CN102091036A - Compound liposome containing anti-tumor drugs and preparation method and application thereof - Google Patents

The invention belongs to a liposome, in particular relates to a compound liposome preparation with high targeting property of tumor tissues and efficient penetrability of tumor cells. The invention is characterized in that hyaluronic acid or sodium hyaluronate, tumor cell selective penetrating peptides and a liposome are used for together constructing a compound liposome. The compound liposome is characterized by utilizing the dual-tumor cell targeting property of the hyaluronic acid and tumor cell selective penetrating peptides to effectively solve the problem that because a conventional liposome and traditional cell penetrating peptides have no selectivity on normal cells and tumor cells, the toxicity is increased when the drug effect is increased in the therapeutic process, thereby fully meeting the clinical requirements of high efficiency and low toxicity when anti-tumor drugs are prepared into preparations for treating diseases.

一种含有抗肿瘤药物的复合脂质体及其制备方法和用途A kind of composite liposome containing antitumor drug and its preparation method and application

技术领域：Technical field:

本发明属于医药技术领域，具体的说是运用透明质酸或透明质酸钠、肿瘤细胞选择性穿膜肽和脂质体共同构建一种复合脂质体。此复合脂质体特征在于利用透明质酸和肿瘤选择性穿膜肽的双重肿瘤细胞靶向性，有效地解决常规脂质体以及传统穿膜肽对正常和肿瘤细胞均无选择性而导致其治疗时药效增加的同时毒性也增加的难题，充分实现肿瘤药物制备成制剂用于治疗时所需的高效、低毒的临床需求。The invention belongs to the technical field of medicine, and specifically uses hyaluronic acid or sodium hyaluronate, tumor cell selective membrane-penetrating peptides and liposomes to jointly construct a composite liposome. This composite liposome is characterized by the dual tumor cell targeting of hyaluronic acid and tumor-selective membrane-penetrating peptide, which effectively solves the problem that conventional liposomes and traditional membrane-penetrating peptides have no selectivity for normal and tumor cells. The problem of increased drug efficacy and increased toxicity during treatment fully realizes the clinical needs of high efficiency and low toxicity required for the preparation of tumor drugs into preparations for treatment.

背景技术：Background technique:

穿膜肽(cell-permeable peptides，CPP)是一类具有较强细胞膜穿透能力的多肽，它们具有高效穿过细胞膜而不损伤细胞膜结构和功能的特点。将穿膜肽通过共价键或非共价键偶合等方式携带各种分子或纳米载体透过细胞膜，已经在介导基因、蛋白质、多肽、小分子、纳米颗粒和脂质体等穿透各种细胞膜方面发挥了巨大作用。Cell-permeable peptides (CPP) are a class of polypeptides with strong cell membrane penetration ability, and they have the characteristics of efficiently passing through the cell membrane without damaging the structure and function of the cell membrane. The membrane-penetrating peptides carry various molecules or nanocarriers through the cell membrane through covalent or non-covalent coupling, and have been mediating genes, proteins, polypeptides, small molecules, nanoparticles and liposomes to penetrate various important role in the cell membrane.

现有研究表明，传统的穿膜肽和普通脂质体(未经修饰的)对正常组织和肿瘤组织一般无选择性，即使主动靶向性脂质体在正常组织也有一定数量的分布，如果将传统的穿膜肽如R₈等用来修饰普通脂质体后，由于穿膜肽的高效穿膜特性，在增加肿瘤细胞内药物量的同时，必然伴随正常细胞中药物量的相应增加，这将导致正常细胞组织的更大伤害(毒副作用明显增加)。因此，如果能够构建一种具有肿瘤细胞选择性的穿膜肽，与采用普通穿膜肽相比，将其通过共价键或非共价键偶合等方式与脂质体等纳米载体相结合并携带其穿过细胞膜，在靶向治疗肿瘤疾病的领域将具有更大的应用前景。Existing studies have shown that traditional membrane-penetrating peptides and ordinary liposomes (unmodified) are generally non-selective to normal tissues and tumor tissues, even active targeting liposomes have a certain amount of distribution in normal tissues, if After using traditional membrane-penetrating peptides such as R ₈ to modify ordinary liposomes, due to the high-efficiency membrane-penetrating properties of membrane-penetrating peptides, while increasing the amount of drugs in tumor cells, it must be accompanied by a corresponding increase in the amount of drugs in normal cells. This will lead to greater damage to normal tissue (significantly increased toxic side effects). Therefore, if a tumor cell-selective membrane-penetrating peptide can be constructed, compared with the use of ordinary membrane-penetrating peptides, it can be combined with nanocarriers such as liposomes through covalent or non-covalent Carrying it through the cell membrane will have greater application prospects in the field of targeted therapy of tumor diseases.

肿瘤细胞选择性穿膜肽(selective cell penetration peptides on tumor cell，SCPPt)为本实验室在经典穿膜肽基础上创新研究出的一种新型穿膜肽，它具有对肿瘤细胞膜有较高的穿透效率，而对正常细胞膜几乎无穿透或穿透效率较低的特点，主要由5～12个精氨酸和3个组氨酸组成。带有本实验室发明的氨基酸序列的穿膜肽在正常pH环境下，仅带有少量正电荷，无法与细胞表面高度结合而穿膜；而当到达肿瘤组织细胞表面时由于其偏酸性的环境使得组氨酸和精氨酸协同表现出带有大量正电荷，从而能够高效地与带负电荷的细胞表面相结合，体现出较强的穿透肿瘤细胞膜的作用。Selective cell penetration peptides on tumor cell (SCPPt) is a new type of penetrating peptide developed by our laboratory on the basis of classic penetrating peptides. It has a high penetrating ability to tumor cell membranes. It has the characteristics of almost no penetration or low penetration efficiency to normal cell membranes, and is mainly composed of 5-12 arginines and 3 histidines. The membrane-penetrating peptide with the amino acid sequence invented by our laboratory has only a small amount of positive charges in a normal pH environment, and cannot be highly combined with the cell surface to penetrate the membrane; when it reaches the surface of tumor tissue cells, due to its acidic environment The combination of histidine and arginine with a large number of positive charges can efficiently combine with the negatively charged cell surface, showing a strong effect of penetrating the tumor cell membrane.

透明质酸(HA)是以D-葡萄糖醛酸-N-乙酰氨基葡萄糖为双糖单位组成的直链高分子多糖，是一种广泛存在于机体多种组织的细胞外基质、细胞表面和细胞内部的阴离子物质，属于体内内源性物质，不诱发免疫反应，安全性高。近年来研究表明，HA可与肿瘤细胞表面高表达的CD44、RHAMM等受体结合，激活针对HA的细胞内信号通路或激活HA的内化作用，而调节细胞的运动等行为，且可被肿瘤细胞表面高度表达的HA酶迅速降解消除。此外，淋巴结内高浓度HA也有利于巨噬细胞游走，发挥吞噬和杀伤肿瘤细胞的作用。据文献报道，HA可借助其粘弹性作用所形成大分子网状结构包绕巨噬细胞，将巨噬细胞与被吞噬细胞或颗粒隔离，从而限制巨噬细胞的吞噬作用，延长其包被载体的血液循环时间，具有长循环效应。HA为电负性，通过静电作用自组装在正电性的脂质体表面，并证明可显著提高脂质体的肿瘤靶向性。鉴于HA具有良好的生物相容性和可降解性、长循环和负电性、无免疫原性、肿瘤细胞高亲和性和抗肿瘤辅助治疗作用等，已成为目前抗肿瘤主动靶向载体的研究热点之一。Hyaluronic acid (HA) is a linear polymer polysaccharide composed of D-glucuronic acid-N-acetylglucosamine as a disaccharide unit, and is a kind of extracellular matrix, cell surface and cell surface widely present in various tissues of the body. The internal anionic substances are endogenous substances in the body, do not induce immune reactions, and are highly safe. Studies in recent years have shown that HA can bind to receptors such as CD44 and RHAMM highly expressed on the surface of tumor cells, activate intracellular signaling pathways for HA or activate the internalization of HA, thereby regulating cell movement and other behaviors, and can be inhibited by tumor cells. The highly expressed HA enzyme on the cell surface is rapidly degraded and eliminated. In addition, the high concentration of HA in lymph nodes is also conducive to the migration of macrophages, which play the role of phagocytosis and killing tumor cells. According to literature reports, HA can use its viscoelasticity to form a macromolecular network structure to envelop macrophages and isolate macrophages from phagocytic cells or particles, thereby limiting the phagocytosis of macrophages and prolonging the encapsulation of macrophages. The blood circulation time has a long circulation effect. HA is electronegative, self-assembles on the surface of positively charged liposomes through electrostatic interaction, and proves to significantly improve the tumor targeting of liposomes. In view of HA's good biocompatibility and degradability, long circulation and negative charge, non-immunogenicity, high affinity for tumor cells and anti-tumor adjuvant therapy effect, etc., it has become the current research of anti-tumor active targeting carrier. One of the hot spots.

发明内容：Invention content:

发明目的purpose of invention

本发明的目的旨在提供一种肿瘤组织高度靶向性、肿瘤细胞高效穿透性的复合脂质体及其制备方法和用途。The purpose of the present invention is to provide a complex liposome with high tumor tissue targeting and high tumor cell penetration, and its preparation method and application.

技术方案Technical solutions

针对上述发明目的，本发明提供以下技术方案：For the purpose of the above invention, the present invention provides the following technical solutions:

一种含有抗肿瘤药物的复合脂质体，其特征在于：它含有抗肿瘤药物、脂质、透明质酸或透明质酸钠、肿瘤细胞选择性穿膜肽。A composite liposome containing antineoplastic drugs is characterized in that it contains antineoplastic drugs, lipids, hyaluronic acid or sodium hyaluronate, and selective membrane-penetrating peptides for tumor cells.

所述的复合脂质体，其特征在于：按质量比，它含有0.01％-30％的抗肿瘤药物，1％-98％的脂质，0.001％-20％透明质酸钠，0.001％-20％的肿瘤细胞选择性穿膜肽，0-20％的醇，0-20％的抗氧剂，0-20％的防腐剂，0-20％的pH调节剂，0-20％的相变调节剂，0-20％的长循环材料，0-20％的等渗调节剂，0-95％的冻干支撑剂，0-95％的水。The composite liposome is characterized in that: by mass ratio, it contains 0.01%-30% antitumor drugs, 1%-98% lipids, 0.001%-20% sodium hyaluronate, 0.001%- 20% tumor cell selective penetrating peptide, 0-20% alcohol, 0-20% antioxidant, 0-20% preservative, 0-20% pH regulator, 0-20% phase Variable adjustment agent, 0-20% long circulation material, 0-20% isotonic adjustment agent, 0-95% freeze-dried proppant, 0-95% water.

所述的复合脂质体，其特征在于所述的抗肿瘤药物选自下列药物中的一种或多种：紫杉醇、多西紫杉醇、放线菌素D、多柔比星、表柔比星、柔红霉素、长春新碱、长春瑞滨、顺铂、奥沙利铂、甲氨蝶呤、氟尿嘧啶、巯嘌呤、阿糖胞苷、脱氧氟尿苷、依托泊苷、替尼泊苷、喜树碱、羟基喜树碱、拓扑替康、伊立替康、米托蒽醌、环磷酰胺、异环磷酰胺、丝裂霉素C、白消安、洛莫司汀、卡莫司汀、司莫司汀、尼莫司汀、雷莫司汀、吉西他滨、卡培他滨、地西他滨、安西他滨、顺铂、博来霉素、平阳霉素、藤黄酸、新藤黄酸以及这些药物的各种药用盐。Described composite liposome is characterized in that described antineoplastic drug is selected from one or more in the following drugs: paclitaxel, docetaxel, actinomycin D, doxorubicin, epirubicin , daunorubicin, vincristine, vinorelbine, cisplatin, oxaliplatin, methotrexate, fluorouracil, mercaptopurine, cytarabine, deoxyfluridine, etoposide, teniposide , camptothecin, hydroxycamptothecin, topotecan, irinotecan, mitoxantrone, cyclophosphamide, ifosfamide, mitomycin C, busulfan, lomustine, carlimus Semustine, Nimustine, Ramustine, Gemcitabine, Capecitabine, Decitabine, Ancitabine, Cisplatin, Bleomycin, Pingyangmycin, Gambogic Acid, Xinteng Xanthic acid and various pharmaceutically acceptable salts of these drugs.

所述的复合脂质体，其特征在于所述的脂质是磷脂和胆固醇，磷脂选自天然磷脂、半合成磷脂和全合成磷脂中的一种或多种。The composite liposome is characterized in that the lipids are phospholipids and cholesterol, and the phospholipids are selected from one or more of natural phospholipids, semi-synthetic phospholipids and fully synthetic phospholipids.

所述的复合脂质体，其特征在于所述的透明质酸或透明质酸钠的相对分子质量从4×10³道尔顿到2×10⁷道尔顿。The composite liposome is characterized in that the relative molecular mass of the hyaluronic acid or sodium hyaluronate ranges from 4×10 ³ Daltons to 2×10 ⁷ Daltons.

所述的复合脂质体，其特征在于所述的肿瘤细胞选择性穿膜肽对于肿瘤细胞或组织有较强的细胞膜穿透作用，对正常细胞或组织的细胞膜穿透作用较弱或不能透过。The composite liposome is characterized in that the tumor cell selective membrane penetrating peptide has a strong cell membrane penetration effect on tumor cells or tissues, and has a weak or impermeable cell membrane penetration effect on normal cells or tissues. Pass.

所述的复合脂质体，其特征在于所述的肿瘤细胞选择性穿膜肽选自下列多肽中的一种或多种：RRRRRHHH(R₅H₃，R在C端或N端)、RRRRRRHHH(R₆H₃，R在C端或N端)、RRRRRRRHHH(R₇H₃，R在C端或N端)、RRRRRRRRHHH(R₈H₃，R在C端或N端)、RRRRRRRRRHHH(R₉H₃，R在C端或N端)、RRRRRRRRRRHHH(R₁₀H₃，R在C端或N端)、RRRRRRRRRRRHHH(R₁₁H₃，R在C端或N端)、RRRRRRRRRRRRHHH(R₁₂H₃，R在C端或N端)，它们的N端被脂肪酸修饰，脂肪酸优选碳链含有8-18个碳原子的，更优选的是硬脂酸。The composite liposome is characterized in that the tumor cell-selective membrane-penetrating peptide is selected from one or more of the following polypeptides: RRRRRHHH (R ₅ H ₃ , R is at the C-terminal or N-terminal), RRRRRHHH (R ₆ H ₃ , R is at the C-terminal or N-terminal), RRRRRRHHH (R ₇ H ₃ , R is at the C-terminal or N-terminal), RRRRRRRRHHH (R ₈ H ₃ , R is at the C-terminal or N-terminal), RRRRRRRRHHH(R ₉ H ₃ , R at the C-terminal or N-terminal), RRRRRRRRRRHHHH (R ₁₀ H ₃ , R at the C-terminal or N-terminal), RRRRRRRRRRHHHH (R ₁₁ H ₃ , R at the C-terminal or N-terminal), RRRRRRRRRRHHHH(R ₁₂ H _3. R is at the C-terminal or N-terminal), and their N-terminals are modified by fatty acids. The fatty acid preferably has a carbon chain containing 8-18 carbon atoms, more preferably stearic acid.

