊泡体外稳定、具有还原敏感性。
实施例十一 制备装载蛋白质或DNA的交联囊泡和靶向交联囊泡
载蛋白质如细胞色素C、颗粒酶B和DNA的交联囊泡的制备同实施例七，只是HEPES溶液中预先溶解有不同浓度的蛋白质或DNA。例如载不同比例(2-30wt.％)的FITC标记的细胞色素C(FITC-CC)的交联聚合物囊泡的粒径在90-108nm,粒径分布在0.13-0.19.得到的囊泡的为FITC-CC-DP8-RCCPs。其荧光分光光度计测定FITC-CC的包裹效率为50％-100％。
颗粒酶B(GrB)和DNA的装载相同。载药靶向囊泡制备同实施例八、九、十，只是HEPES溶液中预先溶解有不同浓度的蛋白质或DNA，得到GrB-DP8-RCCPs和DNA-DP8-RCCPs。
体外释放采用透析法。以FITC-CC的体外释放为例说明，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FITC-CC的交联聚合物囊泡加入10mM DTT的还原环境PB(10Mm,pH 7.4)中；第二组，载FITC-CC的聚合物囊泡在PB(10Mm,Ph7.4)中；载药交联囊泡的浓度为50mg/L,取0.5ml放入透析袋中(MWCO:350kDa)中，每个试管中加入相应的透析介质25ml,在预定时间取5.0ml透析袋外介质用作测试，补加5.0ml新鲜介质。荧光光谱仪测定溶液中药物浓度。附图3A为FITC-CC的累积释放量与时间的关系，可以看出，加入模拟细胞内DTT后，其释放明显要快于没加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。
实施例十二 制备装载DNA的交联囊泡和靶向交联囊泡
通过溶剂交换法可制备包裹DNA的DNA-RCCPs。DNA为pcDEF3-CD8IL-36γ(pIL-36γ)，pcDEF3-CD8IL-12(pIL-12)或小牛胸腺DNA等。例如PEG5k-P(DTC3.0k-TMC15k)-SP制备DNA-RCCPs具体操作为下，100μL PEG5k-P(DTC3.0k-TMC15k)-SP的DMSO溶液(5.0mg/mL)缓慢打入900μL溶解了预定数量的DNA(1mg/mL)的HEPES(10mM,pH 6.8)的混合溶液中，室温放置过夜，透析、加入催化量还原剂二硫苏糖醇(DTT)交联得到DNA-RCCPs。DLS结果显示，DNA-RCCPs粒径随着DNA含量的增加而增大，见表2。以后体内试验用含量为20wt％DNA的样品。
表2 DNA-cNGQ/RCCPs的粒径与DNA含量的关系
DNA/Polymer(wt.％) 0 10 20 30 40 50
Sizea(nm) 96 118 127 141 167 173
PDIb 0.1 0.1 0.1 0.2 0.2 0.3
实施例十三 装载DNA的交联靶向囊泡DNA-cNGQ-RCCPs的凝胶电泳分析
同实施例十二制备DNA-cNGQ/RCCPs。按照重量比4:1混合PEG5k-P(DTC4.4k-TMC19.8k)-SP和cNGQ-PEG7k-P(DTC4.8k-TMC19.2k)。DNA采用了pcDEF3-CD8IL-36γ(pIL-36γ)和pcDEF3-CD8IL-12(pIL-12)。在70mL四溴乙烷(TBE)缓冲溶液中加入0.7g琼脂糖，加热溶解琼脂糖粉末，在冷却后加入1μL溴化乙锭，得到琼脂糖胶待用。DNA-cNGQ-RCCPs或 DNA-RCCPs在DNA和聚合物的重量百分比(wt.％)分别设定为10％、20％、30％、40％、50％。在胶中分别加入20μL的DNA-cNGQ-RCCPs、DNA-RCCPs、自由DNA，在TBE电泳缓冲液中跑胶(100V,30min)。之后，由Molecular Imager FX(Bio-Rad,Hercules，Ex/Em:532/605nm)拍照凝胶图片，通过Quantity One软件(Bio-Rad)分析。琼脂糖凝胶阻留实验结果表明，即使当DNA含量达50wt.％，cNGQ-RCCPs仍可完全紧实包裹DNA，证明DNA-cNGQ-RCCPs稳定性优异。结果显示囊泡可有效复合DNA(pIL-12)。实施例十四制备载siRNA的交联囊泡和靶向交联囊泡siRNA-RCCPs
