WO2022056985A1 - 碳酸钙聚(乳酸-羟基乙酸)复合物微粒及其制备和应用 - Google Patents

碳酸钙聚(乳酸-羟基乙酸)复合物微粒及其制备和应用 Download PDF

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WO2022056985A1
WO2022056985A1 PCT/CN2020/121604 CN2020121604W WO2022056985A1 WO 2022056985 A1 WO2022056985 A1 WO 2022056985A1 CN 2020121604 W CN2020121604 W CN 2020121604W WO 2022056985 A1 WO2022056985 A1 WO 2022056985A1
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glycolic acid
acid
calcium carbonate
tumor
nlg919
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French (fr)
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刘庄
冯良珠
杨志娟
郝钰
朱宇杰
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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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—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
    • A61K31/4164—1,3-Diazoles
    • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • 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
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1611—Inorganic compounds
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the field of tumor treatment preparations, in particular to a calcium carbonate poly(lactic-glycolic acid) composite particle and its preparation and application.
  • Tumor refers to a local mass formed by abnormal proliferation of cells in local tissues under the action of various tumorigenic factors. Malignant tumors are what people call cancer. Chemotherapy remains the mainstay of treatment for various cancers. It is generally believed that chemotherapy works through its own cytotoxic effects. However, increasing evidence suggests that some specific chemotherapeutic drugs not only kill tumor cells through cytotoxicity, but also induce immune responses that play an important role in the overall antitumor activity. Recent studies have shown that the antitumor chemotherapeutic drug doxorubicin (DOX) can induce immunogenic cell death (ICD) through damage-associated molecular patterns (DAMPs), thereby eliciting antitumor immunity. ICD-induced immunogenicity promotes intratumoral infiltration of cytotoxic T lymphocytes (CTLs), which in turn effectively inhibits tumor growth.
  • CTLs cytotoxic T lymphocytes
  • the immune response induced by chemotherapy is affected by the immunosuppressive microenvironment within the tumor, and reversing the tumor immunosuppressive microenvironment can activate the body's antitumor immune response.
  • CTLA-4 immunosuppressive cytotoxic T lymphocyte-associated protein 4
  • PD-1 programmed cell death protein 1
  • IDO-1 indoleamine 2,3-dioxygenase 1
  • Trp intratumoral tryptophan
  • Kyn kynurenine
  • the accumulation of kynurenine in the tumor will not only reduce the infiltration of Th1, CTL and other T cells into the tumor and induce their apoptosis, but also activate the immune regulatory T cells (Tregs) in the tumor, resulting in severe immunosuppression. sexual microenvironment.
  • the object of the present invention is to provide a kind of calcium carbonate poly(lactic-glycolic acid) composite particles and preparation and application thereof, and the calcium carbonate poly(lactic-glycolic acid) composite particles of the present invention can be used as medicines
  • the carrier has good drug encapsulation efficiency and tumor enrichment ability, realizes the co-encapsulation of the chemotherapeutic drug DOX and the IDO inhibitor aNLG919, and can effectively release the chemotherapeutic drug doxorubicin, inhibit the IDO immunosuppressive pathway, and effectively reverse tumor immunity. Inhibit the microenvironment, activate tumor immunity, inhibit tumor growth, and realize tumor chemotherapy or combined chemotherapy and immune therapy.
  • the first object of the present invention is to provide a calcium carbonate poly(lactic-glycolic acid) complex particle, which is modified with alkyl groups NLG919, polylactic-glycolic acid (PLGA) and polylactic-glycolic acid-polyethylene Diol (PLGA-PEG) is the shell layer and calcium carbonate (CaCO 3 ) and doxorubicin (DOX) are the core layers.
  • PLGA polylactic-glycolic acid
  • PLGA-PEG polylactic-glycolic acid-polyethylene Diol
  • CaCO 3 calcium carbonate
  • DOX doxorubicin
  • the particle size of the calcium carbonate poly(lactic acid-glycolic acid) composite particles is 100 to 180 nm.
