WO2022041017A1 - 载小分子药聚合物囊泡及其制备方法与应用 - Google Patents

载小分子药聚合物囊泡及其制备方法与应用 Download PDF

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WO2022041017A1
WO2022041017A1 PCT/CN2020/111532 CN2020111532W WO2022041017A1 WO 2022041017 A1 WO2022041017 A1 WO 2022041017A1 CN 2020111532 W CN2020111532 W CN 2020111532W WO 2022041017 A1 WO2022041017 A1 WO 2022041017A1
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drug
vcr
polymer
amphiphilic block
dar
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PCT/CN2020/111532
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English (en)
French (fr)
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孙欢利
余娜
张翼帆
钟志远
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苏州大学
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Priority to US18/012,410 priority Critical patent/US20240099977A1/en
Priority to PCT/CN2020/111532 priority patent/WO2022041017A1/zh
Publication of WO2022041017A1 publication Critical patent/WO2022041017A1/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
    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
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    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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    • A61K47/6835—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
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    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
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    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
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    • A61K9/51—Nanocapsules; Nanoparticles
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    • A61K9/513—Organic macromolecular compounds; Dendrimers
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    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention belongs to the technical field of polymer nano-drugs, and in particular relates to a reversibly cross-linked degradable polymer vesicle loaded with vincristine sulfate, a preparation method thereof, and an application in tumor targeted therapy.
  • Vincristine sulfate is a water-soluble, potent drug that acts primarily on tubulin to arrest mitosis in metaphase, but its severe neurotoxicity results in lower doses available.
  • the liposomal vincristine sulfate (Marqibo®) nanomedicine approved for marketing in 2012 can prolong the circulation time of VCR and reduce toxic and side effects, the overall improvement is limited. Therefore, how to achieve efficient and stable encapsulation and tumor-targeted delivery of VCR is crucial.
  • the prior art discloses a vincristine sulfate liposome and a preparation method thereof.
  • the vincristine sulfate liposome is composed of vincristine sulfate and nanoliposomes prepared by using sphingomyelin, wherein the vincristine sulfate liposome is Alkali is encapsulated in the nano-liposomes, sphingomyelin is used to prepare nano-liposomes, and vincristine sulfate liposomes are prepared after encapsulating vincristine sulfate, wherein sphingomyelin contains more amide bonds, which can be better It can resist chemical and biological degradation, protect the stability of liposome structure, and improve the drug enrichment of tumor cells, thereby improving the anti-tumor effect.
  • the prior art also discloses a preparation method of a bifunctional nanoparticle preparation encapsulating vincristine sulfate, and encapsulating vincristine sulfate in a PLGA-PEG polymer carrier modified by folic acid/cell penetrating peptide by a double emulsion method
  • the bifunctional nanoparticle preparation was prepared; it showed good pharmacokinetic behavior in vitro and in vivo, and the prepared folic acid/cell penetrating peptide-modified PLGA-PEG bifunctional nanoparticles had a particle size of 287.2 ⁇ 0.8 nm, with high drug loading. quantity and encapsulation efficiency, and good stability.
  • the existing liposome-structured polymer vesicles have a hydrophilic inner cavity, which can be used to load hydrophilic small-molecule drugs.
  • hydrophilic drugs such as VCR
  • the loading efficiency of hydrophilic drugs such as VCR is low, and the stability of the collective internal circulation is still lacking. It has multi-functional characteristics such as tumor-specific targeting, rapid intracellular drug release and excellent biocompatibility.
  • the purpose of the present invention is to disclose amphiphilic block polymers, drug-loaded polymer vesicles and preparation methods and applications thereof, specifically a reversibly cross-linked degradable polymer vesicle loaded with vincristine sulfate (VCR) and Its preparation method and application.
  • VCR vincristine sulfate
  • Drug-loaded polymer vesicles prepared from small molecule drugs, amphiphilic block polymers; or from small molecule drugs, amphiphilic block polymers, functionalized PEG-P (TMC-DTC), targeting monoclonal antibodies preparation.
  • the molecular structural formula of the amphiphilic block polymer is as follows.
  • the molecular weight of PEG is 3000-8000 Da; the molecular weight of the hydrophobic segment is 2.5-6 times the molecular weight of PEG; the molecular weight of the PDTC segment is 8 times the molecular weight of the hydrophobic segment % ⁇ 30%.
  • amphiphilic block polymer of the present invention has hydrophilic segment (n segment), hydrophobic segment (x+y segment), KD z segment (z segment), hydrophobic segment, KD z segment Linked by urethane bonds; the amphiphilic block polymers are denoted PEG-P(TMC-DTC) -KDz , PEG-P(LA-DTC) -KDz , PEG-P(CL-DTC)- KD z .
  • the small molecule drug is vincristine sulfate, doxorubicin hydrochloride, epirubicin hydrochloride, verapamil hydrochloride, irinotecan hydrochloride, requimod, preferably vinblastine sulfate Neobase (VCR);
  • the targeting monoclonal antibody is a monoclonal antibody targeting CD38, such as daratumumab (Dar), isatuximab (Isa) or other monoclonal antibody targeting CD38.
  • the preparation method of the above drug-loaded polymer vesicles is as follows: using small molecule drugs and the amphiphilic block polymer as raw materials, and preparing drug-loaded polymer vesicles by a solvent replacement method; Affinity block polymer, functionalized amphiphilic block polymer, and targeting monoclonal antibody are used as raw materials, and drug-loaded polymer vesicles are prepared by solvent replacement method.
  • the functionalized amphiphilic block polymer is assembled and cross-linked with the amphiphilic block polymer and loaded with a drug, and then reacted with a monoclonal antibody targeting CD38 to prepare a drug-loaded polymer vesicle.
  • the invention discloses the application of the above-mentioned amphiphilic block polymer or drug-loaded polymer vesicles in the preparation of nano-drugs; the nano-drugs are anti-tumor drugs; the tumor is preferably in situ multiple myeloma.
  • VCR vincristine sulfate
  • the drug-loaded vesicles of the present invention are targeting or non-targeting structures, and the targeting molecules of the present invention are monoclonal antibody molecules or monoclonal antibody fragments, and the like, such as daratumumab (Dar), isartor ciximab (Isa) or other mAbs targeting CD38.
  • the targeting molecules of the present invention are monoclonal antibody molecules or monoclonal antibody fragments, and the like, such as daratumumab (Dar), isartor ciximab (Isa) or other mAbs targeting CD38.
  • the amphiphilic block polymer is first combined with the functionalized amphiphilic block polymer as a raw material to prepare drug-carrying vesicles, and then a CD38-targeting monoclonal antibody is connected to obtain CD38-targeting drug-carrying vesicles.
  • the functional groups are derived from PEG initiators, and the resulting polymer PEG ends carry reactive functional groups, such as azide (N 3 ), maleimide (Mal) or N-hydroxysuccinimide (NHS), to amphiphilic
  • the functional block polymer PEG-P(TMC-DTC) as an example, the functionalized amphiphilic block polymer can be N3 - PEG-P(TMC-DTC), Mal-PEG-P(TMC-DTC), NHS-PEG-P (TMC-DTC).
  • the drug-loaded vesicle of the present invention is composed of a drug and a vesicle, and the vesicle is obtained by cross-linking a polymer, and the targeting molecule may or may not be modified; the amphiphilic block polymer PEG-P (TMC-DTC), Taking vincristine sulfate as an example, the preparation method of the drug-loaded vesicles of the present invention can be as follows.
  • the invention discloses the above-mentioned VCR-loaded reversibly cross-linked degradable polymer vesicles and a preparation method thereof.
  • a solution of a PEG-P(TMC-DTC)-KD z polymer is injected into a standing VCR aqueous solution, and dialyzed after stirring.
  • VCR-loaded reversibly cross-linked degradable polymer vesicles Ps-VCR
  • VCR was dissolved in ultrapure water and mixed with HEPES buffer (pH 6.8, 10 mM), and then allowed to stand The DMSO solution of PEG-P(TMC-DTC)-KD z polymer was injected into it, stirred for 3-5 minutes and then dialyzed with HEPES (pH 7.4, 10 mM) to obtain Ps-VCR.
  • the invention also discloses a monoclonal antibody-directed, VCR-loaded, reversibly cross-linked degradable polymer vesicle and a preparation method thereof: a DMSO solution of PEG-P(TMC - DTC) -KDz and a functionalized polymer such as N3 -After the DMSO solution of PEG-P(TMC-DTC) was mixed uniformly, it was injected into the HEPES solution containing VCR, and after stirring for 3-5 minutes, the reversible cross-linked polymer capsules loaded with VCR containing N on the surface were obtained by dialysis vesicles; mAbs modified by dibenzocyclooctyne, such as daratumumab (Dar), isatuximab (Isa), or other CD38-targeting mAbs with azide-functionalized VCR-loaded vesicles
  • the tension-triggered click chemistry reaction of vesicles (N 3 -Ps-VCR
  • the Ab can be easily prepared by the Michael addition reaction between the thiol-functionalized monoclonal antibody molecule and the VCR-loaded vesicles containing Mal on the surface, or the amidation reaction between the monoclonal antibody and the NHS-functionalized VCR-loaded vesicles. -Ps-VCR.
  • KD has good biocompatibility, and combined with the PEG segment and the hydrophobic segment, it can form asymmetric membrane structure vesicles, and realize the efficient and stable encapsulation of small molecule drugs (such as VCR).
  • VCR small molecule drugs
  • the force encapsulates the VCR, and is separated from the outside world by the disulfide cross-linked vesicle membrane, which can avoid the loss and toxic side effects caused by leakage and cell adhesion during the delivery process, and can be efficiently delivered to the lesion and restored in the body.
  • glutathione GSH
  • the polymer vesicles in the present invention are reduction-sensitive reversible cross-linking, intracellular reversible cross-linking and biodegradable polymer vesicles with negatively charged inner membrane;
  • the polymer is PEG-P (TMC-DTC) -KD z , in which the TMC (LA or CL) in the middle block is randomly arranged with DTC;
  • the molecular weight of KD z is 700-2000 Da , which is much smaller than the molecular weight of the PEG segment, and the inner membrane band is obtained after self-assembly and cross-linking
  • the inner shell of the vesicles is KD z for compounding small molecule drugs.
