WO2023104053A1 - 一种氧化还原型纳米颗粒和活细胞载体及其应用 - Google Patents

一种氧化还原型纳米颗粒和活细胞载体及其应用 Download PDF

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WO2023104053A1
WO2023104053A1 PCT/CN2022/137014 CN2022137014W WO2023104053A1 WO 2023104053 A1 WO2023104053 A1 WO 2023104053A1 CN 2022137014 W CN2022137014 W CN 2022137014W WO 2023104053 A1 WO2023104053 A1 WO 2023104053A1
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cells
ilnps
cancer
nanoparticles
immune cells
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French (fr)
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蔡林涛
李文军
孟丹丹
潘宏
徐晓宇
梁振国
张旭
郑金玲
王子茜
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深圳先进技术研究院
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Definitions

  • the invention belongs to the field of nanomedicine, and specifically relates to a novel redox-type nanoparticle and living cell carrier, especially a preparation method of cytokine nanoparticle (ILNPs) and the construction and application of living NK cell carrier (NK-NPs).
  • ILNPs cytokine nanoparticle
  • NK-NPs living NK cell carrier
  • NK cells play an important role in directly killing malignant tumor cells and regulating adaptive immune responses.
  • NK cell immunotherapy can be used as a safe off-the-shelf adoptive cell immunotherapy.
  • NK cells can recognize and clear tumor cells faster without antigen sensitization and MHC restriction, and play a key role in early immune defense.
  • NK cells can regulate the adaptive immune response by secreting various cytokines and chemokines (such as IFN- ⁇ and TNF- ⁇ ), which further stimulate the activation of T cells, leading to a cascade of immune stimulatory responses.
  • cytokines and chemokines such as IFN- ⁇ and TNF- ⁇
  • NK cells are regulated by various signals including cytokines, activating receptors and inhibitory receptors.
  • cytokines such as IL-21, IL-12, IL-15, IL-17, IL-2, etc. activate NK cells and activate transcription factors to regulate autoimmune responses, as well as enhance NK cell toxicity, promote NK cell proliferation and Differentiation, accelerating the maturation of NK cells and the expression of related receptors are crucial.
  • exogenous cytokines cannot be highly enriched around NK cells in vivo to generate a high-concentration region sufficient to activate NK cell functions, which greatly limits the application of cytokines.
  • Nanocarriers are widely used because they can improve the solubility of drugs, enhance the stability of drugs, and achieve anti-tumor effects with low toxicity, high efficiency and sustained release. Nanoparticles are prime candidates for sustained drug release carriers due to their small size and high specific surface area. However, how to enhance the targeting of nanoparticles and improve the response of nanoparticles to the tumor microenvironment, to achieve sustained and controlled release of the drug delivery, so that the nanoparticles maintain structural integrity under normal physiological conditions, and only in the lesion site There is still a lot of room for exploration in terms of targeted release of drugs, thereby reducing damage to normal tissues and maximizing tumor killing effects.
  • NK cells with nanoparticles can realize the nano-engineering of NK cells and endow NK cells with related functions, such as carrying chemotherapy nanoparticles to enhance anti-tumor effect, which is an improved NK cell combined with exogenous cytokines Immunotherapy provides a new clinical basis.
  • NK cells In the prior art, free exogenous cytokine IL-21 was co-incubated with NK cells (Evelyn O Ojo et al., Sci Rep. 2019 Oct 17;9(1):14916. , doi: 10.1038/s41598-019-51287-6), can achieve NK cell expansion in vitro, but free cytokines lack targeting selectivity, are easily cleared by blood circulation, and cannot be The effective stimulating concentration is reached around NK cells, and a large amount of free exogenous cytokines enter the body, which easily triggers a factor storm. With the development of nanocarrier delivery technology, loading NK cells on the delivery carrier provides a solution to the above problems.
  • the way of chemical bonding binds the target object to the particle more stably, and it is not easy to fall off from the surface of the particle, such as CN113004418A.
  • the maleimide group is used to couple with the sulfhydryl group on NK cells to achieve selective modification of NK cells, such as CN113209019A, however, the C-S bond newly generated by this covalent binding method is under physiological conditions If the stability is insufficient, reverse Michael addition will occur, which will cause problems such as instability and low efficiency.
  • the purpose of the present invention is to overcome the inability of the free exogenous cytokine IL-21 to gather around the NK cells at a fixed point, and the method of linking nanoparticles to the surface of the NK cells is unstable.
  • a new type of redox-responsive nanoparticles is provided, as well as a living cell carrier that can "carry" the nanoparticles on the surface of NK cells stably and efficiently.
  • NK cells In the prior art, free exogenous cytokine IL-21 was co-incubated with NK cells (Evelyn O Ojo et al., Sci Rep. 2019 Oct 17;9(1):14916. , doi: 10.1038/s41598-019-51287-6), can achieve NK cell expansion in vitro, but free cytokines lack targeting selectivity, are easily cleared by blood circulation, and cannot be The effective stimulating concentration is reached around NK cells, and a large amount of free exogenous cytokines enter the body, which easily triggers a factor storm. With the development of nanocarrier delivery technology, loading NK cells on the delivery carrier provides a solution to the above problems.
  • the way of chemical bonding binds the target object to the particle more stably, and it is not easy to fall off from the surface of the particle, such as CN113004418A.