所述的复合脂质体其特征在于可以制备治疗肿瘤的药物制剂。The composite liposome is characterized in that it can prepare a pharmaceutical preparation for treating tumors.

有益成果Beneficial results

当该复合脂质体注入静脉后，将首先借助外层透明质酸包被具有的长循环性、肿瘤靶向性以及生物降解性，主动聚集于肿瘤组织及细胞表面并被该部位高浓度透明质酸酶迅速降解，使肿瘤细胞选择性穿膜肽裸露于脂质体表面，充分发挥其对肿瘤细胞的高效穿透作用，携带脂质体更多地进入肿瘤细胞，从而使脂质体所包封的抗肿瘤药物更多地进入时瘤细胞并发挥细胞毒作用。此复合脂质体在解决肿瘤组织药物浸润性差难题的同时，有效降低细胞毒药物对正常细胞的伤害，显著改善常用细胞毒药物和穿膜肽由于无肿瘤细胞靶向性而导致的毒副作用，具有非常广阔的应用前景。When the composite liposome is injected into the vein, it will actively gather on the surface of tumor tissues and cells with the help of the long-circulation, tumor-targeting and biodegradable properties of the outer layer of hyaluronic acid coating and be transparent by the high concentration of this part. Massonidase rapidly degrades tumor cells to selectively expose membrane-penetrating peptides on the surface of liposomes, giving full play to its high-efficiency penetration of tumor cells, and carrying liposomes into tumor cells more, so that the liposomes Encapsulated anti-tumor drugs more enter into tumor cells and exert cytotoxic effect. This complex liposome solves the problem of poor drug infiltration in tumor tissue, effectively reduces the damage of cytotoxic drugs to normal cells, and significantly improves the toxic and side effects of commonly used cytotoxic drugs and membrane-penetrating peptides due to no tumor cell targeting. It has a very broad application prospect.

具体实施方式：Detailed ways:

下列实施例中的R为精氨酸，H为组氨酸R in the following examples is arginine, and H is histidine

实施例1Example 1

以本发明所述及的透明质酸钠、肿瘤细胞选择性穿膜肽R₈H₃与盐酸多柔比星脂质体来说明这一复合脂质体的制备方法。The preparation method of the complex liposome is illustrated by using sodium hyaluronate, tumor cell selective membrane-penetrating peptide R ₈ H ₃ and doxorubicin hydrochloride liposome mentioned in the present invention.

在50mL侧面带支管、支管带有砂板滤芯的圆底烧瓶内加入1.39gRinkamide-MBHA树脂(0.74mmol/g)，加20mL DMF溶胀10分钟，抽滤去掉溶剂。然后加入20mL 20％哌啶/DMF溶液，搅拌30分钟，抽滤。用DMF洗涤树脂6次，抽滤去掉溶剂。向反应瓶中加入2.00g(3.09mmol)Fmoc-Arg(Pbf)-OH(Pbf为2，2，4，6，7-五甲基二氢苯并呋喃-5-磺酰基)、0.64g(3.09mmol)二环己基碳二亚胺和0.42g(3.09mmol)HOBt(1-羟基苯并三唑)和20mLDMF，室温下搅拌进行缩合反应。反应2小时后取少量树脂进行茚三酮显色反应，结果表明缩合反应已完全。抽滤，用DMF洗涤树脂6次，抽滤去掉溶剂。C端的(保护的)氨基酸已联接于树脂上。后面接着联接有C端起的第二个至最后一个(保护的)氨基酸，反应步骤重复上面操作，即从用20％哌啶/DMF溶液脱保护开始，到茚三酮显色试验后用DMF洗涤止。如果茚三酮显色试验表明缩合未完全，则可延长缩合时间直至完全。所有氨基酸的a-氨基都用Fmoc保护，氨基酸依次为：Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH。最后脱掉Fmoc保护，投入硬脂酸与N端氨基完成缩合反应。多肽树脂用甲醇洗第三次，真空干燥。Add 1.39g of Rinkamide-MBHA resin (0.74mmol/g) into a 50mL round-bottomed flask with a branch pipe on the side and a sand plate filter element in the branch pipe, add 20mL DMF to swell for 10 minutes, and remove the solvent by suction filtration. Then add 20mL of 20% piperidine/DMF solution, stir for 30 minutes, and filter with suction. The resin was washed 6 times with DMF, and the solvent was removed by suction filtration. Add 2.00g (3.09mmol) Fmoc-Arg(Pbf)-OH (Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), 0.64g ( 3.09mmol) of dicyclohexylcarbodiimide and 0.42g (3.09mmol) of HOBt (1-hydroxybenzotriazole) and 20mL of DMF were stirred at room temperature for condensation reaction. After 2 hours of reaction, a small amount of resin was taken for ninhydrin color reaction, and the result showed that the condensation reaction was complete. Suction filtration, wash the resin 6 times with DMF, and remove the solvent by suction filtration. The C-terminal (protected) amino acid has been attached to the resin. Followed by the second to the last (protected) amino acid linked from the C-terminus, the reaction steps are repeated above, that is, starting from the deprotection with 20% piperidine/DMF solution, and then using DMF after the ninhydrin color test Wash only. If the ninhydrin color test shows that the condensation is not complete, the condensation time can be extended until it is complete. The a-amino groups of all amino acids are protected by Fmoc, and the amino acids are: Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH , Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-His( Trt)-OH. Finally, the Fmoc protection is removed, and stearic acid and N-terminal amino groups are put in to complete the condensation reaction. The peptide resin was washed a third time with methanol and dried in vacuo.

将上述已接肽的树脂加入到50mL圆底烧瓶中，加入15mL试剂K(TFA/苯甲硫醚/EDT/苯酚/水＝87.5/5.5/2.5/2.5/2.5(v/v))，室温下搅拌3小时。过滤，收集滤液。用三氟乙酸洗三次树脂。合并滤液，将滤液旋蒸除去大部分的三氟乙酸，加入体积为8-10倍的乙醚沉淀接有硬脂酸的多肽，放入冰箱过夜。离心，除去乙醚，真空干燥，得到N端被硬脂酸修饰的RRRRRRRRHHH的粗品。Add the above-mentioned peptide-infused resin into a 50mL round bottom flask, add 15mL of reagent K (TFA/thioanisole/EDT/phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), room temperature Stirring was continued for 3 hours. Filter and collect the filtrate. The resin was washed three times with trifluoroacetic acid. The filtrates were combined, and the filtrate was rotary evaporated to remove most of the trifluoroacetic acid, and 8-10 times the volume of diethyl ether was added to precipitate the polypeptide connected with stearic acid, and put in the refrigerator overnight. Centrifuge, remove ether, and dry in vacuo to obtain a crude product of RRRRRRRRHHH whose N-terminus is modified by stearic acid.

将100mg上述的产物溶于2mL纯水中，用制备用反相HPLC纯化，收集保留主峰，收集液经旋蒸浓缩，然后冷冻干燥得N端被硬脂酸修饰的RRRRRRRRHHH的纯品。质谱表明，其分子量为2143，与计算值相符。最终获得N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₈H₃。Dissolve 100 mg of the above-mentioned product in 2 mL of pure water, purify by preparative reverse-phase HPLC, collect and retain the main peak, concentrate the collected solution by rotary evaporation, and then freeze-dry to obtain the pure product of RRRRRRRHHH whose N-terminus is modified by stearic acid. Mass spectrometry showed that its molecular weight was 2143, which was consistent with the calculated value. Finally, the tumor cell-selective membrane-penetrating peptide R ₈ H ₃ whose N-terminus is modified by stearic acid is obtained.

依处方量称取注射用大豆磷脂和胆固醇于茄型瓶中，加入一定量氯仿使其充分溶解，并加入玻璃珠若干。减压旋蒸1小时，挥除有机溶剂形成药脂薄膜。结束后继续抽真空过夜，以除去痕量有机溶剂。加入柠檬酸溶液(pH4.0)常温手摇水化药脂薄膜，至茄型瓶壁洗净为止，样品呈均匀的乳浊液。冰水浴中探头超声以减小粒径。离心3000rpm以除去探头超声可能掉落的金属屑。取上清液分别过0.45μm，0.22μm聚碳酸酯滤膜各5次进行整粒即可制得空白脂质体。然后在脂质体分散体系中加入盐酸多柔比星，并用1mol/L的Na₂HPO₄将pH值调节至7.0。最后调整体积，使多柔比星浓度为1mg/mL。将此分散体系于55℃孵育20min即得盐酸多柔比星脂质体。Weigh soybean lecithin and cholesterol for injection into an eggplant-shaped bottle according to the prescription, add a certain amount of chloroform to fully dissolve it, and add some glass beads. Rotary evaporation under reduced pressure for 1 hour, the organic solvent was evaporated to form a thin film of drug lipid. Vacuum was continued overnight after completion to remove traces of organic solvent. Add citric acid solution (pH 4.0) to hydrate the lipid film by hand at room temperature until the wall of the eggplant-shaped bottle is washed, and the sample becomes a uniform emulsion. Probe sonication in an ice-water bath to reduce particle size. Centrifuge at 3000 rpm to remove metal shavings that may fall off the probe. Take the supernatant and pass through the 0.45 μm and 0.22 μm polycarbonate filter membranes respectively 5 times for sizing to prepare blank liposomes. Then add doxorubicin hydrochloride to the liposome dispersion system, and adjust the pH value to 7.0 with 1 mol/L Na ₂ HPO ₄ . Finally, adjust the volume so that the concentration of doxorubicin is 1 mg/mL. The dispersion system was incubated at 55° C. for 20 minutes to obtain doxorubicin hydrochloride liposomes.

精密称取8mg N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₈H₃，并将其充分溶解于2mL的双蒸水中。于常温下，将此溶液缓缓滴加到以上制得的盐酸多柔比星脂质体分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min，然后将此分散体系在4℃孵育8小时即可制得肿瘤细胞选择性穿膜肽R₈H₃修饰的盐酸多柔比星脂质体。对修饰前后脂质体的包封率和稳定性的考察结果表明，肿瘤细胞选择性穿膜肽R₈H₃对盐酸多柔比星脂质体的修饰没有对普通盐酸多柔比星脂质体的理化性质产生显著影响，包封率大于90％，常温放置24小时渗漏率小于4％。Precisely weighed 8 mg of tumor cell-selective membrane-penetrating peptide R ₈ H ₃ whose N-terminus was modified by stearic acid, and fully dissolved it in 2 mL of double-distilled water. At normal temperature, slowly add this solution dropwise to the doxorubicin hydrochloride liposome dispersion system prepared above, and keep magnetic stirring continuously during the dropping process. Continue stirring for 30 min after the dropwise addition, and then incubate the dispersion system at 4° C. for 8 hours to prepare doxorubicin hydrochloride liposomes modified with tumor cell-selective membrane-penetrating peptide R ₈ H ₃ . The results of investigation on the encapsulation efficiency and stability of liposomes before and after modification show that the modification of doxorubicin hydrochloride liposomes by tumor cell selective membrane-penetrating peptide R ₈ H ₃ has no effect on common doxorubicin hydrochloride lipids. The physical and chemical properties of the body have a significant impact, the encapsulation rate is greater than 90%, and the leakage rate is less than 4% after 24 hours at room temperature.

精密称取相对分子质量为3×10⁵的透明质酸钠5mg，溶解于4mL的双蒸水中，调节pH值至6.0。于常温下将此溶液缓缓滴加到制得的肿瘤细胞选择性穿膜肽R₈H₃修饰的盐酸多柔比星脂质体的分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min即得以透明质酸钠、肿瘤细胞选择性穿膜肽R₈H₃与盐酸多柔比星脂质体所构建的复合脂质体。该复合脂质体平均粒径为147.8nm，zeta电位为-20.3mV，多分散指数PDI为0.236，盐酸多柔比星包封率为92.7％。以1％的蔗糖为冻干保护剂，将上述复合脂质体制备成冻干粉保存，使用时以5％的葡萄糖溶液复溶供注射使用。Accurately weigh 5 mg of sodium hyaluronate with a relative molecular mass of 3×10 ⁵ , dissolve it in 4 mL of double-distilled water, and adjust the pH to 6.0. Slowly add this solution dropwise to the prepared dispersion system of doxorubicin hydrochloride liposomes modified with selective membrane-penetrating peptide R ₈ H ₃ for tumor cells at room temperature, and keep magnetic stirring continuously during the dropping process. After the dropwise addition, the stirring was continued for 30 minutes to obtain a complex liposome constructed by sodium hyaluronate, tumor cell selective membrane-penetrating peptide R ₈ H ₃ and doxorubicin hydrochloride liposome. The average particle size of the composite liposome is 147.8nm, the zeta potential is -20.3mV, the polydispersity index PDI is 0.236, and the doxorubicin hydrochloride encapsulation efficiency is 92.7%. Using 1% sucrose as a freeze-drying protective agent, the above complex liposomes are prepared as freeze-dried powder for storage, and reconstituted with 5% glucose solution for injection.

实施例2Example 2

以本发明所述及的透明质酸钠、肿瘤细胞选择性穿膜肽R₈H₃与硫酸长春新碱脂质体来说明这一复合脂质体的制备方法。The preparation method of the complex liposome is illustrated by using sodium hyaluronate, tumor cell selective membrane-penetrating peptide R ₈ H ₃ and vincristine sulfate liposome mentioned in the present invention.