  • the second object of the present invention is to provide a preparation method of the above-mentioned calcium carbonate poly(lactic acid-glycolic acid) composite particles, comprising the following steps:
  • step A) the mass ratio of polylactic acid-glycolic acid and polylactic acid-glycolic acid-polyethylene glycol is 1 ⁇ 2:1 ⁇ 2;
  • the alkyl-modified NLG919 is a C2-C20 alkyl-modified NLG919.
  • step B the concentration of NaHCO 3 solution is 0.6-0.7M, and the concentration of calcium chloride in the calcium chloride doxorubicin solution is 1-1.5M.
  • step A) the mass ratio of the sum of polylactic acid-glycolic acid and polylactic acid-glycolic acid-polyethylene glycol, doxorubicin and alkyl-modified NLG919 is (5 ⁇ 10): (0.3 ⁇ 1 ): (0.3 ⁇ 2); In step A), the mass ratio of CaCl 2 and doxorubicin is 25 ⁇ 30:1;
  • step B the volume ratio of the oil phase mixed solution to the NaHCO 3 solution is 3 to 4:1; the volume ratio of the oil phase mixed solution to the calcium chloride doxorubicin solution is 3 to 4:1.
  • step C) the emulsification is phacoemulsification, and the ultrasonic power is 80-120W; the ultrasonic time is 250-350s; in step D), the mass fraction of the polyvinyl alcohol aqueous solution is 1%-2%; The time is 10-14h; the washing is centrifuged at 14000-15000rpm for 20-30min, and washed 2-3 times with ultrapure water.
  • the third object of the present invention is to disclose the application of the above calcium carbonate poly(lactic-glycolic acid) complex particles in the preparation of drugs for tumor chemotherapy or drugs for combined tumor chemotherapy and immunotherapy.
  • the drug is a drug responsive to the acid environment of the tumor, and the pH value of the acid environment is 5.5-6.5.
  • the drug is used to treat solid tumors such as breast cancer and colon cancer.
  • the pharmaceutical dosage form is an injection.
  • the present invention has at least the following advantages:
  • the invention provides calcium carbonate poly(lactic-glycolic acid) composite particles and a preparation method thereof.
  • the particles use alkyl-modified NLG919, PLGA and PLGA - PEG as shell layers, and CaCO3 and DOX as core layers. It has uniform diameter, good dispersion in water, and good stability under physiological conditions. It has good loading efficiency for both hydrophobicized NLG919 and hydrophilic doxorubicin hydrochloride, and is an ideal drug carrier.
  • Kinetic behavior, and the presence of calcium carbonate make the compound preparation have the ability to respond to the acidic microenvironment of the tumor, realize the responsive release of the drug, and reduce the toxic and side effects.
  • the invention also discloses the application of the calcium carbonate poly(lactic acid-glycolic acid) complex particles in the preparation of a drug for tumor chemotherapy or a drug for combined tumor chemotherapy and immunotherapy.
  • the calcium carbonate poly(lactic-glycolic acid) complex particles were injected into mice through the tail vein, and their in vivo pharmacokinetic behavior was monitored by fluorescence imaging technology. It was found that calcium carbonate poly(lactic-glycolic acid) complex preparations It can be effectively enriched in the tumor site, and has a long circulation time in mice, has a good tumor synergistic treatment effect, and can be used for tumor chemotherapy and immunotherapy.
  • Fig. 1 is the transmission electron microscope image of calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 preparation
  • Fig. 2 is the particle size distribution diagram of calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 formulation
  • Fig. 3 is the test result of the drug-loading capacity of calcium carbonate poly(lactic-glycolic acid) complex preparation to different molecules;
  • Fig. 4 is the DOX release curve of calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 preparations under different pH conditions;
  • Figure 5 is an IDO inhibition curve of calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 formulations
  • Figure 6 is a tumor-enriched fluorescence image after intravenous injection of calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 formulation
  • Figure 7 is a biodistribution graph after intravenous injection of calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 formulation
  • Figure 8 is a tumor growth curve of a mouse colon cancer subcutaneous tumor model.
