CN115957342A - Drug/gene-loaded multifunctional nano-delivery platform and its preparation and application - Google Patents
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
Description
技术领域technical field
本发明属于功能性纳米材料及其制备和应用领域，特别涉及一种负载药物/基因的多功能纳米递送平台及其制备和应用。The invention belongs to the field of functional nanomaterials and their preparation and application, in particular to a drug/gene-loaded multifunctional nanometer delivery platform and its preparation and application.
背景技术Background technique
随着纳米技术的不断发展，树状大分子(尤其是聚酰胺-胺类树状大分子，PAMAM)纳米材料在基因递送、药物传递、分子影像和肿瘤诊疗等生物医学领域表现出极大的应用潜力。而基于PAMAM树状大分子构建的核壳结构树状大分子(CSTDs)不但继承了单代树状大分子高度可修饰性、无免疫原性等优点，还克服了单代树状大分子药物负载量、基因传递效率等局限，因此，构建多功能性CSTDs纳米平台，并将其应用于前沿生物医学领域已成为科研工作者研究的最新方向之一。With the continuous development of nanotechnology, dendrimers (especially polyamide-amine dendrimers, PAMAM) nanomaterials have shown great promise in biomedical fields such as gene delivery, drug delivery, molecular imaging, and tumor diagnosis and treatment. application potential. The core-shell structure dendrimers (CSTDs) based on PAMAM dendrimers not only inherit the advantages of single-generation dendrimers, such as high modifiability and non-immunogenicity, but also overcome the disadvantages of single-generation dendrimers. Therefore, constructing a multifunctional CSTDs nano-platform and applying it to the frontier biomedical field has become one of the latest research directions for researchers.
环糊精(CD)和金刚烷(Ad)之间的超分子主客体相互作用已被普遍用于构建超分子结构，如用于构建基于主客体的超分子自组装的CSTDs。在我们前期的工作中，第5代氨基封端的PAMAM树状大分子表面部分修饰β-CD，第3代氨基封端的PAMAM树状大分子表面部分修饰Ad，通过β-CD和Ad的主客体识别，G5-CD/Ad-G3 CSTD可以被成功合成出来。和单独的G5-CD或Ad-G3树状大分子相比，其基因传递效率分别提高了20倍和170倍(Chen F.etal.J.Mater.Chem.B,2017,5,8459)。而且，G5-CD/Ad-G3作为纳米平台还可以负载基因型抑制剂(MicroRNA 21inhibitor，miR 21i)和抗癌药物，实现基因和药物共递送并用于三阴性乳腺癌细胞的基因-化疗的联合治疗(Song C.et al.J.Mater.Chem.B.2020,8,2768-2774)。但遗憾的是，由于纳米平台本身的限制(如表面电势过高、无主动靶向性能等)，该平台负载药物和基因后，没有用于体内联合治疗评价。The supramolecular host-guest interaction between cyclodextrin (CD) and adamantane (Ad) has been commonly used to construct supramolecular structures, such as CSTDs for constructing host-guest-based supramolecular self-assembly. In our previous work, the surface of the 5th generation of amino-terminated PAMAM dendrimers was partially modified with β-CD, and the surface of the 3rd generation of amino-terminated PAMAM dendrimers was partially modified with Ad, through the host-guest of β-CD and Ad identified, G5-CD/Ad-G3 CSTD can be successfully synthesized. Compared with individual G5-CD or Ad-G3 dendrimers, the gene delivery efficiency is increased by 20 times and 170 times respectively (Chen F.etal.J.Mater.Chem.B,2017,5,8459). Moreover, as a nano-platform, G5-CD/Ad-G3 can also be loaded with genotype inhibitors (MiR 21i) and anticancer drugs, so as to realize the co-delivery of genes and drugs and be used for the combination of gene-chemotherapy in triple-negative breast cancer cells. Treatment (Song C. et al. J. Mater. Chem. B. 2020, 8, 2768-2774). Unfortunately, due to the limitations of the nano-platform itself (such as too high surface potential, no active targeting performance, etc.), the platform has not been used for in vivo combined therapy evaluation after loading drugs and genes.
检索国内外相关文献和专利结果表明，改进合成的G5-CD/Ad-G3进行两性离子和甘露糖的修饰，构建出新型的多功能核壳树状大分子纳米递送平台以靶向递送功能性基因YTHDF1siRNA用于激活树突细胞，提高肿瘤免疫治疗效果，目前尚未见报道。将改进合成的G5-CD/Ad-G3进行乙酰化处理构建出新型的乙酰化平台以负载抗癌药物用于基于免疫原性死亡的免疫治疗，尚未见报道。联合使用上述两种多功能纳米平台用于乳腺癌的化疗-免疫治疗，使其具有“双管齐下”和“1+1＞2”的协同治疗效果，目前尚未见报道。Retrieval of relevant literature and patents at home and abroad showed that the improved synthetic G5-CD/Ad-G3 was modified with zwitterions and mannose, and a new multifunctional core-shell dendrimer nano-delivery platform was constructed to deliver functional The gene YTHDF1siRNA is used to activate dendritic cells and improve the effect of tumor immunotherapy, which has not been reported yet. The acetylation treatment of the improved synthetic G5-CD/Ad-G3 to construct a novel acetylation platform to load anticancer drugs for immunotherapy based on immunogenic death has not been reported yet. The combined use of the above two multifunctional nano-platforms for chemotherapy-immunotherapy of breast cancer has a synergistic therapeutic effect of "two-pronged approach" and "1+1>2", which has not been reported so far.
发明内容Contents of the invention
本发明所要解决的技术问题是提供一种负载药物/基因的多功能纳米递送平台及其制备和应用，以填补现有技术的空白。The technical problem to be solved by the present invention is to provide a drug/gene-loaded multifunctional nano-delivery platform and its preparation and application, so as to fill the gap in the prior art.
本发明提供一种多功能纳米递送平台，包括：CBAA-G5/G3-Man多功能纳米平台负载基因YTHDF1 siRNA；所述CBAA-G5/G3-Man多功能纳米平台是由G5-CD和Ad-G3-Man自组装形成的G5-CD/Ad-G3-Man与羧酸甜菜碱CBAA反应得到，其中，G5-CD为第5代氨基封端的PAMAM树状大分子表面修饰β-环糊精β-CD，Ad-G3-Man为第3代氨基封端的PAMAM树状大分子表面修饰金刚烷Ad和甘露糖Man。The present invention provides a multifunctional nano delivery platform, comprising: CBAA-G5/G3-Man multifunctional nano platform loading gene YTHDF1 siRNA; the CBAA-G5/G3-Man multifunctional nano platform is composed of G5-CD and Ad- G5-CD/Ad-G3-Man formed by self-assembly of G3-Man is obtained by reacting with carboxybetaine CBAA, wherein G5-CD is the 5th generation amino-terminated PAMAM dendrimer surface modified β-cyclodextrin β -CD, Ad-G3-Man is the third-generation amino-terminated PAMAM dendrimer surface-modified adamantane Ad and mannose Man.
优选地，所述CBAA-G5/G3-Man多功能纳米平台和YTHDF1 siRNA的氮磷比(N/P)为0.5:1～30:1。Preferably, the nitrogen-to-phosphorus ratio (N/P) of the CBAA-G5/G3-Man multifunctional nano-platform and YTHDF1 siRNA is 0.5:1-30:1.
优选地，还包括负载抗癌药物纳米平台，所述负载抗癌药物纳米平台包括：G5.NHAc-CD/Ad-G3.NHAc乙酰化纳米平台包裹抗癌药物阿霉素；Preferably, it also includes a nano-platform loaded with anti-cancer drugs, the nano-platform loaded with anti-cancer drugs includes: G5.NHAc-CD/Ad-G3.NHAc acetylated nano-platform encapsulates the anti-cancer drug doxorubicin;
所述G5.NHAc-CD/Ad-G3.NHAc乙酰化纳米平台是Ad-G3和G5-CD乙酰化后得到；其中，G5-CD为第5代氨基封端的PAMAM树状大分子表面修饰β-环糊精β-CD，Ad-G3为第3代氨基封端的PAMAM树状大分子表面修饰金刚烷Ad。The G5.NHAc-CD/Ad-G3.NHAc acetylation nano-platform is obtained after the acetylation of Ad-G3 and G5-CD; wherein, G5-CD is the 5th generation amino-terminated PAMAM dendrimer surface modification β -Cyclodextrin β-CD, Ad-G3 is the third-generation amino-terminated PAMAM dendrimer surface-modified adamantane Ad.
优选地，所述G5.NHAc-CD/Ad-G3.NHAc乙酰化纳米平台和抗癌药物阿霉素的摩尔比为1:10～1:20。Preferably, the molar ratio of the G5.NHAc-CD/Ad-G3.NHAc acetylation nano-platform to the anticancer drug doxorubicin is 1:10-1:20.
本发明还提供一种多功能纳米递送平台的制备方法，包括：The present invention also provides a preparation method of a multifunctional nano-delivery platform, comprising:
(1)将金刚烷乙酸Ad-COOH分散在溶剂中，加入EDC.HCl和NHS溶液活化，将活化的Ad-COOH溶液加入到G3.NH2溶液中，反应，透析、冻干处理，得到Ad-G3；(1) Disperse adamantane acetic acid Ad-COOH in a solvent, add EDC.HCl and NHS solution to activate, add the activated Ad-COOH solution to G3.NH 2 solution, react, dialyze and lyophilize to obtain Ad -G3;
(2)将步骤(1)中Ad-G3溶于PBS溶液中，逐滴加入溶于PBS溶液中的甘露糖Man，第反应，透析、冻干处理，得到Ad-G3-Man；(2) Dissolving Ad-G3 in the PBS solution in step (1), adding mannose Man dissolved in the PBS solution dropwise, performing the first reaction, dialysis, and freeze-drying to obtain Ad-G3-Man;
(3)将β-环糊精β-CD分散在溶剂中，逐滴加入N,N'-羰基二咪唑(CDI)溶液活化，将活化的β-CD溶液加入到G5.NH2溶液中，氨羟化反应，，透析、冻干处理，得到G5-CD，然后将G5-CD和步骤(2)中Ad-G3-Man分别用超纯水溶解后混合，超分子自组装反应，透析、冻干处理，得到G5-CD/Ad-G3-Man；(3) Disperse β-cyclodextrin β-CD in the solvent, add N,N'-carbonyldiimidazole (CDI) solution dropwise to activate, and add the activated β-CD solution to the G5.NH 2 solution, Aminohydroxylation reaction, dialysis, and freeze-drying to obtain G5-CD, and then G5-CD and Ad-G3-Man in step (2) were dissolved in ultrapure water and mixed, supramolecular self-assembly reaction, dialysis, Freeze-drying to obtain G5-CD/Ad-G3-Man;
(4)分别将步骤(3)中G5-CD/Ad-G3-Man溶解于溶剂1中，将羧酸甜菜碱CBAA溶解于溶剂2中，将这两种溶液混合，反应，透析、冻干处理，得到CBAA-G5/G3-Man；(4) Dissolve G5-CD/Ad-G3-Man in step (3) in
(5)将步骤(4)中CBAA-G5/G3-Man与YTHDF1 siRNA进行孵育，得到CBAA-G5/G3-Man/YTHDF1 siRNA复合物，即多功能纳米递送平台。(5) Incubate CBAA-G5/G3-Man and YTHDF1 siRNA in step (4) to obtain a CBAA-G5/G3-Man/YTHDF1 siRNA complex, which is a multifunctional nano-delivery platform.
优选地，所述步骤(1)中Ad-COOH、EDC.HCl、NHS和G3.NH2的摩尔比为1～1.5:10:10:1；溶剂为DMSO。Preferably, the molar ratio of Ad-COOH, EDC.HCl, NHS and G3.NH 2 in the step (1) is 1-1.5:10:10:1; the solvent is DMSO.
优选地，所述步骤(1)中活化温度为室温，活化时间为2～4h。Preferably, the activation temperature in the step (1) is room temperature, and the activation time is 2-4 hours.
优选地，所述步骤(1)中反应温度为室温，反应时间为2～4d。Preferably, the reaction temperature in the step (1) is room temperature, and the reaction time is 2-4d.
