WO2023104137A1 - 肿瘤抗原响应性活细胞纳米药物背包的制备方法及应用 - Google Patents

肿瘤抗原响应性活细胞纳米药物背包的制备方法及应用 Download PDF

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WO2023104137A1
WO2023104137A1 PCT/CN2022/137470 CN2022137470W WO2023104137A1 WO 2023104137 A1 WO2023104137 A1 WO 2023104137A1 CN 2022137470 W CN2022137470 W CN 2022137470W WO 2023104137 A1 WO2023104137 A1 WO 2023104137A1
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nano
drug
albumin
responsive
tumor
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蔡林涛
潘宏
郑明彬
罗英梅
陈泽
张保珍
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深圳先进技术研究院
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    • C12N5/0634—Cells from the blood or the immune system
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Definitions

  • the invention relates to the field of nanomedicine, in particular to a preparation method and application of a tumor antigen-responsive living cell nanomedicine backpack.
  • Tumor immunotherapy is a therapeutic strategy that strengthens the patient's immune system to attack tumors, and has shown great potential in the field of anti-tumor immunotherapy and research.
  • chimeric antigen receptor T cells (Chimeric Antigen Receptor T (CAR-T), represented by tumor cell immunotherapy, as a "living drug”, has made an unprecedented breakthrough in the clinical treatment of tumors, and has become a key development direction in the field of medicine.
  • Nanomaterials can improve the stability of drugs, reduce the toxic and side effects of drugs, enable the precise release of antigen tumor drug preparations at the tumor site or achieve the effect of slow release and controlled release, and overcome the low bioavailability and serious adverse reactions of traditional drugs and other shortcomings.
  • Nanoparticles are a better nano-drug loading system, with excellent characteristics such as high drug-loading rate and easy surface modification, and are an emerging strategy for tumor-targeted therapy.
  • Nanoparticles can be passively targeted and accumulated at the tumor site through the high osmotic retention effect (EPR effect), which can improve the tumor microenvironment, enhance the body's anti-tumor immune response, and provide a new strategy for tumor immunotherapy.
  • EPR effect osmotic retention effect
  • a series of new nano-drugs have not only been successfully used to overcome immunosuppression and improve anti-tumor effects, but also provide new ideas for the synergy and joint treatment of tumors between immune cells and nano-drugs.
  • the cell-nanocarrier system combines artificial nanomedicines with natural cells and has the advantages of both. Nanomedicine itself can be independently designed according to different uses, and the drug can be released in a controlled manner. The delivery of target cells enables the system to inherit the unique functional activity of the cells, thereby realizing its 1+1>>2 anti-tumor effect.
  • the present invention proposes a preparation method and application of a tumor antigen-responsive living cell nano drug backpack.
  • Immune lymphocytes were loaded with reduction-responsive albumin nanomedicines by bioorthogonal coupling to obtain living cell nanodrug backpacks with antigen-responsive controlled release. Stimulation of immune lymphocytes by tumor antigens leads to an increase in the reduction potential level on the cell surface, thereby triggering the release of albumin nano-backpacks in response to site-specific reduction.
  • the invention provides an application of a tumor antigen-responsive living cell nano-drug backpack in the preparation of anti-tumor drugs.
  • the tumor antigen-responsive living cell nano-drug backpack includes immune lymphocytes loaded with reducing-responsive Albumin nano-medicine: Tumor antigen stimulates the immune lymphocytes to cause an increase in the reduction potential level on the cell surface, thereby triggering the release of drugs in the living cell nano-medicine backpack in response to site-specific reduction.
  • the reduction-responsive albumin nanomedicine is loaded onto the immune lymphocytes through a biochemical reaction.
  • biochemical reaction is a bioorthogonal reaction.
  • the immune lymphocytes are any one of T cells, B cells, NK cells, CAR-T, and Treg cells.
  • the nano drug is entrapped in the albumin nano particle through hydrophobic interaction.
  • the nano drug is a protein drug or a chemotherapeutic drug.
  • the protein drug is any one or more of cytokines, antibodies, immune factors, and polypeptides
  • the chemotherapy drug is any one or more of doxorubicin, paclitaxel, and alkyl drugs.
  • the present invention also provides a method for preparing a tumor antigen-responsive living cell nano drug backpack, comprising the following steps:
  • the immune lymphocytes in the step (1) embed bio-orthogonal groups on the surface of the cell membrane through cell metabolism to obtain bio-orthogonal group-modified immune lymphocytes; the cells are metabolized into sugars and lipids , Any of the cell metabolism of amino acids.
  • step (2) glutathione is used to open the disulfide bond of albumin, and the drug molecule is encapsulated by ethanol dehydration to prepare reduction-responsive albumin nano drug particles.
  • the bio-orthogonal group-modified immune lymphocytes prepared in the step (1) are co-incubated with the albumin nano drug particles prepared in the step (2).
  • Orthogonal reaction realizes the coupling of albumin nano-medicine particles and immune lymphocytes modified by bio-orthogonal groups, and obtains the living cell nano-medicine backpack.