  • the vesicle membrane is a reversibly cross-linked biodegradable PTMC with good compatibility.
  • the dithiopentane structure of the side chain is similar to the human body's natural antioxidant lipoic acid, which can spontaneously form a reduction-sensitive reversible cross-linking, which not only guarantees the drug Stable and long-term circulation in blood can also achieve rapid intracellular de-crosslinking and rapid release of drugs into target cells.
  • the invention discloses the application of the above-mentioned VCR-loaded tumor-targeted, reversibly cross-linked and degradable polymer vesicles in the preparation of anti-tumor targeted nano-drugs.
  • the tumor is multiple myeloma.
  • the present invention has the following advantages.
  • the present invention designs a new small-molecule hydrophilic drug VCR drug-loaded vesicle and tumor-targeted delivery;
  • Alkane can provide reduction-sensitive reversible cross-linking, which can not only ensure the long-term circulation of drugs in the blood, but also can quickly de-cross-link in cells and release drugs into target cells;
  • the shell is PEG and has targeting molecules such as monoclonal antibodies.
  • the drug-loaded vesicles disclosed in the present invention have significant anti-tumor effects in vitro and in vivo, the polymers have good biocompatibility, can form vesicles with asymmetric membrane structures, and have good drug-carrying effects.
  • the degradable polymer vesicle carrier of the present invention avoids the defects of the existing nanocarriers such as large particle size, poor circulation stability in vivo, low tumor cell selectivity, and slow release of intracellular VCR.
  • the vesicle system of the present invention has many unique advantages, including small size, simple and controllable preparation, excellent biocompatibility, high in vivo circulation stability, strong tumor cell specific selectivity, fast intracellular drug release, tumor The growth inhibitory effect is remarkable and so on. Therefore, this vesicle system is expected to be a simple and all-in-one nanoplatform for efficient and specific targeted delivery of VCR to multiple myeloma cells.
  • Fig. 1 is the nuclear magnetic spectrum of N 3 -PEG-P(TMC-DTC) in Example 1.
  • Fig. 2 is the nuclear magnetic spectrum of PEG-P(TMC-DTC)-NPC in Example 2.
  • Fig. 3 is the nuclear magnetic spectrum of PEG-P(TMC-DTC)-KD 5 in Example 2.
  • FIG. 5 is a graph showing the stability of Dar-Ps-VCR in Example 6 in the presence of high dilution and serum.
  • Figure 6 shows the VCR release behavior of Dar-Ps-VCR under non-reducing conditions and 10 mM GSH in Example 6.
  • Figure 7 shows (A) the endocytosis of Dar-Ps-Cy5 with different targeting densities in LP-1 cells and (B) the incubation of LP-1 cells with Dar 4.4 -Ps-Cy5 and Ps-Cy5 in Example 7 CLSM picture after 4 hours (scale bar: 25 ⁇ m).
  • Figure 8 shows the toxicity of Dar-Ps-VCR, Ps-VCR and free VCR with different targeting densities in LP-1 cells in Example 8.
  • Figure 9 shows the toxicity of Dar-Ps-VCR and Ps-VCR in (A) MV4-11 cells and (B) L929 cells and (C) Dar-Ps and (D) Dar in LP-1 cells in Example 8 toxicity in.
  • Figure 10 is the flow cytometry in Example 9 to determine the apoptosis of LP-1 cells induced by Dar-Ps-VCR, Ps-VCR and free VCR.
  • Figure 11 shows the tumor distribution of LP-1-Luc tumor in various organs, skull and hind leg bones of mice observed by in vitro bioluminescence imaging in Example 10.
  • FIG. 12 is the in vivo fluorescence imaging images of the in situ LP-1-Luc multiple myeloma mice in Example 11 after the tail vein injection of Dar-Ps-Cy5 and Ps-Cy5 at different time points.
  • Figure 13 shows the construction and treatment workflow of the orthotopic LP-1-Luc multiple myeloma mouse transplantation model in Example 12, and the in vivo imaging evaluation of Dar-Ps-VCR's effect on orthotopic LP-1-Luc multiple myeloma. Graph of the therapeutic effect of the mouse model.
  • Fig. 14 is a graph showing the changes in Luc fluorescence signal; changes in body weight and Kaplan-Meier survival curves of mice in different treatment groups in Example 12.
  • Figure 15 is a micro-CT image of the femur (Femur) and tibia (Tibia) of mice in different treatment groups in Example 12.
  • Figure 16 is the analysis of each index of (A) femur and (B) tibia of mice in different treatment groups in Example 12.
  • BMD bone mineral density
  • BV/TV bone volume fraction
  • Tb.N bone trabecular number
  • Tb.Sp bone trabecular separation
  • BS/TV bone surface area
  • Tb.Th bone trabecular thickness.
  • the reversibly cross-linked and degradable polymer vesicles loaded with VCR of the present invention are obtained by self-cross-linking while self-assembly of amphiphilic tri-block polymers;
  • the molecular chains of the tri-block polymers include sequentially connected hydrophilic Segment, hydrophobic segment and KD molecule;
  • the hydrophilic segment is polyethylene glycol (PEG) with a molecular weight of 3000-8000 Da;
  • the hydrophobic segment is a polycarbonate segment, and the molecular weight is a hydrophilic segment 2.1-5.7 times the molecular weight;
  • the molecular weight of KD polypeptide is 15%-50% of the hydrophilic segment of PEG.
  • the PEG-P(TMC-DTC) -KDz polymer of the present invention is prepared by reacting with KDz after activating the terminal hydroxyl group of PEG-P(TMC-DTC) by p-nitrophenyl chloroformate (p-NPC).
  • p-NPC p-nitrophenyl chloroformate
  • step (i) the reaction conditions are anhydrous dichloromethane (DCM), pyridine, 25 oC, 24 hours; in step (ii), the reaction conditions are anhydrous dimethyl sulfoxide (DMSO), KD z , triethylamine, 30 oC, 48 hours.
  • DCM dichloromethane
  • pyridine 25 oC, 24 hours
  • DMSO dimethyl sulfoxide
  • KD z triethylamine
  • the polymer solution was collected and concentrated to a polymer concentration of about 50 mg/mL by rotary evaporation. After precipitation in ice ether, vacuum drying was performed to obtain a white cotton-like polymer PEG-P(TMC-DTC)-KD z .
  • the TMC was routinely replaced with LA or CL to obtain PEG-P(LA-DTC)-KD z and PEG-P(CL-DTC)-KD z .
  • the polymer N 3 -PEG-P (TMC-DTC) is obtained by using DPP as a catalyst and N 3 -PEG-OH as a macromolecular initiator to initiate ring-opening copolymerization of TMC and DTC.
  • TMC 1,4-butane
  • DTC 0.20 g, 1.0 mmol
  • N 3 -PEG-OH was replaced with CH 3 O-PEG-OH with a molecular weight of 5k, and the rest remained unchanged.
  • PEG-P(TMC-DTC) (5.0-(15.0-2.0) kg/mol was obtained ).
  • the synthesis of the polymer PEG-P(TMC-DTC)-KD z is divided into two steps, namely, using p-NPC to activate the terminal hydroxyl group of PEG-P(TMC-DTC) (5.0-(15.0-2.0) kg/mol) , obtained by reacting with KD z polypeptide molecules.
  • PEG-P(TMC-DTC)-KD 5 the specific operation is as follows.
  • PEG-P(TMC-DTC) (1.0 g, 45.5 ⁇ mol) was dissolved in 10 mL of anhydrous DCM under nitrogen atmosphere, It was then transferred to an ice-water bath and pyridine (18.0 mg, 227.5 ⁇ mol) was added, and after stirring for 10 minutes, a solution of p-NPC (48.4 mg, 240.3 ⁇ mol) in DCM (1.0 mL) was added dropwise.
  • KD 5 (60.0 mg, 83.4 ⁇ mol) was weighed and dissolved in 4 mL of anhydrous DMSO and triethylamine (4.2 mg, 41.7 ⁇ mol) was added, and then PEG- Anhydrous DMSO solution (9.0 mL) of P(TMC-DTC)-NPC was added dropwise for 30 minutes.
  • Figures 2 and 3 are hydrogen NMR spectra of PEG-P(TMC-DTC)-NPC and PEG-P(TMC-DTC) -KD5 .
  • the characteristic peaks of p-NPC ⁇ 7.41 and ⁇ 8.30 ppm
  • the characteristic peaks of PEG-P(TMC-DTC) ⁇ 2.03, 2.99, 3.38, 3.63, 4.18 and 4.22 ppm
  • the grafting rate of NPC was about 100%.
  • Figure 3 shows that the characteristic peaks of NPC at ⁇ 7.41 and ⁇ 8.30 ppm disappear, and a new signal peak appears at ⁇ 4.54 ppm, which is the characteristic peak of methine in KD 5 .
  • the degree of substitution of KD 5 was calculated to be ⁇ 100% by comparing the ratio of the peak area at ⁇ 4.54 ppm to the TMC methylene hydrogen peak area at ⁇ 1.95 ppm.
  • the grafting ratio of KD 5 was 100% measured by high performance liquid chromatography (HPLC), demonstrating the successful synthesis of PEG-P(TMC-DTC)-KD 5 , which was used in the following examples.
  • Ps-VCR was prepared by solvent displacement method, in which VCR was encapsulated by electrostatic interaction with KD z . Dissolve PEG-P(TMC-DTC)-KD z in DMSO (40 mg/mL) and dispense 100 ⁇ L into standing 900 ⁇ L HEPES (pH 6.8, 10 mM) containing VCR at 300 rpm After stirring for 3 minutes, Ps-VCR was obtained by dialysis against HEPES (pH 7.4, 10 mM) for 8 hours. The theoretical drug loading of VCR was set at 4.8-11.1 wt.%, and it was found that the particle size of the obtained Ps-VCR was between 26-40 nm and the particle size distribution was 0.05-0.20 (Table 1).