装载siRNA的交联囊泡通过溶剂交换法制备。例如，装载无特异性的对照siRNA(siScramble)得到siRNA-RCCPs的方法。预定数量siRNA的HEPES缓冲溶液(1mg/mL,10mM,pH 7.4)和100μL PEG5k-P(DTC4.4k-TMC19.8k)-SP的DMSO溶液(5.0mg/mL)混合，再打入900μL的HEPES(10mM,pH 6.8)，室温放置过夜、HEPES中透析、孵育4h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。表3为siRNA-cNGQ/RCCPs的粒径与siRNA含量的关系；随着siRNA含量从0增加到50wt.％，siRNA-cNGQ/RCCPs的粒径也由109增长到175nm。以后的细胞实验和体内试验如无特殊说明均实验在siRNA为10wt％的样品。
表3 siRNA-cNGQ/RCCPs的粒径与siScramble含量的关系
Figure PCTCN2017092424-appb-000015
装载荧光标记Cy5-siRNA或荧光素酶基因标记siGL3或治疗性siPLK1的囊泡与此类似。装载不同量siRNA(10％-80％)时，粒径为90-180纳米。
实施例十五 siScramble-cNGQ/RCCPs的凝胶电泳分析
如实施例十四制备siScramble-cNGQ/RCCPs，如实施例十三制备凝胶并跑胶，siRNA-cNGQ/RCCPs或siRNA-RCCPs在siRNA和聚合物的重量百分比(wt.％)分别设定为10％、20％、30％、40％、50％、60％、70％和80％。在胶中分别加入20μL的siRNA-cNGQ/RCCPs、siRNA-RCCPs、自由siRNA，以及用10mM GSH处理20h后的siRNA-cNGQ/RCCPs。实验结果表明，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。
实施例十六 制备载培美曲塞的交联囊泡和靶向交联囊泡PEM-RCCPs
装载生理环境带负电的小分子药物如培美曲塞的交联囊泡通过溶剂交换法制备，与实施例十一中蛋白质的包载类似。将100μL的聚合物PEG5k‐P(DTC4.4k‐TMC19.8k)‐SP的DMSO溶液(5.0mg/mL)打入900μL的含预定数量的培美曲塞的HEPES缓冲溶液(1mg/mL,10mM,pH 6.6)，室温放置过夜、HEPES中透析、孵育4h交联得到PEM‐RCCPs。载20wt％培美曲塞的交联聚合物囊泡的粒径在90nm,粒径分布在0.14.得到的囊泡的为PEM-RCCPs。其紫外分光光度计测定PEM的包裹效率为89％。
实施例十七 MTT法测囊泡对乳腺癌细胞MCF-7的毒性
MTT法使用人乳腺癌癌细胞(MCF-7)，计算细胞存活率。图3B中结果显示，当交联聚合物囊泡的浓度从0.1增到0.5mg/mL时，MCF-7的存活率仍高于85％，说明该交联聚合物囊泡具有良好的生物相容性。
另外制备了表面具有不同靶向分子的交联囊泡，例如，同上MTT法研究了FA体系对卵巢癌SKOV-3细胞、人口腔表皮样癌细胞KB细胞的作用，cNGQ体系对肺癌A549细胞，Gal体系对肝癌细胞HepG2，和cRGD体系对黑色素瘤B16细胞的细胞毒性，结果也说明了这些空交联靶向囊泡具有良好的生物相容性。
实施例十八 MTT法测载GrB的靶向交联囊泡对乳腺癌细胞MCF-7的毒性
测试对象为实施例十一GrB-DP8-RCCPs，由图4A、B中结果可知，载GrB的含30％DP8靶向交联聚合物囊泡对MCF-7细胞的半致死浓度(IC50)为0.188μg/mL，远低于无靶向囊泡的半致死浓度，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，而封端的细胞存活率均在70％以上。另外，载药交联囊泡对细胞表面核仁素表达低的HepG2肝癌细胞的毒性较差，由图4C中结果显示，核仁素在MCF-7细胞表面的特殊表达以及DP8的很好的靶向性。
制备了表面具有不同靶向分子的交联囊泡、装载培美曲塞、甲氨蝶呤、细胞色素C、或是凋亡素等，用MTT法研究FA体系对卵巢癌SKOV-3细胞、人口腔表皮样癌细胞KB细胞的作用，cNGQ体系对肺癌A549细胞，Gal体系对 肝癌细胞HepG2，和cRGD体系对黑色素瘤B16细胞的细胞毒性，均体现了靶向的特异性，载药的靶向聚合物囊泡能更快的进入细胞并发挥效果。
实施例十九 靶向载药囊泡的内吞和细胞内释放实验