在50mL侧面带支管、支管带有砂板滤芯的圆底烧瓶内加入1.39gRinkamide-MBHA树脂(0.74mmol/g)，加20mL DMF溶胀10分钟，抽滤去掉溶剂。然后加入20mL 20％哌啶/DMF溶液，搅拌30分钟，抽滤。用DMF洗涤树脂6次，抽滤去掉溶剂。向反应瓶中加入2.00g(3.09mmol)Fmoc-Arg(Pbf)-OH(Pbf为2，2，4，6，7-五甲基二氢苯并呋喃-5-磺酰基)、0.64g(3.09mmol)二环己基碳二亚胺和0.42g(3.09mmol)HOBt(1-羟基苯并三唑)和20mLDMF，室温下搅拌进行缩合反应。反应2小时后取少量树脂进行茚三酮显色反应，结果表明缩合反应已完全。抽滤，用DMF洗涤树脂6次，抽滤去掉溶剂。C端的(保护的)氨基酸已联接于树脂上。后面接着联接有C端起的第二个至最后一个(保护的)氨基酸，反应步骤重复上面操作，即从用20％哌啶/DMF溶液脱保护开始，到茚三酮显色试验后用DMF洗涤止。如果茚三酮显色试验表明缩合未完全，则可延长缩合时间直至完全。所有氨基酸的a-氨基都用Fmoc保护，氨基酸依次为：Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH。最后脱掉Fmoc保护，投入硬脂酸与N端氨基完成缩合反应。多肽树脂用甲醇洗第三次，真空干燥。Add 1.39g of Rinkamide-MBHA resin (0.74mmol/g) into a 50mL round-bottomed flask with a branch pipe on the side and a sand plate filter element in the branch pipe, add 20mL DMF to swell for 10 minutes, and remove the solvent by suction filtration. Then add 20mL of 20% piperidine/DMF solution, stir for 30 minutes, and filter with suction. The resin was washed 6 times with DMF, and the solvent was removed by suction filtration. Add 2.00g (3.09mmol) Fmoc-Arg(Pbf)-OH (Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), 0.64g ( 3.09mmol) of dicyclohexylcarbodiimide and 0.42g (3.09mmol) of HOBt (1-hydroxybenzotriazole) and 20mL of DMF were stirred at room temperature for condensation reaction. After 2 hours of reaction, a small amount of resin was taken for ninhydrin color reaction, and the result showed that the condensation reaction was complete. Suction filtration, wash the resin 6 times with DMF, and remove the solvent by suction filtration. The C-terminal (protected) amino acid has been attached to the resin. Followed by the second to the last (protected) amino acid linked from the C-terminus, the reaction steps are repeated above, that is, starting from the deprotection with 20% piperidine/DMF solution, and then using DMF after the ninhydrin color test Wash only. If the ninhydrin color test shows that the condensation is not complete, the condensation time can be extended until it is complete. The a-amino groups of all amino acids are protected by Fmoc, and the amino acids are: Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH , Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-His( Trt)-OH. Finally, the Fmoc protection is removed, and stearic acid and N-terminal amino groups are put in to complete the condensation reaction. The peptide resin was washed a third time with methanol and dried in vacuo.

将上述已接肽的树脂加入到50mL圆底烧瓶中，加入15mL试剂K(TFA/苯甲硫醚/EDT/苯酚/水＝87.5/5.5/2.5/2.5/2.5(v/v))，室温下搅拌3小时。过滤，收集滤液。用三氟乙酸洗三次树脂。合并滤液，将滤液旋蒸除去大部分的三氟乙酸，加入体积为8-10倍的乙醚沉淀接有硬脂酸的多肽，放入冰箱过夜。离心，除去乙醚，真空干燥，得到N端被硬脂酸修饰的RRRRRRRRHHH的粗品。Add the above-mentioned peptide-infused resin into a 50mL round bottom flask, add 15mL of reagent K (TFA/thioanisole/EDT/phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), room temperature Stirring was continued for 3 hours. Filter and collect the filtrate. The resin was washed three times with trifluoroacetic acid. The filtrates were combined, and the filtrate was rotary evaporated to remove most of the trifluoroacetic acid, and 8-10 times the volume of diethyl ether was added to precipitate the polypeptide connected with stearic acid, and put in the refrigerator overnight. Centrifuge, remove ether, and dry in vacuo to obtain a crude product of RRRRRRRRHHH whose N-terminus is modified by stearic acid.

将100mg上述的产物溶于2mL纯水中，用制备用反相HPLC纯化，收集保留主峰，收集液经旋蒸浓缩，然后冷冻干燥得N端被硬脂酸修饰的RRRRRRRRHHH的纯品。质谱表明，其分子量为2143，与计算值相符。最终获得N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₈H₃。Dissolve 100 mg of the above-mentioned product in 2 mL of pure water, purify by preparative reverse-phase HPLC, collect and retain the main peak, concentrate the collected solution by rotary evaporation, and then freeze-dry to obtain the pure product of RRRRRRRHHH whose N-terminus is modified by stearic acid. Mass spectrometry showed that its molecular weight was 2143, which was consistent with the calculated value. Finally, the tumor cell-selective membrane-penetrating peptide R ₈ H ₃ whose N-terminus is modified by stearic acid is obtained.

依处方量称取氢化大豆磷脂和胆固醇，溶解于适量的二氯甲烷∶甲醇(体积比1∶1)中，于50℃水浴挥除有机溶剂。减压旋蒸1小时，挥除有机溶剂形成药脂薄膜。结束后继续抽真空过夜，以除去痕量有机溶剂。加入枸橼酸溶液(pH4.3)50℃水浴旋转洗膜，至茄型瓶壁洗净为止，样品呈均匀的乳浊液。冰水浴中探头超声以减小粒径。离心3000rpm以除去探头超声可能掉落的金属屑。取上清液分别过0.45μm，0.22μm聚碳酸酯滤膜各3次进行整粒即可制得空白脂质体。然后在脂质体分散体系中加入硫酸长春新碱溶液和pH9.0的Na₂HPO₄溶液。加入注射用水使外水相调节pH至7.3，在55℃水浴保温15min即得硫酸长春新碱脂质体。Weigh hydrogenated soybean lecithin and cholesterol according to the prescription, dissolve in an appropriate amount of dichloromethane:methanol (volume ratio 1:1), and evaporate the organic solvent in a water bath at 50°C. Rotary evaporation under reduced pressure for 1 hour, the organic solvent was evaporated to form a thin film of drug lipid. Vacuum was continued overnight after completion to remove traces of organic solvent. Add citric acid solution (pH 4.3) to wash the membrane in a water bath at 50°C, until the wall of the eggplant-shaped bottle is washed, and the sample becomes a uniform emulsion. Probe sonication in an ice-water bath to reduce particle size. Centrifuge at 3000 rpm to remove metal shavings that may fall off the probe. Take the supernatant and pass through the 0.45 μm and 0.22 μm polycarbonate filter membranes respectively 3 times for sizing to prepare blank liposomes. Then add vincristine sulfate solution and Na ₂ HPO ₄ solution with pH 9.0 into the liposome dispersion system. Water for injection was added to adjust the pH of the external aqueous phase to 7.3, and the vincristine sulfate liposome was obtained by incubating in a water bath at 55° C. for 15 minutes.

精密称取8mg N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₈H₃，并将其充分溶解于4mL的双蒸水中。于常温下，将此溶液缓缓滴加到以上制得的硫酸长春新碱星脂质体分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min，然后将此分散体系在4℃孵育8小时即可制得肿瘤细胞选择性穿膜肽R₈H₃修饰的硫酸长春新碱脂质体。对修饰前后脂质体的包封率和稳定性的考察结果表明，肿瘤细胞选择性穿膜肽R₈H₃对硫酸长春新碱脂质体的修饰没有对普通硫酸长春新碱脂质体的理化性质产生显著影响，包封率大于80％，常温放置24小时渗漏率小于4％。Precisely weighed 8 mg of tumor cell-selective membrane-penetrating peptide R ₈ H ₃ whose N-terminus was modified by stearic acid, and fully dissolved it in 4 mL of double-distilled water. At normal temperature, this solution is slowly added dropwise to the above-prepared vincristine sulfate liposome dispersion system, and the dropping process is kept under constant magnetic stirring. Continue stirring for 30 min after the dropwise addition, and then incubate the dispersion system at 4° C. for 8 hours to prepare vincristine sulfate liposomes modified with selective membrane-penetrating peptide R ₈ H ₃ for tumor cells. The results of investigation on the encapsulation efficiency and stability of liposomes before and after modification showed that the modification of tumor cell-selective membrane-penetrating peptide R ₈ H ₃ to vincristine sulfate liposomes had no effect on ordinary vincristine sulfate liposomes. The physical and chemical properties have a significant impact, the encapsulation rate is greater than 80%, and the leakage rate is less than 4% after being placed at room temperature for 24 hours.

精密称取相对分子质量为3×10⁵的透明质酸钠8mg，溶解于4mL的双蒸水中，调节pH值至6.0。于常温下将此溶液缓缓滴加到制得的肿瘤细胞选择性穿膜肽R₈H₃修饰的硫酸长春新碱脂质体的分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min即得以透明质酸钠、肿瘤细胞选择性穿膜肽R₈H₃与硫酸长春新碱脂质体所构建的复合脂质体。该复合脂质体平均粒径为203.8nm，zeta电位为-23.7mV，多分散指数PDI为0.156，硫酸长春新碱包封率为86.1％。以一定配比的蔗糖和甘露醇为冻干保护剂，将上述复合脂质体制备成冻干粉保存，使用时以5％的葡萄糖溶液复溶供注射使用。Accurately weigh 8 mg of sodium hyaluronate with a relative molecular mass of 3×10 ⁵ , dissolve it in 4 mL of double-distilled water, and adjust the pH to 6.0. Slowly add this solution dropwise to the prepared vincristine sulfate liposome dispersion system modified with selective membrane-penetrating peptide R ₈ H ₃ for tumor cells at normal temperature, and keep magnetic stirring continuously during the dropping process. After the dropwise addition, the stirring was continued for 30 minutes to obtain a complex liposome constructed by sodium hyaluronate, tumor cell selective membrane-penetrating peptide R ₈ H ₃ and vincristine sulfate liposome. The average particle diameter of the composite liposome is 203.8nm, the zeta potential is -23.7mV, the polydispersity index PDI is 0.156, and the encapsulation rate of vincristine sulfate is 86.1%. Using a certain ratio of sucrose and mannitol as a freeze-drying protection agent, the above-mentioned composite liposome is prepared into a freeze-dried powder for storage, and is reconstituted with 5% glucose solution for injection when used.

实施例3Example 3

以本发明所述及的透明质酸钠、肿瘤细胞选择性穿膜肽R₁₀H₃与甲氨蝶呤脂质体来说明这一复合脂质体的制备方法。The preparation method of this composite liposome is illustrated by using sodium hyaluronate, tumor cell selective membrane-penetrating peptide R ₁₀ H ₃ and methotrexate liposome mentioned in the present invention.

在50mL侧面带支管、支管带有砂板滤芯的圆底烧瓶内加入1.65gRinkamide-MBHA树脂(0.74mmol/g)，加20mL DMF溶胀10分钟，抽滤去掉溶剂。然后加入20mL 20％哌啶/DMF溶液，搅拌30分钟，抽滤。用DMF洗涤树脂6次，抽滤去掉溶剂。向反应瓶中加入2.38g(3.67mmol)Fmoc-Arg(Pbf)-OH(Pbf为2，2，4，6，7-五甲基二氢苯并呋喃-5-磺酰基)、0.76g(3.67mmol)二环己基碳二亚胺和0.50g(3.67mmol)HOBt(1-羟基苯并三唑)和20mLDMF，室温下搅拌进行缩合反应。反应2小时后取少量树脂进行茚三酮显色反应，结果表明缩合反应已完全。抽滤，用DMF洗涤树脂6次，抽滤去掉溶剂。C端的(保护的)氨基酸已联接于树脂上。后面接着联接有C端起的第二个至最后一个(保护的)氨基酸，反应步骤重复上面操作，即从用20％哌啶/DMF溶液脱保护开始，到茚三酮显色试验后用DMF洗涤止。如果茚三酮显色试验表明缩合未完全，则可延长缩合时间直至完全。所有氨基酸的a-氨基都用Fmoc保护，氨基酸依次为：Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH。最后脱掉Fmoc保护，投入硬脂酸与N端氨基完成缩合反应。多肽树脂用甲醇洗第三次，真空干燥。Add 1.65g of Rinkamide-MBHA resin (0.74mmol/g) to a 50mL round-bottomed flask with a branch pipe on the side and a sand plate filter element in the branch pipe, add 20mL DMF to swell for 10 minutes, and remove the solvent by suction filtration. Then add 20mL of 20% piperidine/DMF solution, stir for 30 minutes, and filter with suction. The resin was washed 6 times with DMF, and the solvent was removed by suction filtration. Add 2.38g (3.67mmol) Fmoc-Arg(Pbf)-OH (Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), 0.76g ( 3.67mmol) dicyclohexylcarbodiimide and 0.50g (3.67mmol) HOBt (1-hydroxybenzotriazole) and 20mL DMF were stirred at room temperature for condensation reaction. After 2 hours of reaction, a small amount of resin was taken for ninhydrin color reaction, and the result showed that the condensation reaction was complete. Suction filtration, wash the resin 6 times with DMF, and remove the solvent by suction filtration. The C-terminal (protected) amino acid has been attached to the resin. Followed by the second to the last (protected) amino acid linked from the C-terminus, the reaction steps are repeated above, that is, starting from the deprotection with 20% piperidine/DMF solution, and then using DMF after the ninhydrin color test Wash only. If the ninhydrin color test shows that the condensation is not complete, the condensation time can be extended until it is complete. The a-amino groups of all amino acids are protected by Fmoc, and the amino acids are: Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH , Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His( Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH. Finally, the Fmoc protection is removed, and stearic acid and N-terminal amino groups are put in to complete the condensation reaction. The peptide resin was washed a third time with methanol and dried in vacuo.

将上述已接肽的树脂加入到50mL圆底烧瓶中，加入15mL试剂K(TFA/苯甲硫醚/EDT/苯酚/水＝87.5/5.5/2.5/2.5/2.5(v/v))，室温下搅拌3小时。过滤，收集滤液。用三氟乙酸洗三次树脂。合并滤液，将滤液旋蒸除去大部分的三氟乙酸，加入体积为8-10倍的乙醚沉淀接有硬脂酸的多肽，放入冰箱过夜。离心，除去乙醚，真空干燥，得到N端被硬脂酸修饰的RRRRRRRRRRHHH的粗品。Add the above-mentioned peptide-infused resin into a 50mL round bottom flask, add 15mL of reagent K (TFA/thioanisole/EDT/phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), room temperature Stirring was continued for 3 hours. Filter and collect the filtrate. The resin was washed three times with trifluoroacetic acid. The filtrates were combined, and the filtrate was rotary evaporated to remove most of the trifluoroacetic acid, and 8-10 times the volume of diethyl ether was added to precipitate the polypeptide connected with stearic acid, and put in the refrigerator overnight. Centrifuge, remove ether, and dry in vacuo to obtain a crude product of RRRRRRRRRRHHH whose N-terminus is modified by stearic acid.

将100mg上述产物溶于2mL纯水中，用制备用反相HPLC纯化，收集保留主峰，收集液经旋蒸浓缩，然后冷冻干燥得N端被硬脂酸修饰的RRRRRRRRRRHHH的纯品。纯品的质谱表明，其分子量为2491，与计算值相符。最终获得N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₁₀H₃。Dissolve 100 mg of the above product in 2 mL of pure water, purify by preparative reverse-phase HPLC, collect and retain the main peak, concentrate the collected solution by rotary evaporation, and then freeze-dry to obtain the pure product of RRRRRRRRRHHH whose N-terminus is modified by stearic acid. The mass spectrum of the pure product showed that its molecular weight was 2491, which was consistent with the calculated value. Finally, the tumor cell-selective membrane-penetrating peptide R ₁₀ H ₃ whose N-terminus is modified by stearic acid is obtained.