  • the present invention also provides a tumor antigen-responsive living cell nano drug backpack, which is prepared by the preparation method described in any one of claims 8-11.
  • the immune cell-loaded albumin nano-drug backpack of the present invention can target tumor cells through cellular immune recognition, and deliver nano-drugs to tumor lesions accurately and efficiently.
  • the living cell nano-drug backpack of the present invention targets and recognizes tumor antigens, stimulates and activates immune cells, and enhances the reduction potential of the cell surface, thereby triggering the responsive release of albumin nano-drugs and improving the safety and bioavailability of free drugs in the body Spend.
  • the drug molecules released in a fixed-point response in the living cell nano drug backpack of the present invention can effectively feed back the immune cell carrier, improve the tumor immune microenvironment, and comprehensively improve the anti-tumor immune effect of immune cells.
  • the living cell nano-drug backpack of the present invention can release nano-drugs in response to the reductive environment stimulated by tumor antigens, which can avoid a series of side effects caused by systemic administration of drugs.
  • the preparation method of the present invention is fast and simple, and is easy to operate and popularize.
  • Figure 1 shows the construction of tumor antigen-responsive living cell nano-knapsack and its anti-tumor immune feedback effect.
  • Figure 2 shows the construction, particle size, morphology and drug release process of the original responsive albumin nano-medicine.
  • Figure 3 shows the construction and morphology of living cell nano drug backpack analyzed by confocal imaging, flow cytometry and scanning electron microscopy.
  • Figure 4 shows the tumor antigen-responsive release mechanism of the living cell nano drug backpack.
  • Figure 5 is the anti-tumor immunotherapy effect of living cell nano drug backpack in vivo.
  • the invention provides an application of a living cell nano drug backpack in the preparation of drugs for treating solid tumors.
  • the chemical reporter group is metabolically modified to the surface of lymphocytes, and the paired chemically modified albumin nano-medicine is highly efficient , Specific bio-orthogonal coupling reaction, so as to realize the high-efficiency loading of living cells on albumin nano-medicines.
  • Safe, efficient and synergistic anti-tumor immunotherapy can be achieved through the targeted delivery of immune cells and the release of nano-drugs in response to tumor antigen-dependent stimuli.
  • the preparation and application of living cell nano drug backpack include the following steps:
  • Naturally active immune lymphocytes were co-incubated with sugar/lipid/amino acid derivatives modified by chemical groups for 48 h, and through natural cell sugar, lipid and amino acid metabolism, chemical reports such as -BCN/-Tz/-DBCO/-alkyne
  • the group is embedded on the surface of the cell membrane to obtain bio-orthogonal group-modified immune lymphocytes; the lymphocytes include all immune cells that have antigen stimulation to increase the reduction potential of the cell surface, such as T cells, B cells, NK cells and CAR -T and Treg cells, etc.
  • this type of lymphoid immune cells After being stimulated by antigens and ligands, this type of lymphoid immune cells will increase the level of sulfhydryl groups on the cell surface and enhance the redox potential on the cell surface, which is conducive to the effector release of reduction-responsive nanoparticles.
  • the traditional reduction response release is caused by the tumor microenvironment with high reduction level (such as high glutathione level), the release mechanism of albumin reduction response in the present invention is different from the prior art, and provides a brand new albumin reduction response release path.
  • the disulfide bond of albumin is opened by glutathione (1mM), and the protein or chemotherapeutic drug molecule is dehydrated by ethanol, so as to prepare the reduction-responsive albumin nano drug particle.
  • the preparation method of the albumin nanomedicine comprises the following steps: 0.5-50 mg/ml albumin is reduced by 1-500 M GSH, the disulfide bond is broken, and a large amount of free sulfhydryl groups are exposed; the albumin is dehydrated by ethanol molecules Reconstruction of disulfide bonds causes rearrangement and cross-linking of protein molecules, encapsulating proteins and chemicals.
  • the nanoparticles loaded with drugs can be used for connection with cells by modifying bioorthogonal groups.
  • the purified reduction-responsive albumin nanoparticles loaded with anticancer drugs were obtained by dialysis, and the detailed method can be found in Example 1.
  • the preparation process of the albumin nano-medicine is simple, stable in properties, carrying a chemical reporter group and capable of reduction response release.
  • Protein drugs and chemotherapeutic drugs can be efficiently entrapped in albumin nanoparticles through hydrophobic interaction, forming stable, high encapsulation efficiency, and high loading drug-loaded nanoparticles;
  • albumin nano-drugs have good biocompatibility, Biodegradability can reduce the toxic and side effects of drugs on the body; nano-medicines can quickly respond to the cleavage of the intermolecular disulfide bond of albumin in a reducing environment, and realize the controlled release of drug molecules.
  • the drug molecules include protein drugs such as cytokines, antibodies, immune factors and polypeptides, and chemotherapy drugs such as adriamycin, paclitaxel and alkanes. These proteins and chemotherapy drugs can be effectively encapsulated by albumin and released in response to reduction without affecting the functional activity of the drug molecule itself.