  • the encapsulation efficiency of Ps-VCR was calculated as high as 97.2% by measuring its absorbance at 298 nm by UV-Vis spectroscopy. Based on the same method, under the theoretical drug loading of 4.8%, the encapsulation efficiency of Ps-VCR prepared by PEG-P(LA-DTC)-KD 5 and PEG-P(CL-DTC)-KD 5 was 88.3%, The particle size of the drug-loaded vesicles prepared by PEG-P (TMC-DTC) diblock polymer is about 75 nm, and the encapsulation efficiency of VCR is low, only 14.1%.
  • Example 3 Using a similar method in Example 3, the encapsulation of other drugs such as verapamil hydrochloride (VER), irinotecan hydrochloride (CPT), and requimod (R848) by reversibly cross-linked degradable vesicles was studied. The study found that after encapsulating different drugs, the particle size of the obtained Ps-drug was 20-40 The specific results are shown in Table 2.
  • VER verapamil hydrochloride
  • CPT irinotecan hydrochloride
  • R848 requimod
  • Ab-Ps-VCR was obtained by post-modification of a dibenzocyclooctyne-functionalized monoclonal antibody (Ab-DBCO) on the surface of an azide-functionalized polymersome VCR nanomedicine (N3 - Ps-VCR).
  • N 3 -Ps-VCR is obtained by co-assembly of N 3 -PEG-P(TMC-DTC) and PEG-P(TMC-DTC)-KD z while wrapping VCR, wherein N 3 -PEG-P(TMC-DTC ) content is 1 ⁇ 10 wt.%.
  • N 3 -Ps-VCR containing 2% N 3 -PEG-P(TMC-DTC) 8.0 mg N 3 -PEG-P(TMC-DTC) and 392.0 mg PEG- P(TMC-DTC)-KD 5 (molar ratio 2:98) was dissolved in DMSO (total polymer concentration of 40 mg/mL), while 4.0 mL of VCR in water (5 mg/mL) was added to 90 mL of HEPES (pH 6.8, 10 mM), mix well, pour 10 mL of polymer solution into it under standing, stir for 5 minutes, and then place at 37 oC for 4 hours.
  • DMSO total polymer concentration of 40 mg/mL
  • HEPES pH 6.8, 10 mM
  • N 3 -Ps-VCR The particle size of N 3 -Ps-VCR measured by dynamic light scattering (DLS) was 36 nm, and the distribution was narrow (PDI: 0.11).
  • DLS dynamic light scattering
  • the N 3 -Ps-VCR was then concentrated from 4 mg/mL to 18.6 mg/mL using a tangential flow device to facilitate storage and improve the bonding efficiency of the mAb.
  • the particle size of N 3 -Ps-VCR after concentration was 42 nm and the PDI was 0.07.
  • the particle size remained around 40 nm, the PDI was less than 0.17, and the leakage of VCR was less than 0.6%, indicating that N 3 -Ps-VCR has excellent long-term storage stability (Table 3).
  • Ab-DBCO was prepared by amidation reaction of small molecule NHS-OEG 4 -DBCO with the amino group on the monoclonal antibody, wherein the functionalization degree of DBCO can be adjusted by changing the molar ratio of Ab to NHS-OEG 4 -DBCO.
  • DBCO-functionalized daratumumab (Dar-DBCO) as an example
  • a solution of Dar in PBS (21.7 mg/mL) was diluted to 10 mg/mL with PB (pH 8.5, 10 mM), and 200 ⁇ L of A 3- or 5-fold molar equivalent of NHS-OEG 4 -DBCO in DMSO solution (5 mg/mL) was added under shaking, and the reaction was carried out overnight at 27 oC and 120 rpm in a shaker.
  • the unreacted NHS-OEG 4 -DBCO was removed by ultrafiltration tube centrifugation (MWCO: 10 kDa, 3000 rpm), and washed with PBS (pH 7.4, 10 mM) for ultrafiltration twice to obtain Dar-DBCO.
  • MWCO 10 kDa, 3000 rpm
  • PBS pH 7.4, 10 mM
  • the molar ratio of Dar to NHS-OEG 4 -DBCO was 1:3 and 1:5, 1.5 and 2.8 DBCOs were modified on each Dar by time-of-flight mass spectrometry (MALDI-TOF-MS), respectively (Fig. 4), expressed as Dar-DBCO 1.5 and Dar-DBCO 2.8 .
  • Dar-DBCO 1.5 or other mAbs modified with 1.5-2 DBCOs were used for subsequent experiments.
  • Dar-Ps-VCR can be easily prepared by tension-triggered click chemistry between N 3 on the surface of N 3 -Ps-VCR and Dar-DBCO, and the surface density of Dar can be adjusted by changing the feed ratio.
  • the molar ratios of Dar-DBCO to N3 were set to be 0.25:1, 0.5:1 and 1:1, respectively, that is, 10.4, 20.9 and 41.8 ⁇ L were added to 107.5 ⁇ L of N3 - Ps-VCR (18.6 mg/mL), respectively of Dar-DBCO solution (5.6 mg/mL), then reacted overnight in a shaker at 25 oC, 100 rpm.
  • Unbound Dar-DBCO was removed by ultracentrifugation (58 krpm, 4 oC, 30 min) and washed twice with HEPES (pH 7.4, 10 mM), while Dar-Ps-VCR and supernatant were collected to determine Dar binding combined amount.
  • the unbound Dar-DBCO in the supernatant was determined by HPLC, and the content of Dar per mg of the surface of the polymersomes was calculated to be 28.6, 56.4 and 112.2 ⁇ g, respectively, according to the multi-angle laser light scattering.
  • the preparation methods of other mAb-directed VCR-loaded polymersomes are similar to Dar-Ps-VCR.
  • the particle size is between 40-60 nm, the particle size distribution is narrow (PDI: 0.10-0.30), and the number of mAbs on the surface of each vesicle is 1-10.
  • Example 6 The stability and in vitro drug release of Ab-Ps-VCR targeting polymer vesicle nanomedicine.
  • Dar 4.4 -Ps-VCR containing 4.4 Dar on each vesicle surface was used as a representative to study the stability and in vitro drug release behavior of Ab-Ps-VCR-targeted vesicle nanomedicine.
  • the stability of Dar-Ps-VCR was diluted 50 times with phosphate buffer solution or added with 10% fetal bovine serum, and the particle size changes were detected by dynamic light scattering.
  • Figure 5 is a particle size distribution diagram of Dar-Ps-VCR stability. The results showed that the Dar-Ps-VCR-targeted vesicle nanomedicine kept intact particle size and particle size distribution after being diluted 50 times and adding 10% FBS for 24 hours, with good stability.
  • FIG. 6 is a graph showing the results of in vitro release of Dar-Ps-VCR targeting vesicle nanomedicine. The results showed that Dar-Ps-VCR at 10 mM Under the reducing conditions of GSH, the release of VCR reached more than 85% within 12 hours, while under non-reducing conditions, the cumulative release of VCR within 24 hours was only about 22%.
  • Cy5 is used to label the polymer vesicles.
  • the preparation method of Dar-Ps-Cy5 refers to Example 5, and the preparation method of Ps-Cy5 refers to Example 3; flow cytometry and laser scanning confocal microscopy ( CLSM) to study the uptake of Dar-Ps-Cy5 with different Dar densities in LP-1 cells.
  • CLSM laser scanning confocal microscopy
  • the LP-1 cell suspension was firstly plated in a 6-well plate (2 ⁇ 10 5 cells/well), placed in an incubator for 24 hours, and 200 ⁇ L of Dar-Ps-Cy5 and Ps were added to each well.
  • the endocytic behavior of Dar 4.4 -Ps-Cy5 and Ps-Cy5 in LP-1 cells was further studied by CLSM.
  • the specific experimental steps are as follows.
  • the small discs pretreated with polylysine (300 ⁇ L, 0.1 mg/mL) were placed in a 24-well plate, and LP-1 cell suspension was added (3 ⁇ 10 5 cells/well) , after culturing in the incubator for 24 hours, add 200 ⁇ L Dar 4.4 -Ps-Cy5 and Ps-Cy5 respectively (the concentration of Cy5 in the well is 40 ⁇ g/mL).
  • Figure 7B is a graph showing the uptake results of Dar 4.4 -Ps-Cy5 and Ps-Cy5 in LP-1 cells.
  • the in vitro antitumor activity of Dar-Ps-VCR on LP-1 multiple myeloma cells was determined by CCK-8 kit, and MV4-11 cells were used as control.
  • LP-1 cells were firstly plated in 96-well plates (15,000 cells/well), placed in a 37 oC, 5% CO 2 incubator for 24 hours, and then 20 ⁇ L of cells containing different Dar surface densities were added to each well.
  • Dar-Ps-VCR, Ps-VCR and free VCR, the final concentrations of VCR in the wells were 0.001, 0.01, 0.05, 0.1, 0.5, 1 and 10 ng/mL, respectively.
  • FIG. 8 is a graph showing the cytotoxicity results of Dar-Ps-VCR vesicle nanomedicines with different targeting densities (z is 5) on LP-1 cells.
  • Dar-Ps-VCR refers to Dar 4.4 -Ps-VCR vesicle nanomedicine (z is 5)
  • Dar-Ps-Cy5 refers to Dar 4.4 -Ps-Cy5 (z is 5).
  • LP-1 cells were plated in a 6-well plate at a density of 2 ⁇ 10 5 cells/well. After culturing in the incubator for 24 hours, 200 ⁇ L of Dar-Ps-VCR, Ps-VCR and free VCR (the The concentration in the well was 0.5 ng/mL), and the cells with only PBS were used as a control. After 48 hours of incubation in the incubator, LP-1 cells were collected by centrifugation (800 rpm, 5 min) and washed twice with ice-cold PBS.