以载药囊泡FITC-CC-DP8-RCCPs为例、采用激光共聚焦显微镜(CLSM)跟踪测定。将450μL的MCF-7细胞和HepG2细胞的DMEM(含10％牛血清、100IU/ml青霉素及100μg/ml链霉素)悬浮液铺于24孔培养板(每孔5×104个细胞)中，37℃、5％二氧化碳条件下培养24h。将50μL的FITC-CC-RCCPs和FITC-CC-DP8-RCCPs的PBS溶液加入孔中(FITC终浓度为10μg/ml)，继续孵育4h、8h、12h后，移去培养基并用PBS洗三次、用4％的多聚甲醛溶液200μL固定15min、PBS洗3次；CLSM(TCS SP5)观察拍照。由图5左图结果表明FITC-CC-DP8-RCCPs(A8h、B12h)相对于无靶向FITC-CC-RCCPs(C12h)可以通过介导作用更有效内吞进入MCF-7细胞且FITC-CC在细胞内可以快速释放，引起有效细胞凋亡，而对HepG2细胞基本上没有靶向效果(D)。
制备了表面具有不同靶向分子的载药交联囊泡、利用CLSM实验研究FA体系对卵巢癌SKOV-3细胞、人口腔表皮样癌细胞KB细胞的作用，cNGQ体系对肺癌A549细胞，Gal体系对肝癌细胞HepG2，和cRGD体系对黑色素瘤B16细胞的细胞内吞和细胞内释放行为，结果均表明靶向聚合物物囊泡能更快、更有效地被靶细胞内吞并快速释放药物。
实施例二十 囊泡的血液循环、在荷MCF-7小鼠的体内分布、活体成像
所有动物实验操作符合苏州大学动物实验中心规定严格进行。实验选用体重为18～20克左右、4～6周龄的Balb/C裸鼠。首先用Cy5-NHS和CC通过酰胺化反应制备Cy5标记的蛋白Cy5-CC。囊泡Cy5-CC-DP8-RCCPs(130纳米，粒径分布为0.17)和无靶向Cy5-CC-RCCPs尾静脉注射小鼠体内(Cy5浓度为4μM)，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100μL、浓度为1％的曲拉通和500μL二甲亚砜萃取(其中含有20mM的DTT)；然后离心(20000转/分钟，20分钟)取上层清液，通过荧光光谱仪测得每个时间点Cy5的量。由计算可知，靶向交联囊泡、非靶向交联囊泡在小鼠体内的消除半衰期分别为4.36和3.33小时，所以本发明的交联囊泡在小鼠体内稳定，有较长循环时间，如图5右图所示。
研究了几个具有代表性的样品在小鼠体内的循环时间，结果表明，载药的PEG8k-P(DTC8k-LA30k)-SP交联囊泡、PEG7k-P(DTC4k-LA18k)-SP交联囊泡 和PEG3k-P(DTC0.9k-TMC6k)-SP交联囊泡中药物在小鼠体内的血液循环时间分别为7.56小时、6.51小时和2.18小时。在皮下注射1×107个MCF-7细胞，大约3～4周后，肿瘤大小为100～200mm3时开始实验。将Cy5-CC-DP8-RCCPs、自由蛋白Cy5-CC和非靶向Cy5-CC-RCCPs尾静脉注射小鼠体内(Cy5-CC：0.25mg equiv./kg)，8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二甲亚砜萃取(其中含20mM的DTT)。离心(20000转/分钟，20分钟)取上清液，荧光光谱仪测每个时间点Cy5-CC的量。由图6可知，Cy5-CC-DP8-RCCPs和Cy5-CC-RCCPs注射8小时在肿瘤积累的Cy5-CC量分别为9.5和5.4ID％/g，差1.76倍，说明Cy5-CC-DP8-RCCPs通过主动靶向在肿瘤积累较多。
Cy5-CC-RCCPs和Cy5-CC-DP8-RCCPs尾静脉注射小鼠体内，在时间点4、8、12、24小时用小动物活体成像仪来追踪囊泡的去向。结果由图7可知，Cy5-CC-DP8-RCCPs在肿瘤部位很快积累，且在24小时后荧光仍然很强。说明Cy5-CC-DP8-RCCPs能主动靶向富集到乳腺癌部位，对乳腺癌细胞具有极强的特异性；载药Cy5-CC-RCCPs交联囊泡在进入肿瘤后很快代谢，荧光强度低。
实施例二十一 PEM-CC9-RCCPs在荷H460小鼠中的抑瘤效果、体重变化和存活率
动物选择同实施例二十，在皮下注射1×107个H460人肺癌细胞，大约3～4周后，肿瘤大小为100～200mm3时开始实验。PEM-CC9-RCCPs、PEM-RCCPs、临床注射液Alimta以及PBS在0、4、8和12天通过尾静脉注射小鼠体内(PEM：12.5mg/kg；Alimta：25mg/kg)。在0～18天，每两天称量小鼠的体重，游标卡尺测量肿瘤体积，肿瘤体积计算方法为：V＝(L×W×H)/2，(其中L为肿瘤的长度，W为肿瘤的宽度，H为肿瘤的厚度)。持续观察小鼠的生存到60天。由图8(其中A治疗期间各组肿瘤生长曲线，B为治疗后各组肿瘤图，C为体重变化曲线)中可知，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、Alimta组、PBS组分别在42天、38天、30天全部死亡。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。采用类似的实验方法研究了多种不同载药(凋亡蛋白、植物毒素蛋白、甲氨蝶呤、治疗性DNA和siRNA)的交联囊泡和靶向交联囊泡对荷瘤小鼠的影响，结果表明均可有效抑制肿瘤增长，对小鼠没有毒副作用，还可以延长荷瘤老鼠的生存时间。