  • the maleimide group is used to couple with the sulfhydryl group on NK cells to achieve selective modification of NK cells, such as CN113209019A, however, the C-S bond newly generated by this covalent binding method is under physiological conditions If the stability is insufficient, reverse Michael addition will occur, which will cause problems such as instability and low efficiency.
  • the present invention provides a method for preparing redox-responsive nanoparticles (ILNPs), the method comprising the following steps:
  • the reduction-responsive high-efficiency crosslinking agent is selected from any one or more of the following:
  • NPC-NH-SS-NH-NPC its structural formula is as follows:
  • NPC-O-SS-O-NPC its structural formula is as follows:
  • the reduction-responsive high-efficiency cross-linking agent is NPC-NH-SS-NH-NPC.
  • the cytokine is an interleukin; preferably, the interleukin includes any one or more of the following: IL-21, IL-12, IL-15, IL-17, IL-2; Preferably, the cytokine is IL-21.
  • the recognition group of the living cells includes any one or more of the following: Anti-CD45, Anti-CD56, NHS active ester, Mal (maleimide) active ester; preferably, the active Cell recognition group Anti-CD45.
  • cross-linking agent solution and the cytokine protein solution are mixed according to a molar ratio of 0.1:1-10:1.
  • the stirring time is 10-60 min, preferably the stirring time is 30 min;
  • the ultrafiltration condition is 1-3 times of ultrafiltration through the ultrafiltration tube, 5-25 min each time, preferably ultrafiltration
  • the filtration condition is ultrafiltration of the ultrafiltration tube twice, each time of ultrafiltration for 10 minutes.
  • the particle size of the ILNPs is about 50-300 nm, preferably the particle size of the ILNPs is about 100-200 nm.
  • the present invention also provides redox-responsive nanoparticles (ILNPs) prepared by the above method.
  • ILNPs redox-responsive nanoparticles
  • the present invention also provides a method for constructing a living cell carrier in which nanoparticles are combined with immune cells, comprising the following steps:
  • Immune cells were taken, and a suitable culture medium containing 10% fetal bovine serum was used for expansion and culture;
  • step 2 Co-incubate the immune cells expanded in step 2) with the ILNPs prepared in step 1) in a suitable medium for 1-2 hours, and centrifuge to remove unconnected ILNPs to obtain nanoparticle-bound immune cells living cell carrier.
  • the immune cells include modified immune cells or unmodified isolated immune cells, the modification includes genetic modification, physicochemical modification, and the immune cells include NK cells, DC cells, CIK cells, DC - CIK cells, T cells and/or B cells.
  • the immune cells are NK cells
  • the NK cells include modified NK cells or unmodified isolated NK cells, and the modifications include genetic modification and physicochemical modification, and the medium is 1640 culture.
  • the present invention also provides the living cell carrier of the immune cell combined with the nanoparticle prepared by the above method, preferably the living cell carrier of the nanoparticle combined with the immune cell is a living cell carrier combined with the nanoparticle containing IL-21 and NK cells (NK-NPs), .
  • the living cell carrier of the nanoparticle combined with the immune cell is a living cell carrier combined with the nanoparticle containing IL-21 and NK cells (NK-NPs), .
  • the NK cells include modified NK cells or unmodified isolated NK cells, and the modifications include genetic modification and physicochemical modification.
  • the present invention also provides a composition containing the ILNPs and/or the living cell carrier, preferably, the composition is a pharmaceutical composition, and also contains a medically or pharmaceutically acceptable carrier or adjuvant.
  • the present invention also provides the application of the ILNPs and/or the living cell carrier in the preparation of medicines for treating diseases, preferably, the diseases include tumors, inflammation, Alzheimer's disease and/or cardiovascular and cerebrovascular disease.
  • the ILNPs or the drug or the composition also includes anti-tumor drugs; preferably, the anti-tumor drugs include anti-tumor broad-spectrum drugs and/or anti-tumor targeted drugs; more preferably, the Anti-tumor broad-spectrum drugs are selected from any one or more of camptothecin drugs, doxorubicin drugs, paclitaxel drugs or platinum-based drugs, and the anti-tumor targeted drugs are selected from zanubrutinib, nile Imatinib, Imatinib, Vimodeji, Vemurafenib, Tessirolimus, Sunitinib, Seritinib, Regorafenib, Afatinib, Trametinib, Sodium Axitinib, bortezomib, pazopanib, axitinib, romidepsin, everolimus, ibrutinib, lenvatinib, dabrafen
  • the ILNPs or the drug or the composition also includes a polypeptide substance, and the polypeptide includes an antigen or an antibody, more preferably, the antibody is selected from adalimumab, cetuximab, alternative Ilimomab, trastuzumab, nivolumab, daratumumab, ramucirumab, necituzumab, pembrolizumab, pembrolizumab, ofatumumab, Blinatumumab, bevacizumab, panitumumab, obinutuzumab, burtuximab, denutuximab, tositumomab, elotuzumab Any one or more of , trastuzumab or rituximab.
  • the antibody is selected from adalimumab, cetuximab, alternative Ilimomab, trastuzumab, nivolumab, daratumuma
  • the tumor is selected from basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, nasopharyngeal cancer, Any one or more of pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, and blastoma.
  • the ILNPs or the drug or the composition are in the form of injection; preferably, the injection includes subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, intralymph node injection, intratumoral injection or subfoot injection. one or more.