依处方量称取蛋黄卵磷脂和胆固醇于茄型瓶中，加入4mL乙醚溶解，于冰水浴中一边搅拌一边滴加甲氨蝶呤水溶液2mL。待形成均匀稳定的W/O型乳浊液后，于40℃减压旋蒸蒸去乙醚，得到棕色乳浊液，100W功率探头超声15min，最后0.45μm聚碳酸酯微孔滤膜5次即得甲氨蝶呤脂质体。将甲氨蝶呤脂质体分散体系装入透析袋中，以双蒸水为透析介质，室温下透析24小时除去未包封药物。Weigh egg yolk lecithin and cholesterol in an eggplant-shaped bottle according to the prescription, add 4 mL of ether to dissolve, and add 2 mL of methotrexate solution dropwise while stirring in an ice-water bath. After forming a uniform and stable W/O emulsion, diethyl ether was evaporated under reduced pressure at 40°C to obtain a brown emulsion, ultrasonicated with a 100W power probe for 15 minutes, and finally 0.45 μm polycarbonate microporous filter membrane was filtered 5 times. Get methotrexate liposomes. Put the methotrexate liposome dispersion into a dialysis bag, use double distilled water as the dialysis medium, and dialyze at room temperature for 24 hours to remove the unencapsulated drug.

精密称取10mg N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₁₀H₃，并将其充分溶解于4mL的双蒸水中。于常温下，将此溶液缓缓滴加到以上制得的甲氨蝶呤脂质体分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min，然后将此分散体系在4℃孵育8小时即可制得肿瘤细胞选择性穿膜肽R₁₀H₃修饰的甲氨蝶呤脂质体。对修饰前后脂质体的包封率和稳定性的考察结果表明，肿瘤细胞选择性穿膜肽R₁₀H₃对甲氨蝶呤脂质体的修饰没有对普通甲氨蝶呤脂质体的理化性质产生显著影响，包封率大于70％，常温放置24小时渗漏率小于5％。Precisely weighed 10 mg of the tumor cell-selective membrane-penetrating peptide R ₁₀ H ₃ whose N-terminus was modified by stearic acid, and fully dissolved it in 4 mL of double-distilled water. At normal temperature, slowly add this solution dropwise to the methotrexate liposome dispersion system prepared above, and keep magnetic stirring continuously during the dropping process. Stirring was continued for 30 minutes after the dropwise addition, and then the dispersion system was incubated at 4° C. for 8 hours to prepare methotrexate liposomes modified with tumor cell-selective membrane-penetrating peptide R ₁₀ H ₃ . The results of investigation on the encapsulation efficiency and stability of liposomes before and after modification showed that the modification of methotrexate liposomes by tumor cell-selective membrane-penetrating peptide R ₁₀ H ₃ had no effect on ordinary methotrexate liposomes. The physical and chemical properties have a significant impact, the encapsulation rate is greater than 70%, and the leakage rate is less than 5% after being placed at room temperature for 24 hours.

精密称取相对分子质量为1×10⁵的透明质酸钠8mg，溶解于4mL的双蒸水中，调节pH值至6.0。于常温下将此溶液缓缓滴加到制得的肿瘤细胞选择性穿膜肽R₁₀H₃修饰的甲氨蝶呤脂质体的分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min即得以透明质酸钠、肿瘤细胞选择性穿膜肽R₁₀H₃与甲氨蝶呤脂质体所构建的复合脂质体。该复合脂质体平均粒径为276.4nm，zeta电位为-24.3mV，多分散指数PDI为0.186，甲氨蝶呤包封率为72.3％。以1％的蔗糖为冻干保护剂，将上述复合脂质体制备成冻干粉保存，使用时以5％的葡萄糖溶液复溶供注射使用。Accurately weigh 8 mg of sodium hyaluronate with a relative molecular mass of 1×10 ⁵ , dissolve it in 4 mL of double-distilled water, and adjust the pH to 6.0. Slowly add this solution dropwise to the prepared methotrexate liposome dispersion system modified with selective membrane-penetrating peptide R ₁₀ H ₃ for tumor cells at normal temperature, and keep magnetic stirring continuously during the dropping process. After the dropwise addition, the stirring was continued for 30 minutes to obtain a complex liposome constructed by sodium hyaluronate, tumor cell selective membrane-penetrating peptide R ₁₀ H ₃ and methotrexate liposome. The average particle size of the composite liposome is 276.4nm, the zeta potential is -24.3mV, the polydispersity index PDI is 0.186, and the methotrexate encapsulation efficiency is 72.3%. Using 1% sucrose as a freeze-drying protective agent, the above complex liposomes are prepared as freeze-dried powder for storage, and reconstituted with 5% glucose solution for injection when used.

实施例4Example 4

以本发明所述及的透明质酸钠、肿瘤细胞选择性穿膜肽R₉H₃与新藤黄酸脂质体来说明这一复合脂质体的制备方法。The preparation method of this composite liposome is illustrated by using sodium hyaluronate, tumor cell selective membrane-penetrating peptide R ₉ H ₃ and neogambogic acid liposome mentioned in the present invention.

在50mL侧面带支管、支管带有砂板滤芯的圆底烧瓶内加入1.52gRinkamide-MBHA树脂(0.74mmol/g)，加20mLDMF溶胀10分钟，抽滤去掉溶剂。然后加入20mL 20％哌啶/DMF溶液，搅拌30分钟，抽滤。用DMF洗涤树脂6次，抽滤去掉溶剂。向反应瓶中加入2.19g(3.38mmol)Fmoc-Arg(Pbf)-OH(Pbf为2，2，4，6，7-五甲基二氢苯并呋喃-5-磺酰基)、0.70g(3.38mmol)二环己基碳二亚胺和0.46g(3.38mmol)HOBt(1-羟基苯并三唑)和20mLDMF，室温下搅拌进行缩合反应。反应2小时后取少量树脂进行茚三酮显色反应，结果表明缩合反应已完全。抽滤，用DMF洗涤树脂6次，抽滤去掉溶剂。C端的(保护的)氨基酸已联接于树脂上。后面接着联接有C端起的第二个至最后一个(保护的)氨基酸，反应步骤重复上面操作，即从用20％哌啶/DMF溶液脱保护开始，到茚三酮显色试验后用DMF洗涤止。如果茚三酮显色试验表明缩合未完全，则可延长缩合时间直至完全。所有氨基酸的a-氨基都用Fmoc保护，氨基酸依次为：Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH。最后脱掉Fmoc保护，投入硬脂酸与N端氨基完成缩合反应。多肽树脂用甲醇洗第三次，真空干燥。Add 1.52g of Rinkamide-MBHA resin (0.74mmol/g) into a 50mL round-bottomed flask with a branch pipe on the side and a sand plate filter element in the branch pipe, add 20mL DMF to swell for 10 minutes, and remove the solvent by suction filtration. Then add 20mL of 20% piperidine/DMF solution, stir for 30 minutes, and filter with suction. The resin was washed 6 times with DMF, and the solvent was removed by suction filtration. Add 2.19g (3.38mmol) Fmoc-Arg(Pbf)-OH (Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), 0.70g ( 3.38mmol) of dicyclohexylcarbodiimide and 0.46g (3.38mmol) of HOBt (1-hydroxybenzotriazole) and 20mL of DMF were stirred at room temperature for condensation reaction. After 2 hours of reaction, a small amount of resin was taken for ninhydrin color reaction, and the result showed that the condensation reaction was complete. Suction filtration, wash the resin 6 times with DMF, and remove the solvent by suction filtration. The C-terminal (protected) amino acid has been attached to the resin. Followed by the second to the last (protected) amino acid linked from the C-terminus, the reaction steps are repeated above, that is, starting from the deprotection with 20% piperidine/DMF solution, and then using DMF after the ninhydrin color test Wash only. If the ninhydrin color test shows that the condensation is not complete, the condensation time can be extended until it is complete. The a-amino groups of all amino acids are protected by Fmoc, and the amino acids are: Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH , Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-His( Trt)-OH, Fmoc-His(Trt)-OH. Finally, the Fmoc protection is removed, and stearic acid and N-terminal amino groups are put in to complete the condensation reaction. The peptide resin was washed a third time with methanol and dried in vacuo.

将上述已接肽的树脂加入到50mL圆底烧瓶中，加入15mL试剂K(TFA/苯甲硫醚/EDT/苯酚/水＝87.5/5.5/2.5/2.5/2.5(v/v))，室温下搅拌3小时。过滤，收集滤液。用三氟乙酸洗三次树脂。合并滤液，将滤液旋蒸除去大部分的三氟乙酸，加入体积为8-10倍的乙醚沉淀接有硬脂酸的多肽，放入冰箱过夜。离心，除去乙醚，真空干燥，得到N端被硬脂酸修饰的RRRRRRRRRHHH的粗品。Add the above-mentioned peptide-infused resin into a 50mL round bottom flask, add 15mL of reagent K (TFA/thioanisole/EDT/phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), room temperature Stirring was continued for 3 hours. Filter and collect the filtrate. The resin was washed three times with trifluoroacetic acid. The filtrates were combined, and the filtrate was rotary evaporated to remove most of the trifluoroacetic acid, and 8-10 times the volume of diethyl ether was added to precipitate the polypeptide connected with stearic acid, and put in the refrigerator overnight. Centrifuge, remove ether, and dry in vacuo to obtain a crude product of RRRRRRRRHHH whose N-terminus is modified by stearic acid.

将100mg上述产物溶于2mL纯水中，用制备用反相HPLC纯化，收集保留主峰，收集液经旋蒸浓缩，然后冷冻干燥得N端被硬脂酸修饰的RRRRRRRRRHHH的纯品。纯品的质谱表明，其分子量为2317，与计算值相符。最终获得N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₉H₃。Dissolve 100 mg of the above product in 2 mL of pure water, purify by preparative reverse-phase HPLC, collect and retain the main peak, concentrate the collected solution by rotary evaporation, and then freeze-dry to obtain the pure product of RRRRRRRRRHHH whose N-terminus is modified by stearic acid. The mass spectrum of the pure product showed that its molecular weight was 2317, which was consistent with the calculated value. Finally, the tumor cell-selective membrane-penetrating peptide R ₉ H ₃ whose N-terminus is modified by stearic acid is obtained.

依处方量称取大豆磷脂、胆固醇和新藤黄酸，共溶于适量的氯仿中。将此溶液置于100mL茄型瓶中，40℃恒温水浴减压旋转蒸发，抽除氯仿，1小时后即在瓶壁形成均匀的药脂薄膜。结束后继续抽真空过夜，以除去痕量有机溶剂。加入pH7.4的PBS缓冲溶液，室温下常压旋转洗膜至薄膜完全脱落。采用探头超声仪超声15min以减小粒径，然后3000rpm离心以除去探头超声可能掉落的金属屑。取上清液分别过0.45μm，0.22μm聚碳酸酯滤膜各一次即可制得新藤黄酸脂质体。Weigh soybean lecithin, cholesterol and neogambogic acid according to the prescribed amount, and dissolve them in an appropriate amount of chloroform. Put this solution in a 100mL eggplant-shaped bottle, evaporate under reduced pressure in a constant temperature water bath at 40°C, and remove the chloroform. After 1 hour, a uniform lipid film is formed on the wall of the bottle. Vacuum was continued overnight after completion to remove traces of organic solvent. Add PBS buffer solution with pH 7.4, and spin wash the membrane under normal pressure at room temperature until the membrane falls off completely. Use a probe sonicator to sonicate for 15 minutes to reduce the particle size, and then centrifuge at 3000 rpm to remove metal shavings that may fall from the probe sonication. Take the supernatant and pass through 0.45 μm and 0.22 μm polycarbonate filter membranes respectively once to prepare new gambogic acid liposomes.

精密称取10mg N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₉H₃，并将其充分溶解于4mL的双蒸水中。于常温下，将此溶液缓缓滴加到以上制得的新藤黄酸脂质体分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min，然后将此分散体系在4℃孵育8小时即可制得肿瘤细胞选择性穿膜肽R₉H₃修饰的新藤黄酸脂质体。对修饰前后脂质体的包封率和稳定性的考察结果表明，肿瘤细胞选择性穿膜肽R₉H₃对新藤黄酸脂质体的修饰没有对普通新藤黄酸脂质体的理化性质产生显著影响，包封率大于80％，常温放置24小时渗漏率小于3％。Precisely weighed 10 mg of the tumor cell-selective membrane-penetrating peptide R ₉ H ₃ whose N-terminus was modified by stearic acid, and fully dissolved it in 4 mL of double-distilled water. At normal temperature, slowly add this solution dropwise to the new gambogic acid liposome dispersion system prepared above, and keep magnetic stirring continuously during the dropping process. Stirring was continued for 30 minutes after the dropwise addition, and then the dispersion system was incubated at 4° C. for 8 hours to prepare neogambogic acid liposomes modified with tumor cell-selective membrane-penetrating peptide R ₉ H ₃ . The results of investigation on the encapsulation efficiency and stability of liposomes before and after modification showed that the modification of neogambogic acid liposomes by tumor cell-selective membrane-penetrating peptide R ₉ H ₃ had no physical and chemical properties to ordinary neogambogic acid liposomes. It has a significant impact, the encapsulation rate is greater than 80%, and the leakage rate is less than 3% after being placed at room temperature for 24 hours.

精密称取相对分子质量为5×10⁴的透明质酸钠8mg，溶解于4mL的双蒸水中，调节pH值至6.0。于常温下将此溶液缓缓滴加到制得的肿瘤细胞选择性穿膜肽R₉H₃修饰的新藤黄酸脂质体的分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min即得以透明质酸钠、肿瘤细胞选择性穿膜肽R₉H₃与新藤黄酸脂质体所构建的复合脂质体。该复合脂质体平均粒径为147.6nm，zeta电位为-19.3mV，多分散指数PDI为0.206，新藤黄酸包封率为82.3％。以1％的甘露醇为冻干保护剂，将上述复合脂质体制备成冻干粉保存，使用时以0.9％氯化钠溶液复溶供注射使用。Accurately weigh 8 mg of sodium hyaluronate with a relative molecular mass of 5×10 ⁴ , dissolve it in 4 mL of double-distilled water, and adjust the pH to 6.0. Slowly add this solution dropwise to the prepared tumor cell selective membrane-penetrating peptide R ₉ H ₃ modified neogambogic acid liposome dispersion system at normal temperature, and keep magnetic stirring continuously during the dropping process. Stirring was continued for 30 minutes after the dropwise addition to obtain a composite liposome constructed from sodium hyaluronate, tumor cell selective membrane-penetrating peptide R ₉ H ₃ and neogambogic acid liposome. The average particle diameter of the composite liposome is 147.6nm, the zeta potential is -19.3mV, the polydispersity index PDI is 0.206, and the neogambogic acid encapsulation rate is 82.3%. Using 1% mannitol as a freeze-drying protective agent, the above-mentioned composite liposome is prepared as freeze-dried powder for storage, and reconstituted with 0.9% sodium chloride solution for injection when used.

实施例5Example 5

以肿瘤细胞选择性穿膜肽(R₅H₃、R₆H₃、R₇H₃、R₈H₃、R₉H₃、R₁₀H₃、R₁₁H₃、R₁₂H₃，R在N端或C端)与硫酸长春新碱脂质体所构建的肿瘤细胞选择性穿膜肽修饰的硫酸长春新碱脂质体的体外抑瘤实验为例来说明这一制剂的抗肿瘤功效。Selective membrane-penetrating peptides for tumor cells (R ₅ H ₃ , R ₆ H ₃ , R ₇ H ₃ , R ₈ H ₃ , R ₉ H ₃ , R ₁₀ H ₃ , R ₁₁ H ₃ , R ₁₂ H ₃ , R In vitro tumor inhibition experiments of vincristine sulfate liposomes modified by tumor cell selective membrane-penetrating peptides constructed with vincristine sulfate liposomes at the N-terminal or C-terminal) as an example to illustrate the anti-tumor efficacy of this preparation .