优选地，所述步骤(2)中Ad-G3与Man的摩尔比为1:15～25。Preferably, the molar ratio of Ad-G3 to Man in the step (2) is 1:15-25.
优选地，所述步骤(2)中逐滴加入溶于PBS溶液中的甘露糖Man是在85-95℃条件下。Preferably, the dropwise addition of mannose Man dissolved in PBS solution in the step (2) is at 85-95°C.
优选地，所述步骤(2)中反应温度为85-95℃，反应时间为1～6h。Preferably, the reaction temperature in the step (2) is 85-95°C, and the reaction time is 1-6h.
优选地，所述步骤(1)、(2)、(3)、(4)中透析采用截留分子量为1000-50000的纤维素透析膜，在超纯水中透析2-3天。Preferably, the dialysis in the steps (1), (2), (3), and (4) uses a cellulose dialysis membrane with a molecular weight cut-off of 1000-50000, and dialyzes in ultrapure water for 2-3 days.
优选地，所述步骤(3)中溶剂为DMSO；β-CD、CDI和G5.NH2的摩尔比为25～30:250:1。Preferably, the solvent in the step (3) is DMSO; the molar ratio of β-CD, CDI and G5.NH 2 is 25-30:250:1.
优选地，所述步骤(3)中活化温度为25-35℃，活化时间为5～7h。Preferably, the activation temperature in the step (3) is 25-35° C., and the activation time is 5-7 hours.
优选地，所述步骤(3)中氨羟化反应温度为25-35℃，氨羟化反应时间为58～62h。Preferably, the temperature of the aminohydroxylation reaction in the step (3) is 25-35° C., and the reaction time of the aminohydroxylation is 58-62 hours.
优选地，所述步骤(3)中G5-CD与Ad-G3-Man的摩尔比为1:10～20。Preferably, the molar ratio of G5-CD to Ad-G3-Man in the step (3) is 1:10-20.
优选地，所述步骤(3)中超分子自组装反应温度为室温，超分子自组装反应时间为20～25h。Preferably, the supramolecular self-assembly reaction temperature in the step (3) is room temperature, and the supramolecular self-assembly reaction time is 20-25 hours.
优选地，所述步骤(4)中溶剂1为甲醇；溶剂2为生理盐水。Preferably, the
优选地，所述步骤(4)中CBAA与G5-CD/Ad-G3-Man的摩尔比为200～300:1。Preferably, the molar ratio of CBAA to G5-CD/Ad-G3-Man in the step (4) is 200-300:1.
优选地，所述步骤(4)中反应温度为室温，反应时间为45～50h。Preferably, the reaction temperature in the step (4) is room temperature, and the reaction time is 45-50 h.
优选地，所述步骤(5)中孵育为：将CBAA-G5/G3-Man用焦碳酸二乙酯(DEPC)水稀释，然后用DEPC水稀释YTHDF1 siRNA，再将稀释后的两种溶液混合均匀后于35～40℃孵育20～30min。Preferably, incubating in the step (5) is as follows: CBAA-G5/G3-Man is diluted with diethylpyrocarbonate (DEPC) water, then YTHDF1 siRNA is diluted with DEPC water, and then the diluted two solutions are mixed After uniformity, incubate at 35-40°C for 20-30 minutes.
优选地，所述步骤(5)中YTHDF1 siRNA序列为(5’-3’)GGACAUUGGUACUUGGGAUTT。Preferably, the YTHDF1 siRNA sequence in the step (5) is (5'-3')GGACAUUGGUACUUGGGAUTT.
优选地，所述步骤(5)中CBAA-G5/G3-Man与YTHDF1 siRNA的氮磷比为0.5:1～30:1。Preferably, the nitrogen-to-phosphorus ratio of CBAA-G5/G3-Man to YTHDF1 siRNA in the step (5) is 0.5:1-30:1.
优选地，所述多功能纳米递送平台还包括负载抗癌药物纳米平台；所述负载抗癌药物纳米平台的制备方法包括：Preferably, the multifunctional nano-delivery platform also includes a nano-platform loaded with anticancer drugs; the preparation method of the nano-platform loaded with anti-cancer drugs includes:
(a)将步骤(1)中Ad-G3和步骤(3)中G5-CD分别用超纯水溶解后混合，并逐滴加入三乙胺、醋酸酐溶液，反应，透析、冻干处理，得到G5.NHAc-CD/Ad-G3.NHAc，其中G5-CD、Ad-G3、三乙胺与醋酸酐的摩尔比为1:10～20:100～200:100～200，反应温度为室温，反应时间为20～25h；(a) Dissolve Ad-G3 in step (1) and G5-CD in step (3) respectively and mix them with ultrapure water, and add triethylamine and acetic anhydride solution dropwise, react, dialyze, freeze-dry, Obtain G5.NHAc-CD/Ad-G3.NHAc, wherein the molar ratio of G5-CD, Ad-G3, triethylamine and acetic anhydride is 1:10～20:100～200:100～200, and the reaction temperature is room temperature , the reaction time is 20-25h;
(b)将步骤(a)中G5.NHAc-CD/Ad-G3.NHAc溶于水中，与去质子化的DOX溶液混合，敞口反应，离心处理，取上清液进行冻干处理，得到的G5.NHAc-CD/Ad-G3.NHAc/DOX复合物，即负载抗癌药物纳米平台；其中G5.NHAc-CD/Ad-G3.NHAc与DOX的摩尔比为1:10～20，敞口反应为：避光敞口室温搅拌8-16h。(b) G5.NHAc-CD/Ad-G3.NHAc in step (a) is dissolved in water, mixed with deprotonated DOX solution, exposed to reaction, centrifuged, and the supernatant is lyophilized to obtain The G5.NHAc-CD/Ad-G3.NHAc/DOX complex is a nano-platform loaded with anticancer drugs; the molar ratio of G5.NHAc-CD/Ad-G3.NHAc to DOX is 1:10-20, open The mouth reaction is: avoid light exposure and stir at room temperature for 8-16h.
本发明还提供一种多功能纳米递送平台在制备肿瘤免疫治疗药物或肿瘤免疫治疗和化疗治疗联合治疗药物中的应用。The present invention also provides the application of a multifunctional nano-delivery platform in the preparation of tumor immunotherapy drugs or combination therapy drugs of tumor immunotherapy and chemotherapy.
本发明一方面利用氨基末端的壳组分树状大分子修饰了甘露糖，并通过超分子自组装与核组分树状大分子形成核壳树状大分子，进而表面修饰两性离子并通过静电压缩YTHDF1siRNA构建多功能化的纳米基因复合物用于激活树突细胞，达到免疫治疗原位乳腺癌的目的。另一方面，本发明利用超分子自组装方法让氨基末端的壳组分树状大分子与核组分树状大分子形成核壳树状大分子并作乙酰化处理，进而物理包裹抗癌药物DOX用于原位乳腺癌的化疗并引起肿瘤的免疫原性死亡，进一步地激活小鼠免疫系统，增敏基于YTHDF1 siRNA构建的多功能核壳树状大分子的免疫治疗效果。通过对核壳树状大分子纳米材料的理性设计和应用，该发明克服了树状大分子在应用上的一些缺陷，并且将对临床上的药物副作用和免疫治疗效果有限这两大难题有潜在的指导意义，在实现肿瘤化疗、基因治疗、免疫治疗方面具有潜在的应用前景。On the one hand, the present invention uses the shell component dendrimers at the amino terminal to modify mannose, and forms core-shell dendrimers through supramolecular self-assembly and core component dendrimers, and then surface-modifies zwitterions and passes electrostatic Compressing YTHDF1siRNA to construct a multifunctional nano-gene complex is used to activate dendritic cells and achieve the purpose of immunotherapy for breast cancer in situ. On the other hand, the present invention utilizes the supramolecular self-assembly method to allow the shell component dendrimers at the amino terminal and the core component dendrimers to form a core-shell dendrimer and perform acetylation treatment, thereby physically encapsulating anticancer drugs DOX is used in chemotherapy of orthotopic breast cancer and causes immunogenic death of tumors, further activates the immune system of mice, and sensitizes the immunotherapeutic effect of multifunctional nucleocapsid dendrimers constructed based on YTHDF1 siRNA. Through the rational design and application of core-shell dendrimer nanomaterials, this invention overcomes some defects in the application of dendrimers, and will potentially solve the two major problems of clinical drug side effects and limited immunotherapy effects. It has a potential application prospect in realizing tumor chemotherapy, gene therapy, and immunotherapy.
有益效果Beneficial effect
(1)本发明制备方法具有成本低廉，原料来源商品化、条件温和、易于操作、转染效率高、药物上载率高等优点，在肿瘤化疗及基于基因的免疫治疗方面有良好的应用前景；(1) The preparation method of the present invention has the advantages of low cost, commercial raw material sources, mild conditions, easy operation, high transfection efficiency, high drug loading rate, etc., and has good application prospects in tumor chemotherapy and gene-based immunotherapy;
(2)本发明制备的两性离子和甘露糖修饰的核壳结构树状大分子可以抵抗网状内皮系统的清除和靶向树突细胞，负载基因后可引起树突细胞特定基因的沉默和树突细胞的激活，进而用于肿瘤的免疫治疗。(2) The zwitterion- and mannose-modified nucleocapsid dendritic macromolecule prepared by the present invention can resist the clearance of the reticuloendothelial system and target dendritic cells, and can cause dendritic cell-specific gene silencing and dendritic The activation of dendritic cells can be used in the immunotherapy of tumors.
(3)本发明制备的乙酰化核壳树状大分子具有药物缓释性能，负载药物后可在肿瘤部位缓慢释放药物，持续释放化疗药物并引起肿瘤的免疫原性死亡，用于肿瘤的化疗和增敏免疫治疗。(3) The acetylated core-shell dendrimers prepared by the present invention have sustained-release properties of drugs. After being loaded with drugs, they can slowly release drugs at the tumor site, continuously release chemotherapy drugs and cause immunogenic death of tumors, and are used for chemotherapy of tumors. and sensitizing immunotherapy.
(4)本发明制备的基于核壳树状大分子的两种多功能纳米平台共用具有“双管齐下”和“1+1＞2”的协同治疗效果，并为进一步研究肿瘤联合治疗提供了新思路。(4) The combination of two multifunctional nano-platforms based on core-shell dendrimers prepared by the present invention has synergistic therapeutic effects of "two-pronged approach" and "1+1>2", and provides a new idea for further research on tumor combination therapy .