  • the present invention discloses a method for constructing redox-responsive IL-21 nanoparticles and living cell carriers, so as to realize the reduction-responsive release of nanoparticles in the tumor environment and the role of targeted activation of NK cells, and provides a A method for nanoengineering of NK cells.
  • the IL-21 protein was mixed with the redox-reactive cross-linker NPC-NH-SS-NH-NPC (bis(4-nitrophenyl), 344741-95-3) and stirred.
  • the recognition group Anti-CD45 of living cells was added on the surface of nanoparticles, so that it could be loaded on the surface of NK living cells through antigen-antibody reaction to prepare spherical nanoparticles ILNPs.
  • ILNPs were co-incubated with NK cells to make the hitchhiking nanoparticles on the surface of NK cells to prepare living cell carrier NK-NPs, so as to realize the efficient connection and synchronous transport of NK cells and nanoparticles.
  • NK living cells-nanocarriers Stimulation of the microenvironment at the lesion site (including but not limited to tumors, such as inflammation, Alzheimer's disease and other cardiovascular and cerebrovascular diseases) NK cell surface reducibility increases, resulting in the release of exogenous cytokines (including but not limited to IL-21, such as IL-12, IL-15, IL-17, IL-2, etc.) in redox-responsive nanoparticles , so as to form a high cytokine stimulation area around NK cells, and realize the effect of in situ, controllable and efficient stimulation of NK cell activation.
  • cytokines including but not limited to IL-21, such as IL-12, IL-15, IL-17, IL-2, etc.
  • the present invention has the following beneficial technical effects:
  • the present invention adopts two-step oscillation and stirring method to prepare nanoparticle ILNPs, and adopts a novel cross-linking agent NPC-NH-SS-NH-NPC to directly combine cytokines (including but not limited to IL-21, such as IL-12, IL -15, IL-17, IL-2, etc.) were cross-linked together to form a redox-responsive nanoparticle with remarkably controllable, highly efficient specificity.
  • cytokines including but not limited to IL-21, such as IL-12, IL -15, IL-17, IL-2, etc.
  • the method of constructing nanoparticles based on protein and cross-linking agent has mild reaction conditions, is simple, efficient, fast and has good reproducibility.
  • the preparation of nanoparticles in the present invention is based on the effect of the novel cross-linking agent NPC-NH-SS-NH-NPC, the method of the present invention can be applied to various functional proteins such as different cytokines and small molecules Drugs are entrapped in nanoparticles.
  • NK cell living cell carrier of the present invention is based on the protein specifically expressed on the surface of NK cells, using this feature, we can apply the construction concept of the living cell carrier to other cells, bacteria, viruses and other organisms. Nanoparticle engineered connection transformation.
  • this living cell carrier construction method has significant antibody specificity, which can greatly avoid false signals caused by traditional non-specific labeling based on amino and carboxyl reactions.
  • the labeled product is more stable.
  • the living cell carrier construction method of the present invention is simple and easy to operate and easy to operate and popularize.
  • Figure 1 is a schematic diagram of redox-responsive nanoparticle ILNPs.
  • Figure 2 is a Malvern particle sizer diagram of nanoparticle ILNPs.
  • Figure 3 is a schematic diagram of living cell carrier NK-NPs.
  • Figure 4 is a schematic diagram of the release of nanoparticles.
  • Fig. 5 is a scanning electron microscope image of nanoparticles on the surface of NK cells.
  • Figure 6 is a laser confocal image of the successful construction of live cell vectors by labeling nanoparticles and NK cells.
  • Embodiment 1 the preparation of nanoparticle ILNPs
  • Embodiment 2 Preparation of nanoparticle ILNPs
  • FIG. 1 is a schematic diagram of nanoparticle release.
  • Embodiment 3 the construction of living cell carrier
  • FIG. 3 is a schematic diagram of the living cell carrier, as shown in Figure 4 scanning electron microscope and flow cytometric analysis shown in Figure 5, the reduction-responsive nanoparticles were successfully loaded on the surface of NK cells.
  • Embodiment 4 Construction and confocal observation of living cell carrier
  • NK cells were washed by centrifugation, and the NK cells were uniformly seeded in 8-well culture plates (Lab-Tek® Nunc, USA), replaced with fresh 1640 medium, and 200 mL of medium was added to each well. After 24 hours, replace the 1640 medium containing 2 ⁇ l per well of 10 ⁇ g/mL nanoparticles. After incubation for 1 hour, wash with PBS twice, stain cell nuclei, and observe the 8-well plate under a laser scanning confocal microscope (Leica TCS SP5, Germany).
  • the nanoparticles are distributed on the surface of NK cell membranes, the blue represents the fluorescence of the nucleus Hoechest 33258, and the red represents the fluorescence of ILNPs, indicating that the nanoparticles ILNPs were successfully connected to the surface of NK cells, that is, the NK live cell carrier was successfully constructed.