  • in vitro cultured lymphoid immune cells 0.5-5 ⁇ 10 7
  • phospholipids, amino acids, or oligosaccharide analog molecules at a concentration of 0.1-500 M are placed in a 5% CO 2 incubator at 37°C Incubate for 72 h.
  • the above-mentioned living cell number and the dosage of the lipid/oligosaccharide/amino acid analogue are in a ratio range.
  • modified immune cells were incubated with albumin nanomedicine in a 5% CO 2 incubator at 37°C for 0.5-2 h, centrifuged (1200 rpm, 5 min) to remove free nanoparticles, and the drug loading was determined by enzyme-linked immunosorbent assay.
  • the level of sulfhydryl groups in immune cells is increased, and the surface reduction potential is enhanced, thereby realizing the responsive release of the nano-drug backpack from the cell surface.
  • the living cell nanomedicine cells are mainly aimed at the in vivo treatment model of human-derived solid tumors, and the treatment is performed by intravenous infusion.
  • the anti-tumor treatment method mainly includes the following steps:
  • the immune cells (0.1-10 ⁇ 10 7 ) carrying the albumin nano-drug backpack were injected into the tail vein of a tumor-bearing mouse with a volume of 100 mm 3 to construct a mouse immunotherapy model.
  • the tumor tissue was collected at 24-72 h, and the intratumoral antigen-responsive release of albumin nanomedicine was analyzed by immunofluorescence.
  • the third step is to analyze the functional activity of albumin nanomedicine release on exogenous lymphoid immune cells, and its effect on tumor immunosuppressive microenvironment cells (TAM, MDSC and Treg cells) and immunosuppressive factors (TGF-beta, 1L-10 and IL -4) Etc. regulation, measurement of tumor volume, evaluation of tumor suppression effect.
  • TAM tumor immunosuppressive microenvironment cells
  • TGF-beta immunosuppressive factors
  • the live-cell nano-drug backpack targets and recognizes tumors, and delivers albumin nano-drugs to tumor lesions.
  • Tumor antigen stimulation triggers an increase in the reduction potential of lymphocytes, This triggers the reduction-responsive release of the surface albumin nano-knapsack.
  • the released nano-drugs can feedback and activate immune lymphocytes in situ in tumor lesions, regulate the tumor immune microenvironment, and realize the efficient and synergistic anti-tumor immunotherapy between the two.
  • Preparation of albumin-loaded IL-12 nanoparticles (INS) by disulfide bond reconstruction method First, HSA and reducing agent GSH were co-dissolved in deionized water at a mass ratio of 3:1 (the concentration of HSA was 25 mg/mL ), and stirred at room temperature for 1 h. Subsequently, the reaction solution is removed by dialysis to remove excess GSH molecules, and the reduced HSA solution can be obtained. In the next step, the reduced HSA solution was mixed with the pretreated anti-tumor cytokine IL-12 solution (dissolved in 0.1% HSA in phosphate buffer), so that the mass ratio of HSA and IL-12 was 1000:1.
  • albumin nanomedicine the above IL-12-loaded albumin nanoparticles (INS) were incubated with DBCO-PEG 5 -NHS bioorthogonal reagent at a final concentration of 20 mM for 2 h at room temperature, and then passed The above ultrafiltration dialysis method removes unreacted free bioorthogonal reagents, and prepares DBCO group-modified albumin nanoparticles (DBCO-INSs).
  • DBCO-INSs DBCO group-modified albumin nanoparticles
  • the sulfhydryl group detection kit was used to quantitatively analyze the sulfhydryl group of albumin and its reduction level, and the size and shape of the particles were analyzed and identified by dynamic light scattering particle size analyzer and transmission electron microscope.
  • albumin exhibits controlled release characteristics in response to GSH reduction through the breakage and assembly of disulfide bonds; albumin nanomedicine exhibits good uniformity (particle size about 60 nm) and protein nanostructure morphology .
  • the albumin nanomedicine showed a slow response release process in the GSH reducing environment, in which the protein drug IL-12 was completely released within 15 days and was significantly higher than that of the control group (Figure 2C).
  • Example 2 Construction and functional analysis of living cell nano drug backpack in response to tumor antigen stimulation
  • INS-CAR-T was separated from unbound particles and resuspended in PBS.
  • INS-CAR-T 200 ⁇ L PBS, 5 ⁇ 10 4 /well was added to an 8-well chamber glass cover and incubated until adhered, and acquired by laser confocal Fluorescent images of INS-CAR-T cells.
  • INS-CAR T was incubated with Raji tumor cells at a ratio of 1:1, and the concentration of IL-12 in the supernatant at different time points was detected by ELISA. At the same time, the fluorescent intensity of the Cy5.5-labeled INS-CAR-T suspension was detected by fluorescent staining.
  • the responsive release of particles after incubation of INS-CAR-T with Raji tumor cells for 24 h was analyzed by scanning electron microscopy. Quantitative fluorescence analysis showed that after Raji tumor cells were stimulated for 24 h, the amount of thiols on the surface of CAR-T cells increased by about 5 times ( Figure 4A and 4B).