依处方量称取氢化大豆磷脂和胆固醇，溶解于适量的二氯甲烷∶甲醇(体积比1∶1)中，于50℃水浴挥除有机溶剂。减压旋蒸1小时，挥除有机溶剂形成药脂薄膜。结束后继续抽真空过夜，以除去痕量有机溶剂。加入枸橼酸溶液(pH4.3)50℃水浴旋转洗膜，至茄型瓶壁洗净为止，样品呈均匀的乳浊液。冰水浴中探头超声以减小粒径。离心3000rpm以除去探头超声可能掉落的金属屑。取上清液分别过0.45μm，0.22μm聚碳酸酯滤膜各3次进行整粒即可制得空白脂质体。然后在脂质体分散体系中加入硫酸长春新碱溶液和pH9.0的Na₂HPO₄溶液。加入注射用水使外水相调节pH至7.3，在55℃水浴保温15min即得硫酸长春新碱脂质体。Weigh hydrogenated soybean lecithin and cholesterol according to the prescription, dissolve in an appropriate amount of dichloromethane:methanol (volume ratio 1:1), and evaporate the organic solvent in a water bath at 50°C. Rotary evaporation under reduced pressure for 1 hour, the organic solvent was evaporated to form a thin film of drug lipid. Vacuum was continued overnight after completion to remove traces of organic solvent. Add citric acid solution (pH 4.3) to wash the membrane in a water bath at 50°C, until the wall of the eggplant-shaped bottle is washed, and the sample becomes a uniform emulsion. Probe sonication in an ice-water bath to reduce particle size. Centrifuge at 3000 rpm to remove metal shavings that may fall off the probe. Take the supernatant and pass through the 0.45 μm and 0.22 μm polycarbonate filter membranes respectively 3 times for sizing to prepare blank liposomes. Then add vincristine sulfate solution and Na ₂ HPO ₄ solution with pH 9.0 into the liposome dispersion system. Water for injection was added to adjust the pH of the external aqueous phase to 7.3, and the vincristine sulfate liposome was obtained by incubating in a water bath at 55° C. for 15 minutes.

精密称取8mg N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₅H₃，并将其充分溶解于4mL的双蒸水中。于常温下，将此溶液缓缓滴加到以上制得的硫酸长春新碱星脂质体分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min，然后将此分散体系在4℃孵育8小时即可制得肿瘤细胞选择性穿膜肽R₈H₃修饰的硫酸长春新碱脂质体。对修饰前后脂质体的包封率和稳定性的考察结果表明，肿瘤细胞选择性穿膜肽R₈H₃对硫酸长春新碱脂质体的修饰没有对普通硫酸长春新碱脂质体的理化性质产生显著影响，包封率大于80％，常温放置24小时渗漏率小于4％。Precisely weighed 8 mg of tumor cell-selective membrane-penetrating peptide R ₅ H ₃ whose N-terminus was modified by stearic acid, and fully dissolved it in 4 mL of double-distilled water. At normal temperature, this solution is slowly added dropwise to the above-prepared vincristine sulfate liposome dispersion system, and the dropping process is kept under constant magnetic stirring. Stirring was continued for 30 minutes after the dropwise addition, and then the dispersion system was incubated at 4° C. for 8 hours to prepare vincristine sulfate liposomes modified with selective membrane-penetrating peptide R ₈ H ₃ for tumor cells. The results of investigation on the encapsulation efficiency and stability of liposomes before and after modification showed that the modification of tumor cell selective membrane-penetrating peptide R ₈ H ₃ to vincristine sulfate liposomes had no effect on ordinary vincristine sulfate liposomes. The physical and chemical properties have a significant impact, the encapsulation rate is greater than 80%, and the leakage rate is less than 4% after being placed at room temperature for 24 hours.

其他肿瘤细胞选择性穿膜肽(R₆H₃、R₇H₃、R₈H₃、R₉H₃、R₁₀H₃、R₁₁H₃、R₁₂H₃，R在N端或C端)与硫酸长春新碱脂质体所构建的脂质体剂型可采用相同的方法制备。Other tumor cell-selective penetrating peptides (R ₆ H ₃ , R ₇ H ₃ , R ₈ H ₃ , R ₉ H ₃ , R ₁₀ H ₃ , R ₁₁ H ₃ , R ₁₂ H ₃ , R at N-terminal or C end) and the liposome formulation constructed by vincristine sulfate liposomes can be prepared in the same way.

另外，采用相同的方法制备硫酸长春新碱脂质体，并按上述方法用N端被硬脂酸修饰的穿膜肽R₈制备R₈修饰的硫酸长春新碱脂质体。In addition, the same method was used to prepare vincristine sulfate liposomes, and R ₈ modified vincristine sulfate liposomes were prepared with the penetrating peptide R ₈ modified by stearic acid at the N-terminal as described above.

选择处于对数生长期的人肝癌细胞株HepG2，弃去培养基并加胰酶消化1min，加入适量DMEM培养基终止消化，镜检计算细胞密度，加入一定量培养基制成5×10⁴个/mL的细胞悬液。加细胞悬液至96孔板，每孔200μL。37℃，5％CO₂培养箱培养24小时。配制各种肿瘤细胞选择性穿膜肽修饰的硫酸长春新碱脂质体、R₈修饰的硫酸长春新碱脂质体、未修饰的硫酸长春新碱脂质体和硫酸长春新碱注射液的不同浓度的药液。96孔板弃去培养基，在加入200μL用DMEM培养基稀释的药物溶液，使硫酸长春新碱在板上的终浓度均为：1、3、10、15、30、60、120、180、240、300μg/mL，每个浓度设3个复孔，以不加药组为对照。加药后移入37℃，5％CO₂培养箱培养48小时。然后，弃去孔内液体，加入DMEM培养基190μL/孔和MTT(5mg/mL)10μL/孔。37℃，5％CO₂培养箱中孵育4小时后，弃去各孔内原有液体，加入二甲基亚砜200μL/孔以溶解沉淀。30min后用酶标仪在570nm波长下测定各孔光密度值。计算药物抑制率：药物抑制率＝(1-实验组光密度值/对照组光密度值)×100％。结果如表一所示，与R₈修饰的硫酸长春新碱脂质体、未修饰的硫酸长春新碱脂质体和硫酸长春新碱注射液相比，各种肿瘤细胞选择性穿膜肽修饰的硫酸长春新碱脂质体的细胞毒作用明显增强(P＜0.05)。Select the human liver cancer cell line HepG2 in the logarithmic growth phase, discard the medium and digest with trypsin for 1 min, add an appropriate amount of DMEM medium to stop the digestion, calculate the cell density by microscopic examination, add a certain amount of medium to make 5× ¹⁰ cells /mL of cell suspension. Add cell suspension to 96-well plate, 200 μL per well. Incubate in a 37°C, 5% CO ₂ incubator for 24 hours. Preparation of vincristine sulfate liposomes modified with selective membrane-penetrating peptides for various tumor cells, R ₈ modified vincristine sulfate liposomes, unmodified vincristine sulfate liposomes and vincristine sulfate injection Drugs of different concentrations. Discard the medium in the 96-well plate, and add 200 μL of the drug solution diluted with DMEM medium, so that the final concentrations of vincristine sulfate on the plate are: 1, 3, 10, 15, 30, 60, 120, 180, 240, 300 μg/mL, three replicate wells were set for each concentration, and the no-drug group was used as the control. After dosing, move to 37°C, 5% CO ₂ incubator and cultivate for 48 hours. Then, the liquid in the well was discarded, and 190 μL/well of DMEM medium and 10 μL/well of MTT (5 mg/mL) were added. After incubating in a 5% CO ₂ incubator at 37°C for 4 hours, the original liquid in each well was discarded, and 200 μL/well of dimethyl sulfoxide was added to dissolve the precipitate. After 30 min, the optical density of each well was measured with a microplate reader at a wavelength of 570 nm. Calculation of drug inhibition rate: drug inhibition rate=(1-optical density value of experimental group/optical density value of control group)×100%. The results are shown in Table 1. Compared with R ₈ modified vincristine sulfate liposomes, unmodified vincristine sulfate liposomes and vincristine sulfate injection, various tumor cells were selectively modified with membrane-penetrating peptides. The cytotoxic effect of vincristine sulfate liposomes was significantly enhanced (P<0.05).

表一 实验组各制剂对人肝癌细胞株HepG2的半数抑制浓度(IC₅₀)Table 1 The half maximal inhibitory concentration (IC ₅₀ ) of each preparation of the experimental group on the human liver cancer cell line HepG2

制剂                                  IC₅₀(μg/mL)Formulation _IC50 (μg/mL)

硫酸长春新碱注射液                    46.3±2.7Vincristine Sulfate Injection 46.3±2.7

未修饰的硫酸长春新碱脂质体            36.1±3.0Unmodified vincristine sulfate liposomes 36.1±3.0

R₈修饰的硫酸长春新碱脂质体            30.8±1.9R ₈ modified vincristine sulfate liposomes 30.8 ± 1.9

R₅H₃修饰的硫酸长春新碱脂质体          25.3±2.7R ₅ H ₃ modified vincristine sulfate liposomes 25.3±2.7

R₆H₃修饰的硫酸长春新碱脂质体          23.3±2.3R ₆ H ₃ modified vincristine sulfate liposomes 23.3±2.3

R₇H₃修饰的硫酸长春新碱脂质体          21.2±2.1R ₇ H ₃ modified vincristine sulfate liposomes 21.2±2.1

R₈H₃修饰的硫酸长春新碱脂质体          19.8±2.4R ₈ H ₃ modified vincristine sulfate liposome 19.8±2.4

R₉H₃修饰的硫酸长春新碱脂质体          22.0±1.7R ₉ H ₃ modified vincristine sulfate liposomes 22.0±1.7

R₁₀H₃修饰的硫酸长春新碱脂质体         23.1±1.2R ₁₀ H ₃ modified vincristine sulfate liposomes 23.1±1.2

R₁₁H₃修饰的硫酸长春新碱脂质体         24.2±2.6R ₁₁ H ₃ modified vincristine sulfate liposomes 24.2±2.6

R₁₂H₃修饰的硫酸长春新碱脂质体         25.1±2.4R ₁₂ H ₃ modified vincristine sulfate liposomes 25.1±2.4

实施例6Example 6

以本发明所述及的透明质酸钠、肿瘤细胞选择性穿膜肽R₈H₃与紫杉醇脂质体所构建的复合脂质体在荷瘤小鼠体内的组织分布为例来说明其肿瘤靶向性。Taking the tissue distribution of the complex liposome constructed by sodium hyaluronate, tumor cell-selective membrane-penetrating peptide _R8H3 and paclitaxel liposome in the _body of tumor-bearing mice as an example to illustrate its tumor targeting.

在50mL侧面带支管、支管带有砂板滤芯的圆底烧瓶内加入1.39gRinkamide-MBHA树脂(0.74mmol/g)，加20mLDMF溶胀10分钟，抽滤去掉溶剂。然后加入20mL 20％哌啶/DMF溶液，搅拌30分钟，抽滤。用DMF洗涤树脂6次，抽滤去掉溶剂。向反应瓶中加入2.00g(3.09mmol)Fmoc-Arg(Pbf)-OH(Pbf为2，2，4，6，7-五甲基二氢苯并呋喃-5-磺酰基)、0.64g(3.09mmol)二环己基碳二亚胺和0.42g(3.09mmol)HOBt(1-羟基苯并三唑)和20mLDMF，室温下搅拌进行缩合反应。反应2小时后取少量树脂进行茚三酮显色反应，结果表明缩合反应已完全。抽滤，用DMF洗涤树脂6次，抽滤去掉溶剂。C端的(保护的)氨基酸已联接于树脂上。后面接着联接有C端起的第二个至最后一个(保护的)氨基酸，反应步骤重复上面操作，即从用20％哌啶/DMF溶液脱保护开始，到茚三酮显色试验后用DMF洗涤止。如果茚三酮显色试验表明缩合未完全，则可延长缩合时间直至完全。所有氨基酸的a-氨基都用Fmoc保护，氨基酸依次为：Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH。最后脱掉Fmoc保护，投入硬脂酸与N端氨基完成缩合反应。多肽树脂用甲醇洗第三次，真空干燥。Add 1.39g of Rinkamide-MBHA resin (0.74mmol/g) into a 50mL round-bottomed flask with a branch pipe on the side and a sand plate filter element in the branch pipe, add 20mL DMF to swell for 10 minutes, and remove the solvent by suction filtration. Then add 20mL of 20% piperidine/DMF solution, stir for 30 minutes, and filter with suction. The resin was washed 6 times with DMF, and the solvent was removed by suction filtration. Add 2.00g (3.09mmol) Fmoc-Arg(Pbf)-OH (Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), 0.64g ( 3.09mmol) of dicyclohexylcarbodiimide and 0.42g (3.09mmol) of HOBt (1-hydroxybenzotriazole) and 20mL of DMF were stirred at room temperature for condensation reaction. After 2 hours of reaction, a small amount of resin was taken for ninhydrin color reaction, and the result showed that the condensation reaction was complete. Suction filtration, wash the resin 6 times with DMF, and remove the solvent by suction filtration. The C-terminal (protected) amino acid has been attached to the resin. Followed by the second to the last (protected) amino acid linked from the C-terminus, the reaction steps are repeated above, that is, starting from the deprotection with 20% piperidine/DMF solution, and then using DMF after the ninhydrin color test Wash only. If the ninhydrin color test shows that the condensation is not complete, the condensation time can be extended until it is complete. The a-amino groups of all amino acids are protected by Fmoc, and the amino acids are: Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH , Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-His( Trt)-OH. Finally, the Fmoc protection is removed, and stearic acid and N-terminal amino groups are put in to complete the condensation reaction. The peptide resin was washed a third time with methanol and dried in vacuo.

将上述已接肽的树脂加入到50mL圆底烧瓶中，加入15mL试剂K(TFA/苯甲硫醚/EDT/苯酚/水＝87.5/5.5/2.5/2.5/2.5(v/v))，室温下搅拌3小时。过滤，收集滤液。用三氟乙酸洗三次树脂。合并滤液，将滤液旋蒸除去大部分的三氟乙酸，加入体积为8-10倍的乙醚沉淀接有硬脂酸的多肽，放入冰箱过夜。离心，除去乙醚，真空干燥，得到N端被硬脂酸修饰的修饰的RRRRRRRRHHH的粗品。Add the above-mentioned peptide-infused resin into a 50mL round bottom flask, add 15mL of reagent K (TFA/thioanisole/EDT/phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), room temperature Stirring was continued for 3 hours. Filter and collect the filtrate. The resin was washed three times with trifluoroacetic acid. The filtrates were combined, and the filtrate was rotary evaporated to remove most of the trifluoroacetic acid, and 8-10 times the volume of diethyl ether was added to precipitate the polypeptide connected with stearic acid, and put in the refrigerator overnight. Centrifuge, remove ether, and dry in vacuo to obtain a crude product of modified RRRRRRRRHHH whose N-terminus is modified with stearic acid.