  • Example 1 Preparation of calcium carbonate poly(lactic acid-glycolic acid) complex-DOX-NLG919 preparation
  • NLG919 was dissolved in dichloromethane solution, and N,N-diisopropylethylamine (DIPEA) was used as base, and dodecyl chloride was added to the reaction system under ice bath conditions to react to generate alkyl-modified NLG919.
  • DIPEA N,N-diisopropylethylamine
  • the product aNGL919 was obtained by liquid chromatography column purification.
  • Emulsion A and Emulsion B were prepared by ultrasonic emulsification.
  • the aqueous phase of Emulsion A was NaHCO 3 solution
  • the oil phase was dichloromethane solution of PLGA, PLGA-PEG and aNLG919
  • the aqueous phase of Emulsion B was DOX dissolved in CaCl 2 solution
  • the oil phase is also the dichloromethane solution of PLGA, PLGA-PEG and aNLG919.
  • the emulsions A and B were mixed, and the emulsion C was obtained by continuing ultrasonication.
  • Example 2 Property detection of calcium carbonate poly(lactic acid-glycolic acid) complex-DOX-NLG919 preparation
  • the calcium carbonate poly(lactic acid-glycolic acid) complex-DOX-NLG919 preparation prepared in Example 1 was qualitatively detected, and transmission electron microscopy and dynamic light scattering were respectively performed. Among them, the results of transmission electron microscopy are shown in Figure 1. The results show that the calcium carbonate poly(lactic acid-glycolic acid) complex-DOX-NLG919 preparation prepared in Example 1 is distributed in a monodispersed state in water, indicating that the preparation of the present invention The obtained calcium carbonate poly(lactic acid-glycolic acid) complex-DOX-NLG919 preparation has uniform particle size and good dispersion in water.
  • Example 3 Detection of drug loading capacity of calcium carbonate poly(lactic acid-glycolic acid) complex preparation
  • Calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 preparations were qualitatively tested to detect their ability to encapsulate small molecule drugs. Try to use calcium carbonate poly(lactic-glycolic acid) complex preparation to encapsulate drugs such as doxorubicin (DOX), chlorphene e6 (Ce6), mitoxantrone (Mitoxantrone), bovine serum albumin (BSA), etc. The method is the same as that of Example 1, except that DOX is replaced with equimolar Ce6, Mitoxantrone or BSA respectively in the aqueous phase of emulsion B.
  • DOX doxorubicin
  • Ce6 chlorphene e6
  • Mitoxantrone mitoxantrone
  • BSA bovine serum albumin
  • PLGA-PEG nanoparticles which are mixed nanoparticles of PLGA and PLGA-PEG, were used as a comparison.
  • the drug content in the prepared calcium carbonate poly(lactic-glycolic acid) complex formulation was measured by UV-VIS to test the entrapment ability of the calcium carbonate poly(lactic-glycolic acid) complex formulation for various drugs.
  • the test results are shown in Figure 3.
  • the results show that the prepared calcium carbonate poly(lactic acid-glycolic acid) complex preparation (CaNPs in the figure) prepared by the present invention has a good encapsulation capacity for a variety of small molecules, and is a A good drug carrier.
  • Example 4 Detection of acid-responsive drug release ability of calcium carbonate poly(lactic acid-glycolic acid) complex-DOX-NLG919 preparations.
  • the calcium carbonate poly(lactic acid-glycolic acid) complex-DOX-NLG919 preparation prepared in Example 1 was qualitatively tested to detect its acid-responsive drug release ability.
  • the calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 preparation was transferred into a dialysis bag and immersed in 10 mL of different pH buffer solutions (i.e., pH 7.4 solution, pH 6.5 solution, pH 5.5 solution) at room temperature. At predetermined times, the external solution was collected and the DOX concentration was determined using a UV-Vis spectrophotometer.
  • the test results are shown in Figure 4. The results show that the calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 preparation prepared in Example 6 shows better drug release ability under acidic conditions.