  • Example 3 Anti-tumor therapy of living cell nano-drug backpacks in response to tumor antigen stimulation
  • the present invention discloses a preparation method and application of a tumor antigen-responsive living cell nano drug backpack.
  • Immune lymphocytes were loaded with reduction-responsive albumin nanomedicines by bioorthogonal coupling to obtain living cell nanodrug backpacks with antigen-responsive controlled release. Stimulation of immune lymphocytes by tumor antigens leads to an increase in the reduction potential level on the cell surface, thereby triggering the release of albumin nano-backpacks in response to site-specific reduction.
  • the released nano-drugs can activate immune lymphocytes in situ at tumor lesions, feedback enhances the anti-tumor effect of immune lymphocytes, realizes efficient and synergistic tumor immunotherapy, and enhances the safety and bioavailability of free drugs in vivo.

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Abstract

公开了一种肿瘤抗原响应性活细胞纳米药物背包的制备方法及应用。通过生物正交偶联实现免疫淋巴细胞负载还原响应的白蛋白纳米药物(Nano Backpacks)，获得具有抗原响应控释的活细胞纳米药物背包(Living Cellular Nano Backpacks,LCNBs)。肿瘤抗原刺激免疫淋巴细胞引发细胞表面还原电势水平上升，从而触发白蛋白纳米背包定点还原响应释放。释放的纳米药物可以在肿瘤病灶原位激活免疫淋巴细胞，反馈增强免疫淋巴细胞的抗肿瘤效应，实现高效协同的肿瘤免疫治疗，并增强游离药物在体内的安全性与生物利用度

Description

肿瘤抗原响应性活细胞纳米药物背包的制备方法及应用 技术领域
本发明涉及纳米医学领域，特别涉及一种肿瘤抗原响应性活细胞纳米药物背包的制备方法及应用。
 背景技术
肿瘤免疫治疗是一种通过增强患者免疫系统攻击肿瘤的治疗策略，在抗肿瘤免疫治疗与研究领域展现出巨大的潜力。其中以嵌合抗原受体T细胞（Chimeric Antigen Receptor T，CAR-T）为代表的肿瘤细胞免疫疗法作为一种“活的药物”，在肿瘤临床治疗方面取得了前所未有的突破，已成为医药领域的重点发展方向。
尽管细胞免疫治疗在基础研究和临床应用上取得了系列的突破，但作为一种单一疗法，细胞免疫治疗实体瘤在安全性、有效性及普适性方面仍存在严重不足。研究表明，肿瘤细胞在机体免疫选择压力下，凭借自身的高突变特性逃避免疫监视,逐步建立起免疫抑制微环境，以抵抗和抑制机体抗肿瘤免疫反应。在肿瘤微环境中，免疫细胞的浸润、增殖、活化被阻碍，免疫抑制已成为细胞治疗实体瘤的主要障碍。因此，针对实体瘤可能需要一种协同、安全、有效的细胞免疫联合疗法，改善肿瘤免疫微环境，增强淋巴细胞的趋化、浸润与活性，是细胞免疫疗法亟待解决的关键问题，这对肿瘤免疫治疗与抗肿瘤药物研发具有重要意义。