  • Binding buffer 200 ⁇ L was added to each sample to resuspend the cells ( The cell density is approximately 10 6 cells/mL). After pipetting evenly, 100 ⁇ L was added to the flow tube, followed by adding 5 ⁇ L AnnexinV-FITC and 10 ⁇ L PI solution. After staining at room temperature for 15 minutes in the dark, 400 ⁇ L PBS was added to mix evenly, and flow cytometry was used for measurement within 1 hour.
  • FIG. 10 is a graph showing the results of Dar-Ps-VCR-induced apoptosis of LP-1 cells. The results showed that Dar-Ps-VCR could effectively induce cell apoptosis.
  • VCR concentration of VCR was 0.5 ng/mL
  • the concentration of VCR was 0.5 ng/mL
  • mice were firstly myeloablated by intraperitoneal injection of 10 mg/mL cyclophosphamide solution for two consecutive days, each injection per mouse 2 mg, LP-1-Luc cells (8 ⁇ 10 6 cells/mouse) were injected into mice through the tail vein on the third day, in vivo imaging and treatment began on the 10th day after inoculation, and the mice were weighed at the same time.
  • FIG. 11 is a graph showing the results of in vitro bioluminescence imaging of each organ, skull and hind leg bones of LP-1-Luc in situ multiple myeloma-bearing mice.
  • Example 11 In vivo imaging experiment of Dar-Ps-Cy5 in LP-1-Luc orthotopic multiple myeloma mice.
  • VCR VCR equiv./kg
  • the dose of VCR is 0.25 mg/kg, one injection for 4 days, a total of 4 injections, expressed as Dar-Ps-VCR (0.25 mg VCR equiv ./kg, Q4d); the other is a VCR dose of 0.50 mg/kg, 8 days for one injection, a total of 2 injections, expressed as Dar-Ps-VCR (0.50 mg VCR equiv./kg, Q8d).
  • the same VCR dose of Ps-VCR and free VCR, the equivalent of Dar-Ps and PBS were used as controls.
  • mice There were 10 tumor-bearing mice in each treatment group, of which 4 were used for bioluminescence imaging and 6 were used to monitor body weight and observe survival.
  • the study found that the LP-1-Luc cells of the mice in the PBS group continued to grow rapidly, and the onset of the disease started when the bioluminescence intensity reached 1.0 ⁇ 10 9 p/sec/cm 2 /sr 37-45 days after inoculation, manifested as paralysis of both legs, Weight loss and death occurred (Fig. 13).
  • the Luc signal of the mice in the two Dar-Ps-VCR administration groups did not increase significantly or even decreased slightly, indicating that they can effectively inhibit the Spread and proliferation of LP-1-Luc cells in mice.
  • the tumors of the mice in the non-targeted Ps-VCR and free VCR groups began to recur, and the Luc bioluminescence signal increased rapidly, increasing by 157 and 53 times, respectively, on the 39th day.
  • mice injected with Dar-Ps-VCR in the tail vein could still sustainably inhibit the growth of LP-1-Luc tumor, and no obvious Luc signal was detected even on the 39th day after inoculation, and its fluorescence value was the same as that of healthy mice.
  • the background signals were comparable, 235- and 114-fold lower than the Ps-VCR and free VCR groups, respectively, and about 5000-fold lower than the untreated PBS group.
  • the tumor growth trend of mice in the empty vector Dar-Ps group was similar to that in the PBS group (Fig. 14A).
  • Osteolytic lesions are one of the common clinical manifestations of MM patients, so micro-CT was used to evaluate the related indexes of the femur and tibia of mice in each treatment group.
  • the results showed that there were severe osteoclasts in the hind leg bones of the PBS group and Dar-Ps group, and a large number of bone trabeculae were lost.
  • the situation in mice was similar (Fig. 15).
  • we found that the bone mineral density, bone volume fraction, number of trabecular bone, trabecular bone separation, and bone mineral density of the femur and tibia of mice in the Dar-Ps-VCR targeted therapy group under the two administration methods were found.

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Abstract

载小分子药聚合物囊泡及其制备方法与应用，载小分子药聚合物囊泡由两亲性嵌段聚合物与小分子药物组装制备得到；或者由两亲性嵌段聚合物与官能化两亲性嵌段聚合物组装交联并负载小分子药物，再与靶向单抗反应得到。该囊泡体系拥有许多独特的优点，包括尺寸小、制备简单可控、生物相容性优异、体内循环稳定性高、肿瘤细胞特异选择性强、细胞内药物释放速度快、肿瘤生长抑制效果显著等。因此，该囊泡体系有望成为简单且集多功能于一身的纳米平台，用于高效及特异性靶向递送硫酸长春新碱至多发性骨髓瘤细胞。

Description

载小分子药聚合物囊泡及其制备方法与应用 技术领域
本发明属于聚合物纳米药物技术领域，具体涉及一种负载硫酸长春新碱的可逆交联可降解聚合物囊泡及其制备方法与在肿瘤靶向治疗中的应用。
 背景技术