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Abstract

提供一种氧化还原响应型纳米颗粒和活细胞载体，以实现纳米颗粒在肿瘤等环境中还原响应释放和定点激活NK细胞等免疫细胞的作用。提供的NK细胞纳米工程化改造的方法可简便高效地产生大量稳定的纳米颗粒，并使活NK细胞表面负载纳米颗粒，成功地解决了外源性细胞囚子不能移植到NK细胞周围产生高剂量蓄积以激活NK细胞的难题。

Description

一种氧化还原型纳米颗粒和活细胞载体及其应用 技术领域
本发明属于纳米医药领域，具体涉及一种新型氧化还原型纳米颗粒和活细胞载体，尤其是细胞因子纳米颗粒（ILNPs）的制备方法及活NK细胞载体（NK-NPs）的构建与应用。
 背景技术
自然杀伤（NK）细胞在直接杀伤恶性肿瘤细胞和调节适应性免疫反应中起着重要作用。NK细胞免疫疗法可以作为一种安全的现成过继细胞免疫治疗。NK细胞可以在没有抗原致敏和MHC限制的情况下更快地识别和清除肿瘤细胞，在早期免疫防御中起着关键作用。同时，NK细胞可通过分泌多种细胞因子和趋化因子（如IFN-γ和TNF-α）来调节适应性免疫反应，进一步刺激T细胞活化，导致级联免疫刺激反应。然而，在实体瘤的治疗过程中，NK细胞面临扩增，持久性和活化不足等问题，使得理论上有效的NK免疫治疗效果大打折扣。
NK细胞的发育和功能受到细胞因子、活化性受体和抑制性受体等多种信号调控。其中细胞因子如IL-21，IL-12，IL-15，IL-17，IL-2等对激活NK细胞并启动转录因子来调节自身免疫反应，以及增强NK细胞毒性，促进NK细胞的增殖和分化，加速NK细胞的成熟和相关受体的表达等至关重要。然而，在体内外源性细胞因子无法高度富集在NK细胞周围以产生足以激活NK细胞功能的高浓度区域，这大大限制了细胞因子的应用。
目前，联合多种药物、以不同机制对肿瘤进行治疗的“ 鸡尾酒疗法”已成为治疗癌症的有效手段，而纳米技术的快速发展也为治疗癌症提供了新材料和新思路。
纳米载体因其能够提高药物的溶解度，增强药物的稳定性，实现低毒高效缓释的抗肿瘤效果而被广泛应用。纳米颗粒由于其较小的尺寸和较高的比表面积成为药物缓释载体的首要候选者。然而，如何增强纳米颗粒的靶向性，以及改善纳米粒对肿瘤微环境的响应，实现对递送药物的缓释和控释，使纳米颗粒在正常的生理条件下维持结构完整，仅在病灶部位定点释放药物，进而减少了对正常组织的损伤和发挥最大的肿瘤杀伤效果等方面，依然有很大的探索空间。
将NK细胞与纳米颗粒连接在一起可实现NK细胞的纳米工程化改造，赋予NK细胞相关功能，如携带化疗纳米颗粒增强抗肿瘤效果，这为细胞与外源性细胞因子相结合的改良NK细胞免疫疗法提供了新的临床基础。
现有技术中，将游离的外源性细胞因子IL-21与NK细胞共孵育（Evelyn O Ojo等，Sci Rep. 2019 Oct 17;9(1):14916. ， doi: 10.1038/s41598-019-51287-6），能够实现NK细胞体外扩增，但是游离的细胞因子缺乏靶向选择性，容易被血液循环清除，无法在NK细胞周围达到有效刺激浓度，且大量的游离的外源性细胞因子进入体内，容易引发因子风暴。随着纳米载体递送技术的发展，将NK细胞负载与递送载体上为上述问题提供了解决方案，一般来说，纳米颗粒表面修饰方法有两种，一种是静电吸附，另一种是化学键合。相比于静电吸附，化学键合的方式将靶向物与颗粒结合更稳定，不容易从颗粒表面脱落，如CN113004418A。而马来酰亚胺基团被用于与NK细胞上的巯基偶联，以实现对NK细胞的选择性修饰，如CN113209019A，然而，由该共价结合方式新生成的C-S键在生理条件下稳定性不足，会发生逆迈克尔加成，进而造成不稳定和效率低等问题。
为了解决上述现有技术长期存在而未能解决的技术问题，本发明目的在于克服游离的外源性细胞因子IL-21无法定点聚集在NK细胞周围、纳米颗粒连接在NK细胞表面方法不稳定，效率低，特异性差等缺点，提供一种新型氧化还原性响应型纳米颗粒，以及能够使纳米颗粒稳定高效的“背负”在NK细胞表面的活细胞载体。
 技术问题
现有技术中，将游离的外源性细胞因子IL-21与NK细胞共孵育（Evelyn O Ojo等，Sci Rep. 2019 Oct 17;9(1):14916. ， doi: 10.1038/s41598-019-51287-6），能够实现NK细胞体外扩增，但是游离的细胞因子缺乏靶向选择性，容易被血液循环清除，无法在NK细胞周围达到有效刺激浓度，且大量的游离的外源性细胞因子进入体内，容易引发因子风暴。随着纳米载体递送技术的发展，将NK细胞负载与递送载体上为上述问题提供了解决方案，一般来说，纳米颗粒表面修饰方法有两种，一种是静电吸附，另一种是化学键合。相比于静电吸附，化学键合的方式将靶向物与颗粒结合更稳定，不容易从颗粒表面脱落，如CN113004418A。而马来酰亚胺基团被用于与NK细胞上的巯基偶联，以实现对NK细胞的选择性修饰，如CN113209019A，然而，由该共价结合方式新生成的C-S键在生理条件下稳定性不足，会发生逆迈克尔加成，进而造成不稳定和效率低等问题。