将100mg上述产物溶于2mL纯水中，用制备用反相HPLC纯化，收集保留主峰，收集液经旋蒸浓缩，然后冷冻干燥得N端被硬脂酸修饰的RRRRRRRRHHH的纯品。纯品的质谱表明，其分子量为2143，与计算值相符。最终获得N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₈H₃。Dissolve 100 mg of the above product in 2 mL of pure water, purify by preparative reverse-phase HPLC, collect and retain the main peak, concentrate the collected solution by rotary evaporation, and then freeze-dry to obtain the pure product of RRRRRRRHHH whose N-terminus is modified by stearic acid. The mass spectrum of the pure product showed that its molecular weight was 2143, which was consistent with the calculated value. Finally, the tumor cell-selective membrane-penetrating peptide R ₈ H ₃ whose N-terminus is modified by stearic acid is obtained.

依处方量称取注射用大豆磷脂、胆固醇和紫杉醇于茄型瓶中，加入一定量的氯仿使其充分溶解，并加入玻璃珠若干。减压旋蒸1小时，挥除有机溶剂形成药脂薄膜。结束后继续抽真空过夜，以除去痕量有机溶剂。10mL纯水常温手摇水合，至茄型瓶壁洗净为止，样品呈均匀的乳浊液。冰水浴中探头超声以减小粒径。然后3000rpm离心以除去探头超声可能掉落的金属屑。取上清液分别过0.45μm，0.22μm聚碳酸酯滤膜各一次即可制得紫杉醇脂质体。Weigh soybean lecithin for injection, cholesterol and paclitaxel according to the prescribed amount into an eggplant-shaped bottle, add a certain amount of chloroform to fully dissolve it, and add some glass beads. Rotary evaporation under reduced pressure for 1 hour, the organic solvent was evaporated to form a thin film of drug lipid. Vacuum was continued overnight after completion to remove traces of organic solvent. Hydrate with 10mL pure water by hand shaking at room temperature until the eggplant-shaped bottle wall is washed, and the sample becomes a uniform emulsion. Probe sonication in an ice-water bath to reduce particle size. Then centrifuge at 3000rpm to remove metal shavings that may be dropped by probe ultrasound. Take the supernatant and pass through the 0.45 μm and 0.22 μm polycarbonate filter membranes respectively once to prepare paclitaxel liposomes.

精密称取8mg N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₈H₃，并将其充分溶解于2mL的双蒸水中。于常温下，将此溶液缓缓滴加到制得的紫杉醇脂质体分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min，然后将此分散体系在4℃孵育8小时即可制得肿瘤细胞选择性穿膜肽修饰的紫杉醇脂质体。对修饰前后脂质体的包封率和稳定性的考察结果表明，肿瘤细胞选择性穿膜肽R₈H₃对紫杉醇脂质体的修饰没有对普通紫杉醇脂质体的理化性质产生显著影响，包封率大于96％，常温放置24小时渗漏率小于3％。Precisely weighed 8 mg of tumor cell-selective membrane-penetrating peptide R ₈ H ₃ whose N-terminus was modified by stearic acid, and fully dissolved it in 2 mL of double-distilled water. At normal temperature, this solution was slowly added dropwise into the prepared paclitaxel liposome dispersion system, and the dropping process was kept under constant magnetic stirring. Stirring was continued for 30 minutes after the dropwise addition, and then the dispersion system was incubated at 4° C. for 8 hours to prepare tumor cell-selective membrane-penetrating peptide-modified paclitaxel liposomes. The results of investigation on the encapsulation efficiency and stability of liposomes before and after modification showed that the modification of paclitaxel liposomes by tumor cell-selective membrane-penetrating peptide R ₈ H ₃ did not significantly affect the physicochemical properties of ordinary paclitaxel liposomes. The encapsulation rate is greater than 96%, and the leakage rate is less than 3% after being placed at room temperature for 24 hours.

精密称取相对分子质量为10⁴的透明质酸钠10mg，溶解于4mL的双蒸水中，调节pH值至6.0。于常温下将此溶液缓缓滴加到制得的肿瘤细胞选择性穿膜肽R₈H₃修饰的紫杉醇脂质体的分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min即得以透明质酸钠、肿瘤细胞选择性穿膜肽R₈H₃与紫杉醇脂质体所构建的复合脂质体。该复合脂质体平均粒径为168.4nm，zeta电位为-18.7mV，多分散指数PDI为0.288，紫杉醇包封率为96.5％。以1％的蔗糖为冻干保护剂，将上述复合脂质体制备成冻干粉保存，使用时以5％的葡萄糖溶液复溶供注射使用。Accurately weigh 10 mg of sodium hyaluronate with a relative molecular mass of 10 ⁴ , dissolve it in 4 mL of double-distilled water, and adjust the pH to 6.0. Slowly add this solution dropwise to the prepared tumor cell selective membrane-penetrating peptide R ₈ H ₃ modified paclitaxel liposome dispersion system at normal temperature, and keep magnetic stirring continuously during the dropping process. After the dropwise addition, the stirring was continued for 30 minutes to obtain a complex liposome constructed by sodium hyaluronate, tumor cell selective membrane-penetrating peptide R ₈ H ₃ and paclitaxel liposome. The average particle diameter of the composite liposome is 168.4nm, the zeta potential is -18.7mV, the polydispersity index PDI is 0.288, and the paclitaxel encapsulation efficiency is 96.5%. Using 1% sucrose as a freeze-drying protective agent, the above complex liposomes are prepared as freeze-dried powder for storage, and reconstituted with 5% glucose solution for injection.

以市售的紫杉醇脂质体为对照，对上述方法制备的紫杉醇复合脂质体在荷瘤小鼠体内的组织分布进行考察。选取腋下接种有S180肉瘤的荷瘤小鼠90只(n＝3)，禁食12小时。按20mg/kg剂量分别尾静脉注射市售紫杉醇脂质体和紫杉醇复合脂质体，给药后分别于5，15，30min，1，2，4，8，12，24，48小时眼眶取血后，断颈处死，取荷瘤小鼠的心、肝、脾、肺、肾、肿瘤样品，生理盐水冲洗，滤纸吸干其水分。分别精密称重，加入生理盐水1.5mL制成匀浆液。精密量取0.8mL(肝取0.5mL)匀浆液，血浆取150μL，分别加入不同浓度的地西泮内标液50μL，混匀后，加入叔丁基甲醚4mL，涡旋5min，3000r/min离心10min，取有机相3mL，于40℃减压挥干，残渣用流动相200μL溶解，涡旋1min，10000r/min离心10min，取上清液20μL进样，按体内色谱条件进行分析。Using the commercially available paclitaxel liposome as a control, the tissue distribution of the paclitaxel complex liposome prepared by the above method in tumor-bearing mice was investigated. 90 tumor-bearing mice (n=3) inoculated with S180 sarcoma in the armpit were selected and fasted for 12 hours. Inject commercially available paclitaxel liposomes and paclitaxel complex liposomes into the tail vein at a dose of 20 mg/kg, and collect blood from the orbit at 5, 15, 30 minutes, 1, 2, 4, 8, 12, 24, and 48 hours after administration. Afterwards, they were killed by neck dislocation, and the heart, liver, spleen, lung, kidney, and tumor samples of tumor-bearing mice were taken, rinsed with normal saline, and blotted dry with filter paper. They were weighed precisely, and 1.5 mL of normal saline was added to make a homogenate. Precisely measure 0.8mL (0.5mL for liver) homogenate, 150μL for plasma, add 50μL of diazepam internal standard solution of different concentrations, mix well, add 4mL of tert-butyl methyl ether, vortex for 5min, and centrifuge at 3000r/min for 10min , take 3 mL of the organic phase, evaporate to dryness at 40°C under reduced pressure, dissolve the residue with 200 μL of mobile phase, vortex for 1 min, centrifuge at 10,000 r/min for 10 min, take 20 μL of the supernatant and inject it, and analyze it according to the in vivo chromatographic conditions.

给药系统的靶向性评价采用靶向效率(Te)作为评价参数。Te值表示药物制剂对靶器官和非靶器官的选择性。Te值越大，选择性越强。本实验考查各器官的AUC与血浆AUC的比值，即Te＝AUC_器官/AUC_血浆。其结果如表二所示。Targeting evaluation of the drug delivery system uses targeting efficiency (Te) as an evaluation parameter. The Te value indicates the selectivity of the drug formulation to target organs and non-target organs. The larger the Te value, the stronger the selectivity. In this experiment, the ratio of AUC of each organ to plasma AUC was investigated, namely Te=AUC _organ /AUC _plasma . The results are shown in Table II.

表二 靶向性评价Table 2 Targeted evaluation

由表二的结果可知，紫杉醇复合脂质体对肿瘤的靶向效率Te明显高于市售紫杉醇脂质体，而对心脏、肾脏等器官的靶向效率Te明显低于市售紫杉醇脂质体。由此可见，紫杉醇复合脂质体对肿瘤具有较高的靶向性，而且降低了对心脏、肾脏等器官的毒性。As can be seen from the results in Table 2, the targeting efficiency Te of paclitaxel composite liposomes to tumors is significantly higher than that of commercially available paclitaxel liposomes, while the targeting efficiency Te of the heart, kidney and other organs is significantly lower than that of commercially available paclitaxel liposomes. . It can be seen that the paclitaxel complex liposome has a high targeting ability to tumors, and reduces the toxicity to organs such as the heart and kidney.

实施例7Example 7

以本发明所述及的透明质酸钠、肿瘤细胞选择性穿膜肽R₆H₃与紫杉醇脂质体所构建的复合脂质体在荷瘤小鼠体内的组织分布为例来说明其肿瘤靶向性。Taking the tissue distribution of the complex liposome constructed by sodium hyaluronate, tumor cell selective membrane-penetrating peptide _R6H3 and paclitaxel liposome in the _body of tumor-bearing mice as an example to illustrate its tumor targeting.

在50mL侧面带支管、支管带有砂板滤芯的圆底烧瓶内加入1.13gRinkamide-MBHA树脂(0.74mmol/g)，加20mLDMF溶胀10分钟，抽滤去掉溶剂。然后加入20mL 20％哌啶/DMF溶液，搅拌30分钟，抽滤。用DMF洗涤树脂6次，抽滤去掉溶剂。向反应瓶中加入1.63g(2.51mmol)Fmoc-Arg(Pbf)-OH(Pbf为2，2，4，6，7-五甲基二氢苯并呋喃-5-磺酰基)、0.52g(2.51mmol)二环己基碳二亚胺和0.34g(2.51mmol)HOBt(1-羟基苯并三唑)和20mLDMF，室温下搅拌进行缩合反应。反应2小时后取少量树脂进行茚三酮显色反应，结果表明缩合反应已完全。抽滤，用DMF洗涤树脂6次，抽滤去掉溶剂。C端的(保护的)氨基酸已联接于树脂上。后面接着联接有C端起的第二个至最后一个(保护的)氨基酸，反应步骤重复上面操作，即从用20％哌啶/DMF溶液脱保护开始，到茚三酮显色试验后用DMF洗涤止。如果茚三酮显色试验表明缩合未完全，则可延长缩合时间直至完全。所有氨基酸的a-氨基都用Fmoc保护，氨基酸依次为：Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-His(Trt)-OH。最后脱掉Fmoc保护，投入硬脂酸与N端氨基完成缩合反应。多肽树脂用甲醇洗第三次，真空干燥。Add 1.13g of Rinkamide-MBHA resin (0.74mmol/g) into a 50mL round-bottomed flask with a branch pipe on the side and a sand plate filter element in the branch pipe, add 20mL DMF to swell for 10 minutes, and remove the solvent by suction filtration. Then add 20mL of 20% piperidine/DMF solution, stir for 30 minutes, and filter with suction. The resin was washed 6 times with DMF, and the solvent was removed by suction filtration. Add 1.63g (2.51mmol) Fmoc-Arg(Pbf)-OH (Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), 0.52g ( 2.51mmol) of dicyclohexylcarbodiimide and 0.34g (2.51mmol) of HOBt (1-hydroxybenzotriazole) and 20mL of DMF were stirred at room temperature for condensation reaction. After 2 hours of reaction, a small amount of resin was taken for ninhydrin color reaction, and the result showed that the condensation reaction was complete. Suction filtration, wash the resin 6 times with DMF, and remove the solvent by suction filtration. The C-terminal (protected) amino acid has been attached to the resin. Followed by the second to the last (protected) amino acid linked from the C-terminus, the reaction steps are repeated above, that is, starting from the deprotection with 20% piperidine/DMF solution, and then using DMF after the ninhydrin color test Wash only. If the ninhydrin color test shows that the condensation is not complete, the condensation time can be extended until it is complete. The a-amino groups of all amino acids are protected by Fmoc, and the amino acids are: Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH , Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH. Finally, the Fmoc protection is removed, and stearic acid and N-terminal amino groups are put in to complete the condensation reaction. The peptide resin was washed a third time with methanol and dried in vacuo.

将上述已接肽的树脂加入到50mL圆底烧瓶中，加入15mL试剂K(TFA/苯甲硫醚/EDT/苯酚/水＝87.5/5.5/2.5/2.5/2.5(v/v))，室温下搅拌3小时。过滤，收集滤液。用三氟乙酸洗三次树脂。合并滤液，将滤液旋蒸除去大部分的三氟乙酸，加入体积为8-10倍的乙醚沉淀接有硬脂酸的多肽，放入冰箱过夜。离心，除去乙醚，真空干燥，得到N端被硬脂酸修饰的RRRRRRHHH的粗品。Add the above-mentioned peptide-infused resin into a 50mL round bottom flask, add 15mL of reagent K (TFA/thioanisole/EDT/phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), room temperature Stirring was continued for 3 hours. Filter and collect the filtrate. The resin was washed three times with trifluoroacetic acid. The filtrates were combined, and the filtrate was rotary evaporated to remove most of the trifluoroacetic acid, and 8-10 times the volume of diethyl ether was added to precipitate the polypeptide connected with stearic acid, and put in the refrigerator overnight. Centrifuge to remove ether, and dry in vacuo to obtain the crude product of RRRRRRHHH whose N-terminus is modified by stearic acid.

将100mg上述产物溶于2mL纯水中，用制备用反相HPLC纯化，收集保留主峰，收集液经旋蒸浓缩，然后冷冻干燥得N端被硬脂酸修饰的RRRRRRHHH的纯品。纯品的质谱表明，其分子量为1794，与计算值相符。最终获得N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₆H₃。Dissolve 100 mg of the above product in 2 mL of pure water, purify by preparative reverse-phase HPLC, collect and retain the main peak, concentrate the collected solution by rotary evaporation, and then freeze-dry to obtain the pure product of RRRRRHHH whose N-terminus is modified by stearic acid. The mass spectrum of the pure product showed that its molecular weight was 1794, which was consistent with the calculated value. Finally, the tumor cell-selective membrane-penetrating peptide R ₆ H ₃ whose N-terminus is modified by stearic acid is obtained.