附图说明Description of drawings
图1为本发明制备载体/siRNA复合物(A)和载体/药物复合物(B)的工艺流程示意图；Fig. 1 is the schematic diagram of the technological process of preparation carrier/siRNA complex (A) and carrier/drug complex (B) of the present invention;
图2为本发明制备的Ad-G3.NH2(A)和G5.NH2-CD(B)的1H NMR图谱；Fig. 2 is the 1 H NMR spectrum of Ad-G3.NH 2 (A) and G5.NH 2 -CD (B) prepared in the present invention;
图3为本发明制备的Ad-G3-Man(A)、G5-CD/Ad-G3-Man(B)和CBAA-G5-CDFig. 3 is Ad-G3-Man (A), G5-CD/Ad-G3-Man (B) and CBAA-G5-CD prepared by the present invention
/Ad-G3-Man(C)的1H NMR图谱； 1 H NMR spectrum of /Ad-G3-Man(C);
图4为本发明制备的G5.NH2-CD/Ad-G3.NH2(A)和G5.NHAc-CD/Ad-G3.NHAc(B)的1HNMR图谱；Fig. 4 is the 1 HNMR spectrum of G5.NH 2 -CD/Ad-G3.NH 2 (A) and G5.NHAc-CD/Ad-G3.NHAc (B) prepared in the present invention;
图5为本发明制备的G5.NHAc-CD/Ad-G3.NHAc的原子力显微镜(AFM)图片(A)和高度图(B)、本发明制备的G5.NHAc-CD/Ad-G3.NHAc/DOX复合物的原子力显微镜(AFM)图片(C)和高度图(D)；Figure 5 is the atomic force microscope (AFM) picture (A) and height map (B) of G5.NHAc-CD/Ad-G3.NHAc prepared by the present invention, and the G5.NHAc-CD/Ad-G3.NHAc prepared by the present invention Atomic force microscopy (AFM) image (C) and height map (D) of the /DOX complex;
图6为本发明制备的CBAA-G5/G3-Man的原子力显微镜(AFM)图片(A)和高度图(B)；Figure 6 is an atomic force microscope (AFM) picture (A) and height map (B) of CBAA-G5/G3-Man prepared in the present invention;
图7为本发明制备的CBAA-G5/G3-Man的7天水动力学直径变化图(A)和CBAA-G5/G3-Man在不同质量条件下的抗蛋白吸附实验数据图(B)；Figure 7 is the 7-day hydrodynamic diameter change diagram (A) of CBAA-G5/G3-Man prepared by the present invention and the anti-protein adsorption experiment data diagram (B) of CBAA-G5/G3-Man under different quality conditions;
图8为本发明制备的CBAA-G5/G3-Man/YTHDF1 siRNA复合物的凝胶阻滞试验电泳图，其中泳道1为marker 2000，泳道2为裸YTHDF1 siRNA，泳道3-8分别对应0.5、1、2、4、6、8：1的氮磷(N/P)比下的纳米复合物；Fig. 8 is the gel retardation test electrophoresis figure of the CBAA-G5/G3-Man/YTHDF1 siRNA complex prepared by the present invention, wherein
图9为本发明制备的CBAA-G5/G3-Man/YTHDF1 siRNA在不同N/P比下的水动力学直径图(A)和表面电势图(B)；Fig. 9 is the hydrodynamic diameter diagram (A) and the surface potential diagram (B) of CBAA-G5/G3-Man/YTHDF1 siRNA prepared by the present invention under different N/P ratios;
图10为本发明制备的CBAA-G5/G3-Man/YTHDF1 siRNA在不同G5/G3浓度条件下处理树突(DC)细胞24小时后的细胞活力图；Fig. 10 is the cell viability figure after CBAA-G5/G3-Man/YTHDF1 siRNA prepared by the present invention is treated with dendritic (DC) cells under different G5/G3 concentration conditions for 24 hours;
图11为本发明制备的CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA复合物在不同N/P比下处理DC细胞4小时后，流式细胞仪检测DC细胞吞噬能力的直方图和荧光强度定量数据图；Figure 11 is the histogram and fluorescence intensity of the phagocytosis of DC cells detected by flow cytometry after the CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA complex prepared by the present invention treated DC cells at different N/P ratios for 4 hours Quantitative data graph;
图12为本发明制备的CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA复合物在最佳N/P比下处理DC细胞4小时后，共聚焦显微镜检测DC细胞的形貌图；Figure 12 is the morphology of DC cells detected by confocal microscope after the CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA complex prepared by the present invention treated DC cells for 4 hours at the optimal N/P ratio;
图13为本发明制备的CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA复合物在最佳N/P比下处理DC细胞4小时后，对DC荧光强度分析的流式细胞直方图(A)和荧光强度定量数据图。其中，PBS、free siRNA、DC细胞提前通过Man处理后再加入CBAA-G5/G3-Man/Cy3-YTHDF1siRNA复合物作为对照组；Figure 13 is the flow cytometry histogram (A) of DC fluorescence intensity analysis for the CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA complex prepared by the present invention after treating DC cells for 4 hours under the optimal N/P ratio and fluorescence intensity quantification data graph. Among them, PBS, free siRNA, and DC cells were treated with Man in advance, and then CBAA-G5/G3-Man/Cy3-YTHDF1siRNA complex was added as the control group;
图14为本发明制备的CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA复合物对DC细胞中YTHDF1蛋白表达影响的蛋白质印迹(Westernblot)试验结果图；Fig. 14 is the Western blot (Westernblot) test result figure that the CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA complex prepared by the present invention affects the expression of YTHDF1 protein in DC cells;
图15为本发明制备的CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA复合物在最佳N/P比下处理DC细胞24小时后，利用CD80和CD86荧光抗体进行流式检测DC细胞成熟的数据图；Figure 15 is the CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA complex prepared by the present invention, after treating DC cells for 24 hours at the optimal N/P ratio, using CD80 and CD86 fluorescent antibodies to detect DC cell maturation by flow cytometry data graph;
图16为本发明制备的G5.NHAc-CD/Ad-G3.NHAc/DOX复合物的体外药物释放图(A)和G5.NHAc-CD/Ad-G3.NHAc/DOX复合物处理4T1细胞24小时后的细胞活力图(B)；Figure 16 is the in vitro drug release profile of the G5.NHAc-CD/Ad-G3.NHAc/DOX complex prepared by the present invention (A) and the treatment of 4T1 cells with the G5.NHAc-CD/Ad-G3.NHAc/
图17为本发明制备的G5.NHAc-CD/Ad-G3.NHAc/DOX复合物在不同DOX浓度条件下处理4T1细胞6小时后，流式细胞仪检测4T1细胞吞噬能力的直方图(A)和荧光强度定量数据图(B)；Figure 17 is a histogram (A) of the phagocytosis of 4T1 cells detected by flow cytometry after the G5.NHAc-CD/Ad-G3.NHAc/DOX complexes prepared in the present invention treated 4T1 cells for 6 hours under different DOX concentrations and fluorescence intensity quantitative data graph (B);
图18为本发明制备的G5.NHAc-CD/Ad-G3.NHAc/DOX复合物及其对照组在不同DOX浓度条件下处理4T1细胞24小时后，共聚焦显微镜检测4T1细胞表面CRT表达情况图；Figure 18 is a diagram of the expression of CRT on the surface of 4T1 cells detected by confocal microscope after the G5.NHAc-CD/Ad-G3.NHAc/DOX complex prepared by the present invention and its control group were treated with 4T1 cells under different DOX concentrations for 24 hours ;
图19为本发明制备的G5.NHAc-CD/Ad-G3.NHAc/DOX复合物在不同DOX浓度条件下处理4T1细胞24小时后，利用CD80和CD86荧光抗体进行流式检测DC细胞成熟的数据图；Figure 19 shows the data of flow cytometric detection of DC cell maturation using CD80 and CD86 fluorescent antibodies after the G5.NHAc-CD/Ad-G3.NHAc/DOX complex prepared by the present invention treated 4T1 cells under different DOX concentrations for 24 hours picture;
图20为淋巴结注射本发明制备的CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA复合物和瘤内注射G5.NHAc-CD/Ad-G3.NHAc/DOX复合物后，小鼠原位乳腺癌模型提取心、肝、脾、肺、肾后实施的生物安全性评价实验图(即对各组织切片后进行苏木精-伊红(HE)染色的效果图)；Figure 20 shows the orthotopic breast cancer in mice after lymph node injection of CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA complex prepared by the present invention and intratumoral injection of G5.NHAc-CD/Ad-G3.NHAc/DOX complex The biosafety evaluation experiment diagram after extracting the heart, liver, spleen, lung and kidney from the model (that is, the effect diagram of hematoxylin-eosin (HE) staining after each tissue section);
图21为淋巴结注射本发明制备的CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA复合物和瘤内注射G5.NHAc-CD/Ad-G3.NHAc/DOX复合物后，小鼠原位乳腺癌模型的抗肿瘤效果图，包括肿瘤体积变化图(A)和小鼠体重变化图(B)；Figure 21 shows the orthotopic breast cancer in mice after lymph node injection of CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA complex prepared by the present invention and intratumoral injection of G5.NHAc-CD/Ad-G3.NHAc/DOX complex The anti-tumor effect diagram of the model, including the tumor volume change diagram (A) and the mouse body weight change diagram (B);
图22为淋巴结注射本发明制备的CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA复合物和瘤内注射G5.NHAc-CD/Ad-G3.NHAc/DOX复合物后，小鼠原位乳腺癌模型提取脾脏组织并利用荧光标记的CD4和CD8抗体进行检测CD4+/CD8+T细胞的流式图(A)和对应统计的CD4+T细胞占比统计图(B)以及对应统计的CD8+T细胞占比统计图(C)，其中Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ分别代表PBS组、DOX.HCl组、G5.NHAc/G3.NHAc/DOX组、CBAA-G5/G3-Man/YTHDF1 siRNA组、DOX.HCl+CBAA-G5/G3-Man/YTHDF1 siRNA组、和G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1siRNA组。Figure 22 shows the orthotopic breast cancer in mice after lymph node injection of CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA complex prepared by the present invention and intratumoral injection of G5.NHAc-CD/Ad-G3.NHAc/DOX complex The model extracts spleen tissue and uses fluorescently labeled CD4 and CD8 antibodies to detect CD4+/CD8+ T cells. Statistical chart of cell ratio (C), where Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ represent PBS group, DOX.HCl group, G5.NHAc/G3.NHAc/DOX group, CBAA-G5/G3-Man/ YTHDF1 siRNA group, DOX.HCl+CBAA-G5/G3-Man/YTHDF1 siRNA group, and G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1 siRNA group.
图23为淋巴结注射本发明制备的CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA复合物和瘤内注射G5.NHAc-CD/Ad-G3.NHAc/DOX复合物后，小鼠原位乳腺癌模型提取肿瘤组织后的肿瘤切片TUNEL(TdT-mediated dUTP Nick-End Labeling)图，其中Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ分别代表PBS组、DOX.HCl组、G5.NHAc/G3.NHAc/DOX组、CBAA-G5/G3-Man/YTHDF1siRNA组、DOX.HCl+CBAA-G5/G3-Man/YTHDF1 siRNA组、和G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1siRNA组。Figure 23 shows the orthotopic breast cancer in mice after lymph node injection of CBAA-G5/G3-Man/Cy3-YTHDF1 siRNA complex prepared by the present invention and intratumoral injection of G5.NHAc-CD/Ad-G3.NHAc/DOX complex Tumor slice TUNEL (TdT-mediated dUTP Nick-End Labeling) diagram after the tumor tissue was extracted from the model, where Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ represent PBS group, DOX.HCl group, G5.NHAc/G3.NHAc /DOX group, CBAA-G5/G3-Man/YTHDF1 siRNA group, DOX.HCl+CBAA-G5/G3-Man/YTHDF1 siRNA group, and G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/ YTHDF1 siRNA panel.
具体实施方式Detailed ways
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
以下各实施例中，除非特殊说明，否则所有化学试剂都是可商购的，无需进一步纯化即可直接使用。其中，第三代氨基端聚酰胺-胺树状大分子G3.NH2和第五代氨基端聚酰胺-胺树状大分子G5.NH2购自Dendritech公司(Midland，MI)、甘露糖Man购买于Sigma-Aldrich(圣路易斯，密苏里)。YTHDF1 siRNA由上海吉玛制药技术有限公司合成，序列为(5’-3’)GGACAUUGGUACUUGGGAUTT；而其余如无特别说明的原料或处理技术，则表明其均为本领域的常规市售原料或常规处理技术。In the following examples, unless otherwise specified, all chemical reagents were commercially available and used directly without further purification. Among them, the third-generation amino-terminal polyamido-amine dendrimer G3.NH 2 and the fifth-generation amino-terminal polyamido-amine dendrimer G5.NH 2 were purchased from Dendritech (Midland, MI), Mannose Man Purchased from Sigma-Aldrich (St. Louis, MO). YTHDF1 siRNA was synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd., the sequence is (5'-3')GGACAUUGGUACUUGGGAUTT; and the rest, if there is no special description of raw materials or processing techniques, it means that they are conventional commercially available raw materials or conventional processing in this field technology.
实施例1Example 1
负载基因或药物的多功能化核壳树状大分子纳米平台及其复合物的制备方法，参见图1所示，具体包括以下步骤：The preparation method of the multifunctional core-shell dendrimer nano-platform and its complexes loaded with genes or drugs, as shown in Figure 1, specifically includes the following steps:
(1)分别称取8.44mg Ad-COOH，83.24mg EDC.HCl，43.96mg NHS，分别溶于5mL的DMSO溶液中，然后将EDC.HCl和NHS溶液逐滴加入到Ad-COOH溶液中，室温搅拌3h。然后称取200mg G3.NH2溶于5mL的DMSO溶液，将活化的Ad-COOH溶液逐滴加入G3.NH2溶液中，继续反应3天，将得到的产物转移至截留分子量为1000的透析袋中、在蒸馏水中透析三天(2L×3)，进行冷冻干燥处理，得到Ad-G3粉末，-20℃保存备用。(1) Weigh 8.44mg Ad-COOH, 83.24mg EDC.HCl, and 43.96mg NHS respectively, dissolve them in 5mL of DMSO solution, then add the EDC.HCl and NHS solutions dropwise to the Ad-COOH solution, and keep at room temperature Stir for 3h. Then weigh 200mg G3.NH2 and dissolve it in 5mL of DMSO solution, add the activated Ad-COOH solution dropwise into the G3.NH2 solution, continue the reaction for 3 days, and transfer the obtained product to a dialysis bag with a molecular weight cut-off of 1000 Neutralization and dialysis in distilled water for three days (2L×3), and freeze-drying to obtain Ad-G3 powder, which was stored at -20°C for future use.
