CN102552144A

Movatterモバイル変換

Info

Publication number: CN102552144A
Application number: CN2012100099624A
Authority: CN
Inventors: 张烜; 王欣; 张强
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2012-01-13
Filing date: 2012-01-13
Publication date: 2012-07-11

Abstract

本发明属于靶向给药系统领域，具体涉及一种靶向热敏脂质体组合物，其特征在于所述组合物包括表面修饰有肿瘤归巢肽CREKA或具有CREKA核心序列的肽段的热敏脂质体，所述热敏脂质体中包含有抗肿瘤药物。本发明还涉及所述靶向热敏脂质体组合物的制备方法和所述靶向热敏脂质体组合物在制备用于治疗恶性肿瘤的药物中的用途。本发明的靶向热敏脂质体具有很好的靶向肿瘤的作用及抗肿瘤效果。The invention belongs to the field of targeted drug delivery systems, and in particular relates to a targeted thermosensitive liposome composition, which is characterized in that the composition includes a thermogenic liposome whose surface is modified with a tumor-homing peptide CREKA or a peptide segment having a CREKA core sequence. Sensitive liposomes contain antitumor drugs in the thermosensitive liposomes. The present invention also relates to the preparation method of the targeted thermosensitive liposome composition and the use of the targeted thermosensitive liposome composition in the preparation of medicines for treating malignant tumors. The targeted thermosensitive liposome of the present invention has good tumor-targeting effect and anti-tumor effect.

Description

Translated fromChinese

一种靶向热敏脂质体A targeted thermosensitive liposome

技术领域technical field

本发明涉及一种靶向给药系统，特别是涉及一种肿瘤归巢肽修饰的靶向热敏脂质体组合物。本发明还涉及所述靶向热敏脂质体组合物的制备方法和所述靶向热敏脂质体组合物在制备用于治疗恶性肿瘤的药物中的用途。The invention relates to a targeted drug delivery system, in particular to a targeted thermosensitive liposome composition modified with a tumor homing peptide. The present invention also relates to the preparation method of the targeted thermosensitive liposome composition and the use of the targeted thermosensitive liposome composition in the preparation of medicines for treating malignant tumors.

背景技术Background technique

脂质体(liposomes)在过去四十年中作为化疗药物给药系统的载体，尤其是在肿瘤治疗方面，得到了广泛的发展。近年来，结合了物理化学靶向和脉冲释放性能的热敏脂质体在抗肿瘤领域越来越引起重视。Liposomes have been extensively developed over the past forty years as carriers of chemotherapeutic drug delivery systems, especially in tumor therapy. In recent years, thermosensitive liposomes that combine physicochemical targeting and pulse release properties have attracted more and more attention in the field of antitumor.

热敏脂质体(thermosensitive liposomes，TSL)是一类由相变温度略高于正常组织温度的温敏材料制成的，能在局部高温条件下迅速将所载药物释放的脂质体。在正常的体温下，脂质体膜呈致密排列的胶晶态，亲水性药物很难透过脂质体膜而扩散出来。当脂质体随血液循环经过被加热的靶器官时，局部的高温使磷脂分子运动加强，脂质体膜的结构发生变化，原来排列整齐致密的胶晶态磷脂双分子层在较高温度下变成疏松混乱的液晶态，导致脂质体迅速释放内含药物。Thermosensitive liposomes (thermosensitive liposomes, TSL) are a class of liposomes made of thermosensitive materials whose phase transition temperature is slightly higher than normal tissue temperature, and can rapidly release the loaded drug under local high temperature conditions. At normal body temperature, the liposome membrane is in a densely arranged colloidal crystal state, and it is difficult for hydrophilic drugs to diffuse out through the liposome membrane. When the liposome passes through the heated target organ with the blood circulation, the local high temperature strengthens the movement of the phospholipid molecules, and the structure of the liposome membrane changes. It becomes a loose and chaotic liquid crystal state, causing the liposome to release the drug contained in it rapidly.