  • Example 5 Detection of IDO inhibitory properties of calcium carbonate poly(lactic acid-glycolic acid) complex-DOX-NLG919 preparations.
  • Calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 preparations were qualitatively tested to detect their IDO inhibitory effect.
  • Different calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 formulations were prepared according to the method of Example 1, the difference was that the concentration of aNLG919 in the oil phase of emulsion A was different.
  • NLG919 (Free NLG919) and the prepared calcium carbonate poly(lactic acid-glycolic acid) complex-DOX-NLG919 preparation were added to CT26 cells with IFN- ⁇ and incubated for 48h, and then 30% trichloroacetic acid was added.
  • Example 6 In vivo behavior of calcium carbonate poly(lactic acid-glycolic acid) complex-DOX-NLG919 formulation in mice after tail vein injection.
  • Calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919-Dir preparations were labeled with the fluorescent dye DiR and then injected into mice via the tail vein for real-time acquisition of pictures at different time points on a small animal imaging system , to observe the enrichment of calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919-Dir preparations at tumor sites, and the results are shown in Figure 6.
  • the selected excitation light source was 748 nm, and the exposure time was 50 ms.
  • the results in Figure 6 show that the fluorescence signal of the calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919-Dir preparation in the tumor region gradually increased over time, and its enrichment at the tumor site increased with time.
  • Example 7 Combined chemotherapy-immunotherapy for tumors
  • mice with colon cancer subcutaneous tumor model were divided into 5 groups, including: the first group, the control group (injected with normal saline only, control); the second group, injected with blank calcium carbonate poly(lactic-glycolic acid) complex