纳米技术的迅猛发展使得肿瘤的靶向治疗和药物递送有了一个飞跃的发展。纳米材料作为药物载体，可以提高药物的稳定性，降低药物的毒副作用，使抗原肿瘤药物制剂在肿瘤部位精确释放或者达到缓释和控制释放的效果，克服传统药物生物利用度低，不良反应严重等缺点。纳米颗粒是较好的纳米载药体系，具有载药率高，表面易修饰等优异的特性，是一种新兴的肿瘤靶向治疗策略。常规纳米颗粒可通过高渗透滞留效应（EPR效应）的被动靶向聚积在肿瘤部位，可改善肿瘤微环境，增强机体的抗肿瘤免疫反应，为肿瘤免疫治疗提供了新策略。随着纳米技术的蓬勃发展，一系列新型纳米药物不但成功地用于克服免疫抑制，提高抗肿瘤效应，也为免疫细胞与纳米药物的协同、联合治疗肿瘤提供新思路。研究表明，细胞-纳米载体系统将人工纳米药物与天然细胞结合起来，兼有两者的优势。纳米药物本身可根据用途的不同自主设计、可控释放药物，运载靶细胞又使该系统继承了细胞独特的功能活性，从而实现其1+1>>2的抗肿瘤功效。
然而，纳米药物治疗的主动靶向与有效性较差，导致的生物安全性问题已成为人们关注的焦点。为了提高药物在肿瘤中的累积，通常在纳米颗粒表面修饰主动靶向的配体分子，例如抗体，多肽、适配体等，通过配体-受体的连接实现抗肿瘤药物在肿瘤部位的高度富集，减少毒副作用。虽然在纳米颗粒表面修饰靶向基团等来增加颗粒在肿瘤的靶向累积的技术已经发展的较为成熟，但是纳米颗粒功能化修饰不仅增加了制备工艺的复杂程度，同时也给纳米药物生物医学应用带来潜在不可控因素，难以普及与推广。
近年来，以免疫细胞为代表的“活细胞药物”不仅能很好靶向识别肿瘤细胞，还能有效的杀伤肿瘤组织，在生物医药领域引起了广泛的关注。然而，单一的免疫细胞疗法在实体瘤治疗过程中仍然面临着免疫抑制、安全性和疗效不佳等诸多挑战。通过将纳米药物与细胞药物的协同联合治疗不仅可以将纳米药靶向递送至肿瘤病灶，而且药物可以增强免疫细胞的功能，实现纳米药物与细胞的协同联合治疗。
 技术问题
如何控制纳米药物在肿瘤病灶的靶向可控释放，提高药物的安全性与生物利用度依然是纳米药物与细胞联合治疗的关键难题。针对这些技术瓶颈，现急需开发出一种天然、安全、有效的活细胞纳米药物治疗体系，提高纳米药物的靶向、控释，促进免疫细胞功能活性，协同增强两者的抗肿瘤免疫治疗疗效与安全性。
 技术解决方案
针对现有技术中的缺陷，本发明提出了一种肿瘤抗原响应性活细胞纳米药物背包的制备方法及应用。通过生物正交偶联实现免疫淋巴细胞负载还原响应的白蛋白纳米药物，获得具有抗原响应控释的活细胞纳米药物背包。肿瘤抗原刺激免疫淋巴细胞引发细胞表面还原电势水平上升，从而触发白蛋白纳米背包定点还原响应释放。
本发明提供一种肿瘤抗原响应性活细胞纳米药物背包在制备抗肿瘤药物中的应用，所述肿瘤抗原响应性活细胞纳米药物背包包括免疫淋巴细胞，所述免疫淋巴细胞上负载具有还原响应的白蛋白纳米药物；肿瘤抗原刺激所述免疫淋巴细胞引发细胞表面还原电势水平上升，从而触发所述活细胞纳米药物背包中药物的定点还原响应释放。
进一步的，所述具有还原响应的白蛋白纳米药物通过生物化学反应负载到所述免疫淋巴细胞上。
进一步的，所述生物化学反应为生物正交反应。
进一步的，所述免疫淋巴细胞为T细胞、B细胞、NK细胞、CAR-T、Treg细胞中的任意一种。
进一步的，所述纳米药物通过疏水作用包载在白蛋白纳米颗粒内。
进一步的，所述纳米药物为蛋白药物或化疗药物。
进一步的，所述蛋白药物为细胞因子、抗体、免疫因子、多肽中的任意一种或多种；所述化疗药物为阿霉素、紫杉醇、烷类药物中的任意一种或多种。
本发明还提供一种肿瘤抗原响应性活细胞纳米药物背包的制备方法，包括如下步骤：
（1）免疫淋巴细胞的化学基团修饰；
（2）还原响应白蛋白纳米药物颗粒的制备；
（3）活细胞纳米药物背包的制备。
进一步的，所述步骤（1）中所述免疫淋巴细胞通过细胞代谢，将生物正交基团嵌入细胞膜表面，获得生物正交基团修饰的免疫淋巴细胞；所述细胞代谢为糖、脂类、氨基酸的细胞代谢中的任意一种。
进一步的，所述步骤（2）中通过谷胱甘肽打开白蛋白的二硫键，通过乙醇脱水包裹药物分子，制备得到还原响应的白蛋白纳米药物颗粒。
进一步的，所述步骤（3）中将所述步骤（1）制备得到的生物正交基团修饰的免疫淋巴细胞与所述步骤（2）制备得到的白蛋白纳米药物颗粒共孵育，通过生物正交反应实现白蛋白纳米药物颗粒与生物正交基团修饰的免疫淋巴细胞的偶联，获得所述的活细胞纳米药物背包。
本发明还提供一种肿瘤抗原响应性活细胞纳米药物背包，由权利要求8~11任一项所述的制备方法制备得到。
 有益效果
综上，与现有技术相比，本发明达到了以下技术效果：
（1）本发明的免疫细胞负载白蛋白纳米药物背包，可通过细胞免疫识别靶向肿瘤细胞，将纳米药物精准、高效地递送至肿瘤病灶。