Claims (10)

  1. 一种内膜具有正电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、疏水链段以及精胺分子；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2000-8000Da；疏水链段的分子量为亲水链段分子量的2.3-8.4倍。
  2. 根据权利要求1所述内膜具有正电的可逆交联生物可降解聚合物囊泡，其特征在于：所述聚合物的化学结构式如下：
    Figure PCTCN2017092424-appb-100001
    其中，R1选自以下基团中的一种：
    Figure PCTCN2017092424-appb-100002
    R2选自以下基团中的一种：
    Figure PCTCN2017092424-appb-100003
    所述聚合物中，PEG的分子量为2000-8000Da；PTMC或PLA的总分子量为PEG分子量的2-6倍；PDTC的总分子量为PTMC或PLA总分子量的15％-40％。
  3. 根据权利要求2所述内膜具有正电的可逆交联生物可降解聚合物囊泡，其特征在于：PEG的分子量为4000-8000Da；PTMC或PLA的总分子量为PEG分子量的2.5-5倍；PDTC的总分子量为PTMC或PLA总分子量的18％-38％。
  4. 权利要求1-3所述任意一项内膜具有正电的可逆交联生物可降解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(LA-DTC)的端羟基通过羟基活化剂活化，再与精胺反应制得PEG-P(TMC-DTC)-SP或者PEG-P(LA-DTC)-SP；
    (2)在PEG-P(TMC-DTC)-SP或者PEG-P(LA-DTC)-SP的PEG端偶联肿瘤特异性靶向分子，得到靶向PEG-P(TMC-DTC)-SP或者靶向PEG-P(LA-DTC)-SP；
    (3)以PEG-P(TMC-DTC)-SP、PEG-P(LA-DTC)-SP、靶向 PEG-P(TMC-DTC)-SP、靶向PEG-P(LA-DTC)-SP中的一种或几种为原料，通过溶剂置换法制备内膜具有正电的可逆交联生物可降解聚合物囊泡。
  5. 根据权利要求4所述内膜具有正电的可逆交联生物可降解聚合物囊泡的制备方法，其特征在于，步骤(1)的活化在干燥的溶剂中反应，然后沉淀、过滤、真空干燥；与精胺反应后透析、沉淀、抽滤、真空干燥得到PEG-P(TMC-DTC)-SP或者PEG-P(LA-DTC)-SP；步骤(2)为将步骤(1)的产物和溶于有机溶剂的靶向分子反应得到靶向PEG-P(TMC-DTC)-SP或者靶向PEG-P(LA-DTC)-SP；步骤(3)为将原料溶液中加入非离子缓冲溶液中，室温放置后透析、交联，得到内膜具有正电的可逆交联生物可降解聚合物囊泡。
  6. 一种抗肿瘤药物，由权利要求1-3所述任意一项内膜具有正电荷的可逆交联聚合物囊泡装载药物得到；所述药物为蛋白质、核酸或生理环境带负电的小分子药物。
  7. 权利要求6所述抗肿瘤药物的制备方法，其特征在于，将囊泡原料溶液与药物、非离子缓冲液混合，室温放置，然后透析、孵育交联得到抗肿瘤药物。
  8. 权利要求1-3所述任意一项内膜具有正电的可逆交联生物可降解聚合物囊泡作为药物载体的应用；所述药物为蛋白质、核酸或生理环境下带负电的小分子药物。
  9. 权利要求1-3所述任意一项内膜具有正电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。
  10. 一种聚合物，其特征在于，所述聚合物的化学结构式如下：
    Figure PCTCN2017092424-appb-100004
PCT/CN2017/092424 2016-07-15 2017-07-10 内膜具有正电的可逆交联生物可降解聚合物囊泡及其制备方法与在制备抗肿瘤药物中的应用 WO2018010624A1 (zh)

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