(2)分别称取100mg Ad-G3.NH2，50.60mg Man，分别溶于5mL的PBS溶液中，然后在90℃条件下将Man溶液逐滴加入到Ad-G3.NH2溶液中，搅拌2h。然后将得到的产物转移至截留分子量为1000的透析袋中、在蒸馏水中透析1天(2L×3)，进行冷冻干燥处理，得到Man-G3-Ad粉末，-20℃保存备用。(2) Weigh 100mg Ad-G3.NH 2 and 50.60mg Man respectively, dissolve them in 5mL of PBS solution, then add the Man solution dropwise to the Ad-G3.NH 2 solution at 90°C, stir 2h. Then the obtained product was transferred to a dialysis bag with a molecular weight cut-off of 1000, dialyzed in distilled water for 1 day (2L×3), and freeze-dried to obtain Man-G3-Ad powder, which was stored at -20°C for future use.
(3)分别称取43.64mgβ-CD，62.34mg CDI，分别溶于5mL的DMSO溶液中，然后将CDI溶液逐滴加入到β-CD溶液中，室温搅拌6h。称取40mg G5.NH2溶于5mL的DMSO溶液，将得到的活化的β-CD溶液逐滴加入G5.NH2溶液中，继续反应60h，将得到的产物转移至截留分子量为5000的透析袋中、在蒸馏水中透析三天(2L×3)，然后进行冷冻干燥处理，得到G5-CD，-20℃保存备用。(3) Weigh 43.64 mg β-CD and 62.34 mg CDI, respectively, and dissolve them in 5 mL of DMSO solution, then add the CDI solution dropwise into the β-CD solution, and stir at room temperature for 6 h. Weigh 40 mg of G5.NH 2 and dissolve in 5 mL of DMSO solution, add the obtained activated β-CD solution into the G5.NH 2 solution drop by drop, continue the reaction for 60 h, and transfer the obtained product to a dialysis bag with a molecular weight cut-off of 5000 Neutralization and dialysis in distilled water for three days (2L×3), and then freeze-drying to obtain G5-CD, which was stored at -20°C for future use.
(4)分别称取61.67mg上述步骤(2)中得到的Man-G3-Ad和22.60mg上述步骤(3)中得到的G5-CD，分别溶于5mL的DMSO溶液中，然后将Man-G3-Ad溶液逐滴加入到G5-CD溶液中，继续反应24h，将得到的产物转移至截留分子量为50000的透析袋中、在蒸馏水中透析三天(2L×3)，然后进行冷冻干燥处理，得到G5-CD/Ad-G3-Man粉末，即G5/G3-Man粉末，-20℃保存备用。(4) Weigh 61.67 mg of the Man-G3-Ad obtained in the above step (2) and 22.60 mg of the G5-CD obtained in the above step (3), respectively, dissolve them in 5 mL of DMSO solution, and then mix the Man-G3 -Ad solution was added dropwise to the G5-CD solution, and the reaction was continued for 24 hours. The obtained product was transferred to a dialysis bag with a molecular weight cut-off of 50,000, dialyzed in distilled water for three days (2L×3), and then freeze-dried. The obtained G5-CD/Ad-G3-Man powder, that is, the G5/G3-Man powder, was stored at -20°C for future use.
(5)分别称取40mg上述步骤(1)中得到的G3-Ad和15.16mg上述步骤(3)中得到的G5-CD，分别溶于5mL的DMSO溶液中，然后将G3-Ad溶液逐滴加入到G5-CD溶液中，继续搅拌24h后逐滴加入三乙胺400μL，半小时后逐滴加入乙酸酐200μL，继续搅拌24h后将得到的产物转移至截留分子量为50000的透析袋中、在蒸馏水中透析三天(2L×3)，然后进行冷冻干燥处理，得到G5.NHAc-CD/Ad-G3.NHAc粉末，即G5.NHAc/G3.NHAc粉末，-20℃保存备用。(5) Weigh 40 mg of the G3-Ad obtained in the above step (1) and 15.16 mg of the G5-CD obtained in the above step (3), respectively, dissolve them in 5 mL of DMSO solution, and then add the G3-Ad solution dropwise Add it into the G5-CD solution, continue to stir for 24 hours, then add 400 μL of triethylamine dropwise, half an hour later, add 200 μL of acetic anhydride dropwise, continue to stir for 24 hours, then transfer the obtained product to a dialysis bag with a molecular weight cut-off of 50,000. Distilled water was dialyzed for three days (2L×3), and then freeze-dried to obtain G5.NHAc-CD/Ad-G3.NHAc powder, that is, G5.NHAc/G3.NHAc powder, which was stored at -20°C for future use.
(6)分别称取20mg上述步骤(4)中得到的G5/G3-Man溶解于5mL甲醇中，称取10mgCBAA溶于5mL生理盐水中，然后将CBAA逐滴加入到G5/G3-Man溶液中，室温搅拌2天，将得到的产物转移至截留分子量为1000的透析袋中、在蒸馏水中透析三天(2L×3)，然后进行冷冻干燥处理，得到CBAA-G5/G3-Man粉末，-20℃保存备用。(6) Weigh 20 mg of G5/G3-Man obtained in the above step (4) and dissolve them in 5 mL of methanol, weigh 10 mg of CBAA and dissolve them in 5 mL of normal saline, and then add CBAA dropwise to the G5/G3-Man solution , stirred at room temperature for 2 days, the obtained product was transferred to a dialysis bag with a molecular weight cut-off of 1000, dialyzed in distilled water for three days (2L×3), and then freeze-dried to obtain CBAA-G5/G3-Man powder,- Store at 20°C for later use.
(7)将步骤(6)中得到的CBAA-G5/G3-Man用DEPC水溶解，配置成2mg/mL的水溶液，然后用DEPC水溶解YTHDF1 siRNA，配制成264μg/mL的水溶液，按照不同N/P比(0.5、1、2、4、6、8、10、15、30)将CBAA-G5/G3-Man水溶液与1μg YTHDF1 siRNA进行混合后放入37℃培养箱进行孵育，20～30min后得到CBAA-G5/G3-Man/YTHDF1 siRNA复合物，现配现用。(7) Dissolve the CBAA-G5/G3-Man obtained in step (6) with DEPC water, and configure it into a 2 mg/mL aqueous solution, then dissolve YTHDF1 siRNA with DEPC water, and prepare a 264 μg/mL aqueous solution. /P ratio (0.5, 1, 2, 4, 6, 8, 10, 15, 30) Mix CBAA-G5/G3-Man aqueous solution with 1 μg YTHDF1 siRNA and put it in a 37°C incubator for 20-30min The CBAA-G5/G3-Man/YTHDF1 siRNA complex was obtained afterward, and it is prepared and used now.
(8)分别称取1.389mg抗癌药物DOX.HCl和20mg步骤(5)得到的G5.NHAc/G3.NHAc。随后将G5.NHAc/G3.NHAc溶解于5mL超纯水中，DOX.HCl溶解于500μL甲醇中并加入5μL三乙胺以得到去质子化的DOX。随后将DOX溶液和G5.NHAc/G3.NHAc溶液混合，混合溶液在避光条件下经过敞口过夜搅拌以蒸发掉其中的甲醇，将得到的混合溶液通过离心的方式(7000rpm，10min)除去未络合上的去质子的DOX沉淀，之后进行冻干上清液以获得G5.NHAc/G3.NHAc/DOX，-20℃保存备用。(8) Weigh 1.389 mg of anticancer drug DOX.HCl and 20 mg of G5.NHAc/G3.NHAc obtained in step (5). Subsequently, G5.NHAc/G3.NHAc was dissolved in 5 mL of ultrapure water, DOX.HCl was dissolved in 500 μL of methanol and 5 μL of triethylamine was added to obtain deprotonated DOX. Then the DOX solution and the G5.NHAc/G3.NHAc solution were mixed, and the mixed solution was stirred overnight under the condition of avoiding light to evaporate the methanol therein, and the obtained mixed solution was removed by centrifugation (7000rpm, 10min). The complexed deprotonated DOX was precipitated, and then the supernatant was freeze-dried to obtain G5.NHAc/G3.NHAc/DOX, which was stored at -20°C for future use.
实施例2Example 2
对实施例1步骤(1)～步骤(6)中制备的各个材料进行核磁表征，1H NMR表征结果如说明书附图2、图3及图4所示：图2A中的2.2-3.4ppm是G3.NH2的特征质子峰，1.6-1.9ppm是Ad的特征质子峰，根据他们的积分面积之比，计算出每个G3连接了1.2个Ad分子；图2B中的2.2-3.4ppm是G5的特征质子峰，3.4-4.0ppm及5.0ppm是β-CD的特征质子峰，根据他们的积分面积之比，计算出每个G5连接了14.0个CD分子；图3A中的2.2-3.4ppm是G3的特征质子峰，3.5-3.8ppm是Man的特征质子峰，根据他们的积分面积之比，计算出每个Ad-G3连接了2.4个Man分子；图3B中的1.6-1.9ppm是Ad的特征质子峰，5.0ppm是β-CD的特征质子峰，根据他们的积分面积之比，计算出每个β-CD对应0.91个Ad，进而计算出平均每个G5-CD连接了12.8个Ad-G3-Man；图3C中的2.0ppm是CBAA的特征质子峰，5.0ppm是β-CD的特征质子峰，根据他们的积分面积之比，计算出平均每个CD对应着2.8个CBAA，进而计算出平均每个G5-CD/Ad-G3-Man连接了39.2个CBAA；图4A中的1.6-1.9ppm是Ad的特征质子峰，3.4-4.0ppm及5.0ppm是β-CD的特征质子峰，根据他们的积分面积之比，计算出每个CD对应0.94个Ad，进而计算出平均每个G5-CD连接了13.2个Ad-G3；图4B中的1.9ppm是乙酰基的甲基质子峰,这表明材料G5/G3表面剩余氨基被成功乙酰化。Carry out NMR characterization of each material prepared in step (1) to step (6) of Example 1, and the 1 H NMR characterization results are shown in Figure 2, Figure 3 and Figure 4 of the specification: 2.2-3.4ppm in Figure 2A is The characteristic proton peak of G3.NH 2 , 1.6-1.9ppm is the characteristic proton peak of Ad, according to the ratio of their integral areas, it is calculated that each G3 is connected with 1.2 Ad molecules; 2.2-3.4ppm in Figure 2B is G5 The characteristic proton peaks of 3.4-4.0ppm and 5.0ppm are the characteristic proton peaks of β-CD. According to the ratio of their integral areas, it is calculated that each G5 is connected with 14.0 CD molecules; 2.2-3.4ppm in Fig. 3A is The characteristic proton peak of G3, 3.5-3.8ppm is the characteristic proton peak of Man. According to the ratio of their integral areas, it is calculated that each Ad-G3 is connected with 2.4 Man molecules; 1.6-1.9ppm in Figure 3B is Ad. The characteristic proton peak, 5.0ppm is the characteristic proton peak of β-CD. According to their integral area ratio, it is calculated that each β-CD corresponds to 0.91 Ad, and then calculates that each G5-CD is connected with 12.8 Ad- G3-Man; 2.0ppm in Figure 3C is the characteristic proton peak of CBAA, and 5.0ppm is the characteristic proton peak of β-CD. According to the ratio of their integral areas, it is calculated that each CD corresponds to 2.8 CBAAs on average, and then calculated On average, 39.2 CBAAs are connected to each G5-CD/Ad-G3-Man; 1.6-1.9ppm in Figure 4A is the characteristic proton peak of Ad, 3.4-4.0ppm and 5.0ppm are the characteristic proton peaks of β-CD, According to the ratio of their integral areas, it is calculated that each CD corresponds to 0.94 Ad, and then it is calculated that each G5-CD is connected with 13.2 Ad-G3; 1.9ppm in Figure 4B is the methyl proton peak of the acetyl group, This indicated that the remaining amino groups on the surface of material G5/G3 were successfully acetylated.