依处方量称取注射用大豆磷脂、胆固醇和紫杉醇于茄型瓶中，加入一定量的氯仿使其充分溶解，并加入玻璃珠若干。减压旋蒸1小时，挥除有机溶剂形成药脂薄膜。结束后继续抽真空过夜，以除去痕量有机溶剂。10mL纯水常温手摇水合，至茄型瓶壁洗净为止，样品呈均匀的乳浊液。冰水浴中探头超声以减小粒径。然后3000rpm离心以除去探头超声可能掉落的金属屑。取上清液分别过0.45μm，0.22μm聚碳酸酯滤膜各一次即可制得脂质体紫杉醇。Weigh soybean lecithin for injection, cholesterol and paclitaxel according to the prescribed amount into an eggplant-shaped bottle, add a certain amount of chloroform to fully dissolve it, and add some glass beads. Rotary evaporation under reduced pressure for 1 hour, the organic solvent was evaporated to form a thin film of drug lipid. Vacuum was continued overnight after completion to remove traces of organic solvent. Hydrate with 10mL pure water by hand shaking at room temperature until the eggplant-shaped bottle wall is washed, and the sample becomes a uniform emulsion. Probe sonication in an ice-water bath to reduce particle size. Then centrifuge at 3000rpm to remove metal shavings that may be dropped by probe ultrasound. Liposome paclitaxel can be prepared by taking the supernatant and passing through 0.45 μm and 0.22 μm polycarbonate filter membranes respectively once.

精密称取10mg N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₆H₃，并将其充分溶解于2mL的双蒸水中。于常温下，将此溶液缓缓滴加到制得的紫杉醇脂质体分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min，然后将此分散体系在4℃孵育8小时即可制得肿瘤细胞选择性穿膜肽修饰的紫杉醇脂质体。对修饰前后脂质体的包封率和稳定性的考察结果表明，肿瘤细胞选择性穿膜肽R₆H₃对紫杉醇脂质体的修饰没有对普通紫杉醇脂质体的理化性质产生显著影响，包封率大于96％，常温放置24小时渗漏率小于3％。Precisely weighed 10 mg of the tumor cell-selective membrane-penetrating peptide R ₆ H ₃ whose N-terminus was modified by stearic acid, and fully dissolved it in 2 mL of double-distilled water. At normal temperature, this solution was slowly added dropwise into the prepared paclitaxel liposome dispersion system, and the dropping process was kept under constant magnetic stirring. Stirring was continued for 30 minutes after the dropwise addition, and then the dispersion system was incubated at 4° C. for 8 hours to prepare tumor cell-selective membrane-penetrating peptide-modified paclitaxel liposomes. The results of investigation on the encapsulation efficiency and stability of liposomes before and after modification showed that the modification of paclitaxel liposomes by tumor cell-selective membrane-penetrating peptide R ₆ H ₃ did not significantly affect the physicochemical properties of ordinary paclitaxel liposomes. The encapsulation rate is greater than 96%, and the leakage rate is less than 3% after being placed at room temperature for 24 hours.

精密称取相对分子质量为10⁴的透明质酸钠10mg，溶解于4mL的双蒸水中，调节pH值至6.0。于常温下将此溶液缓缓滴加到制得的肿瘤细胞选择性穿膜肽R₆H₃修饰的紫杉醇脂质体的分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min即得以透明质酸钠、肿瘤细胞选择性穿膜肽R₆H₃与紫杉醇脂质体所构建的复合脂质体。该复合脂质体平均粒径为160.7nm，zeta电位为-21.3mV，多分散指数PDI为0.278，紫杉醇包封率为97.0％。以1％的蔗糖为冻干保护剂，将上述复合脂质体制备成冻干粉保存，使用时以5％的葡萄糖溶液复溶供注射使用。Accurately weigh 10 mg of sodium hyaluronate with a relative molecular mass of 10 ⁴ , dissolve it in 4 mL of double-distilled water, and adjust the pH to 6.0. Slowly add this solution dropwise to the prepared tumor cell selective membrane-penetrating peptide R ₆ H ₃ modified paclitaxel liposome dispersion system at room temperature, and keep magnetic stirring continuously during the dropping process. After the dropwise addition, the stirring was continued for 30 minutes to obtain a complex liposome constructed by sodium hyaluronate, tumor cell selective membrane penetrating peptide R ₆ H ₃ and paclitaxel liposome. The average particle diameter of the composite liposome is 160.7nm, the zeta potential is -21.3mV, the polydispersity index PDI is 0.278, and the paclitaxel encapsulation efficiency is 97.0%. Using 1% sucrose as a freeze-drying protective agent, the above complex liposomes are prepared as freeze-dried powder for storage, and reconstituted with 5% glucose solution for injection.

以市售的紫杉醇脂质体为对照，对上述方法制备的紫杉醇复合脂质体在荷瘤小鼠体内的组织分布进行考察。选取腋下接种有H22肝癌的荷瘤小鼠90只(n＝3)，禁食12小时。按20mg/kg剂量分别尾静脉注射市售紫杉醇脂质体和紫杉醇复合脂质体，给药后分别于5，15，30min，1，2，4，8，12，24，48小时眼眶取血后，断颈处死，取荷瘤小鼠的心、肝、脾、肺、肾、肿瘤样品，生理盐水冲洗，滤纸吸干其水分。分别精密称重，加入生理盐水1.5mL制成匀浆液。精密量取0.8mL(肝取0.5mL)匀浆液，血浆取150μL，分别加入不同浓度的地西泮内标液50μL，混匀后，加入叔丁基甲醚4mL，涡旋5min，3000r/min离心10min，取有机相3mL，于40℃减压挥干，残渣用流动相200μL溶解，涡旋1min，10000r/min离心10min，取上清液20μL进样，按体内色谱条件进行分析。Using the commercially available paclitaxel liposome as a control, the tissue distribution of the paclitaxel complex liposome prepared by the above method in tumor-bearing mice was investigated. 90 tumor-bearing mice (n=3) inoculated with H22 liver cancer in the armpit were selected and fasted for 12 hours. Inject commercially available paclitaxel liposomes and paclitaxel complex liposomes into the tail vein at a dose of 20 mg/kg, and collect blood from the orbit at 5, 15, 30 minutes, 1, 2, 4, 8, 12, 24, and 48 hours after administration. Afterwards, they were killed by neck dislocation, and the heart, liver, spleen, lung, kidney, and tumor samples of tumor-bearing mice were taken, rinsed with normal saline, and blotted dry with filter paper. They were weighed precisely, and 1.5 mL of normal saline was added to make a homogenate. Precisely measure 0.8mL (0.5mL for liver) homogenate, 150μL for plasma, add 50μL of diazepam internal standard solution of different concentrations, mix well, add 4mL of tert-butyl methyl ether, vortex for 5min, and centrifuge at 3000r/min for 10min , take 3 mL of the organic phase, evaporate to dryness at 40°C under reduced pressure, dissolve the residue with 200 μL of mobile phase, vortex for 1 min, centrifuge at 10,000 r/min for 10 min, take 20 μL of the supernatant and inject it, and analyze it according to the in vivo chromatographic conditions.

给药系统的靶向性评价采用靶向效率(Te)作为评价参数。Te值表示药物制剂对靶器官和非靶器官的选择性。Te值越大，选择性越强。本实验考查各器官的AUC与血浆AUC的比值，即Te＝AUC_器官/AUC_血浆。其结果如表三所示。Targeting evaluation of the drug delivery system uses targeting efficiency (Te) as an evaluation parameter. The Te value indicates the selectivity of the drug formulation to target organs and non-target organs. The larger the Te value, the stronger the selectivity. In this experiment, the ratio of AUC of each organ to plasma AUC was investigated, namely Te=AUC _organ /AUC _plasma . The results are shown in Table 3.

表三 靶向性评价Table 3 Targeted evaluation

由表三的结果可知，紫杉醇复合脂质体对肿瘤的靶向效率Te明显高于市售紫杉醇脂质体，而对心脏、肾脏等器官的靶向效率Te明显低于市售紫杉醇脂质体。由此可见，紫杉醇复合脂质体对肿瘤具有较高的靶向性，而且降低了对心脏、肾脏等器官的毒性。As can be seen from the results in Table 3, the targeting efficiency Te of paclitaxel composite liposomes to tumors is significantly higher than that of commercially available paclitaxel liposomes, while the targeting efficiency Te of the heart, kidney and other organs is significantly lower than that of commercially available paclitaxel liposomes. . It can be seen that the paclitaxel complex liposome has a high targeting ability to tumors, and reduces the toxicity to organs such as the heart and kidney.

实施例8Example 8

以透明质酸钠、肿瘤细胞选择性穿膜肽(R₅H₃、R₆H₃、R₇H₃、R₈H₃、R₉H₃、R₁₀H₃、R₁₁H₃、R₁₂H₃，R在N端或C端)与羟基喜树碱脂质体所构建的复合脂质体在荷瘤小鼠体内的抑瘤效果为例来说明这一制剂的抗肿瘤功效。Sodium hyaluronate, tumor cell selective membrane penetrating peptide (R ₅ H ₃ , R ₆ H ₃ , R ₇ H ₃ , R ₈ H ₃ , R ₉ H ₃ , R ₁₀ H ₃ , R ₁₁ H ₃ , R ₁₂ H ₃ , R at the N-terminus or C-terminus) and hydroxycamptothecin liposomes, the anti-tumor effect in tumor-bearing mice was taken as an example to illustrate the anti-tumor efficacy of this preparation.

依处方量称取注射用卵磷脂、胆固醇和羟基喜树碱于茄型瓶中，加入一定量的乙醇使其充分溶解，并加入玻璃珠若干。减压旋蒸1小时，挥除有机溶剂形成药脂薄膜。结束后继续抽真空过夜，以除去痕量有机溶剂。10mL磷酸盐缓冲溶液(pH4.5)常温手摇水合，至茄型瓶壁洗净为止，样品呈均匀的乳浊液。采用实验型高压均质机以减小粒径。然后3000rpm离心以除去探头超声可能掉落的金属屑。取上清液分别过0.45μm，0.22μm聚碳酸酯滤膜各一次即可制得羟基喜树碱脂质体。Weigh lecithin for injection, cholesterol and hydroxycamptothecin according to the prescribed amount into an eggplant-shaped bottle, add a certain amount of ethanol to fully dissolve it, and add some glass beads. Rotary evaporation under reduced pressure for 1 hour, the organic solvent was evaporated to form a thin film of drug lipid. Vacuum was continued overnight after completion to remove traces of organic solvent. 10mL of phosphate buffer solution (pH4.5) was hydrated by hand at room temperature until the eggplant-shaped bottle wall was washed, and the sample was a uniform emulsion. An experimental high-pressure homogenizer was used to reduce the particle size. Then centrifuge at 3000rpm to remove metal shavings that may be dropped by probe ultrasound. Take the supernatant and pass through 0.45 μm and 0.22 μm polycarbonate filter membranes respectively once to prepare hydroxycamptothecin liposomes.

精密称取一定量的N端被硬脂酸修饰的肿瘤细胞选择性穿膜肽R₅H₃(R在C端或N端)，并将其充分溶解于2mL的双蒸水中。于常温下，将此溶液缓缓滴加到制得的羟基喜树碱脂质体分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min，然后将此分散体系在4℃孵育8小时即可制得肿瘤细胞选择性穿膜肽R₅H₃(R在C端或N端)修饰的羟基喜树碱脂质体。对修饰前后脂质体的包封率和稳定性的考察结果表明，肿瘤细胞选择性穿膜肽R₅H₃(R在C端或N端)对羟基喜树碱脂质体的修饰没有对普通羟基喜树碱脂质体的理化性质产生显著影响，包封率大于90％，常温放置24小时渗漏率小于3％。Precisely weigh a certain amount of tumor cell-selective membrane-penetrating peptide R ₅ H ₃ (R is at the C-terminus or N-terminus) modified by stearic acid at the N-terminus, and fully dissolve it in 2 mL of double-distilled water. At normal temperature, this solution is slowly added dropwise into the prepared hydroxycamptothecin liposome dispersion system, and the dropping process is kept under constant magnetic stirring. Continue to stir for 30 minutes after the dropwise addition, and then incubate the dispersion system at 4°C for 8 hours to prepare tumor cell-selective membrane-penetrating peptide R ₅ H ₃ (R is at the C-terminal or N-terminal) modified hydroxycamptothecin resin plastid. The results of investigation on the encapsulation efficiency and stability of liposomes before and after modification showed that the modification of tumor cell selective membrane-penetrating peptide R ₅ H ₃ (R at the C-terminus or N-terminus) had no effect on the modification of hydroxycamptothecin liposomes. The physical and chemical properties of ordinary hydroxycamptothecin liposomes have a significant impact, the encapsulation rate is greater than 90%, and the leakage rate is less than 3% after being placed at room temperature for 24 hours.

精密称取相对分子质量为10⁴的透明质酸钠10mg，溶解于4mL的双蒸水中，调节pH值至6.0。于常温下将此溶液缓缓滴加到制得的肿瘤细胞选择性穿膜肽R₅H₃(R在C端或N端)修饰的羟基喜树碱脂质体的分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min即得以透明质酸钠、肿瘤细胞选择性穿膜肽R₅H₃(R在C端或N端)与羟基喜树碱脂质体所构建的复合脂质体。该复合脂质体平均粒径为187.5nm，zeta电位为-20.4mV，多分散指数PDI为0.259，紫杉醇包封率为92.5％。以1％的蔗糖为冻干保护剂，将上述复合脂质体制备成冻干粉保存，使用时以5％的葡萄糖溶液复溶供注射使用。Accurately weigh 10 mg of sodium hyaluronate with a relative molecular mass of 10 ⁴ , dissolve it in 4 mL of double-distilled water, and adjust the pH to 6.0. Slowly add this solution dropwise to the dispersion system of hydroxycamptothecin liposomes modified by tumor cell selective membrane-penetrating peptide R ₅ H ₃ (R is at the C-terminal or N-terminal) at room temperature, dropwise The process is maintained with constant magnetic stirring. Stirring was continued for 30 minutes after the dropwise addition to obtain complex liposomes constructed of sodium hyaluronate, tumor cell selective membrane-penetrating peptide R ₅ H ₃ (R at the C-terminal or N-terminal) and hydroxycamptothecin liposomes. The average particle diameter of the composite liposome is 187.5nm, the zeta potential is -20.4mV, the polydispersity index PDI is 0.259, and the paclitaxel encapsulation efficiency is 92.5%. Using 1% sucrose as a freeze-drying protective agent, the above complex liposomes are prepared as freeze-dried powder for storage, and reconstituted with 5% glucose solution for injection.