 技术解决方案
针对以上问题，本发明提供了一种氧化还原响应型纳米颗粒（ILNPs）的制备方法，所述方法包括如下步骤：
1）将还原响应性的高效交联剂溶解在二甲亚砜（DMSO）溶液中，得到交联剂溶液；
2）将细胞因子蛋白粉末溶解在磷酸盐缓冲溶液（PBS）中，得到细胞因子蛋白溶液；
3）将所述的交联剂溶液与细胞因子蛋白溶液混合，得到混合液，取适量所述混合液并用PBS缓冲液将混合物定量，然后在20℃-35℃，500-2500rpm下旋转搅拌，再将活细胞的识别基团加入上述溶液中，并在20℃-35℃，500-2500rpm下再旋转搅拌，超滤后即得氧化还原响应性纳米颗粒（ILNPs）；
所述的还原响应性的高效交联剂选自以下任一种或多种：
A. NPC-NH-SS-NH-NPC，其结构式如下：
Figure dest_path_image001
B.  NPC-O-SS-O-NPC，其结构式如下：
Figure dest_path_image002
C.  NHS-SS-NHS，其结构式如下：
Figure dest_path_image003
；
较佳地，所述的还原响应性的高效交联剂为NPC-NH-SS-NH-NPC。
进一步地，所述细胞因子为白细胞介素；优选地，所述白细胞介素包括以下任一种或多种：IL-21、IL-12、IL-15、IL-17、IL-2；较佳地，所述细胞因子为IL-21。
进一步地，所述活细胞的识别基团包括以下任意一种或多种：Anti-CD45、Anti-CD56、NHS活性酯、Mal（马来酰亚胺）活性酯；较佳地，所述活细胞的识别基团Anti-CD45。
进一步地，所述交联剂溶液和所述细胞因子蛋白溶液按照摩尔比为0.1:1-10:1的比例混合。
进一步地，所述搅拌时间为10-60 min，较佳地搅拌时间为30min；所述超滤条件为超滤管超滤1-3次，每次超滤5-25 min，较佳地超滤条件为超滤管超滤2次，每次超滤10分钟。
进一步地，所述ILNPs的粒径约为50-300 nm，较佳地所述ILNPs的粒径约为100-200nm。
本发明还提供了由上述方法制备的氧化还原响应型纳米颗粒（ILNPs）。
本发明还提供了一种纳米颗粒结合免疫细胞的活细胞载体的构建方法，包括如下步骤：
1）ILNPs的制备
按上述的制备方法获得ILNPs；
2）免疫细胞的培养与扩增
取免疫细胞,使用含10%胎牛血清的合适的培养基进行扩增培养；
3）免疫细胞的活细胞载体的构建
在合适的培养基中将所述步骤2）扩增得到的免疫细胞与所述步骤1）制备的ILNPs按比例共孵育1-2小时，离心去除未连接的ILNPs，即得到纳米颗粒结合免疫细胞的活细胞载体。
进一步地，所述免疫细胞包括经修饰的免疫细胞或或未经修饰的分离的免疫细胞，所述修饰包括遗传修饰、物理化学修饰，所述免疫细胞包括NK细胞、DC细胞、CIK细胞、DC-CIK细胞、T细胞和/或B细胞。
进一步地，所述免疫细胞为NK细胞，所述NK细胞包括经修饰的NK细胞或未经修饰的分离的NK细胞，所述修饰包括遗传修饰、物理化学修饰，所述培养基为1640培养。
本发明还提供了由上述方法制备的纳米颗粒结合的免疫细胞的活细胞载体，优选为所述纳米颗粒结合免疫细胞的活细胞载体为含IL-21的纳米颗粒与NK细胞结合的活细胞载体（NK-NPs），。
进一步地，所述NK细胞包括经修饰的NK细胞或未经修饰的分离的NK细胞，所述修饰包括遗传修饰、物理化学修饰。
本发明还提供了一种含有所述ILNPs和/或所述活细胞载体的组合物，优选地，所述组合物为药物组合物，还含有医学或药学上可接受的载体或辅料。
本发明还提供了所述ILNPs和/或所述活细胞载体在制备用于治疗疾病的药物中的应用，优选地，所述疾病包括肿瘤、炎症、阿尔兹海默症和/或心脑血管疾病。
进一步地，所述ILNPs或所述药物或所述组合物还包括抗肿瘤药物；优选地，所述抗肿瘤药物包括抗肿瘤广谱药物和/或抗肿瘤靶向药物；更优选地，所述抗肿瘤广谱药物选自喜树碱类药物、阿霉素类药物、紫杉醇类药物或铂类药物中任一种或多种，所述抗肿瘤靶向药物选自泽布替尼、尼罗替尼、伊马替尼、维莫德吉、维罗非尼、替西罗莫司、舒尼替尼、赛立替尼、瑞格非尼、阿法替尼、曲美替尼、普钠替尼、硼替佐米、帕唑帕尼、阿西替尼、罗米地辛、依维莫司、依鲁替尼、乐伐替尼、达拉菲尼、克唑替尼、卡非佐米、奥斯替尼、卡博替尼、卡比替尼、吉非替尼、伏立诺他、凡德他尼、艾乐替尼、狄诺塞麦、索尼德吉、索拉非尼、博舒替尼、贝利司他、奥拉帕尼、阿柏西普、拉帕替尼、达沙替尼、帕博西尼、帕比司他或厄洛替尼中任一种或多种。