实施例3Example 3
对实施例1步骤(5)、步骤(6)以及步骤(8)中制备的材料G5.NHAc/G3.NHAc、CBAA-G5/G3-Man以及G5.NHAc/G3.NHAc/DOX用原子力显微镜(AFM)进行表征，结果如说明书附图5及图6所示，G5.NHAc/G3.NHAc在AFM上测试的平均高度为11.8±0.32nm(图5A-B)，G5.NHAc/G3.NHAc/DOX为14.4±0.53nm(图5C-D)，CBAA-G5/G3-Man为5.7±0.50nm(图6)。To the material G5.NHAc/G3.NHAc, CBAA-G5/G3-Man and G5.NHAc/G3.NHAc/DOX prepared in
对实施例1步骤(6)中制备的CBAA-G5/G3-Man进行稳定性实验。配制成2mg/mL的水溶液后室温放置七天，并在第1，3，5，7天时测试其粒径变化。结果如图7A所示，在不同的时间条件下，CBAA-G5/G3-Man的水动力学粒径在207～244nm之间。这说明，制备的CBAA-G5/G3-Man总体粒径呈现稳定状态，材料粒径的稳定性有利于进行下一步的基因转染实验。A stability test was performed on the CBAA-G5/G3-Man prepared in step (6) of Example 1. After being formulated into a 2 mg/mL aqueous solution, it was placed at room temperature for seven days, and the particle size change was tested on the 1st, 3rd, 5th, and 7th days. The results are shown in Figure 7A, under different time conditions, the hydrodynamic particle size of CBAA-G5/G3-Man is between 207nm and 244nm. This shows that the overall particle size of the prepared CBAA-G5/G3-Man is in a stable state, and the stability of the particle size of the material is conducive to the next step of gene transfection experiments.
对实施例1步骤(6)中制备的CBAA-G5/G3-Man进行抗蛋白吸附实验。将牛血清白蛋白水溶液(2mg/mL)按照1:1的体积比分别与不同浓度(0.25、1和4mg/mL)的G5/G3水溶液进行混合，在37℃条件下放置4小时后离心(8000rpm，5min)收集上清液，并根据标准规范使用BCA定量试剂盒测量上清液中蛋白的含量。结果如图7B所示，没有CBAA修饰的G5/G3-Man组，上清液中蛋白的含量随着材料浓度的增加而减少，这说明由于表面氨基基团的存在，G5/G3-Man吸附非特异性蛋白的能力随着材料浓度的增加而增加。而CBAA修饰后，CBAA-G5/G3-Man组上清液中蛋白的含量一直高于0.8mg/mL，而且蛋白含量随着材料浓度的增加还略有增加，这说明CBAA-G5/G3-Man由于CBAA的修饰而具有很好的抗非特异性蛋白吸附的能力。Anti-protein adsorption experiments were performed on the CBAA-G5/G3-Man prepared in step (6) of Example 1. Bovine serum albumin aqueous solution (2 mg/mL) was mixed with G5/G3 aqueous solutions of different concentrations (0.25, 1 and 4 mg/mL) according to the volume ratio of 1:1, and centrifuged after 4 hours at 37 ° C ( 8000rpm, 5min) to collect the supernatant, and use the BCA quantitative kit to measure the protein content in the supernatant according to the standard specification. The results are shown in Figure 7B. In the G5/G3-Man group without CBAA modification, the protein content in the supernatant decreased with the increase of the material concentration, which indicated that due to the presence of surface amino groups, G5/G3-Man adsorption The capacity for non-specific proteins increases with increasing material concentration. After CBAA modification, the protein content in the supernatant of the CBAA-G5/G3-Man group was always higher than 0.8 mg/mL, and the protein content increased slightly with the increase of the material concentration, which indicated that the CBAA-G5/G3-Man Man has a good ability to resist non-specific protein adsorption due to the modification of CBAA.
实施例4Example 4
对实施例1步骤(6)中制备的CBAA-G5/G3-Man进行定氮实验。配置成2mg/mL的水溶液，遵循标准规范使用初级氨基氮(NOPA)检测试剂盒，最终测出每个CBAA-G5/G3-Man表面氨基数平均为88个。Nitrogen determination experiments were carried out on the CBAA-G5/G3-Man prepared in step (6) of Example 1. It was configured into a 2mg/mL aqueous solution, followed the standard specification and used the primary amino nitrogen (NOPA) detection kit, and finally measured the average number of amino groups on the surface of each CBAA-G5/G3-Man to be 88.
对实施案例1步骤(7)中制备的CBAA-G5/G3-Man/YTHDF1 siRNA复合物进行凝胶阻滞实验。配制8孔含有核酸染料的琼脂糖凝胶(1.0％w/v)，室温放置待琼脂糖凝胶凝固后放置于电泳槽中进行加样操作。其中，样品包括CBAA-G5/G3-Man溶液(2mg/mL)按照不同N/P比(0.5、1、2、4、6、8)与YTHDF1 siRNA(1μg/孔)混合并孵育20～30min后配制成CBAA-G5/G3-Man/YTHDF1 siRNA复合物，以及DNA marker和单独(裸)的YTHDF1 siRNA。在电压80V，时间40min的条件下进行电泳，然后用凝胶成像仪对CBAA-G5/G3-Man/YTHDF1siRNA复合物在凝胶中的迁移能力进行分析。结果如图8所示，在N/P比大于等于1的时候，CBAA-G5/G3-Man能够完全将YTHDF1 siRNA压缩，阻滞YTHDF1 siRNA的迁移。Gel retardation experiments were performed on the CBAA-G5/G3-Man/YTHDF1 siRNA complex prepared in step (7) of Example 1. An 8-well agarose gel (1.0% w/v) containing a nucleic acid dye was prepared, placed at room temperature until the agarose gel was solidified, and placed in an electrophoresis tank for sample loading. Among them, samples including CBAA-G5/G3-Man solution (2mg/mL) were mixed with YTHDF1 siRNA (1μg/well) according to different N/P ratios (0.5, 1, 2, 4, 6, 8) and incubated for 20-30min Afterwards, it is prepared into CBAA-G5/G3-Man/YTHDF1 siRNA complex, as well as DNA marker and separate (naked) YTHDF1 siRNA. Electrophoresis was performed under the conditions of voltage 80V and time 40min, and then the migration ability of the CBAA-G5/G3-Man/YTHDF1siRNA complex in the gel was analyzed with a gel imager. The results are shown in Figure 8. When the N/P ratio is greater than or equal to 1, CBAA-G5/G3-Man can completely compress YTHDF1 siRNA and block the migration of YTHDF1 siRNA.
实施例5Example 5
对实施例1步骤(7)中制备的CBAA-G5/G3-Man/YTHDF1 siRNA复合物进行水动力学直径和表面电势表征，即制备不同N/P比(1、5、10、15、20、30)的CBAA-G5/G3-Man/YTHDF1siRNA复合物，并用DEPC水进行稀释至终体积为1mL，用马尔文激光粒度仪(Malvern,ΜK，633nm激光)进行表征。结果如图9所示，在不同的N/P比条件下，复合物的水动力学粒径都大致在160～176nm之间(图9A)，表面电势都在32～37mV之间(图9B)，这说明一定范围内的N/P比的改变并不能明显改变复合物的粒径和电势，粒径和电势总体上呈现稳定状态且都处于合适的基因传递范围，有利于被细胞吸附、内吞以及细胞内基因的传递。The CBAA-G5/G3-Man/YTHDF1 siRNA complex prepared in step (7) of Example 1 was characterized by hydrodynamic diameter and surface potential, that is, preparation of different N/P ratios (1, 5, 10, 15, 20 , 30) CBAA-G5/G3-Man/YTHDF1siRNA complex, and diluted with DEPC water to a final volume of 1 mL, characterized by a Malvern laser particle size analyzer (Malvern, MK, 633nm laser). The results are shown in Figure 9. Under different N/P ratio conditions, the hydrodynamic particle size of the composites is roughly between 160-176nm (Figure 9A), and the surface potential is between 32-37mV (Figure 9B ), which shows that the change of the N/P ratio within a certain range cannot significantly change the particle size and potential of the complex. The particle size and potential are generally in a stable state and are in a suitable range for gene transfer, which is conducive to being adsorbed by cells, Endocytosis and intracellular gene delivery.
实施例6Example 6
以树突细胞为模型细胞，检测CBAA-G5/G3-Man和CBAA-G5/G3-Man/YTHDF1 siRNA复合物在不同浓度条件下的细胞活力。将树突细胞以5×103个/孔的细胞密度接种于96孔板中，每孔添加100μL含有100U/mL青霉素、100U/mL链霉素和10％ FBS的DMEM培养基(命名为DMEM++)。细胞放置于5％ CO2，37℃的培养箱中培养过夜后，将培养基换成100μL含有不同G5/G3摩尔浓度的DMEM++，其中G5/G3浓度分别为0、50、100、500、1000、2000和3000nM，YTHDF1 siRNA添加量均为1μg/孔，然后继续培养细胞24h。弃去培养液，按照说明书程序使用CCK-8试剂盒进行细胞活性的检测。结果如图10所示，随着材料浓度的增加，细胞存活率略有下降，但即使在浓度高达3000nM的情况下，细胞的存活率仍在80％以上，说明了CBAA-G5/G3-Man良好的细胞相容性。与此同时，CBAA-G5/G3-Man与YTHDF1siRNA复合后的毒性在一定程度上还有所降低，进而也验证了复合物良好的细胞相容性。Using dendritic cells as model cells, the cell viability of CBAA-G5/G3-Man and CBAA-G5/G3-Man/YTHDF1 siRNA complexes at different concentrations was detected. Dendritic cells were seeded in a 96-well plate at a cell density of 5× 103 /well, and 100 μL of DMEM medium containing 100 U/mL penicillin, 100 U/mL streptomycin and 10% FBS (named DMEM++) was added to each well. ). Cells were cultured overnight in a 5% CO 2 , 37°C incubator, and the medium was replaced with 100 μL of DMEM++ containing different G5/G3 molar concentrations, where the G5/G3 concentrations were 0, 50, 100, 500, 1000 , 2000 and 3000nM, the amount of YTHDF1 siRNA added was 1 μg/well, and then the cells were cultured for 24h. Discard the culture medium, and use the CCK-8 kit to detect cell viability according to the instructions. The results are shown in Figure 10, with the increase of the material concentration, the cell survival rate decreased slightly, but even in the case of a concentration as high as 3000nM, the cell survival rate was still above 80%, indicating that CBAA-G5/G3-Man Good cell compatibility. At the same time, the toxicity of CBAA-G5/G3-Man combined with YTHDF1siRNA was reduced to a certain extent, which also verified the good cytocompatibility of the complex.