  • the third group was injected with calcium carbonate poly(lactic-glycolic acid) complex-NLG919 preparation treatment group (NCaNPs); the fourth group was injected with calcium carbonate poly(lactic-glycolic acid) complex- The NLG919 preparation treatment group (DCaNPs); the fifth group, the calcium carbonate poly(lactic-glycolic acid) complex-DOX-NLG919 preparation treatment group (DNCaNPs).

Abstract

一种碳酸钙聚(乳酸-羟基乙酸)复合物微粒及其制备和应用。碳酸钙聚(乳酸-羟基乙酸)复合物微粒，以烷基修饰的NLG919、聚乳酸-羟基乙酸和聚乳酸-羟基乙酸-聚乙二醇为壳层，以碳酸钙和阿霉素为核层。还公开了碳酸钙聚(乳酸-羟基乙酸)复合物微粒在制备肿瘤化疗的药物或肿瘤化疗和免疫联合治疗的药物中的应用。复合物微粒可作为药物载体，具有良好的药物包载效率和肿瘤富集能力，实现化疗药物DOX和IDO抑制剂aNLG919的共包载，且可有效释放化疗药物阿霉素，并抑制IDO免疫抑制通路，有效扭转肿瘤免疫抑制微环境，激活肿瘤免疫，抑制肿瘤生长，实现肿瘤的化疗或化疗与免疫联合治疗。

Description

碳酸钙聚(乳酸-羟基乙酸)复合物微粒及其制备和应用 技术领域
本发明涉及一种肿瘤治疗制剂领域，尤其涉及一种碳酸钙聚(乳酸-羟基乙酸)复合物微粒及其制备和应用。
背景技术
肿瘤是指机体在各种致瘤因素作用下，局部组织的细胞异常增生而形成的局部肿块。恶性肿瘤就是人们所说的癌症。化疗仍然是各种癌症的主要治疗手段。一般认为，化疗是通过其本身的细胞毒性效应起作用的。但是越来越多的证据表明，一些特定的化疗药物不仅能通过细胞毒性杀伤肿瘤细胞，其诱导的免疫反应也在整体抗肿瘤活性中发挥重要作用。最近的研究表明，抗肿瘤化疗药物阿霉素(DOX)可以通过损伤相关的分子模式(DAMPs)诱导免疫原性细胞死亡(ICD)，从而引发抗肿瘤免疫。ICD诱导的免疫原性促进细胞毒性T淋巴细胞(CTL)的肿瘤内浸润，进而有效抑制肿瘤生长。
然而，化疗引起的免疫反应受到肿瘤内免疫抑制性微环境的影响，而逆转肿瘤免疫抑制性微环境可以激活机体的抗肿瘤免疫反应。目前，临床实践结果表明，通过利用特异性的抗体来阻断肿瘤内免疫抑制性的细胞毒性T淋巴细胞相关蛋白4(CTLA-4)和程序性细胞死亡蛋白1(PD-1)可实现对多种肿瘤生长的有效抑制。除此之外，多种实体瘤会高表达吲哚胺2,3-双加氧酶1(IDO-1)其能够催化瘤内色氨酸(Trp)的降解成犬尿氨酸(Kyn)，而肿瘤内犬尿氨酸的积累不但会减少Th1、CTL等T细胞向瘤内浸润并诱导其凋亡，还会激活瘤内免疫调节性T细胞(Tregs)，进而造成了严重的免疫抑制性微环境。
因此，开发一种能够提高抗肿瘤药物在肿瘤区域富集的效果和药物的酸响应靶向释放，实现肿瘤的化疗与免疫联合治疗的药物是非常必要的。
发明内容
为解决上述技术问题，本发明的目的是提供一种碳酸钙聚(乳酸-羟基乙酸)复合物微粒及其制备和应用，本发明的碳酸钙聚(乳酸-羟基乙酸)复合物微粒可作为药物载体，具有良好的药物包载效率和肿瘤富集能力，实现化疗药物DOX和IDO抑制剂aNLG919的共包载，且可有效释放化疗药物阿霉素，并抑制IDO免疫抑制通路，有效扭转肿瘤免疫抑制微环境，激活肿瘤免疫，抑制肿瘤生长，实现肿瘤的化疗或化疗与免疫联合治疗。
本发明的第一个目的是提供一种碳酸钙聚(乳酸-羟基乙酸)复合物微粒，该微粒以烷基修饰的NLG919、聚乳酸-羟基乙酸(PLGA)和聚乳酸-羟基乙酸-聚乙二醇(PLGA-PEG)为壳层，以碳酸钙(CaCO 3)和阿霉素(DOX)为核层。
进一步地，碳酸钙聚(乳酸-羟基乙酸)复合物微粒的粒径为100～180nm。
本发明的第二个目的是提供一种上述碳酸钙聚(乳酸-羟基乙酸)复合物微粒的制备方法，包括以下步骤：
A)将聚乳酸-羟基乙酸、聚乳酸-羟基乙酸-聚乙二醇和烷基修饰的NLG919在有机溶剂中混合，得到油相混合溶液；
将CaCl 2、阿霉素和水混合，得到氯化钙阿霉素溶液；
B)将油相混合溶液与NaHCO 3溶液混合，乳化，得到第一乳液；
将油相混合溶液与氯化钙阿霉素溶液混合，乳化，得到第二乳液；
C)将第一乳液和第二乳液混合，乳化，得到第三乳液；
D)将第三乳液分散于聚乙烯醇水溶液中，搅拌、洗涤得到碳酸钙聚(乳酸-羟基乙酸)复合物微粒。
进一步地，在步骤A)中，聚乳酸-羟基乙酸和聚乳酸-羟基乙酸-聚乙二醇的质量比为1～2:1～2；
烷基修饰的NLG919为C2～C20的烷基修饰的NLG919。
进一步地，在步骤B)中，NaHCO 3溶液浓度为0.6～0.7M，氯化钙阿霉素溶液中氯化钙的浓度为1～1.5M。