进一步地，所述ILNPs或所述药物或所述组合物还包括多肽类物质，所述多肽包括抗原或抗体，更优选地，所述抗体选自阿达木单抗、西妥昔单抗、替伊莫单抗、曲妥珠单抗、纳武单抗、达雷木单抗雷莫芦单抗、耐昔妥珠单抗、派姆单抗、派姆单抗、奥法木单抗、博纳吐单抗、贝伐珠单抗、帕尼单抗、奥宾尤妥珠单抗、本妥昔单抗、地努图希单抗、托西莫单抗、埃罗妥珠单抗、曲妥珠单抗或利妥昔单抗中任一种或多种。
进一步地，所述肿瘤选自基底细胞癌、鳞状细胞癌、食管癌、恶性胶质瘤、膀胱癌、宫颈癌、乳腺癌、肺癌、肝癌、胃癌、结肠癌、直肠癌、鼻咽癌、胰腺癌、甲状腺癌、前列腺癌、白血病、淋巴瘤、肾脏肿瘤、肉瘤、母细胞瘤中任一种或多种。
进一步地，所述ILNPs或所述药物或所述组合物通过注射剂型；优选地，所述注射包括皮下注射、肌肉注射、腹腔注射、静脉注射、淋巴结内注射、瘤内注射或足下注射中任一种或多种。
由上，本发明公开了一种氧化还原响应型IL-21纳米颗粒和活细胞载体的构建方法，以实现纳米颗粒在肿瘤环境中的还原响应释放和定点激活NK细胞的作用，提供了一种NK细胞纳米工程化改造的方法。将IL-21蛋白与氧化还原反应性交联剂NPC-NH-SS-NH-NPC（双（4-硝基苯基），344741-95-3）混合搅拌。接着，在纳米颗粒表面添加活细胞的识别基团Anti-CD45，使其能够通过抗原抗体反应负载于NK活细胞表面，以制备球形纳米颗粒ILNPs。这种方法可简便高效地产生大量稳定的纳米颗粒。随后，将ILNPs与NK细胞共孵育，使NK细胞表面“背”上搭便车的纳米颗粒，制备得到活细胞载体NK-NPs，从而实现NK细胞和纳米颗粒的高效连接与同步转运。
本发明的NK活细胞-纳米载（NK-NPs）体在病变部位的释放：在病变部位（包括但不限于肿瘤，如炎症、阿尔兹海默症等心脑血管疾病）微环境的刺激作用下，NK细胞表面还原性增高，导致氧化还原响应性纳米颗粒中释放外源性细胞因子（包括但不限于IL-21，例如IL-12，IL-15，IL-17，IL-2等），从而在NK细胞周围形成高细胞因子刺激区域，实现原位、可控、高效的刺激NK细胞活化的作用。这成功地解决了外源性细胞因子不能移植到NK细胞周围产生高剂量蓄积以激活NK细胞的难题。
 有益效果
本发明相比于现有技术，具有以下有益的技术效果：
1. 本发明采用两步震荡搅拌法制备纳米颗粒ILNPs，采用新型的交联剂NPC-NH-SS-NH-NPC，直接将细胞因子（包括但不限于IL-21，例如IL-12，IL-15，IL-17，IL-2等）交联在一起，形成了一个氧化还原响应性的纳米颗粒，具有明显的可控，高效的特异性。
2. 基于蛋白与交联剂的纳米颗粒的构建方法，反应条件温和，简便，高效，快速，重现性好。
3. 由于本发明中的纳米颗粒制备是基于新型交联剂NPC-NH-SS-NH-NPC的作用，因此，本发明的方法可以适用于将不同的细胞因子等各种功能蛋白和小分子药物进行纳米颗粒包载。
4. 由于本发明的NK细胞活细胞载体的构建是基于NK细胞表面特异表达的蛋白，利用此特征，我们可以将活细胞载体这一构建理念应用到对其它细胞，细菌，病毒等生物体的纳米颗粒工程化连接改造。
5. 对于相对复杂的生物体系如血液、人体组织等体系中，这种活细胞载体构建方法具有显著的抗体特异性，可以大大避免传统基于氨基和羧基反应的非特异性标记带来的伪信号。同时相对正负电荷吸附方法，由于是抗原抗体特异性结合，因此标记产物更加稳定。
6. 本发明的活细胞载体构建方法简便易行，便于操作推广。
 附图说明
图1为氧化还原响应型纳米颗粒ILNPs的示意图。
图2为纳米颗粒ILNPs的马尔文粒径仪图。
图3为活细胞载体NK-NPs的示意图。
图4为纳米颗粒的释放示意图。
图5为纳米颗粒在NK细胞表面的扫描电镜图。
图6为通过标记纳米颗粒和NK细胞确定活细胞载体成功构建的激光共聚焦图。
 本发明的实施方式
以下通过具体实施例对本发明作进一步详细说明，以使本领域技术人员能够更好地理解本发明并予以实施，但实施例并不作为本发明的限定。
以下实施例中所使用的实验方法如无特殊说明，均为常规方法。所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：纳米颗粒ILNPs的制备
将0.05 µmol NPC-NH-SS-NH-NPC溶解在25 µl二甲亚砜（DMSO）溶液，再0.0335 µmol 的IL-21蛋白粉末溶解于50 µl PBS溶液中。将两者置于同一个EP管中，并用PBS缓冲液将混合物定量至1 ml，然后在25℃，1200rpm下旋转搅拌30min。再将10µl 1mg/ml的Anti-CD45加入上述溶液中，并在25℃，1200rpm下再旋转搅30min，超滤管超滤2次，每次超滤10分钟，即得氧化还原响应性纳米颗粒ILNPs，其中粒径约为100-200nm。如图1和图2所示，纳米颗粒的示意图以及纳米颗粒的水合粒径。