实施例7Example 7
以树突细胞作为模型细胞，选用Cy3(一种荧光染料)标记的YTHDF1 siRNA，通过流式细胞仪检测不同N/P比条件下CBAA-G5/G3-Man/YTHDF1 siRNA复合物在细胞中递送YTHDF1 siRNA的效率。将树突细胞以1×105个/孔的细胞密度接种于12孔板中，每孔添加1mL DMEM++培养基，并将孔板置于5％ CO2，37℃的培养箱中孵育过夜。然后培养基换成1mL含有不同N/P比的CBAA-G5/G3-Man/YTHDF1 siRNA复合物的DMEM++培养基，其中N/P比分别为0、5、10、15、20和30，YTHDF1 siRNA添加量均为1μg/孔。孵育4h后，用PBS缓冲溶液清洗细胞2遍，利用胰酶对细胞进行消化，离心(1000rpm，5min)收集细胞后用适量PBS重悬。最后，用流式细胞仪检测细胞的荧光强度，结果如图11所示，和正常细胞组(对照组)相比，裸YTHDF1 siRNA组荧光强度略有上升，但仍然很低。而材料组荧光强度随着N/P比的增加而增强，在N/P比为15的时候，红色荧光强度最强。因此，在实验设计的N/P比条件下，CBAA-G5/G3-Man显示出了良好的递送YTHDF1 siRNA的效率，且在N/P比为15时递送效率最高。Using dendritic cells as model cells, YTHDF1 siRNA labeled with Cy3 (a fluorescent dye) was selected, and the delivery of CBAA-G5/G3-Man/YTHDF1 siRNA complexes in cells under different N/P ratio conditions was detected by flow cytometry Efficiency of YTHDF1 siRNA. Dendritic cells were seeded in a 12-well plate at a cell density of 1×10 5 cells/well, 1 mL of DMEM++ medium was added to each well, and the well plate was incubated overnight in an incubator at 5% CO 2 at 37°C. Then the medium was replaced with 1 mL of DMEM++ medium containing CBAA-G5/G3-Man/YTHDF1 siRNA complexes with different N/P ratios of 0, 5, 10, 15, 20 and 30, YTHDF1 The amount of siRNA added was 1 μg/well. After incubation for 4 h, the cells were washed twice with PBS buffer solution, digested with trypsin, collected by centrifugation (1000 rpm, 5 min), and resuspended with an appropriate amount of PBS. Finally, the fluorescence intensity of the cells was detected by flow cytometry, and the results are shown in Figure 11. Compared with the normal cell group (control group), the fluorescence intensity of the naked YTHDF1 siRNA group increased slightly, but was still very low. The fluorescence intensity of the material group increases with the increase of the N/P ratio, and the red fluorescence intensity is the strongest when the N/P ratio is 15. Therefore, under the N/P ratio conditions designed by the experiment, CBAA-G5/G3-Man showed a good delivery efficiency of YTHDF1 siRNA, and the delivery efficiency was the highest when the N/P ratio was 15.
以树突细胞作为模型细胞，选用Cy3(一种荧光染料)标记的YTHDF1 siRNA，通过激光共聚焦显微镜检测最佳N/P比(N/P＝15)条件下CBAA-G5/G3-Man/YTHDF1 siRNA复合物在细胞中递送YTHDF1 siRNA的效率。将树突细胞以1×105个/孔的细胞密度接种于共聚焦显微镜专用皿中，每皿添加1mL DMEM++培养基，并将皿置于5％ CO2、37℃的培养箱中孵育过夜。然后培养基换成1mL含有N/P＝15的CBAA-G5/G3-Man/YTHDF1 siRNA复合物的DMEM++培养基，其中YTHDF1 siRNA添加量均为1μg/皿。孵育4h后，用PBS缓冲溶液清洗细胞2遍，并通过标准规范用戊二醛固定细胞和用DAPI对细胞核进行染色。最后，每孔添加300μL PBS，用激光共聚焦显微镜观察细胞的吞噬情况。结果如图12所示，与对照组相比，裸YTHDF1 siRNA组未见明显红色荧光，CBAA-G5/G3-Man/YTHDF1 siRNA复合物组有很强的红色荧光，且大部分在细胞核周围或内部。因此，CBAA-G5/G3-Man能够成功将YTHDF1 siRNA传递到细胞质和细胞核中，有利于后续基因沉默的实现。Dendritic cells were used as model cells, Cy3 (a fluorescent dye)-labeled YTHDF1 siRNA was selected, and CBAA-G5/G3-Man/CBAA-G5/G3-Man/ Efficiency of YTHDF1 siRNA complexes to deliver YTHDF1 siRNA in cells. Seed dendritic cells at a cell density of 1×10 5 cells/well in confocal microscope dishes, add 1 mL DMEM++ medium to each dish, and incubate the dish overnight in an incubator with 5% CO 2 and 37°C . Then the medium was replaced with 1 mL of DMEM++ medium containing CBAA-G5/G3-Man/YTHDF1 siRNA complex with N/P=15, wherein the addition amount of YTHDF1 siRNA was 1 μg/dish. After 4 h of incubation, cells were washed twice with PBS buffer solution, fixed with glutaraldehyde and stained with DAPI for nuclei by standard procedures. Finally, 300 μL of PBS was added to each well, and the phagocytosis of cells was observed with a confocal laser microscope. The results are shown in Figure 12. Compared with the control group, there was no obvious red fluorescence in the naked YTHDF1 siRNA group, but there was strong red fluorescence in the CBAA-G5/G3-Man/YTHDF1 siRNA complex group, most of which were around the nucleus or internal. Therefore, CBAA-G5/G3-Man can successfully deliver YTHDF1 siRNA into the cytoplasm and nucleus, which is conducive to the realization of subsequent gene silencing.
以树突细胞作为模型细胞，选用Cy3(一种荧光染料)标记的YTHDF1 siRNA，通过流式细胞仪检测最佳N/P比条件下CBAA-G5/G3-Man/YTHDF1 siRNA复合物靶向DC细胞递送YTHDF1 siRNA的能力。将树突细胞以1×105个/孔的细胞密度接种于12孔板中，每孔添加1mL DMEM++培养基，并置于5％ CO2、37℃的培养箱中孵育过夜。然后将孔板分为随机分成4组，每组3个孔，分别为对照(PBS)组、裸YTHDF1 siRNA组、CBAA-G5/G3-Man/YTHDF1siRNA(N/P＝15)组、Man+CBAA-G5/G3-Man/YTHDF1 siRNA组。然后各组培养基换成1mL含有相应组别含量的DMEM++培养基，其中YTHDF1 siRNA添加量均为1μg/孔，Man+CBAA-G5/G3-Man/YTHDF1siRNA组需提前添加Man(5μM/孔)4h后再添加CBAA-G5/G3-Man/YTHDF1 siRNA。孵育4h后，用PBS缓冲溶液清洗细胞2遍，利用胰酶对细胞进行消化，离心(1000rpm，5min)，收集细胞后用适量PBS重悬。最后，用流式细胞仪检测细胞的荧光强度。结果如图13所示，与对照组相比，裸YTHDF1 siRNA组未见明显红色荧光，CBAA-G5/G3-Man/YTHDF1 siRNA复合物组的红色荧光最强，而且经提前添加Man进行阻断后CBAA-G5/G3-Man/YTHDF1 siRNA复合物的红色荧光有所下降。这说明，CBAA-G5/G3-Man/YTHDF1 siRNA因为Man而具有靶向DC细胞的能力，这更有利于后续基因沉默的实现。Using dendritic cells as model cells, YTHDF1 siRNA labeled with Cy3 (a fluorescent dye) was selected, and the CBAA-G5/G3-Man/YTHDF1 siRNA complex targeting DC was detected by flow cytometry under the condition of optimal N/P ratio. Ability of cells to deliver YTHDF1 siRNA. The dendritic cells were seeded in a 12-well plate at a cell density of 1×10 5 cells/well, 1 mL of DMEM++ medium was added to each well, and incubated overnight in an incubator with 5% CO 2 and 37°C. Then the orifice plate was randomly divided into 4 groups with 3 wells in each group, respectively control (PBS) group, naked YTHDF1 siRNA group, CBAA-G5/G3-Man/YTHDF1 siRNA (N/P=15) group, Man+ CBAA-G5/G3-Man/YTHDF1 siRNA panel. Then the medium of each group was replaced with 1 mL of DMEM++ medium containing the corresponding group content, in which the amount of YTHDF1 siRNA was 1 μg/well, and the Man+CBAA-G5/G3-Man/YTHDF1siRNA group needed to add Man in advance (5 μM/well). Add CBAA-G5/G3-Man/YTHDF1 siRNA 4h later. After incubation for 4 h, the cells were washed twice with PBS buffer solution, digested with trypsin, centrifuged (1000 rpm, 5 min), collected and resuspended with an appropriate amount of PBS. Finally, the fluorescence intensity of the cells was detected by flow cytometry. The results are shown in Figure 13. Compared with the control group, there was no obvious red fluorescence in the naked YTHDF1 siRNA group, and the red fluorescence in the CBAA-G5/G3-Man/YTHDF1 siRNA complex group was the strongest, and it was blocked by adding Man in advance. After that, the red fluorescence of CBAA-G5/G3-Man/YTHDF1 siRNA complex decreased. This shows that CBAA-G5/G3-Man/YTHDF1 siRNA has the ability to target DC cells because of Man, which is more conducive to the realization of subsequent gene silencing.
实施例8Example 8
以树突细胞作为模型细胞，通过Western Blot来检测细胞内YTHDF1蛋白的表达情况。将树突细胞以2×105个/孔的细胞密度接种于6孔板中，每孔添加2mL DMEM++培养基，并将孔板置于5％ CO2，37℃的培养箱中孵育过夜。然后将每孔培养基换成含PBS缓冲液、裸YTHDF1 siRNA、CBAA-G5/G3-Man和CBAA-G5/G3-Man/YTHDF1 siRNA的培养液，其中复合物的N/P比均为15，YTHDF1 siRNA添加量均为1μg/孔，每孔添加1mL培养液，孵育4h。随后弃掉培养液，每孔加入1mL新鲜DMEM++培养基，在培养箱中继续培养48h。用PBS缓冲溶液清洗细胞2遍，利用胰酶对细胞进行消化，离心(1000rpm，5min)收集细胞后按照标准规范进行WesternBlot实验。结果如图14所示，以PBS为空白对照组，GADPH作为内参蛋白，YTHDF1蛋白在空白对照组和CBAA-G5/G3-Man组中的表达都很正常，CBAA-G5/G3-Man/YTHDF1 siRNA组YTHDF1蛋白的表达下调，蛋白下调效率为44％。Using dendritic cells as model cells, the expression of YTHDF1 protein in cells was detected by Western Blot. Dendritic cells were seeded in a 6-well plate at a cell density of 2×10 5 cells/well, 2 mL of DMEM++ medium was added to each well, and the well plate was incubated overnight in an incubator at 5% CO 2 at 37°C. Then the culture medium in each well was replaced with a culture medium containing PBS buffer, naked YTHDF1 siRNA, CBAA-G5/G3-Man and CBAA-G5/G3-Man/YTHDF1 siRNA, and the N/P ratio of the complexes was 15 , the amount of YTHDF1 siRNA added was 1 μg/well, and 1 mL of culture solution was added to each well, and incubated for 4 hours. Then the culture medium was discarded, and 1 mL of fresh DMEM++ medium was added to each well, and culture was continued for 48 h in the incubator. Wash the cells twice with PBS buffer solution, digest the cells with trypsin, collect the cells by centrifugation (1000 rpm, 5 min), and perform Western Blot experiments according to standard specifications. The results are shown in Figure 14. With PBS as the blank control group and GADPH as the internal reference protein, the expression of YTHDF1 protein in both the blank control group and the CBAA-G5/G3-Man group was normal. CBAA-G5/G3-Man/YTHDF1 The expression of YTHDF1 protein was down-regulated in the siRNA group, and the protein down-regulation efficiency was 44%.