（2）本发明的活细胞纳米药物背包靶向识别肿瘤抗原，刺激、激活免疫细胞，增强细胞表面还原电势，从而引发白蛋白纳米背包的响应释放，提高游离药物在体内的安全性与生物利用度。
（3）本发明的活细胞纳米药物背包中定点响应释放的药物分子可有效反馈作用免疫细胞载体，改善肿瘤免疫微环境，全面提高免疫细胞的抗肿瘤免疫效应。
（4）本发明的活细胞纳米药物背包可利用肿瘤抗原刺激的还原性环境响应释放纳米药物，能够避免药物全身施用而引起一系列副作用。
（5）本发明的制备方法快速简便，便于操作推广。
 附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为肿瘤抗原响应性活细胞纳米背包的构建及其抗肿瘤免疫反馈效应。
图2为原响应性白蛋白纳米药物的构建、粒径、形态及其药物释放过程。
图3为共聚焦成像、流式细胞与扫描电镜分析活细胞纳米药物背包的构建及其形态。
图4为活细胞纳米药物背包的肿瘤抗原响应性释放机制。
图5为体内活细胞纳米药物背包的抗肿瘤免疫治疗效果。
 本发明的实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
本发明提供一种活细胞纳米药物背包在制备治疗实体肿瘤的药物中的应用。通过-N 3、-BCN、-Tz与-炔基等化学基团间的生物正交偶联反应，将化学报告基团代谢修饰至淋巴细胞表面与经配对化学修饰的白蛋白纳米药物发生高效、特异的生物正交偶联反应，从而实现活细胞对白蛋白纳米药物的高效负载。通过免疫细胞的靶向递送与肿瘤抗原依赖的刺激响应释放纳米药物，实现安全、高效、协同的抗肿瘤免疫治疗。活细胞纳米药物背包的制备和应用包括以下步骤：
（1）免疫淋巴细胞的化学基团修饰
天然活性免疫淋巴细胞经过化学基团修饰的糖/脂/氨基酸类衍生物共孵育48 h，通过天然细胞糖、脂与氨基酸代谢，将-BCN/-Tz/-DBCO/-炔基等化学报告基团嵌入细胞膜表面，获得生物正交基团修饰的免疫淋巴细胞；所述的淋巴细胞包括所有具有抗原刺激引发细胞表面还原电势增加的免疫细胞，例如，T细胞、B细胞、NK细胞以及CAR-T与Treg细胞等。这一类淋巴免疫细胞在受到抗原、配体刺激后会上升细胞表面巯基水平，增强细胞表面的氧化还原电势，从而有利于效应释放还原响应的纳米颗粒。传统的还原响应释放是由还原水平高的肿瘤微环境（如谷胱甘肽水平高）引起，本发明的白蛋白还原响应释放机理不同于现有技术，提供了一种全新的白蛋白还原响应释放路径。
（2）还原响应白蛋白纳米药物颗粒的制备
通过谷胱甘肽（1mM）打开白蛋白的二硫键，通过乙醇脱水包裹蛋白或化疗药物分子，从而制备得到还原响应的白蛋白纳米药物颗粒。
所述白蛋白纳米药物的制备方法包括以下步骤：0.5-50 mg/ml白蛋白经过1-500 M的GSH还原，二硫键断裂，暴露大量的游离巯基；通过乙醇分子脱水作用使白蛋白发生二硫键重构引起蛋白质分子间发生重排、交联，将蛋白与化学药物包裹其中。其中负载药物的纳米颗粒可通过修饰生物正交基团供与细胞连接使用。最后，通过透析得到纯化的负载抗癌药物的还原响应性白蛋白纳米颗粒，具体详细方法可参见实施例1。
所述白蛋白纳米药物的制备工艺简单、性质稳定、携带化学报告基团并具有还原响应释放。蛋白药物、化疗药物可以通过疏水作用高效地包载在白蛋白纳米颗粒内，形成稳定的、高包封率、高负载量的载药纳米颗粒；白蛋白纳米药物具有良好的生物相容性、生物可降解性，可降低药物对机体的毒副作用；在还原环境下纳米药物可快速响应发生白蛋白的分子间二硫键裂解，实现药物分子响应控制释放。所述的药物分子包括：细胞因子、抗体、免疫因子、多肽等蛋白药物以及阿霉素、紫杉醇和烷类等化疗药物。这类蛋白与化疗药物均能有效的被白蛋白包裹、还原响应释放，并不影响药物分子自身的功能活性。
将上述配对的化学基团修饰至白蛋白纳米颗粒表面（共孵育48 h），从而获得化学基团功能化的白蛋白纳米药物；
（3）活细胞纳米药物背包的制备
将上述（1）与（2）化学功能基团修饰的免疫淋巴细胞与白蛋白纳米药物在37℃孵育1小时，通过高效、特异的生物正交反应实现纳米颗粒与活细胞的偶联，获得活细胞纳米药物背包；
具体包括以下步骤：将在体外培养淋巴免疫细胞（0.5-5×10 7个）与浓度为0.1-500 M的磷脂、氨基酸、或寡糖类似物分子在37℃的5% CO 2培养箱中孵育72 h。其中上述活细胞数与脂类/寡糖/氨基酸类似物的用量是一个比例范围。上述修饰免疫细胞与白蛋白纳米药物在37℃的5% CO 2培养箱孵育0.5-2 h，离心（1200 rpm, 5 min）去除游离纳米颗粒，并用酶联免疫法测定药物负载量。