实施例2：纳米颗粒ILNPs的制备
将1 µmol NPC-NH-SS-NH-NPC溶解在25 µl二甲亚砜（DMSO）溶液，再将0.05 µmol 的IL-21蛋白粉末溶解于50 µl PBS溶液中。将两者置于同一EP管中，并用PBS缓冲液将混合物定量至2ml，然后在25℃，1200rpm下旋转搅拌60min。再将 5µl 1mg/ml的Anti-CD45加入上述溶液中，并在25℃，1200rpm下再旋转搅60min，超滤管超滤1次，每次超滤5分钟，即得氧化还原响应性纳米颗粒ILNPs ，如图3为纳米颗粒释放的示意图。
实施例3:活细胞载体的构建
取50万NK细胞离心清洗，换新鲜的1640培养基，然后加入准备好的纳米颗粒（10µl，10μg/mL），共孵育1小时，然后离心去除未连接的纳米颗粒，即得到活细胞载体NK-NPs。如图3为活细胞载体的示意图，如图4扫描电镜和图5所示的流式细胞分析所示，还原响应性纳米颗粒成功地负载在NK细胞表面。
实施例4:活细胞载体的构建与共聚焦观察
取100万NK细胞离心清洗，将NK细胞均匀接种在8孔培养板中（Lab-Tek® Nunc，美国），换新鲜的1640培养基，每孔各加入200mL培养基。24小时后，更换每孔含2µl，10μg/mL 纳米颗粒的1640培养基。孵育1小时后，PBS洗涤清洗2次，染细胞核，将8孔板置于激光扫描共聚焦显微镜下观察（Leica TCS SP5，德国）。如图6所示，纳米颗粒分布于NK细胞膜表面，蓝色代表细胞核 Hoechest 33258的荧光，红色代表ILNPs的荧光，表明纳米颗粒ILNPs成功连接在NK细胞表面，即成功构建了NK活细胞载体。
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。

Claims (18)

  1. 一种氧化还原响应型纳米颗粒（ILNPs）的制备方法，所述方法包括如下步骤：
    1）将还原响应性的高效交联剂溶解在二甲亚砜（DMSO）溶液中，得到交联剂溶液；
    2）将细胞因子蛋白粉末溶解在磷酸盐缓冲溶液（PBS）中，得到细胞因子蛋白溶液；
    3）将所述的交联剂溶液与细胞因子蛋白溶液混合，得到混合液，取适量所述混合液并用PBS缓冲液将混合物定量，然后在20℃-35℃，500-2500rpm下旋转搅拌，再将活细胞的识别基团加入上述溶液中，并在20℃-35℃，500-2500rpm下再旋转搅拌，超滤后即得氧化还原响应性纳米颗粒（ILNPs）；
    所述的还原响应性的高效交联剂选自以下任一种或多种：
    A. NPC-NH-SS-NH-NPC，其结构式如下：
    Figure dest_path_image001
    B.  NPC-O-SS-O-NPC，其结构式如下：
    Figure dest_path_image002
    C.  NHS-SS-NHS，其结构式如下：
    Figure dest_path_image003
    ；
    较佳地，所述的还原响应性的高效交联剂为NPC-NH-SS-NH-NPC。
  2. 根据权利要求1所述的方法，所述细胞因子为白细胞介素；优选地，所述白细胞介素包括以下任一种或多种：IL-21、IL-12、IL-15、IL-17、IL-2；较佳地，所述细胞因子为IL-21。
  3. 根据权利要求1所述的方法，所述活细胞的识别基团包括以下任意一种或多种：Anti-CD45、Anti-CD56、NHS活性酯、Mal（马来酰亚胺）活性酯；较佳地，所述活细胞的识别基团Anti-CD45。
  4. 根据权利要求1所述的方法，所述交联剂溶液和所述细胞因子蛋白溶液按照摩尔比为0.1:1-10:1的比例混合。
  5. 根据权利要求1所述的方法，所述搅拌时间为10-60 min，较佳地搅拌时间为30min；所述超滤条件为超滤管超滤1-3次，每次超滤5-25 min，较佳地超滤条件为超滤管超滤2次，每次超滤10分钟。
  6. 根据权利要求1所述的方法，所述ILNPs的粒径约为50-300 nm；较佳地所述ILNPs的粒径约为100-200nm。
  7. 根据权利要求1-6任一项所述的方法制备的氧化还原响应型纳米颗粒（ILNPs）。
  8. 一种纳米颗粒结合免疫细胞的活细胞载体的构建方法，包括如下步骤：
    1）ILNPs的制备
    按权利要求1-6任一项所述的制备方法获得ILNPs；
    2）免疫细胞的培养与扩增
    取免疫细胞,使用含10%胎牛血清的合适的培养基进行扩增培养；
    3）免疫细胞的活细胞载体的构建