实施例9Example 9
以树突细胞和乳腺癌细胞(4T1)作为模型细胞，利用荧光标记的CD80和CD86抗体，通过流式细胞仪检测最佳N/P比条件下CBAA-G5/G3-Man/YTHDF1 siRNA复合物提高DC细胞抗原递呈的效率。将4T1细胞以1×105个/孔的细胞密度接种于transwell-12孔板(0.4μm)的上室中,每孔添加1mL DMEM++培养基，同时，将树突细胞以1×105个/孔的细胞密度接种于transwell-12孔板的下室中，并将接种了上下两室的细胞分开放置，于5％ CO2、37℃的培养箱中培养。培养过夜后，将上、下室合并在一起培养，并将下室培养基换成1mL含有PBS或Free YTHDF1 siRNA或CBAA-G5/G3-Man/NC siRNA复合物或CBAA-G5/G3-Man/YTHDF1siRNA复合物的DMEM++培养基，其中N/P比为15，YTHDF1siRNA或NC siRNA添加量为1μg/孔。孵育4h后，下室换新鲜的DMEM++培养基继续培养24小时，然后用PBS缓冲溶液清洗下室DC细胞2遍，利用胰酶对DC细胞进行消化，离心(1000rpm，5min)收集细胞后用适量PBS重悬。根据标准规范，利用CD80和CD86抗体或同型对照进行标记，通过流式细胞仪检测细胞表面两种荧光的强度。结果如图15所示，通过和其他组对比荧光强度，可知CBAA-G5/G3-Man/YTHDF1siRNA复合物组中CD80和CD86的表达显著上调，这表明在癌细胞存在下CBAA-G5/G3-Man/YTHDF1 siRNA复合物仍具有可促进DC细胞的交叉递呈抗原能力。Using dendritic cells and breast cancer cells (4T1) as model cells, fluorescently labeled CD80 and CD86 antibodies were used to detect CBAA-G5/G3-Man/YTHDF1 siRNA complexes under optimal N/P ratio conditions by flow cytometry Improve the efficiency of DC cell antigen presentation. 4T1 cells were seeded in the upper chamber of a transwell-12-well plate (0.4 μm) at a cell density of 1×10 5 cells/well, and 1 mL of DMEM++ medium was added to each well, while dendritic cells were seeded at a density of 1×10 5 The cell density per well was inoculated in the lower chamber of a transwell-12 well plate, and the cells inoculated in the upper and lower chambers were placed separately and cultured in an incubator with 5% CO 2 and 37°C. After culturing overnight, combine the upper and lower chambers for culture, and replace the medium in the lower chamber with 1 mL containing PBS or Free YTHDF1 siRNA or CBAA-G5/G3-Man/NC siRNA complex or CBAA-G5/G3-Man DMEM++ medium of /YTHDF1siRNA complex, wherein the N/P ratio is 15, and the addition amount of YTHDF1siRNA or NC siRNA is 1 μg/well. After incubation for 4 hours, replace the lower chamber with fresh DMEM++ medium and continue culturing for 24 hours, then wash the DC cells in the lower chamber twice with PBS buffer solution, digest the DC cells with trypsin, collect the cells by centrifugation (1000rpm, 5min) and use an appropriate amount of Resuspended in PBS. Labeling with CD80 and CD86 antibodies or isotype controls was performed according to standard protocols, and the intensities of the two fluorophores on the cell surface were measured by flow cytometry. The results are shown in Figure 15. By comparing the fluorescence intensity with other groups, it can be seen that the expressions of CD80 and CD86 in the CBAA-G5/G3-Man/YTHDF1 siRNA complex group were significantly up-regulated, which indicated that in the presence of cancer cells, CBAA-G5/G3- The Man/YTHDF1 siRNA complex still has the ability to promote the cross-presentation antigen of DC cells.
实施例10Example 10
对实施案例1步骤(8)中制备的G5.NHAc/G3.NHAc/DOX复合物进行载药量以及缓释效果的表征。将实施案例1步骤(8)中离心得到的去质子的DOX沉淀收集起来并溶解在甲醇中做紫外分光光度计(UV-vis)测试以得到其在490nm处的吸光值。相应测出DOX在甲醇中的标准曲线，进而计算出DOX沉淀的量，然后用反应中DOX的加入量减去DOX沉淀量得到G5.NHAc/G3.NHAc/DOX复合物中DOX的负载量。结果显示，材料对DOX的包封率为78.5％，进一步计算可知，平均1个G5.NHAc/G3.NHAc中包裹了11.8个DOX。The drug loading and sustained release effect of the G5.NHAc/G3.NHAc/DOX complex prepared in step (8) of Example 1 were characterized. The deprotonated DOX precipitate obtained by centrifugation in step (8) of Example 1 was collected and dissolved in methanol for an ultraviolet spectrophotometer (UV-vis) test to obtain its absorbance at 490 nm. Correspondingly measure the standard curve of DOX in methanol, and then calculate the amount of DOX precipitation, and then subtract the amount of DOX precipitation from the amount of DOX added in the reaction to obtain the loading amount of DOX in the G5.NHAc/G3.NHAc/DOX complex. The results showed that the DOX encapsulation efficiency of the material was 78.5%. Further calculation showed that 11.8 DOX were encapsulated in an average of 1 G5.NHAc/G3.NHAc.
将实施例1步骤(8)中制备的G5.NHAc/G3.NHAc/DOX复合物溶解在水(2mg，1mL)中，分别置于分子量为14000的纤维素透析膜中，扎紧后悬浮于9mL的PBS(pH＝7.4)或醋酸缓冲液(pH＝5.5)中，然后将其置于37℃恒温摇床中振荡。在每个时间点下将1mL的缓冲外液取出并用UV-vis测试。与此同时，将1mL对应pH值的新鲜缓冲液加入其中。作为对比，对应浓度的DOX·HCl溶解在水中并置于PBS(pH＝7.4)的外液中，测试其缓释效果。与此同时，利用UV-vis得到DOX在pH＝5.0和pH＝7.4条件下的标准曲线，并将取得的不同pH条件下(pH＝5.5和pH＝7.4)的缓冲外液经UV-vis测试得到吸光值，由标准曲线计算得到缓释出的DOX的浓度，从而统计出G5.NHAc/G3.NHAc/DOX复合物在不同pH条件下(pH＝5.5和pH＝7.4)的缓释曲线。结果如图16A所示，与单独的DOX相比，G5.NHAc/G3.NHAc/DOX复合物中的DOX释放速率明显减缓，这证明了载体优良的药物缓释特性。而且pH＝5.5时，G5.NHAc/G3.NHAc/DOX复合物中的DOX释放速度比pH＝7.4要快，这说明了酸性微环境中更利于药物的释放。Dissolve the G5.NHAc/G3.NHAc/DOX complex prepared in step (8) of Example 1 in water (2 mg, 1 mL), place them in cellulose dialysis membranes with a molecular weight of 14000, and suspend them in 9 mL of PBS (pH = 7.4) or acetate buffer (pH = 5.5), and then place it in a constant temperature shaker at 37°C for shaking. At each
实施例11Example 11
以鼠源乳腺癌(4T1)细胞作为模型细胞，以G5.NHAc/G3.NHAc和DOX为对照，检测G5.NHAc/G3.NHAc/DOX复合物在不同DOX浓度条件下的细胞毒性。将4T1细胞以2×103个/孔的细胞密度接种于96孔板中，每孔添加100μL DMEM++培养基。细胞放置于5％ CO2，37℃的培养箱中培养过夜后，将培养基换成100μL含有不同DOX摩尔浓度材料的DMEM++，其中DOX浓度分别为0、0.25、0.5、2.5、5、10、25和50μg/mL，然后继续培养细胞24h。弃去培养液，按照说明书程序使用CCK-8试剂盒进行细胞活性的检测。结果如图16B所示，随着DOX浓度的增加，和自由DOX组一样，G5.NHAc/G3.NHAc/DOX复合物组细胞活力下降，这说明负载了DOX的G5.NHAc/G3.NHAc材料具有抑制癌细胞增殖的效果，而未载药的G5.NHAc/G3.NHAc材料组对细胞活力不产生影响。Using murine breast cancer (4T1) cells as model cells and G5.NHAc/G3.NHAc and DOX as controls, the cytotoxicity of G5.NHAc/G3.NHAc/DOX complexes under different DOX concentrations was detected. 4T1 cells were seeded in a 96-well plate at a cell density of 2×10 3 cells/well, and 100 μL of DMEM++ medium was added to each well. After the cells were cultured overnight in a 5% CO 2 , 37°C incubator, the medium was replaced with 100 μL of DMEM++ containing materials with different DOX molar concentrations, where the DOX concentrations were 0, 0.25, 0.5, 2.5, 5, 10, 25 and 50 μg/mL, and then continue to culture the cells for 24h. Discard the culture medium, and use the CCK-8 kit to detect cell viability according to the instructions. The results are shown in Figure 16B. As the DOX concentration increased, the cell viability in the G5.NHAc/G3.NHAc/DOX complex group decreased as in the free DOX group, which indicated that the DOX-loaded G5.NHAc/G3.NHAc material It has the effect of inhibiting the proliferation of cancer cells, while the non-drug-loaded G5.NHAc/G3.NHAc material group has no effect on cell viability.
实施例12Example 12
以4T1细胞作为模型细胞，利用DOX自身的红色荧光，通过流式细胞仪研究评价G5.NHAc/G3.NHAc负载DOX后被细胞吞噬的效率。以1×105/孔的密度将4T1接种于12孔板中，每孔添加1mL DMEM++培养基。细胞放置于5％ CO2、37℃的培养箱中培养过夜后，将培养基换成1mL含有不同DOX摩尔浓度材料的DMEM++，其中DOX浓度分别为2.5、5、10和20μg/mL。孵育4h后，用PBS缓冲溶液清洗细胞2遍，利用胰酶对细胞进行消化，离心(1000rpm，5min)收集细胞后用适量PBS重悬。最后，用流式细胞仪检测细胞的荧光强度，结果如图17所示。和正常细胞组(对照组)相比，G5.NHAc/G3.NHAc/DOX组荧光强度随着DOX浓度的增加而增强，这说明制备的G5.NHAc/G3.NHAc/DOX复合物能被细胞吞噬，而且呈剂量依赖性。Using 4T1 cells as model cells, the red fluorescence of DOX was used to evaluate the phagocytosis efficiency of G5.NHAc/G3.NHAc loaded with DOX by flow cytometry. 4T1 was seeded in a 12-well plate at a density of 1×10 5 /well, and 1 mL of DMEM++ medium was added to each well. After the cells were cultured overnight in a 5% CO 2 , 37°C incubator, the medium was replaced with 1 mL of DMEM++ containing materials with different DOX molar concentrations, where the DOX concentrations were 2.5, 5, 10 and 20 μg/mL. After incubation for 4 h, the cells were washed twice with PBS buffer solution, digested with trypsin, collected by centrifugation (1000 rpm, 5 min), and resuspended with an appropriate amount of PBS. Finally, the fluorescence intensity of the cells was detected by flow cytometry, and the results are shown in FIG. 17 . Compared with the normal cell group (control group), the fluorescence intensity of the G5.NHAc/G3.NHAc/DOX group increased with the increase of the DOX concentration, which indicated that the prepared G5.NHAc/G3.NHAc/DOX complex could be absorbed by the cells. Phagocytosis, and was dose-dependent.
实施例13Example 13
以4T1细胞作为模型细胞，利用钙网蛋白(CRT)抗体实施免疫荧光染色，通过激光共聚焦显微镜研究评价G5.NHAc/G3.NHAc负载DOX后诱导的免疫原性死亡效应。将4T1细胞以1×105个/孔的细胞密度接种于共聚焦显微镜专用皿中，每皿添加500μL DMEM++培养基，并将皿置于5％ CO2、37℃的培养箱中孵育过夜。然后培养基换成500μL含有DOX浓度为10μg/mL的G5.NHAc/G3.NHAc/DOX复合物(IV)的DMEM++培养基，并设PBS组(I)、对应浓度的G5.NHAc/G3.NHAc组(II)、对应浓度的DOX组(III)为对照。细胞培养12h后，用PBS缓冲溶液清洗细胞2遍，并通过标准规范用戊二醛固定细胞，然后按照标准流程用免疫染色封闭缓冲液处理60min，再用抗CRT(一抗)孵育60min，用PBS洗涤，并与FITC标记的二抗孵育60min。最后将细胞用DAPI在37℃下染色15min，用激光共聚焦显微镜观察CRT表达。结果如图18所示，和PBS组、G5.NHAc/G3.NHAc组相比，DOX组和G5.NHAc/G3.NHAc/DOX复合物组的CRT表达都有所增加，这说明由于DOX的存在，制备的G5.NHAc/G3.NHAc/DOX复合物能够引起癌细胞的免疫原性死亡效应。Using 4T1 cells as model cells, immunofluorescent staining was performed with calreticulin (CRT) antibody, and the immunogenic death effect induced by G5.NHAc/G3.NHAc loaded with DOX was evaluated by confocal laser microscopy. 4T1 cells were seeded in confocal microscope dishes at a cell density of 1×10 5 cells/well, 500 μL of DMEM++ medium was added to each dish, and the dishes were incubated overnight in an incubator with 5% CO 2 and 37°C. Then the medium was replaced with 500 μL of DMEM++ medium containing the G5.NHAc/G3.NHAc/DOX complex (IV) with a DOX concentration of 10 μg/mL, and the PBS group (I) and the G5.NHAc/G3. The NHAc group (II) and the DOX group (III) with corresponding concentrations were used as controls. After the cells were cultured for 12 hours, the cells were washed twice with PBS buffer solution, and the cells were fixed with glutaraldehyde according to standard specifications, and then treated with immunostaining blocking buffer for 60 minutes according to the standard procedure, and then incubated with anti-CRT (primary antibody) for 60 minutes, and then used Wash with PBS and incubate with FITC-labeled secondary antibody for 60 min. Finally, the cells were stained with DAPI for 15 min at 37°C, and the expression of CRT was observed with a laser confocal microscope. The results are shown in Figure 18. Compared with the PBS group and the G5.NHAc/G3.NHAc group, the expression of CRT in the DOX group and the G5.NHAc/G3.NHAc/DOX complex group has increased, which shows that due to the presence of DOX There, the prepared G5.NHAc/G3.NHAc/DOX complex can cause the immunogenic death effect of cancer cells.