进一步地，在步骤A)中，聚乳酸-羟基乙酸和聚乳酸-羟基乙酸-聚乙二醇总和、阿霉素和烷基修饰的NLG919的质量比为(5～10):(0.3～1):(0.3～2)；在步骤A)中，CaCl 2和阿霉素的质量比为25～30:1；
在步骤B)中，油相混合溶液与NaHCO 3溶液的体积比为3～4:1；油相混合溶液与氯化钙阿霉素溶液的体积比为3～4:1。
进一步地，在步骤C)中，乳化为超声乳化，超声功率为80～120W；超声时间为250～350s；在步骤D)中，聚乙烯醇水溶液的质量分数为1％～2％；搅拌的时间为10～14h；洗涤为14000～15000rpm的条件下离心20～30min，用超纯水洗涤2～3次。
本发明的第三个目的是公开上述碳酸钙聚(乳酸-羟基乙酸)复合物微粒在制备肿瘤化疗的药物或肿瘤化疗和免疫联合治疗的药物中的应用。
进一步地，药物为肿瘤酸环境响应性药物，酸环境的pH值为5.5～6.5。
进一步地，药物用于治疗乳腺癌与结肠癌等实体肿瘤。
进一步地，药物剂型为注射剂。
借由上述方案，本发明至少具有以下优点：
本发明提供了碳酸钙聚(乳酸-羟基乙酸)复合物微粒及其制备方法，该微粒以烷基修饰的NLG919、PLGA和PLGA-PEG为壳层，以CaCO 3和DOX为核层，其粒径均一，在水中分散良好，在生理条件下具有良好的稳定性，其对疏水化的NLG919以及亲水性的盐酸阿霉素都具有良好的装载效率，是理想的药物载体，具有良好的药代动力学行为，并且碳酸钙的存在使得该复合制剂具有对肿瘤酸性微环境的响应能力，实现药物的响应性释放，降低毒副作用。
本发明还公开碳酸钙聚(乳酸-羟基乙酸)复合物微粒在制备肿瘤化疗的药物或肿瘤化疗和免疫联合治疗的药物中的应用。将碳酸钙聚(乳酸-羟基乙酸)复合物微粒对小鼠通过尾静脉注射，并由荧光成像技术对其体内药代动力学行为进行监测发现，碳酸钙聚(乳酸-羟基乙酸)复合物制剂能够有效富集在肿瘤部位，且在小鼠体内有较长的循环时间，具有良好的肿瘤协同治疗效果，能够用于肿瘤化疗免疫联合治疗。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1是碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂的透射电镜图；
图2是碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂的粒径分布图；
图3是碳酸钙聚(乳酸-羟基乙酸)复合物制剂对不同分子的载药量测试结果；
图4是碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂在不同pH条件下的DOX释放曲线；
图5是碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂的IDO抑制曲线；
图6是静脉注射碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂之后的肿瘤富集荧光成像图；
图7是静脉注射碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂之后的生物分布图；
图8是小鼠结肠癌皮下肿瘤模型的肿瘤生长曲线。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本 发明，但不用来限制本发明的范围。
实施例一：碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂的制备
将NLG919溶于二氯甲烷溶液中，以N,N-二异丙基乙胺(DIPEA)做碱，冰浴条件下将十二烷基酰氯加入反应体系，反应生成烷基修饰的NLG919，通过液相色谱柱纯化得到产物aNGL919。
采用超声乳化法制备W/O乳液A和乳液B，乳液A水相为NaHCO 3溶液，油相为PLGA、PLGA-PEG与aNLG919的二氯甲烷溶液；乳液B水相为DOX溶于CaCl 2溶液，油相同样为PLGA、PLGA-PEG与aNLG919的二氯甲烷溶液。分别超声乳化后将乳液A、B混合，继续超声得到乳液C，最后分散与1％PVA水溶液中，得到W1/O/W2微乳体系，搅拌过夜待挥发二氯甲烷后得到碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂。
实施例二：碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂的性质检测
对实施例一制得的碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂进行定性检测，分别进行透射电镜检测、动态光散射。其中，透射电镜检测的结果如图1所示，结果显示，实施例一制得的碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂在水中呈单分散状态分布，表明本发明制得的碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂的粒径均一，在水中分散良好。