硫酸长春新碱（VCR）是一种水溶性的强效药物，主要作用于微管蛋白，使有丝分裂停止于中期，但由于其存在严重的神经毒性，导致可用剂量较低。2012年批准上市的脂质体硫酸长春新碱（Marqibo®）纳米药物虽然可以延长VCR的循环时间、降低毒副作用，但整体改善较为有限。因此，如何实现VCR的高效稳定包裹及肿瘤靶向递送至关重要。现有技术公开了一种硫酸长春新碱脂质体及其制备方法，该硫酸长春新碱脂质体由硫酸长春新碱以及使用鞘磷脂制备的纳米脂质体组成，其中所述硫酸长春新碱包裹在所述纳米脂质体中，使用鞘磷脂来制备纳米脂质体，并包裹硫酸长春新碱后制得硫酸长春新碱脂质体，其中鞘磷脂含有较多的酰胺键能够更好地抵抗化学和生物的降解，保护脂质体结构的稳定，提高肿瘤细胞的药物富集量，从而提高抗肿瘤效果。现有技术还公开了包载硫酸长春新碱的双功能纳米粒制剂的制备方法，通过复乳法将硫酸长春新碱包封于叶酸/细胞穿透肽修饰的PLGA-PEG聚合物载体中，制得双功能纳米粒制剂；在体内外表现出良好的药代动力学行为，制备的叶酸/细胞穿透肽修饰的PLGA-PEG双功能纳米粒粒径287.2±0.8 nm，具有较高载药量和包封率，以及良好的稳定性。现有类脂质体结构的聚合物囊泡具有一个亲水内腔，可用于装载亲水性小分子药物，然而对VCR等亲水性药物的装载效率较低，且尚缺乏集体内循环稳定性、肿瘤特异靶向性、细胞内药物快速释放及生物相容性优异等多功能于一体的特性。
 技术问题
本发明的目的是公开两亲性嵌段聚合物、载药聚合物囊泡及其制备方法与应用，具体为一种负载硫酸长春新碱（VCR）的可逆交联可降解聚合物囊泡及其制备方法和应用。
 技术解决方案
为达到上述发明目的，本发明采用如下技术方案。
载药聚合物囊泡，由小分子药物、两亲性嵌段聚合物制备；或者由小分子药物、两亲性嵌段聚合物、官能团化PEG-P(TMC-DTC)、靶向单抗制备。
所述两亲性嵌段聚合物的分子结构式如下一种。
Figure 480018dest_path_image001
。
其中，z为5～15。
本发明中，所述两亲性嵌段聚合物中，PEG的分子量为3000～8000 Da；疏水链段的分子量为PEG分子量的2.5～6倍；PDTC链段的分子量为疏水链段分子量的8%～30%。本发明的两亲性嵌段聚合物有亲水链段（n链段）、疏水链段（x+y链段）、KD z链段（z链段），疏水链段、KD z链段通过氨酯键连接；所述两亲性嵌段聚合物表示为PEG-P(TMC-DTC)-KD z、PEG-P(LA-DTC)-KD z、PEG-P(CL-DTC)-KD z。
本发明中，所述小分子药物为硫酸长春新碱、阿霉素盐酸盐、表阿霉素盐酸盐、盐酸维拉帕米、盐酸伊立替康、瑞喹莫德，优选为硫酸长春新碱（VCR）；所述靶向单抗为靶向CD38单抗，如达雷木单抗（Dar）、艾沙妥昔单抗（Isa）或其它靶向CD38的单抗。
上述载药聚合物囊泡的制备方法为，以小分子药物、所述两亲性嵌段聚合物为原料，通过溶剂置换法制备载药聚合物囊泡；或者以小分子药物、所述两亲性嵌段聚合物、官能团化两亲性嵌段聚合物、靶向单抗为原料，通过溶剂置换法制备载药聚合物囊泡。优选的，将官能团化两亲性嵌段聚合物与所述两亲性嵌段聚合物组装交联并负载药物，然后与靶向CD38的单抗反应，制备载药聚合物囊泡。
本发明公开了上述两亲性嵌段聚合物或者载药聚合物囊泡在制备纳米药物中的应用；纳米药物为抗肿瘤药物；所述肿瘤优选为原位多发性骨髓瘤。
本发明负载硫酸长春新碱（VCR）的可逆交联可降解聚合物囊泡，由两亲性嵌段聚合物组装并交联后得到，其具有不对称膜结构，外壳为聚乙二醇（PEG），膜层为可逆交联的疏水聚碳酸酯，内壳为KD z，可以实现VCR的高效装载。本发明的载药囊泡为靶向或者非靶向结构，本发明的靶向分子为单抗分子或单抗片段等，所述单抗分子如达雷木单抗（Dar）、艾沙妥昔单抗（Isa）或其它靶向CD38的单抗。
本发明采用两亲性嵌段聚合物先与官能团化两亲性嵌段聚合物，作为原料制备载药囊泡，然后再连接靶向CD38的单抗，得到CD38靶向载药囊泡。官能团来自PEG引发剂，得到的聚合物PEG端带有可反应性官能团，比如叠氮（N 3），马来酰亚胺（Mal）或N-羟基琥珀酰亚胺（NHS），以两亲性嵌段聚合物PEG-P(TMC-DTC)为例，官能团化两亲性嵌段聚合物可以为N 3-PEG-P(TMC-DTC)、Mal-PEG-P(TMC-DTC)、NHS-PEG-P(TMC-DTC)。
本发明的载药囊泡由药物与囊泡组成，囊泡由聚合物交联得到，可以修饰也可以不修饰靶向分子；以两亲性嵌段聚合物PEG-P(TMC-DTC)、硫酸长春新碱为例，本发明载药囊泡的制备方法可以如下。
（1）将PEG-P(TMC-DTC)的端羟基通过氯甲酸对硝基苯酯活化，再与KD z反应制得PEG-P(TMC-DTC)-KD z。
（2）在PEG-P(TMC-DTC)的PEG端引入N 3、Mal或者NHS等官能团，得到官能化的PEG-P(TMC-DTC)。
（3）以硫酸长春新碱、PEG-P(TMC-DTC)-KD z为原料，通过溶剂置换法制备负载VCR的可逆交联可降解聚合物囊泡；或者以硫酸长春新碱、PEG-P(TMC-DTC)-KD z和官能化的PEG-P(TMC-DTC)为原料，通过溶剂置换法制备表面含有可反应性官能团的、负载VCR的、可逆交联、可降解聚合物囊泡，进而与单抗反应制备单抗导向的负载VCR的多功能囊泡。
本发明公开了上述负载VCR的可逆交联可降解聚合物囊泡及其制备方法，将PEG-P(TMC-DTC)-KD z聚合物的溶液注射入静置的VCR水溶液中，搅拌后透析，即得到负载VCR的可逆交联可降解聚合物囊泡（Ps-VCR）；具体为将VCR溶于超纯水中并与HEPES缓冲液（pH 6.8，10 mM）混合均匀，然后在静置下向其中注入PEG-P(TMC-DTC)-KD z聚合物的DMSO溶液，搅拌3-5分钟后用HEPES（pH 7.4，10 mM）透析，即得到Ps-VCR。
本发明还公开了单抗导向、负载VCR的、可逆交联可降解聚合物囊泡及其制备方法：将PEG-P(TMC-DTC)-KD z的DMSO溶液和官能化聚合物如N 3-PEG-P(TMC-DTC)的DMSO溶液混合均匀后，再注入含有VCR的HEPES溶液中，搅拌3-5分钟后，透析即可得到表面含有N 3的负载VCR的可逆交联聚合物囊泡；通过二苯并环辛炔修饰的单抗，如达雷木单抗（Dar）、艾沙妥昔单抗（Isa）或其它靶向CD38的单抗与叠氮官能化的载VCR囊泡（N 3-Ps-VCR）发生张力触动的点击化学反应，可在温和条件下制备得到单抗导向的负载VCR囊泡（Ab-Ps-VCR）。采用同样的方法，通过巯基官能化的单抗分子与表面含有Mal的载VCR囊泡发生迈克尔加成反应，或者单抗与NHS官能化的载VCR囊泡发生酰胺化反应也可简单制备得到Ab-Ps-VCR。
本发明的聚合物中，KD生物相容性好，结合PEG链段与疏水链段，可以形成不对称膜结构囊泡，实现小分子药物（如VCR）的高效稳定包载；本发明通过静电作用力包载VCR，同时被双硫交联的囊泡膜与外界分隔，可避免在输送过程中泄漏及被细胞黏附而造成的损失和毒副作用，能够高效送至病灶部位，并在体内还原剂谷胱甘肽（GSH）的作用下，快速释放VCR，有效杀伤肿瘤细胞。
本发明中的聚合物囊泡为内膜带负电荷的还原敏感可逆交联、细胞内可解交联且生物可降解的聚合物囊泡；所述聚合物为PEG-P(TMC-DTC)-KD z，其中中间嵌段的TMC（LA或者CL）与DTC呈无规排列；KD z的分子量为700-2000 Da ，远小于PEG段的分子量，在自组装、交联后得到内膜带有负电荷的可逆交联聚合物囊泡，囊泡的内壳为KD z用于复合小分子药物。囊泡膜为可逆交联的生物可降解且相容性好的PTMC，侧链的二硫戊烷结构类似人体天然的抗氧化剂硫辛酸，可自发形成还原敏感的可逆交联，不但可保证药物在血液中的稳定长循环，还可实现细胞内快速解交联，快速释放药物到靶细胞内。
本发明公开了上述负载VCR的肿瘤靶向、可逆交联可降解聚合物囊泡在制备抗肿瘤靶向纳米药物中的应用。优选的，肿瘤为多发性骨髓瘤。
 有益效果
与现有技术相比，本发明具有如下优点。
1. 本发明设计了新的小分子亲水药物VCR载药囊泡及肿瘤靶向递送；囊泡膜为可逆交联的生物可降解且生物相容性好的PTMC，侧链的二硫戊烷可提供还原敏感的可逆交联，不但可保证药物在血液中的长循环，还可在细胞内快速解交联，释放药物到靶细胞内；外壳为PEG同时具有单抗等靶向分子，可特异性结合癌细胞；囊泡的小尺寸以及肿瘤特异性靶向使得囊泡可高效输送VCR至肿瘤细胞内。
2. 本发明公开的载药囊泡体内外具有显著的抗肿瘤效果，聚合物生物相容性好，可形成不对称膜结构的囊泡，具有良好的药物包载效果。
3. 本发明的可降解聚合物囊泡载体避免了现有纳米载体粒径大、体内循环稳定性差、肿瘤细胞选择性低、细胞内VCR释放缓慢等缺陷。
4. 本发明的囊泡体系拥有许多独特的优点，包括尺寸小、制备简单可控、生物相容性优异、体内循环稳定性高、肿瘤细胞特异选择性强、细胞内药物释放速度快、肿瘤生长抑制效果显著等。因此，该囊泡体系有望成为简单且集多功能于一身的纳米平台，用于高效及特异性靶向递送VCR至多发性骨髓瘤细胞。
 附图说明
图1为实施例一中N 3-PEG-P(TMC-DTC)的核磁谱图。
图2为实施例二中PEG-P(TMC-DTC)-NPC的核磁谱图。
图3为实施例二中PEG-P(TMC-DTC)-KD 5的核磁谱图。
图4为实施例五中Dar与Dar-DBCO的大分子质谱图。
图5为实施例六中Dar-Ps-VCR在高倍稀释及血清存在下的稳定性图。
图6为实施例六中Dar-Ps-VCR在非还原条件及10 mM GSH下的VCR释放行为。
图7为实施例七中（A）不同靶向密度的Dar-Ps-Cy5在LP-1细胞中的内吞情况以及（B）LP-1细胞与Dar 4.4-Ps-Cy5和Ps-Cy5孵育4小时后的CLSM图片（标尺：25 μm）。
图8为实施例八中不同靶向密度的Dar-Ps-VCR、Ps-VCR和游离VCR在LP-1细胞中的毒性。
图9为实施例八中Dar-Ps-VCR和Ps-VCR在（A）MV4-11细胞和（B）L929细胞中的毒性以及（C）Dar-Ps和（D）Dar在LP-1细胞中的毒性。