透明质酸钠、其他肿瘤细胞选择性穿膜肽(R₆H₃、R₇H₃、R₈H₃、R₉H₃、R₁₀H₃、R₁₁H₃、R₁₂H₃，R在N端或C端)与羟基喜树碱脂质体所构建的复合脂质体可采用相同的方法制备。Sodium hyaluronate, other tumor cell-selective penetrating peptides (R ₆ H ₃ , R ₇ H ₃ , R ₈ H ₃ , R ₉ H 3 , R ₁₀ H ₃ , R ₁₁ H ₃ , R ₁₂ H ₃ , _R Composite liposomes constructed with hydroxycamptothecin liposomes at the N-terminal or C-terminal) can be prepared in the same manner.

另外，采用相同的方法制备羟基喜树碱脂质体，并按上述方法用N端被硬脂酸修饰的穿膜肽R₈和透明质酸钠一起制备R₈修饰的羟基喜树碱脂质体。In addition, use the same method to prepare hydroxycamptothecin liposomes, and prepare R ₈ modified hydroxycamptothecin lipids together with the penetrating peptide R ₈ modified by stearic acid at the N-terminus and sodium hyaluronate according to the above method body.

以小鼠艾氏腹水瘤(EAC)为模型：取出生长良好的艾氏腹水瘤细胞接种于雌性NIH品系小白鼠腹腔内(每毫升约5×10⁶个，每只0.2mL)，8天后取腹水，用生理盐水稀释，重新接种于另一只健康小鼠腹腔内，共传3代。将60只雌性NIH品系小白鼠称重，随机分为12组，每组5只；从传代小鼠腹腔中抽出腹水瘤并进行计数，稀释至每毫升4×10⁶个，接种于小鼠右侧背部皮下(每只0.2mL)；将制得的各种羟基喜树碱复合脂质体、R₈修饰的羟基喜树碱脂质体、普通羟基喜树碱脂质体和羟基喜树碱注射液按5mg/kg羟基喜树碱，于接种后第7天单次给药，空白对照组注射等体积的生理盐水(空白对照)。接种两周后将小鼠断颈处死，局部用75％乙醇消毒，用剪刀取皮下瘤块，剥去纤维组织并称重，肿瘤抑制率按抑制率％＝(空白对照组肿瘤的平均重量-给药组肿瘤的平均重量)/空白对照组肿瘤的平均重量×100％，瘤重比较如表四所示。结果显示，肿瘤细胞接种后第7天给予羟基喜树碱复合脂质体5mg/kg剂量泊疗，肿瘤抑制率显著高于R₈修饰的羟基喜树碱脂质体、羟基喜树碱的普通脂质体和注射液剂型给药组的抑制率(P＜0.05)。由此可知，以透明质酸钠、肿瘤细胞选择性穿膜肽与羟基喜树碱脂质体所构建的复合脂质体在荷瘤小鼠体内的抑瘤效果明显强于R₈修饰的羟基喜树碱脂质体、羟基喜树碱的普通脂质体和注射液剂型。Taking mouse Ehrlich's ascites tumor (EAC) as a model: take out well-growing Ehrlich's ascites tumor cells and inoculate them into the abdominal cavity of female NIH strain mice (about 5×10 ⁶ cells per milliliter, 0.2 mL per mouse), and take them 8 days later. The ascites was diluted with normal saline and reinoculated into the peritoneal cavity of another healthy mouse for 3 passages. Weighed 60 female NIH strain mice, randomly divided them into 12 groups, 5 in each group; extracted and counted ascites tumors from the abdominal cavity of passaged mice, diluted them to 4× ¹⁰⁶ per ml, and inoculated them on the right side of mice. Side back subcutaneous (0.2mL each); various hydroxycamptothecin complex liposomes, R ₈ modified hydroxycamptothecin liposomes, common hydroxycamptothecin liposomes and hydroxycamptothecin The injection was administered at 5 mg/kg hydroxycamptothecin once on the 7th day after inoculation, and the blank control group was injected with an equal volume of normal saline (blank control). Two weeks after the inoculation, the mice were killed by neck dislocation, and the local area was sterilized with 75% ethanol. The subcutaneous tumor mass was taken with scissors, and the fibrous tissue was stripped off and weighed. The average weight of the tumors in the administration group)/the average weight of the tumors in the blank control group×100%, and the comparison of tumor weights is shown in Table 4. The results showed that the hydroxycamptothecin complex liposome 5 mg/kg dose was administered on the 7th day after tumor cell inoculation, and the tumor inhibition rate was significantly higher than that of _R8 -modified hydroxycamptothecin liposome and hydroxycamptothecin. Inhibition rate of liposome and injection dosage form administration group (P<0.05). It can be seen that the composite liposome constructed with sodium hyaluronate, tumor cell-selective membrane-penetrating peptide and hydroxycamptothecin liposome has a significantly stronger tumor-inhibiting effect in tumor-bearing mice than the hydroxyl group modified by _R8 . Common liposome and injection formulations of camptothecin liposome and hydroxycamptothecin.

表四 各给药组的瘤体称重(g)和肿瘤抑制率(％)，n＝5，

Table 4 Tumor body weight (g) and tumor inhibition rate (%) of each administration group, n=5,

实施例9Example 9

以透明质酸钠、肿瘤细胞选择性穿膜肽(R₅H₃、R₆H₃、R₇H₃、R₈H₃、R₉H₃、R₁₀H₃、R₁₁H₃、R₁₂H₃，R在N端或C端)与紫杉醇脂质体所构建的复合脂质体在荷瘤小鼠体内的抑瘤效果为例来说明这一制剂的抗肿瘤功效。Sodium hyaluronate, tumor cell selective membrane penetrating peptide (R ₅ H ₃ , R ₆ H ₃ , R ₇ H ₃ , R ₈ H ₃ , R ₉ H ₃ , R ₁₀ H ₃ , R ₁₁ H ₃ , R ₁₂ H ₃ , R at the N-terminus or C-terminus) and paclitaxel liposomes, the anti-tumor effect in tumor-bearing mice was taken as an example to illustrate the anti-tumor efficacy of this preparation.

依处方量称取注射用大豆磷脂、胆固醇和紫杉醇于茄型瓶中，加入一定量的氯仿使其充分溶解，并加入玻璃珠若干。减压旋蒸1小时，挥除有机溶剂形成药脂薄膜。结束后继续抽真空过夜，以除去痕量有机溶剂。10mL纯水常温手摇水合，至茄型瓶壁洗净为止，样品呈均匀的乳浊液。冰水浴中探头超声以减小粒径。然后3000rpm离心以除去探头超声可能掉落的金属屑。取上清液分别过0.45μm，0.22μm聚碳酸酯滤膜各一次即可制得脂质体紫杉醇。Weigh soybean lecithin for injection, cholesterol and paclitaxel according to the prescribed amount into an eggplant-shaped bottle, add a certain amount of chloroform to fully dissolve it, and add some glass beads. Rotary evaporation under reduced pressure for 1 hour, the organic solvent was evaporated to form a thin film of drug lipid. Vacuum was continued overnight after completion to remove traces of organic solvent. Hydrate with 10mL pure water by hand shaking at room temperature until the eggplant-shaped bottle wall is washed, and the sample becomes a uniform emulsion. Probe sonication in an ice-water bath to reduce particle size. Then centrifuge at 3000rpm to remove metal shavings that may be dropped by probe ultrasound. Liposome paclitaxel can be prepared by taking the supernatant and passing through 0.45 μm and 0.22 μm polycarbonate filter membranes respectively once.

精密称取10mg N端被便脂酸修饰的肿瘤细胞选择性穿膜肽R₅H₃(R在C端或N端)，并将其充分溶解于2mL的双蒸水中。于常温下，将此溶液缓缓滴加到制得的紫杉醇脂质体分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min，然后将此分散体系在4℃孵育8小时即可制得肿瘤细胞选择性穿膜肽修饰的紫杉醇脂质体。对修饰前后脂质体的包封率和稳定性的考察结果表明，肿瘤细胞选择性穿膜肽R₅H₃对紫杉醇脂质体的修饰没有对普通紫杉醇脂质体的理化性质产生显著影响，包封率大于96％，常温放置24小时渗漏率小于3％。Precisely weigh 10 mg tumor cell-selective membrane-penetrating peptide R ₅ H ₃ (R is at the C-terminus or N-terminus) whose N-terminus is modified by fatty acid, and fully dissolve it in 2 mL of double-distilled water. At normal temperature, this solution was slowly added dropwise into the prepared paclitaxel liposome dispersion system, and the dropping process was kept under constant magnetic stirring. Stirring was continued for 30 minutes after the dropwise addition, and then the dispersion system was incubated at 4° C. for 8 hours to prepare tumor cell-selective membrane-penetrating peptide-modified paclitaxel liposomes. The results of investigation on the encapsulation efficiency and stability of liposomes before and after modification showed that the modification of paclitaxel liposomes by tumor cell-selective membrane-penetrating peptide R ₅ H ₃ did not significantly affect the physicochemical properties of ordinary paclitaxel liposomes. The encapsulation rate is greater than 96%, and the leakage rate is less than 3% after being placed at room temperature for 24 hours.

精密称取相对分子质量为10⁴的透明质酸钠10mg，溶解于4mL的双蒸水中，调节pH值至6.0。于常温下将此溶液缓缓滴加到制得的肿瘤细胞选择性穿膜肽R₅H₃修饰的紫杉醇脂质体的分散体系中，滴加过程保持不断地磁力搅拌。滴加结束后继续搅拌30min即得以透明质酸钠、肿瘤细胞选择性穿膜肽R₅H₃与紫杉醇脂质体所构建的复合脂质体。该复合脂质体平均粒径为162.4nm，zeta电位为-19.3mV，多分散指数PDI为0.265，紫杉醇包封率为98.4％。以1％的蔗糖为冻干保护剂，将上述复合脂质体制备成冻干粉保存，使用时以5％的葡萄糖溶液复溶供注射使用。Accurately weigh 10 mg of sodium hyaluronate with a relative molecular mass of 10 ⁴ , dissolve it in 4 mL of double-distilled water, and adjust the pH to 6.0. Slowly add this solution dropwise to the prepared tumor cell selective membrane-penetrating peptide R ₅ H ₃ modified paclitaxel liposome dispersion system at normal temperature, and keep magnetic stirring continuously during the dropping process. After the dropwise addition, the stirring was continued for 30 minutes to obtain a complex liposome constructed by sodium hyaluronate, tumor cell selective membrane-penetrating peptide R ₅ H ₃ and paclitaxel liposome. The average particle diameter of the composite liposome is 162.4nm, the zeta potential is -19.3mV, the polydispersity index PDI is 0.265, and the paclitaxel encapsulation efficiency is 98.4%. Using 1% sucrose as a freeze-drying protective agent, the above complex liposomes are prepared as freeze-dried powder for storage, and reconstituted with 5% glucose solution for injection.

透明质酸钠、其他肿瘤细胞选择性穿膜肽(R₆H₃、R₇H₃、R₈H₃、R₉H₃、R₁₀H₃、R₁₁H₃、R₁₂H₃，R在N端或C端)与紫杉醇脂质体所构建的复合脂质体可采用相同的方法制备。Sodium hyaluronate, other tumor cell-selective penetrating peptides (R ₆ H ₃ , R ₇ H ₃ , R ₈ H ₃ , R ₉ H 3 , R ₁₀ H ₃ , R ₁₁ H ₃ , R ₁₂ H ₃ , _R Composite liposomes constructed with paclitaxel liposomes at the N-terminal or C-terminal) can be prepared using the same method.

另外，采用相同的方法制备紫杉醇脂质体，并按上述方法用N端被硬脂酸修饰的穿膜肽R₈和透明质酸钠一起制备R₈修饰的紫杉醇脂质体。In addition, paclitaxel liposomes were prepared using the same method, and R ₈ modified paclitaxel liposomes were prepared together with the penetrating peptide R ₈ modified by stearic acid at the N-terminal and sodium hyaluronate according to the above method.

取生长良好的人卵巢癌细胞株COC1细胞接种于BABL/C裸鼠背部皮下。经过5周后，接种部位肿瘤开始生长，第8周时肿瘤直径达1.5cm左右，可以用于实验。上述方法成功接种肿瘤的裸鼠共60只，随机分为12组，每组5只。Well-growing human ovarian cancer cell line COC1 cells were inoculated subcutaneously in the back of BABL/C nude mice. After 5 weeks, the tumor at the inoculation site began to grow, and the diameter of the tumor reached about 1.5 cm at the 8th week, which could be used for experiments. A total of 60 nude mice successfully inoculated with tumors by the above method were randomly divided into 12 groups with 5 mice in each group.

将制得的各种紫杉醇复合脂质体、R₈修饰的紫杉醇脂质体、普通紫杉醇脂质体和紫杉醇注射液按10mg/kg紫杉醇，单次尾静脉注射给药，空白对照组注射等体积的生理盐水(空白对照)。给药一周后将小鼠断颈处死，局部用75％乙醇消毒，用剪刀取皮下瘤块，剥去纤维组织并称重，肿瘤抑制率按抑制率％＝(空白对照组肿瘤的平均重量-给药组肿瘤的平均重量)/空白对照组肿瘤的平均重量×100％，瘤重比较如表五所示。结果显示，肿瘤细胞接种后给予紫杉醇复合脂质体10mg/kg剂量治疗，肿瘤抑制率显著高于R₈修饰的紫杉醇脂质体、紫杉醇普通脂质体和紫杉醇注射液剂型给药组的抑制率(P＜0.05)。由此可知，以透明质酸钠、肿瘤细胞选择性穿膜肽与紫杉醇脂质体所构建的复合脂质体在荷瘤小鼠体内的抑瘤效果明显强于R₈修饰的紫杉醇脂质体、紫杉醇普通脂质体和紫杉醇注射液剂型。Various paclitaxel complex liposomes, R ₈ modified paclitaxel liposomes, common paclitaxel liposomes and paclitaxel injections were administered by 10 mg/kg paclitaxel in a single tail vein injection, and the blank control group was injected with equal volumes. normal saline (blank control). One week after the administration, the mice were killed by neck breaking, and the local area was sterilized with 75% ethanol. The subcutaneous tumor mass was taken with scissors, and the fibrous tissue was stripped off and weighed. The average weight of the tumors in the administration group)/the average weight of the tumors in the blank control group×100%, and the comparison of tumor weights is shown in Table 5. The results showed that after inoculation of tumor cells, paclitaxel complex liposome 10 mg/kg was administered, and the tumor inhibition rate was significantly higher than that of the R ₈ modified paclitaxel liposome, paclitaxel ordinary liposome and paclitaxel injection dosage form administration group. (P<0.05). It can be seen that the composite liposome constructed with sodium hyaluronate, tumor cell-selective membrane-penetrating peptide and paclitaxel liposome has significantly stronger tumor-inhibiting effect in tumor-bearing mice than the paclitaxel liposome modified by R ₈ , paclitaxel ordinary liposome and paclitaxel injection dosage forms.

表五 各给药组的瘤体称重(g)和肿瘤抑制率(％)，n＝5，

Table 5 Tumor body weight (g) and tumor inhibition rate (%) of each administration group, n=5,