动态光散射检测的结果如图2所示，结果显示，实施例一制得的碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂在水中粒径主要集中在100nm左右，粒径分布较为均匀。
实施例三：碳酸钙聚(乳酸-羟基乙酸)复合物制剂的药物包载能力检测
对碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂进行定性检测，检测其对小分子药物的包载能力。尝试使用碳酸钙聚(乳酸-羟基乙酸)复合物制剂包载阿霉素(DOX)、二氢卟吩e6(Ce6)、米托蒽醌(Mitoxantrone)、牛血清白蛋白(BSA)等药物，其方法与实施例一的相同，不同之处在于，在乳液B水相时，将DOX分别替换为等摩尔的Ce6、Mitoxantrone或BSA。同时利用PLGA-PEG nanoparticles作为对比，其为PLGA和PLGA-PEG的混合纳米粒子。通过UV-VIS测量所制得的碳酸钙聚(乳酸-羟基乙酸)复合物制剂中药物含量以测试碳酸钙聚(乳酸-羟基乙酸)复合物制剂对各种药物的包载能力。测试结果如图3所示，结果显示，本发明制得的制得的碳酸钙聚(乳酸-羟基乙酸)复合物制剂(图中CaNPs)对多种小分子都具有良好的包载能力，是一种良好的药物载体。
实施例四：碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂的酸响应药物释放能力检测。
对实施例一制得的碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂进行定性检测，检测其酸响应药物释放能力。将碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂转移到透析袋中，室温下浸入10mL不同pH缓冲溶液(即pH 7.4溶液、pH 6.5溶液、pH 5.5溶液)中。在预定的时间，收集外部溶液，用紫外-可见分光光度计测定DOX浓度。测试结果如图4所示，结果显示，实施例六制得的碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂在酸性条件下显示出更好的药物释放能力。
实施例五：碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂的IDO抑制性质检测。
对碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂进行定性检测，检测其IDO抑制效果。按照实施例一的方法制备不同的碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂，不同之处在于，乳液A油相中aNLG919的浓度不同。将NLG919(Free NLG919)与制得的碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂分别与IFN-γ同时加入CT26细胞孵育48h，再加入30％三氯乙酸，在50℃下孵育6h，将甲酰犬尿氨酸水解为犬尿氨酸。最后加入埃利希试剂，室温显色10min，测490nm波长处吸收。测试结果如图5所示，结果显示，与未经修饰的NLG919相比，碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂的IDO抑制能力并不会在修饰及制备过程中损失，依然显示出几乎不减弱的IDO抑制能力，这为后续的联合治疗策略提供了基础。
实施例六：碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂尾静脉注射后在小鼠的体内行为。
将碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919-Dir制剂用荧光染料DiR标记，然后通过尾静脉注射到小鼠体内，在小动物成像系统上在不同的时间点进行实时采集图片，观察碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919-Dir制剂在肿瘤部位的富集量，结果见图6。选用的激发光源是748nm，曝光时间是50ms。图6结果显示随着时间的推移，肿瘤区域碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919-Dir制剂的荧光信号逐渐增强，其在肿瘤部位的富集随时间增加。
将碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919-DiR制剂通过尾静脉注射到小鼠体 内，24小时后将小鼠主要器官取出后，测量其荧光强度，根据荧光强度的数值定量分析出脂质体-奥沙利铂前药-NLG919在不同器官中分布情况，结果见图7，图7为实施例六中小鼠注射碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919DiR制剂24h后，在体内各个器官分布的荧光强度统计图；从图6-7可以看出，碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919-DiR制剂在肿瘤(图7f)部位的富集低于肝(图7a)、脾(图7b)，但显著高于肾(图7c)、心(图7d)、肺(图7e)，表明碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919-DiR制剂有较好的肿瘤富集能力。
实施例七：肿瘤的化疗-免疫联合治疗
将带有结肠癌皮下肿瘤模型的小鼠分为5组，其中包括：第一组，对照组(仅注射生理盐水，control)；第二组，注射空白碳酸钙聚(乳酸-羟基乙酸)复合物制剂治疗组(CaNPs)；第三组，注射碳酸钙聚(乳酸-羟基乙酸)复合物-NLG919制剂治疗组(NCaNPs)；第四组，注射碳酸钙聚(乳酸-羟基乙酸)复合物-NLG919制剂治疗组(DCaNPs)；第五组，注射碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂治疗组(DNCaNPs)。对小鼠进行相应的治疗后，测量其肿瘤的生长，结果见于图8。
从图8可以看出，相比较对照组，第二组、第三组、第四组的肿瘤生长仅得到了部分抑制，而第五组的肿瘤生长则得到了有效的抑制。表明碳酸钙聚(乳酸-羟基乙酸)复合物-DOX-NLG919制剂能够实现对肿瘤的化疗与免疫联合治疗。
以上仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (10)

  1. 一种碳酸钙聚(乳酸-羟基乙酸)复合物微粒，其特征在于，包括核层和包裹于所述核层外的壳层，所述核层包括碳酸钙和阿霉素，所述壳层包括烷基修饰的NLG919、聚乳酸-羟基乙酸和聚乳酸-羟基乙酸-聚乙二醇。
  2. 根据权利要求1所述的碳酸钙聚(乳酸-羟基乙酸)复合物微粒，其特征在于，所述碳酸钙聚(乳酸-羟基乙酸)复合物微粒的粒径为100～180nm。
  3. 一种权利要求1或2所述的碳酸钙聚(乳酸-羟基乙酸)复合物微粒的制备方法，其特征在于，包括以下步骤：
    A)将聚乳酸-羟基乙酸、聚乳酸-羟基乙酸-聚乙二醇和烷基修饰的NLG919在有机溶剂中混合，得到油相混合溶液；
    将CaCl 2、阿霉素和水混合，得到氯化钙阿霉素溶液；
    B)将所述油相混合溶液与NaHCO 3溶液混合，乳化，得到第一乳液；
    将所述油相混合溶液与氯化钙阿霉素溶液混合，乳化，得到第二乳液；
    C)将所述第一乳液和第二乳液混合，乳化，得到第三乳液；
    D)将所述第三乳液分散于聚乙烯醇水溶液中，搅拌、洗涤得到所述碳酸钙聚(乳酸-羟基乙酸)复合物微粒。
  4. 根据权利要求3所述的制备方法，其特征在于，在步骤A)中，所述聚乳酸-羟基乙酸和聚乳酸-羟基乙酸-聚乙二醇的质量比为1～2:1～2；
    所述烷基修饰的NLG919为C2～C20的烷基修饰的NLG919。
  5. 根据权利要求3所述的制备方法，其特征在于，在步骤B)中，所述NaHCO 3溶液浓度为0.6～0.7M，所述氯化钙阿霉素溶液中氯化钙的浓度为1～1.5M。
  6. 根据权利要求3所述的制备方法，其特征在于，在步骤A)中，所述聚乳酸-羟基乙酸和聚乳酸-羟基乙酸-聚乙二醇总和、阿霉素和烷基修饰的NLG919的质量比为(5～10):(0.3～1):(0.3～2)；在步骤A)中，所述CaCl 2和阿霉素的质量比为25～30:1；
    在步骤B)中，所述油相混合溶液与NaHCO 3溶液的体积比为3～4:1；所述油相混合溶液与氯化钙阿霉素溶液的体积比为3～4:1。
  7. 根据权利要求3所述的制备方法，其特征在于，在步骤C)中，所述乳化为超声乳化，超声功率为80～120W；超声时间为250～350s；在步骤D)中，所述聚乙烯醇水溶液的质量分数为1％～2％；搅拌的时间为10～14h；洗涤为14000～15000rpm的条件下离心20～30min，用超纯水洗涤2～3次。
  8. 权利要求1或2所述的碳酸钙聚(乳酸-羟基乙酸)复合物微粒在制备肿瘤化疗的药物或 肿瘤化疗和免疫联合治疗的药物中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述药物为肿瘤酸环境响应性药物，酸环境的pH值为5.5～6.5。
  10. 根据权利要求8所述的应用，其特征在于，所述药物用于治疗实体肿瘤。
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