图10为实施例九中流式细胞仪测定Dar-Ps-VCR、Ps-VCR和游离VCR诱导LP-1细胞凋亡的情况。
图11为实施例十中离体生物发光成像观察LP-1-Luc肿瘤在小鼠各器官、头颅以及后腿骨的肿瘤分布情况。
图12为实施例十一中荷原位LP-1-Luc多发性骨髓瘤小鼠在尾静脉注射Dar-Ps-Cy5和Ps-Cy5不同时间点后的活体荧光成像图。
图13为实施例十二中原位LP-1-Luc多发性骨髓瘤小鼠移植模型的构建和治疗工作流程以及活体成像评价Dar-Ps-VCR对原位LP-1-Luc多发性骨髓瘤小鼠模型的治疗效果图。
图14为实施例十二中不同治疗组小鼠的Luc荧光信号变化；体重变化以及Kaplan-Meier生存曲线图。
图15为实施例十二中不同治疗组小鼠股骨（Femur）和胫骨（Tibia）的micro-CT图。
图16为实施例十二中不同治疗组小鼠（A）股骨和（B）胫骨的各指标分析。（BMD：骨矿物密度；BV/TV：骨体积分数；Tb.N：骨小梁数目；Tb.Sp：骨小梁分离度；BS/TV：骨表面积；Tb.Th：骨小梁厚度。
 本发明的实施方式
本发明负载VCR的可逆交联可降解聚合物囊泡，由两亲性三嵌段聚合物自组装的同时发生自交联得到；所述三嵌段聚合物的分子链包括依次连接的亲水链段、疏水链段以及KD分子；所述亲水链段为聚乙二醇（PEG），分子量为3000-8000 Da；所述疏水链段为聚碳酸酯链段，分子量为亲水链段分子量的2.1-5.7倍；KD多肽的分子量为PEG亲水链段的15%-50%。
本发明PEG-P(TMC-DTC)-KD z聚合物通过氯甲酸对硝基苯酯（p-NPC）活化PEG-P(TMC-DTC)的末端羟基后，与KD z反应制得，合成路线如下。
Figure 181127dest_path_image002
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其中，在步骤（i）中，反应条件为无水二氯甲烷（DCM），吡啶，25 ºC，24小时；在步骤（ii）中，反应条件为无水二甲亚砜（DMSO），KD z，三乙胺，30 ºC，48小时。
具体合成步骤如下。
（1）在冰水浴中，向PEG-P(TMC-DTC)的无水DCM溶液中加入吡啶，搅拌10分钟后向其中缓慢滴加p-NPC的DCM溶液。滴加完成后（约30分钟）继续在室温下反应24小时，然后抽滤除去吡啶盐，收集聚合物溶液旋蒸浓缩至~100 mg/mL，经冰乙醚沉淀、真空干燥，得到产物PEG-P(TMC-DTC)-NPC。
（2）在氮气保护下，称取KD z多肽置于双颈圆底烧瓶中并加入无水DMSO使其完全溶解，在搅拌下加入三乙胺，然后向其中逐滴加入PEG-P(TMC-DTC)-NPC的无水DMSO溶液，30分钟滴加完成。在30 ºC下反应2天后，先用含有5%无水甲醇的DMSO透析36小时（更换4~5次介质）以除去未反应的KD z和反应生成的对硝基苯酚，再用DCM透析6小时，然后收集聚合物溶液并旋蒸浓缩至聚合物浓度约为50 mg/mL，在冰乙醚中沉淀后真空干燥，即得到白色棉絮状的聚合物PEG-P(TMC-DTC)-KD z。将TMC常规更换为LA或者CL，得到PEG-P(LA-DTC)-KD z、PEG-P(CL-DTC)-KD z。
本发明涉及的原料为现有市售原料，具体的制备方法以及测试方法为本领域常规技术；下面结合实施例和附图对本发明作进一步描述。
实施例一 合成聚合物N 3-PEG-P(TMC-DTC)。
聚合物N 3-PEG-P(TMC-DTC)是以DPP为催化剂，N 3-PEG-OH为大分子引发剂，引发TMC和DTC开环共聚合得到。首先，在手套箱氮气环境下称取N 3-PEG-OH（ M n = 7.9 kg/mol，0.79 g，0.1 mmol），TMC（1.50 g，14.8 mmol）和DTC（0.20 g，1.0 mmol）于密闭反应器中，加入5.0 mL无水DCM溶解，然后加入DPP（0.25 g，1.2 mmol），并密封好反应器转移出手套箱，置于30 ºC下反应四天。反应结束后，用冰乙醚沉淀两次，真空干燥后得到白色絮状聚合物N 3-PEG-P(TMC-DTC)，产率：85.4%。附图1中可以看到δ 3.38 和3.63 ppm处N 3-PEG的特征峰，δ 2.03和4.18 ppm处TMC的特征峰，以及δ 2.99和4.22 ppm处DTC的特征峰。通过δ 2.03和δ 2.99 ppm处的亚甲基氢积分面积与δ 3.63 ppm处PEG亚甲基氢积分面积比值可计算得到N 3-PEG-P(TMC-DTC)聚合物的分子量为7.9-(15.0-2.0) kg/mol，GPC测得其分子量分布为1.1，用于以下实施例。
将N 3-PEG-OH更换为分子量为5k的CH 3O-PEG-OH，其余不变，参照以上制备方法，得到PEG-P(TMC-DTC)（5.0-(15.0-2.0) kg/mol）。
实施例二 合成聚合物PEG-P(TMC-DTC)-KD z。
聚合物PEG-P(TMC-DTC)-KD z的合成分为两步，即采用p-NPC活化PEG-P(TMC-DTC)（5.0-(15.0-2.0) kg/mol）的末端羟基后，与KD z多肽分子反应得到。以PEG-P(TMC-DTC)-KD 5的合成为例，具体操作如下，在氮气氛围下将PEG-P(TMC-DTC)（1.0 g，45.5 μmol）溶解于10 mL无水DCM中，然后转移至冰水浴中并加入吡啶（18.0 mg，227.5 μmol），搅拌10分钟后向其中滴加p-NPC（48.4 mg，240.3 μmol）的DCM溶液（1.0 mL）。3 0分钟滴加完成后继续在室温下反应24小时，接着抽滤除去吡啶盐，收集聚合物溶液旋蒸浓缩至~100 mg/mL，经冰乙醚沉淀、真空干燥，得到产物PEG-P(TMC-DTC)-NPC，产率：90.0%。随后，在氮气保护下，称取KD 5（60.0 mg，83.4 μmol）溶解于4 mL无水DMSO中并加入三乙胺（4.2 mg，41.7 μmol），然后在搅拌下向其中逐滴加入PEG-P(TMC-DTC)-NPC的无水DMSO溶液（9.0 mL），30分钟滴加完成。在30 ºC下反应2天后，用含有5%无水甲醇的DMSO透析36小时（更换4~5次介质）以除去未反应的KD 5和反应生成的对硝基苯酚，再用DCM透析6小时，然后收集聚合物溶液并旋蒸浓缩至聚合物浓度为50 mg/mL，在冰乙醚中沉淀并真空干燥，即得到白色棉絮状的聚合物PEG-P(TMC-DTC)-KD 5，产率：91.0%。附图2和3是PEG-P(TMC-DTC)-NPC和PEG-P(TMC-DTC)-KD 5的核磁氢谱图。从附图2中可以看到p-NPC的特征峰（δ 7.41和δ 8.30 ppm）以及PEG-P(TMC-DTC)的特征峰（δ 2.03、2.99、3.38、3.63、4.18和4.22 ppm），根据p-NPC特征峰的积分面积与δ 3.38 ppm处PEG甲基氢峰面积比值计算得到NPC的接枝率约为100%。附图3可以看到δ 7.41和δ 8.30 ppm处NPC的特征峰消失，且在δ 4.54 ppm处出现了一个新的信号峰，即为KD 5中次甲基的特征峰。通过比较δ 4.54 ppm处峰面积与δ 1.95 ppm处TMC亚甲基氢峰面积的比值计算得到KD 5的取代度为~100%。此外，通过高效液相色谱（HPLC）测得KD 5的接枝率为100%，证明PEG-P(TMC-DTC)-KD 5的成功合成，用于以下实施例。
实施例三 负载VCR的可逆交联生物可降解囊泡（Ps-VCR）的制备。
Ps-VCR通过溶剂置换法制备，其中VCR通过与KD z之间的静电相互作用进行包裹。将PEG-P(TMC-DTC)-KD z溶解于DMSO中（40 mg/mL），取100 µL打入静置的含有VCR的900 µL HEPES（pH 6.8，10 mM）中，在300 rpm下搅拌3分钟后，用HEPES（pH 7.4，10 mM）透析8小时即得到Ps-VCR。其中VCR的理论载药量设定为4.8-11.1 wt.%，研究发现所得Ps-VCR的粒径在26-40 nm之间，粒径分布在0.05-0.20（表1）。通过紫外可见光谱测定其在298 nm波长下的吸光值计算得到Ps-VCR的包封率高达97.2%。基于同样的方法，理论载药量4.8%下，PEG-P(LA-DTC)-KD 5、PEG-P(CL-DTC)-KD 5制备的Ps-VCR的包封率分别为88.3%、83.9%；而采用PEG-P(TMC-DTC)两嵌段聚合物制备的载药囊泡粒径在75 nm左右，且VCR的包封率较低，仅有14.1%。
Figure 310757dest_path_image003
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实施例四 负载其它药物的可逆交联生物可降解囊泡（Ps-drug）的制备。
采用实施例三中类似的方法，研究了可逆交联可降解囊泡对其它药物如盐酸维拉帕米（VER）、盐酸伊立替康（CPT）、瑞喹莫德（R848）的包载。研究发现在包载不同药物后，所得Ps-drug的粒径在20-40 nm之间，具体结果见表2。
Figure 921867dest_path_image004
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实施例五 负载VCR的单抗导向聚合物囊泡（Ab-Ps-VCR）的制备。