所述的纳米药物背包在受到肿瘤抗原刺激后，引发免疫细胞巯基水平上升、表面还原电势增强，从而实现纳米药物背包从细胞表面响应释放。
（4）活细胞纳米药物背包的抗肿瘤免疫治疗
所述的活细胞纳米药物细胞主要针对人源实体肿瘤的体内治疗模型，并以静脉输入的给药方式进行治疗。所述的抗肿瘤治疗方法主要包括以下步骤：
第一步，将携带白蛋白纳米药物背包的免疫细胞（0.1-10×10 7个），尾静脉注射体积为100 mm 3荷瘤小鼠体内，构建小鼠免疫治疗模型。
第二步，在24-72 h 收集肿瘤组织，免疫荧光分析白蛋白纳米药物的瘤内抗原响应释放情况。
第三步，分析白蛋白纳米药物释放对外源淋巴免疫细胞的功能活性，及其对肿瘤免疫抑制微环境细胞（TAM, MDSC和Treg细胞）与免疫抑制因子（TGF-beta, 1L-10和IL-4）等调控，测量肿瘤体积，评估抑瘤效果。
在小鼠治疗模型中，通过活细胞纳米药物尾静脉给药治，活细胞纳米药物背包靶向识别肿瘤，并将白蛋白纳米药物递送至肿瘤病灶，肿瘤抗原刺激引发淋巴细胞还原电势升高，从而触发表面白蛋白纳米背包还原响应释放。释放的纳米药物可以在肿瘤病灶原位反馈激活免疫淋巴细胞，调控肿瘤免疫微环境，实现两者高效协同的抗肿瘤免疫治疗。
实施例1  携带化学报告基团并具有还原响应释放白蛋白纳米药物的制备
二硫键重构法制备白蛋白负载的IL-12纳米颗粒（INS）：首先，将HSA与还原剂GSH按3：1的质量比共溶于去离子水中（HSA的浓度为25 mg/mL），在室温下搅拌反应1 h。随后，反应溶液采用透析法除去过量的GSH分子，即可获得还原后的HSA溶液。下一步，将还原后的HSA溶液与预处理的抗肿瘤细胞因子IL-12溶液（溶于0.1%HSA的磷酸缓冲液）混合，使HSA、IL-12的质量比为1000：1。溶液充分混合后，将其pH调节至7.8，然后在剧烈搅拌的条件下，迅速加入1.5倍体积的无水乙醇溶液，室温搅拌反应30 min后，反应溶液通过超滤法（超滤膜截留分子量为100 kD，离心转速为4500 rpm）除去乙醇、游离的IL-12和HSA，即得包载IL-12的白蛋白纳米颗粒（INS）。
白蛋白纳米药物的表面基团修饰与功能表征：上述包载IL-12的白蛋白纳米颗粒（INS）与终浓度20 mM的DBCO-PEG 5-NHS生物正交试剂室温孵育2 h，然后通过上述超滤透析法去除未反应的游离生物正交试剂，制备获得DBCO基团修饰白蛋白纳米颗粒（DBCO-INSs）。同时，采用巯基检测试剂盒对白蛋白的巯基及其还原水平进行定量分析，并通过动态光散射粒度仪与透射电镜对颗粒的大小与形貌进行分析鉴定。如图2所示，白蛋白通过二硫键的断裂与组装，展现出GSH还原响应的可控释放特性；白蛋白纳米药物展现出良好的均一性（粒径约60 nm）与蛋白纳米结构形态。同时，白蛋白纳米药物在GSH还原环境下呈现出一种缓慢响应释放过程，其中在15天内蛋白药物IL-12即可释放完全并显著高于对照组（图2C）。
实施例2  肿瘤抗原刺激响应的活细胞纳米药物背包构建与功能分析
在CAR-T细胞培养液中加入50 μM叠氮糖，孵育48 h，将1 mL N 3-CAR-T溶液(10 7个CAR-T细胞/mL)与100 μL DBCO-INS在37℃下孵育60 min，发生生物正交连接反应。最后，将INS-CAR-T与未结合颗粒分离并在PBS中重悬。为了评估INS与CAR-T细胞的结合，将INS-CAR-T (200 μL PBS, 5×10 4/孔)加入到8孔的腔室玻璃盖中并孵育至粘附，采用激光共聚焦获取INS-CAR-T细胞的荧光图像。用扫描电镜对CAR-T细胞、INS+CAR-T细胞和INS-CAR-T细胞的形态和偶联情况进行结构形态表征，并采用流式细胞术分析INS+CAR-T细胞和INS-CAR-T细胞的偶联效率。
共聚焦荧光成像与流式定量分析结果表明，DBCO-INS与未标记CAR-T细胞相比，大量白蛋白纳米颗粒与叠氮CAR-T细胞表面颗粒偶联，并几乎从未被内化（图3）。此外，流式细胞术分析也显示，负载INS颗粒的阳性细胞率高达99%（图3），进一步证实白蛋白纳米颗粒可以有效地偶联至CAR-T细胞表面。同时，扫描电子显微镜（SEM）图像显示DBCO-INS颗粒成功结合在叠氮CAR-T细胞表面。