实施例14Example 14
以4T1作为模型细胞，利用荧光标记的CD80和CD86抗体，通过流式细胞仪检测癌细胞培养液中添加G5.NHAc/G3.NHAc/DOX复合物后，ICD刺激后，DC细胞的抗原递呈能力。将4T1细胞以1×105个/孔的细胞密度接种于transwell-12孔板(0.4μm)的上室中,每孔添加0.5mL DMEM++培养基，同时，将树突细胞以1×105个/孔的细胞密度接种于transwell-12孔板的下室中，并将接种了上下两室的细胞分开放置，于5％ CO2、37℃的培养箱中培养。培养过夜后，将上室培养基换成0.5mL含有PBS、5μg/mL DOX的G5.NHAc/G3.NHAc/DOX复合物或50μg/mL DOX的G5.NHAc/G3.NHAc/DOX复合物的DMEM++培养基。将上、下室合并在一起培养24h。然后用PBS缓冲溶液清洗树突细胞2遍，利用胰酶对细胞进行消化，离心(1000rpm，5min)收集细胞后用适量PBS重悬。根据标准规范，利用CD80和CD86抗体或同型对照进行标记，通过流式细胞仪检测细胞表面两种荧光的强度。结果如图19所示，通过和其他组对比荧光强度，可知G5.NHAc/G3.NHAc/DOX复合物组中CD80和CD86的表达显著上调，而且DOX浓度越高，上调越明显，这表明G5.NHAc/G3.NHAc/DOX复合物可促进DC细胞的抗原递呈能力。Using 4T1 as a model cell, using fluorescently labeled CD80 and CD86 antibodies, the antigen presentation of DC cells after ICD stimulation was detected by flow cytometry after the G5.NHAc/G3.NHAc/DOX complex was added to the cancer cell culture medium ability. 4T1 cells were seeded in the upper chamber of a transwell-12-well plate (0.4 μm) at a cell density of 1×10 5 cells/well, and 0.5 mL of DMEM++ medium was added to each well, while dendritic cells were seeded at a density of 1×10 5 Cells per well were seeded in the lower chamber of a transwell-12 well plate, and the cells inoculated in the upper and lower chambers were placed separately and cultured in an incubator with 5% CO 2 and 37°C. After culturing overnight, replace the medium in the upper chamber with 0.5 mL of G5.NHAc/G3.NHAc/DOX complex containing PBS, 5 μg/mL DOX or G5.NHAc/G3.NHAc/DOX complex with 50 μg/mL DOX DMEM++ medium. The upper and lower chambers were combined and incubated for 24 hours. Then the dendritic cells were washed twice with PBS buffer solution, digested with trypsin, collected by centrifugation (1000 rpm, 5 min), and resuspended with an appropriate amount of PBS. Labeling with CD80 and CD86 antibodies or isotype controls was performed according to standard protocols, and the intensities of the two fluorophores on the cell surface were measured by flow cytometry. The results are shown in Figure 19. By comparing the fluorescence intensity with other groups, it can be seen that the expressions of CD80 and CD86 in the G5.NHAc/G3.NHAc/DOX complex group were significantly up-regulated, and the higher the DOX concentration, the more obvious the up-regulation, which indicated that the G5.NHAc/G3.NHAc/DOX complex group .NHAc/G3.NHAc/DOX complex can promote the antigen presenting ability of DC cells.
实施例15Example 15
选用4-6周的雌性ICR健康小白鼠(购自上海斯莱克实验动物中心)，并根据制定的方案进行注射给药(所有动物实验均严格按照动物保护协会标准进行)以评估设计的多功能核壳树状大分子负载药物或基因后的生物安全性。首先，选用ICR小鼠18只，随机分成六组，分别为PBS组、DOX.HCl组、G5.NHAc/G3.NHAc/DOX组、CBAA-G5/G3-Man/YTHDF1 siRNA组、DOX.HCl+CBAA-G5/G3-Man/YTHDF1 siRNA组、G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1 siRNA组，其中，含DOX的组别需保证为静脉注射，且DOX的注射剂量为5mg/kg；含YTHDF1 siRNA的组别需保证为腹股沟淋巴结注射，且YTHDF1siRNA的注射剂量为0.3mg/kg(约10μg/只)。注射15天后，对小鼠进行安乐死处理，并通过解剖提取主要器官进行切片和H&E染色。结果如图20所示，除了含有游离DOX的组别具有明显的心脏毒性外，其他组别的制剂没有产生任何明显的心脏毒性、肝肾损伤、肺毒性和脾脏浸润。这说明运用多功能核壳树状大分子制备的基因或药物复合物具有良好的生物相容性。4-6 weeks old female ICR healthy mice (purchased from Shanghai Slack Experimental Animal Center) were selected, and injected according to the established protocol (all animal experiments were carried out in strict accordance with the standards of the Animal Protection Association) to evaluate the multifunctionality of the design Biosafety of core-shell dendrimers loaded with drugs or genes. First, 18 ICR mice were selected and randomly divided into six groups: PBS group, DOX.HCl group, G5.NHAc/G3.NHAc/DOX group, CBAA-G5/G3-Man/YTHDF1 siRNA group, DOX.HCl group +CBAA-G5/G3-Man/YTHDF1 siRNA group, G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1 siRNA group, among them, the group containing DOX needs to be injected intravenously, and DOX The injection dose of YTHDF1 siRNA is 5mg/kg; the group containing YTHDF1 siRNA needs to be guaranteed to be injected into the inguinal lymph nodes, and the injection dose of YTHDF1 siRNA is 0.3mg/kg (about 10μg/mouse). Fifteen days after injection, mice were euthanized and major organs were dissected for sectioning and H&E staining. The results are shown in Figure 20, except that the group containing free DOX had obvious cardiotoxicity, the preparations of other groups did not produce any obvious cardiotoxicity, liver and kidney damage, pulmonary toxicity and spleen infiltration. This shows that the gene or drug complex prepared by using the multifunctional core-shell dendrimer has good biocompatibility.
实施例16Example 16
以4T1作为模型细胞，构建原位乳腺癌小鼠模型，根据制定的方案进行注射给药以评估设计的多功能核壳树状大分子负载药物或基因后的治疗效果。首先培养并收集细胞，以1×106 4T1细胞/鼠的剂量在小白鼠乳房垫处注射肿瘤细胞，当肿瘤体积达到约350mm3时，选用模型小鼠36只，随机分成六组，分别为PBS组(I)、DOX.HCl组(II)、G5.NHAc/G3.NHAc/DOX组(III)、CBAA-G5/G3-Man/YTHDF1 siRNA组(IV)、DOX.HCl+CBAA-G5/G3-Man/YTHDF1 siRNA组(V)、G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1 siRNA组(VI)。其中，含DOX的组别需保证为瘤内注射，且DOX的注射剂量为5mg/kg；含YTHDF1 siRNA的组别需保证为腹股沟淋巴结注射，且YTHDF1 siRNA的注射剂量为0.3mg/kg(约10μg/只)。同时，肿瘤体积达到约350mm3时记作实验的第1天，在第1天瘤内注射含有DOX的组别，在第2天腹股沟淋巴结注射含YTHDF1 siRNA的组别。每组间隔3天给药一次，总共给药5次，肿瘤体积每2天测量一次，小鼠重量每2天称一次。结果如图21所示，与空白对照组相比，各组小鼠的肿瘤体积均有减小，其中尤以G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1 siRNA组治疗效果最为明显。小鼠体重在治疗期间变化不大，仅单独的DOX组在治疗周期后体重略有下降。这说明运用多功能核壳树状大分子制备的基因或药物复合物具有良好的治疗效果。Using 4T1 as a model cell, an orthotopic breast cancer mouse model was constructed, and injected according to the established protocol to evaluate the therapeutic effect of the designed multifunctional core-capsid dendrimer loaded with drugs or genes. First, cells were cultured and collected, and tumor cells were injected into the breast pads of mice at a dose of 1×10 6 4T1 cells/mouse. When the tumor volume reached about 350 mm 3 , 36 model mice were selected and randomly divided into six groups, respectively: PBS group (I), DOX.HCl group (II), G5.NHAc/G3.NHAc/DOX group (III), CBAA-G5/G3-Man/YTHDF1 siRNA group (IV), DOX.HCl+CBAA-G5 /G3-Man/YTHDF1 siRNA group (V), G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1 siRNA group (VI). Among them, the group containing DOX needs to ensure intratumoral injection, and the injection dose of DOX is 5 mg/kg; the group containing YTHDF1 siRNA needs to ensure inguinal lymph node injection, and the injection dose of YTHDF1 siRNA is 0.3 mg/kg (about 10μg/piece). At the same time, when the tumor volume reached about 350mm3 , it was recorded as the first day of the experiment, the group containing DOX was injected into the tumor on the first day, and the group containing YTHDF1 siRNA was injected into the inguinal lymph nodes on the second day. Each group was administered once at intervals of 3 days, a total of 5 administrations, the tumor volume was measured every 2 days, and the weight of the mice was weighed every 2 days. The results are shown in Figure 21. Compared with the blank control group, the tumor volume of mice in each group was reduced, especially in the G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1 siRNA group The treatment effect is most obvious. The body weight of the mice did not change much during the treatment period, only the DOX group alone had a slight decrease in body weight after the treatment cycle. This shows that the gene or drug complex prepared by using the multifunctional core-shell dendrimer has good therapeutic effect.
治疗15天后，对小鼠进行安乐死处理，并通过解剖提取小鼠脾脏和肿瘤部位。利用荧光标记的CD4+和CD8+抗体，通过流式细胞仪检测各组脾脏组织中各类T细胞含量，评估T细胞激活程度；另外，提取的肿瘤部位进行切片和免疫荧光染色以及TUNEL实验，即利用CD4+和CD8+抗体，通过免疫荧光染色评估激活的T细胞浸润情况；利用TUNEL实验，通过荧光显微镜评估肿瘤部位的细胞凋亡情况。结果如图22所示，G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1 siRNA组的脾脏中CD4+和CD8+T细胞的比例都是最高的，这说明G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1 siRNA组脾脏中的T细胞被显著激活。TUNEL染色结果显示(图23)，G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1siRNA组肿瘤部位的细胞凋亡程度最为明显。这些现象也充分说明了运用多功能核壳树状大分子制备的基因或药物复合物进行化疗-免疫治疗的联合治疗，可以达到增强治疗效果的目的。After 15 days of treatment, the mice were euthanized, and the mouse spleen and tumor sites were extracted by dissection. Fluorescently labeled CD4 + and CD8 + antibodies were used to detect the contents of various T cells in the spleen tissues of each group by flow cytometry to evaluate the degree of T cell activation; in addition, the extracted tumor sites were sectioned, immunofluorescent stained and TUNEL experiments were carried out. That is, using CD4 + and CD8 + antibodies, the infiltration of activated T cells was evaluated by immunofluorescence staining; the apoptosis of tumor sites was evaluated by fluorescence microscopy by using TUNEL assay. The results are shown in Figure 22, the proportions of CD4 + and CD8 + T cells in the spleen of the G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1 siRNA group are the highest, which shows that the G5.NHAc T cells in the spleen of the /G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1 siRNA group were significantly activated. The results of TUNEL staining ( FIG. 23 ) showed that the degree of cell apoptosis at the tumor site was the most obvious in the G5.NHAc/G3.NHAc/DOX+CBAA-G5/G3-Man/YTHDF1siRNA group. These phenomena also fully demonstrate that the use of gene or drug complexes prepared by multifunctional core-shell dendrimers for combined chemotherapy-immunotherapy can achieve the purpose of enhancing the therapeutic effect.
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