Ab-Ps-VCR通过在叠氮官能化的聚合物囊泡VCR纳米药物（N 3-Ps-VCR）表面后修饰二苯并环辛炔官能化的单抗（Ab-DBCO）得到。N 3-Ps-VCR由N 3-PEG-P(TMC-DTC)和PEG-P(TMC-DTC)-KD z共组装的同时包裹VCR而得到，其中N 3-PEG-P(TMC-DTC)的含量为1~10 wt.%。具体地，以含有2% N 3-PEG-P(TMC-DTC)的N 3-Ps-VCR的制备为例，称取8.0 mg N 3-PEG-P(TMC-DTC)和392.0 mg PEG-P(TMC-DTC)-KD 5（摩尔比2∶98）溶解于DMSO中（聚合物总浓度为40 mg/mL），同时将4.0 mL VCR的水溶液（5 mg/mL）加入到90 mL HEPES（pH 6.8，10 mM）中混合均匀，在静置下向其中注入10 mL聚合物溶液，搅拌5分钟后，置于37 ºC静置4小时。用HEPES（pH 7.4，10 mM）透析（MWCO：14 kDa）8小时除去有机溶剂后，采用纳滤系统除去游离的VCR，得N 3-Ps-VCR。动态光散射（DLS）测得N 3-Ps-VCR的粒径为36 nm，且分布较窄（PDI：0.11）。当VCR的理论载药量为4.8 wt.%时，包封率高达97.2%，载药量为4.6 wt.%。为了高效地键合单抗，随后采用切向流装置将N 3-Ps-VCR由4 mg/mL浓缩到18.6 mg/mL，以方便储存并提高单抗的键合效率。浓缩后N 3-Ps-VCR的粒径为42 nm，PDI为0.07。其在4 ºC储存180天期间粒径均保持在40 nm左右，PDI小于0.17，且VCR的泄漏量低于0.6%，说明N 3-Ps-VCR具有优异的长期储存稳定性（表3）。
Figure 478750dest_path_image005
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Ab-DBCO通过小分子NHS-OEG 4-DBCO与单抗上的氨基发生酰胺化反应制备得到，其中DBCO的官能化度可通过改变Ab与NHS-OEG 4-DBCO的摩尔比进行调节。以DBCO官能化达雷木单抗（Dar-DBCO）的制备为例，用PB（pH 8.5，10 mM）将Dar的PBS溶液（21.7 mg/mL）稀释到10 mg/mL，取200 μL在振荡下向其中加入3或5倍摩尔当量的NHS-OEG 4-DBCO的DMSO溶液（5 mg/mL），置于27 ºC，120 rpm摇床中反应过夜。反应结束后，用超滤管离心（MWCO：10 kDa，3000 rpm）除去未反应的NHS-OEG 4-DBCO，并用PBS（pH 7.4，10 mM）洗涤超滤两次，得到Dar-DBCO。当Dar与NHS-OEG 4-DBCO的摩尔比为1∶3和1∶5时，通过飞行时间质谱（MALDI-TOF-MS）测得每个Dar上分别修饰了1.5和2.8个DBCO（附图4），表示为Dar-DBCO 1.5和Dar-DBCO 2.8。为了最大程度地保持单抗的靶向性及生物学活性，后续均采用Dar-DBCO 1.5或者修饰有1.5-2个DBCO的其它单抗进行实验。
通过N 3-Ps-VCR表面的N 3与Dar-DBCO之间发生张力触动的点击化学反应可简单制备得到Dar-Ps-VCR，Dar的表面密度可通过改变投料比进行调节。设定Dar-DBCO与N 3的摩尔比分别为0.25∶1、0.5∶1和1∶1，即在107.5 μL N 3-Ps-VCR（18.6 mg/mL）中分别加入10.4、20.9和41.8 μL的Dar-DBCO溶液（5.6 mg/mL），然后在25 ºC、100 rpm摇床中反应过夜。采用超速离心（58 krpm，4 ºC、30分钟）除去未键合的Dar-DBCO，并用HEPES（pH 7.4，10 mM）洗涤两次，同时收集Dar-Ps-VCR和上清以测定Dar的键合量。上清中未键合的Dar-DBCO通过HPLC测定，从而计算出每毫克聚合物囊泡表面Dar的含量分别为28.6、56.4和112.2 μg，根据多角度激光光散射测得的聚合物囊泡的绝对分子量（1.15×10 7g/mol）和聚集数（523个）计算可知每个Dar-Ps-VCR表面分别键合有2.2、4.4和8.7个Dar（表4）。随着Dar密度的增加，Dar-Ps-VCR的粒径略有增加（43-49 nm），粒径分布较窄（PDI：0.14-0.21），接了单抗后，包封结果与本实施例N 3-Ps-VCR一样。
Figure 417887dest_path_image006
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其它单抗导向的负载VCR聚合物囊泡，如Isa-Ps-VCR和Anti-CD38-Ps-VCR的制备方法均与Dar-Ps-VCR类似。其粒径在40-60 nm之间，粒径分布较窄（PDI：0.10-0.30），每个囊泡表面单抗的个数为1-10个。
现有技术CN110229323A表7公开的载皂草素蛋白（SAP）非靶向囊泡（KD 5）经过超速离心（58 krpm，4 ºC、30分钟）后，DLE由68.3%下降至23%，药物大量泄漏，说明其无法接靶向单抗。
实施例六 Ab-Ps-VCR靶向聚合物囊泡纳米药物的稳定性及体外药物释放。
采用每个囊泡表面含有4.4个Dar的Dar 4.4-Ps-VCR为代表，研究Ab-Ps-VCR靶向囊泡纳米药物的稳定性及体外药物释放行为。Dar-Ps-VCR的稳定性分别采用磷酸缓冲溶液稀释50倍或者加入10%的胎牛血清，并通过动态光散射检测其粒径变化。附图5为Dar-Ps-VCR稳定性的粒径分布图。结果表明，Dar-Ps-VCR靶向囊泡纳米药物在稀释50倍以及加入10% FBS 24小时后均保持完好的粒径和粒径分布，具有良好的稳定性。
Dar-Ps-VCR的体外药物释放行为采用透析法研究，其中有2种释放介质，分别为HEPES（pH 7.4，10 mM）和含有10 mM GSH的HEPES溶液（氮气环境）。首先将0.5 mL Dar-Ps-VCR（0.5 mg/mL）装进释放袋（MWCO：14 kDa）中，然后置于20 mL相应的释放介质中，于37 ºC、100 rpm摇床中进行。在设定的时间点（0、1、2、4、6、8、10、12、24 h）取出5 mL透析液，并补加5 mL新鲜介质。透析液中VCR的含量通过HPLC（流动相为甲醇：水（加入15%三乙胺，再用磷酸调节pH为7.0）= 70：30）测定。附图6为Dar-Ps-VCR靶向囊泡纳米药物的体外释放结果图。结果表明，Dar-Ps-VCR在10 mM GSH的还原条件下，12小时内VCR的释放量达到85%以上，而在非还原条件下，24小时内VCR的累积释放量只有22%左右。
实施例七 Dar-Ps-VCR靶向聚合物囊泡纳米药物的细胞内吞行为。
由于VCR本身无荧光，采用Cy5标记聚合物囊泡，Dar-Ps-Cy5的制备方法参考实施例五，Ps-Cy5的制备方法参考实施例三；通过流式细胞仪和激光扫描共聚焦显微镜（CLSM）研究不同Dar密度的Dar-Ps-Cy5在LP-1细胞中的摄取情况。流式实验中，首先将LP-1细胞悬液铺在6孔板中（2×10 5个/孔），置于培养箱孵育24小时后，每孔加入200 μL Dar-Ps-Cy5和Ps-Cy5（Cy5孔内浓度为2.0 μg/mL），用PBS组作为对照。继续孵育4小时后，离心（800 rpm，5分钟）收集细胞，并用PBS清洗两次，最后用500 μL PBS分散并置于流式管中进行测定。测试结果显示，Dar-Ps-Cy5在LP-1细胞中的内吞量明显高于Ps-Cy5，其中与Dar 4.4-Ps-Cy5孵育的细胞具有最高的荧光强度，其荧光强度是Ps-Cy5对照组的6.4倍（附图7A），表明Dar的引入可显著增强Ps-Cy5的细胞摄取，且当每个囊泡表面键合4.4个Dar时靶向性最佳。
随后采用CLSM进一步研究了Dar 4.4-Ps-Cy5和Ps-Cy5在LP-1细胞中的内吞行为。具体实验步骤如下，将多聚赖氨酸（300 μL，0.1 mg/mL）预处理的小圆片置于24孔板中，并加入LP-1细胞悬液（3×10 5个/孔），于培养箱中培养24小时后，分别加入200 μL Dar 4.4-Ps-Cy5和Ps-Cy5（Cy5孔内浓度为40 μg/mL）。继续孵育4小时后小心移去培养基，用PBS洗3次，接着用4%多聚甲醛溶液固定15分钟，用PBS洗3次，再用DAPI染细胞核3分钟，用PBS清洗3次，最后采用甘油封片并用CLSM（Leica，TCS SP5）进行观察和拍摄。附图7B为Dar 4.4-Ps-Cy5和Ps-Cy5在LP-1细胞中的摄取结果图。结果表明，当LP-1细胞与Dar 4.4-Ps-Cy5孵育4小时后，细胞核周围呈现出明显的红色荧光，而与Ps-Cy5孵育的细胞中荧光较为微弱，表明Dar-Ps-Cy5具备优异的靶向性及高效快速的细胞内吞。
实施例八 Dar-Ps-VCR靶向聚合物囊泡纳米药物的细胞毒性实验。
Dar-Ps-VCR对LP-1多发性骨髓瘤细胞的体外抗肿瘤活性采用CCK-8试剂盒进行测定，以MV4-11细胞作为对照。先将LP-1细胞铺于96孔板中（15000个/孔），置于37 ºC、含5% CO 2的培养箱中培养24小时后，向每孔加入20 μL含有不同Dar表面密度的Dar-Ps-VCR、Ps-VCR和游离的VCR，孔内VCR的最终浓度分别为0.001、0.01、0.05、0.1、0.5、1和10 ng/mL。在37 ºC孵育48小时后，每孔加入10 μL CCK-8溶液继续孵育4小时，最后用酶标仪测试其在492 nm处的吸光度值。细胞存活率通过实验组吸光度值与加入PBS培养的细胞的吸光度值的比值计算得到，实验平行进行四组（mean ± SD，z = 4）。附图8为不同靶向密度的Dar-Ps-VCR囊泡纳米药物（z为5）对LP-1细胞的细胞毒性结果图。结果表明，当每个囊泡表面键合4.4个Dar时（Dar 4.4-Ps-VCR）细胞毒性最强，其半致死浓度（IC 50）低至0.07 ng/mL，相比游离VCR（IC 50：1.38 ng/mL）和非靶向对照组Ps-VCR（z为5，IC 50：0.85 ng/mL）分别降低了20和12倍，说明Dar的引入显著增加了VCR的靶向递送和细胞内快速释放。