    在合适的培养基中将所述步骤2）扩增得到的免疫细胞与所述步骤1）制备的ILNPs按比例共孵育1-2小时，离心去除未连接的ILNPs，即得到纳米颗粒结合免疫细胞的活细胞载体。
  9. 根据权利要求8所述的方法，所述免疫细胞包括经修饰的免疫细胞或或未经修饰的分离的免疫细胞，所述修饰包括遗传修饰、物理化学修饰，所述免疫细胞包括NK细胞、DC细胞、CIK细胞、DC-CIK细胞、T细胞和/或B细胞。
  10. 根据权利要求8所述的方法，所述免疫细胞为NK细胞，所述NK细胞包括经修饰的NK细胞或未经修饰的分离的NK细胞，所述修饰包括遗传修饰、物理化学修饰，所述培养基为1640培养。
  11. 根据权利要求8-10任一项所述的方法制备的纳米颗粒结合免疫细胞的活细胞载体。
  12. 根据权利要求11所述的活细胞载体，所述纳米颗粒结合免疫细胞的活细胞载体为含IL-21的纳米颗粒与NK细胞结合的活细胞载体（NK-NPs），所述NK细胞包括经修饰的NK细胞或未经修饰的分离的NK细胞，所述修饰包括遗传修饰、物理化学修饰。
  13. 一种含有权利要求1-7任一项所述的ILNPs和/或权利要求8-12任一项所述活细胞载体的组合物；优选地，所述组合物为药物组合物，还含有医学或药学上可接受的载体或辅料。
  14. 权利要求1-7任一项所述的ILNPs和/或权利要求8-12任一项所述的活细胞载体在制备用于治疗疾病的药物中的应用；优选地，所述疾病包括肿瘤、炎症、阿尔兹海默症和/或心脑血管疾病；更优选地，所述疾病为肿瘤。
  15. 根据权利要求13所述的组合物或权利要求14所述的应用，所述组合物或所述药物还包括抗肿瘤药物；优选地，所述抗肿瘤药物包括抗肿瘤广谱药物和/或抗肿瘤靶向药物；更优选地，所述抗肿瘤广谱药物选自喜树碱类药物、阿霉素类药物、紫杉醇类药物或铂类药物中任一种或多种，所述抗肿瘤靶向药物选自泽布替尼、尼罗替尼、伊马替尼、维莫德吉、维罗非尼、替西罗莫司、舒尼替尼、赛立替尼、瑞格非尼、阿法替尼、曲美替尼、普钠替尼、硼替佐米、帕唑帕尼、阿西替尼、罗米地辛、依维莫司、依鲁替尼、乐伐替尼、达拉菲尼、克唑替尼、卡非佐米、奥斯替尼、卡博替尼、卡比替尼、吉非替尼、伏立诺他、凡德他尼、艾乐替尼、狄诺塞麦、索尼德吉、索拉非尼、博舒替尼、贝利司他、奥拉帕尼、阿柏西普、拉帕替尼、达沙替尼、帕博西尼、帕比司他或厄洛替尼中任一种或多种。
  16. 根据权利要求13所述的组合物或权利要求14所述的应用，所述组合物或所述药物还包括多肽类物质；优选地，所述多肽包括抗原或抗体；更优选地，所述抗体选自阿达木单抗、西妥昔单抗、替伊莫单抗、曲妥珠单抗、纳武单抗、达雷木单抗雷莫芦单抗、耐昔妥珠单抗、派姆单抗、派姆单抗、奥法木单抗、博纳吐单抗、贝伐珠单抗、帕尼单抗、奥宾尤妥珠单抗、本妥昔单抗、地努图希单抗、托西莫单抗、埃罗妥珠单抗、曲妥珠单抗或利妥昔单抗中任一种或多种。
  17. 根据权利要求14-16任一项所述的应用，所述肿瘤选自基底细胞癌、鳞状细胞癌、食管癌、恶性胶质瘤、膀胱癌、宫颈癌、乳腺癌、肺癌、肝癌、胃癌、结肠癌、直肠癌、鼻咽癌、胰腺癌、甲状腺癌、前列腺癌、白血病、淋巴瘤、肾脏肿瘤、肉瘤、母细胞瘤中任一种或多种。
  18. 根据权利要求7所述的ILNPs或权利要求13所述的组合物或权利要求14所述的应用，所述ILNPs或所述药物或所述组合物是注射剂；优选地，所述注射包括皮下注射、肌肉注射、腹腔注射、静脉注射、淋巴结内注射、瘤内注射或足下注射中任一种或多种。
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ZHAO SONGBO, DUAN JIAZHI, LOU YALIN, GAO RUYUN, YANG SHANSHAN, WANG PIMING, WANG CHUNHUA, HAN LIN, LI MINGHUAN, MA CHUNHONG, LIANG: "Surface specifically modified NK-92 cells with CD56 antibody conjugated superparamagnetic Fe 3 O 4 nanoparticles for magnetic targeting immunotherapy of solid tumors", NANOSCALE, ROYAL SOCIETY OF CHEMISTRY, UNITED KINGDOM, vol. 13, no. 45, 25 November 2021 (2021-11-25), United Kingdom , pages 19109 - 19122, XP093072571, ISSN: 2040-3364, DOI: 10.1039/D1NR03329H *

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