肿瘤抗原刺激对活细胞纳米药物的响应释放功能分析：INS-CAR T分别与Raji肿瘤细胞以1:1的比例孵育，ELISA法检测不同时间点上清液中IL-12的浓度。同时，用荧光染色法检测Cy5.5标记INS-CAR-T悬浮液的荧光强度。此外，通过扫描电镜观察分析INS-CAR-T与Raji肿瘤细胞孵育24 h后颗粒的响应性释放。荧光定量分析显示Raji肿瘤细胞刺激24 h后，CAR-T细胞表面的硫醇量增加约5倍（图4A和4B）。同时，扫描电镜成像显示，与对照组相比，经肿瘤细胞抗原刺激后所有与CAR-T细胞偶联的颗粒均已消失，IL-12已释放至细胞上清（图4C和4D），表明白蛋白纳米药物从CAR-T细胞表面响应释放。
实施例3  肿瘤抗原刺激响应的活细胞纳米药物背包抗肿瘤治疗
6-8周龄NOD-SCID小鼠皮下注射5 × 10 6 Raji细胞构建小鼠肿瘤模型。荷瘤后第8天（肿瘤体积约100 mm 3）分别尾静脉注射PBS、CAR-T细胞、IL-12 + CAR-T细胞或INS-CAR-T（100 μL, 1 × 10 7细胞/只）。在治疗期间连续测量小鼠肿瘤体积（肿瘤体积=长×宽 2/2），并计算小鼠存活率。其中根据动物伦理要求，当小鼠肿瘤大小超过1500 mm 3默认为死亡。
小鼠肿瘤体积测量结果表明，与单独细胞或白蛋白纳米药物相比，活细胞纳米药物背（INS-CAR-T）治疗组显著抑制了肿瘤生长，同时荷瘤小鼠存活率提高至100%，而其他对照治疗组小鼠出现了不同程度的死亡（图5）。
综合以上实施例，本发明公开了一种肿瘤抗原响应性活细胞纳米药物背包的制备方法及应用。通过生物正交偶联实现免疫淋巴细胞负载还原响应的白蛋白纳米药物，获得具有抗原响应控释的活细胞纳米药物背包。肿瘤抗原刺激免疫淋巴细胞引发细胞表面还原电势水平上升，从而触发白蛋白纳米背包定点还原响应释放。释放的纳米药物可以在肿瘤病灶原位激活免疫淋巴细胞，反馈增强免疫淋巴细胞的抗肿瘤效应，实现高效协同的肿瘤免疫治疗，并增强游离药物在体内的安全性与生物利用度。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (12)

  1. 一种肿瘤抗原响应性活细胞纳米药物背包在制备抗肿瘤药物中的应用，其特征在于，所述肿瘤抗原响应性活细胞纳米药物背包包括免疫淋巴细胞，所述免疫淋巴细胞上负载具有还原响应的白蛋白纳米药物；肿瘤抗原刺激所述免疫淋巴细胞引发细胞表面还原电势水平上升，从而触发所述活细胞纳米药物背包中药物的定点还原响应释放。
  2. 根据权利要求1所述的应用，其特征在于，所述具有还原响应的白蛋白纳米药物通过生物化学反应负载到所述免疫淋巴细胞上。
  3. 根据权利要求2所述的应用，其特征在于，所述生物化学反应为生物正交反应。
  4. 根据权利要求1所述的应用，其特征在于，所述免疫淋巴细胞为T细胞、B细胞、NK细胞、CAR-T、Treg细胞中的任意一种。
  5. 根据权利要求1所述的应用，其特征在于，所述纳米药物通过疏水作用包载在白蛋白纳米颗粒内。
  6. 根据权利要求1所述的应用，其特征在于，所述纳米药物为蛋白药物或化疗药物。
  7. 根据权利要求6所述的应用，其特征在于，所述蛋白药物为细胞因子、抗体、免疫因子、多肽中的任意一种或多种；所述化疗药物为阿霉素、紫杉醇、烷类药物中的任意一种或多种。
  8. 一种肿瘤抗原响应性活细胞纳米药物背包的制备方法，其特征在于，包括如下步骤：
    （1）免疫淋巴细胞的化学基团修饰；
    （2）还原响应白蛋白纳米药物颗粒的制备；
    （3）活细胞纳米药物背包的制备。
  9. 根据权利要求8所述的制备方法，其特征在于，所述步骤（1）中所述免疫淋巴细胞通过细胞代谢，将生物正交基团嵌入细胞膜表面，获得生物正交基团修饰的免疫淋巴细胞；所述细胞代谢为糖、脂类、氨基酸的细胞代谢中的任意一种。
  10. 根据权利要求8所述的制备方法，其特征在于，所述步骤（2）中通过谷胱甘肽打开白蛋白的二硫键，通过乙醇脱水包裹药物分子，制备得到还原响应的白蛋白纳米药物颗粒。
  11. 根据权利要求8所述的制备方法，其特征在于，所述步骤（3）中将所述步骤（1）制备得到的生物正交基团修饰的免疫淋巴细胞与所述步骤（2）制备得到的白蛋白纳米药物颗粒共孵育，通过生物正交反应实现白蛋白纳米药物颗粒与生物正交基团修饰的免疫淋巴细胞的偶联，获得所述的活细胞纳米药物背包。
  12. 一种肿瘤抗原响应性活细胞纳米药物背包，其特征在于，由权利要求8~11任一项所述的制备方法制备得到。
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