将MV4-11细胞（12000个/孔）和L929成纤维细胞（3000个/孔）分别铺于96孔板中培养24小时，然后向每孔加入20 μL Dar 4.4-Ps-VCR（z为5）和Ps-VCR（z为5），孔内VCR的最终浓度为0.0001-100 ng/mL。MV4-11细胞在37 ºC孵育48小时后，每孔加入10 μL CCK-8溶液继续孵育4小时，并用酶标仪测试其在492 nm处的吸光度值。L929细胞在37 ºC孵育48小时后，向每孔中加入10 μL MTT的PBS溶液（5 mg/mL）孵育4小时，随后小心移除培养基并加入150 μL DMSO溶解产生的甲瓒结晶，用酶标仪测试其在570 nm处的吸光度；结果显示，在MV4-11细胞中，IC 50是LP-1细胞中的20倍之高（附图9A）。更为有趣的是，对于L929正常细胞而言，即使在VCR浓度高达100 ng/mL时，Dar 4.4-Ps-VCR和Ps-VCR也没有表现出明显的毒性，细胞存活率均接近100%（附图9B）。这些结果综合表明Dar-Ps-VCR可选择性靶向并高效杀伤多发性骨髓瘤细胞，而对正常细胞毒性较小。
此外，采用同样的方法测试Dar-Ps和Ps空囊泡以及游离Dar对LP-1细胞的毒性，结果表明即使在Ps浓度高达30 μg/mL（附图9C），Dar浓度为9 μg/mL时（附图9D），细胞存活率均接近100%，没有明显的细胞毒性。
以下实施例中Dar-Ps-VCR均是指Dar 4.4-Ps-VCR囊泡纳米药物（z为5），Dar-Ps-Cy5均为Dar 4.4-Ps-Cy5（z为5）。
实施例九 Dar-Ps-VCR靶向聚合物囊泡纳米药物诱导细胞凋亡的情况。
Dar-Ps-VCR的细胞凋亡实验通过荧光染料AnnexinV-FITC/PI双染处理，然后用流式细胞仪进行测试。首先将LP-1细胞以2×10 5个/孔的密度铺在6孔板中，培养箱中培养24小时后，分别加入200 μL Dar-Ps-VCR、Ps-VCR和游离VCR（VCR的孔内浓度为0.5 ng/mL），只加PBS的细胞作为对照。放入培养箱孵育48小时后，离心（800 rpm，5分钟）收集LP-1细胞，并用冰PBS洗涤两遍，最后向每个样品中加入200 μL结合缓冲液（Binding buffer）重悬细胞（细胞密度大约为10 6个/mL）。吹打均匀后取100 μL至流式管内依次加入5 μL AnnexinV-FITC和10 μL PI溶液，室温避光染色15分钟后，再加入400 μL PBS混合均匀，1小时内用流式细胞仪测定。其中以放入50 ºC水浴锅中处理5分钟及用4%多聚甲醛固定5分钟的PBS组样品分别作为早凋组和晚凋组，并分别加入5 μL AnnexinV-FITC溶液和10 μL PI溶液染色15分钟。附图10为Dar-Ps-VCR诱导LP-1细胞凋亡的结果图。结果表明，Dar-Ps-VCR能够有效诱导细胞凋亡，当VCR浓度为0.5 ng/mL时，可引起60.8%的细胞凋亡，细胞凋亡率明显高于非靶向对照Ps-VCR组（43.4%）和游离VCR组（31.4%），且各组晚期凋亡的细胞数要显著多于早期凋亡。
实施例十 荷LP-1-Luc原位多发性骨髓瘤小鼠模型的构建。
所有动物实验及操作均获得苏州大学实验动物中心和苏州大学动物护理和使用委员会的批准。原位MM肿瘤模型的建立：采用6周龄的ZOD/SCID雌性小鼠，首先连续两天通过腹腔注射10 mg/mL的环磷酰胺溶液对小鼠进行清髓，每只小鼠每次注射2 mg，第三天将LP-1-Luc细胞（8×10 6个/只）通过尾静脉注射到小鼠体内，接种后第10天开始活体成像和治疗，同时对小鼠进行称重。为了研究LP-1-Luc原位多发性骨髓瘤小鼠移植模型的肿瘤分布情况，在接种后第35天，通过小鼠腹腔注射荧光素钾盐，8分钟后解剖收集小鼠心、肝、脾、肺、肾、肠、头颅以及后腿骨进行荧光成像。附图11为荷LP-1-Luc原位多发性骨髓瘤小鼠各器官、头颅以及后腿骨的离体生物发光成像结果图。从图片中可以看出接种35天后，LP-1-Luc肿瘤主要集中在小鼠的后腿及头颅上，心、肝、脾、肺、肾等器官中没有明显的肿瘤Luc信号分布情况。
实施例十一 Dar-Ps-Cy5在荷LP-1-Luc原位多发性骨髓瘤小鼠中的活体成像实验。
Dar-Ps-Cy5在LP-1-Luc原位多发性骨髓瘤小鼠体内的分布情况通过小鼠活体成像分析得到。在接种后第37天（小鼠即将发病时），将200 μL Dar-Ps-Cy5和Ps-Cy5溶液（250 µg Cy5 equiv./kg）通过尾静脉分别注射到小鼠体内，注射后分别在1、2、4、6、8、10、12、24小时采用异氟烷麻醉小鼠进行活体荧光成像，采用Lumia II软件对小鼠成像性骨髓瘤小鼠中的生物分布图（附图12）。结果显示，Dar-Ps-Cy5能够高效靶向富集到肿瘤部位，其在小鼠腿部及头颅的荧光信号显著高于非靶向的Ps-Cy5组。
实施例十二 Dar-Ps-VCR在荷LP-1-Luc原位多发性骨髓瘤小鼠中的抗肿瘤效果。
为了研究Dar-Ps-VCR对荷原位LP-1-Luc多发性骨髓瘤小鼠的抗肿瘤效果，在接种后第10天生物发光强度达到1.2×10 6 p/sec/cm 2/sr时开始治疗实验。其给药方案有两种，保持VCR的总给药量相同：一种是VCR剂量为0.25 mg/kg，4天给一针，共4针，表示为Dar-Ps-VCR（0.25 mg VCR equiv./kg，Q4d）；另一种是VCR剂量为0.50 mg/kg，8天给一针，共2针，表示为Dar-Ps-VCR（0.50 mg VCR equiv./kg，Q8d）。依据第一种给药方案，采用同等VCR剂量的Ps-VCR和游离VCR，等当量的Dar-Ps以及PBS作为对照。每个治疗组均有10只荷瘤小鼠，其中4只用于生物发光成像，6只用于监测体重及观察生存期。研究发现PBS组小鼠的LP-1-Luc细胞持续快速增长，在接种后37-45天生物发光强度达到1.0×10 9 p/sec/cm 2/sr时开始发病，表现为双腿瘫痪、体重下降并发生死亡（附图13）。在给药治疗期间（10-22天），Dar-Ps-VCR两种给药方式组、Ps-VCR及游离VCR组小鼠的Luc信号无明显增长甚至稍有下降，表明它们可有效抑制小鼠体内LP-1-Luc细胞的扩散和增殖。给药结束后，非靶向Ps-VCR和游离VCR组小鼠的肿瘤开始复发，Luc生物发光信号快速增长，在第39天时即分别增加了157和53倍。然而尾静脉注射Dar-Ps-VCR的两组小鼠仍可持续抑制LP-1-Luc肿瘤的生长，即使在接种后第39天时也没有检测到明显的Luc信号，其荧光值与健康小鼠的背景信号相当，相比Ps-VCR和游离VCR组分别降低了235和114倍，相比未治疗的PBS组则降低了5000倍左右。空载体Dar-Ps组小鼠的肿瘤生长趋势与PBS组相似（附图14A）。这些结果表明Dar-Ps-VCR可高效靶向递送VCR至肿瘤部位，短期内完全消灭了肿瘤。值得注意的是，所有治疗组小鼠在给药期间体重无明显变化，直至小鼠发病时会出现体重下降，表明它们的毒副作用相对较低（附图14B）。此外，Dar-Ps-VCR治疗组小鼠的生存期得到了显著延长（附图14C），其中0.25 mg VCR equiv./kg，Q4d 和0.50 mg VCR equiv./kg，Q8d 两种给药方式下，小鼠的中位生存期分别为156和154天，相比PBS组（43天）、Dar-Ps组（49.5天）、Ps-VCR（51天）和游离VCR组（52天）延长了3.0-3.6倍。这些结果综合表明Dar的引入显著增加了Ps-VCR的选择性靶向，从而高效抑制了原位多发性骨髓瘤的生长。
溶骨性病变是MM患者的常见临床表现之一，因此采用micro-CT评价了各治疗组小鼠股骨和胫骨的相关指标。结果发现PBS组和Dar-Ps组小鼠后腿骨存在严重的破骨现象，骨小梁大量缺失，而经Dar-Ps-VCR治疗后，小鼠的溶骨性病变得到明显改善，与健康小鼠的情况相似（附图15）。随后通过进一步的定量分析，发现两种给药方式下的Dar-Ps-VCR靶向治疗组小鼠股骨和胫骨的骨矿物密度、骨体积分数、骨小梁数目、骨小梁分离度以及骨表面积等指标相近，与未治疗的PBS组之间存在显著性差异，而与健康小鼠的各项指标之间无明显差异（附图16）。此外，有些指标如骨体积分数等与非靶向Ps-VCR对照组之间也存在着显著性差异，进一步表明Dar的引入显着增加了Ps-VCR对原位多发性骨髓瘤的疗效，有效抑制了溶骨性病变。

Claims (10)

  1. 载药聚合物囊泡，其特征在于，由小分子药物、两亲性嵌段聚合物制备；或者由小分子药物、两亲性嵌段聚合物、官能团化两亲性嵌段聚合物、靶向分子制备；
    所述两亲性嵌段聚合物的分子结构式如下一种：
    Figure 922360dest_path_image001
    其中，z为5～15。
  2. 根据权利要求1所述载药聚合物囊泡，其特征在于，所述两亲性嵌段聚合物中，PEG链段的分子量为3000～8000 Da；疏水链段的分子量为PEG分子量的2.5～6倍；PDTC链段的分子量为疏水链段分子量的8%～30%。
  3. 根据权利要求1所述载药聚合物囊泡，其特征在于，所述小分子药物为硫酸长春新碱、阿霉素盐酸盐、表阿霉素盐酸盐、盐酸维拉帕米、盐酸伊立替康、瑞喹莫德。
  4. 根据权利要求1所述载药聚合物囊泡，其特征在于，所述靶向分子为靶向单抗。
  5. 根据权利要求4所述载药聚合物囊泡，其特征在于，所述靶向单抗为靶向CD38单抗。
  6. 权利要求1所述载药聚合物囊泡在制备抗骨髓瘤药物中的应用。
  7. 权利要求1所述载药聚合物囊泡的制备方法，其特征在于，包括以下步骤，以小分子药物、所述两亲性嵌段聚合物为原料，通过溶剂置换法制备载药聚合物囊泡；或者以小分子药物、所述两亲性嵌段聚合物、官能团化两亲性嵌段聚合物、靶向单抗为原料，通过溶剂置换法制备载药聚合物囊泡。
  8. 根据权利要求7所述载药聚合物囊泡的制备方法，其特征在于，将官能团化两亲性嵌段聚合物与所述两亲性嵌段聚合物组装交联并负载药物，然后与靶向单抗反应，制备载药聚合物囊泡；所述官能团化两亲性嵌段聚合物中的官能团为N 3-、Mal-或者NHS-。
  9. 权利要求1所述两亲性嵌段聚合物在制备抗骨髓瘤纳米药物中的应用，其特征在于，所述纳米药物的活性成分为小分子药物。
  10. 权利要求1所述两亲性嵌段聚合物、官能团化两亲性嵌段聚合物和靶向分子；在制备抗骨髓瘤纳米药物中的应用，其特征在于，所述纳米药物的活性成分为小分子药物。
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