目前抗肿瘤药物热敏脂质体研究中的最大问题是如何提高其在肿瘤部位的浓度，降低抗肿瘤药物对内皮网状系统及正常组织的毒性。虽然热敏脂质体已经集被动靶向性(从间隙比较大的肿瘤血管中泄漏进入肿瘤部位，实现在肿瘤中的蓄积)和物理化学靶向性(在高于相变温度时，将所包封的药物释放于加热部位)于一身，但是有文献报道，这两种靶向作用的效果在对抗肿瘤药效的提高上并不明显。因此，如何显著提高热敏脂质体的靶向性，增强热敏脂质体的抗肿瘤效果，是本领域亟待解决的问题。At present, the biggest problem in the research of anti-tumor drug thermosensitive liposome is how to increase its concentration in the tumor site and reduce the toxicity of anti-tumor drugs to the endothelial reticular system and normal tissues. Although thermosensitive liposomes have integrated passive targeting (leaking from tumor blood vessels with relatively large gaps into the tumor site to achieve accumulation in the tumor) and physical and chemical targeting (when the temperature is higher than the phase transition temperature, the The encapsulated drug is released at the heating site), but it has been reported in the literature that the effect of these two targeting effects is not obvious in the improvement of the anti-tumor drug effect. Therefore, how to significantly improve the targeting of thermosensitive liposomes and enhance the antitumor effect of thermosensitive liposomes is an urgent problem to be solved in this field.

肿瘤归巢肽(tumor homing peptide)由于特异性强、生物相容性好、无毒、无免疫原性，是用于主动靶向的良好配体。所谓肿瘤归巢肽，是一类由几个到几十个氨基酸组成的肽链序列，能以高度的亲和力和特异性结合到肿瘤细胞或者肿瘤血管/淋巴管。除此之外，很多肿瘤归巢肽能被肿瘤细胞内化，在肿瘤细胞内以很高的浓度积累。Tumor homing peptide (tumor homing peptide) is a good ligand for active targeting due to its strong specificity, good biocompatibility, non-toxicity, and non-immunogenicity. The so-called tumor homing peptide is a kind of peptide chain sequence composed of several to dozens of amino acids, which can bind to tumor cells or tumor blood vessels/lymphatic vessels with high affinity and specificity. In addition, many tumor-homing peptides can be internalized by tumor cells and accumulate in high concentrations in tumor cells.

CREKA(cys-arg-glu-lys-ala)是一种由半胱氨酸、精氨酸、谷氨酸、赖氨酸和丙氨酸组成的五肽，其作用于肿瘤微环境，对乳腺癌、前列腺癌、非小细胞肺癌、黑色素瘤等多种癌细胞均有靶向作用，没有肿瘤选择性。CREKA在体内发挥靶向作用的机制是：1.识别肿瘤血管和间隙中特异性存在的凝固的血浆蛋白，选择性归巢到肿瘤部位；2、不仅归巢到肿瘤部位，同时能够自扩大化归巢作用，即CREKA在肿瘤部位积累后，能够诱导额外的局部凝固，因而产生出更多的CREKA结合位点，类似于血小板的自扩大效应。CREKA (cys-arg-glu-lys-ala) is a pentapeptide composed of cysteine, arginine, glutamic acid, lysine and alanine, which acts on the tumor microenvironment, Cancer, prostate cancer, non-small cell lung cancer, melanoma and many other cancer cells have targeting effect, and there is no tumor selectivity. The mechanism by which CREKA plays a targeting role in vivo is: 1. Recognize coagulated plasma proteins that specifically exist in tumor blood vessels and spaces, and selectively home to tumor sites; 2. Not only home to tumor sites, but also self-expand The homing effect, that is, the accumulation of CREKA at the tumor site can induce additional local coagulation, thereby generating more CREKA binding sites, similar to the self-expanding effect of platelets.

目前，国内外尚没有利用CREKA对包含抗肿瘤药物的热敏脂质体进行修饰的报道。At present, there is no report of using CREKA to modify thermosensitive liposomes containing antitumor drugs at home and abroad.

发明内容Contents of the invention

本发明的创新点在于将热敏脂质体与主动靶向技术相结合，实现多种靶向技术的配合，以提高热敏脂质体的靶向性，增强热敏脂质体的抗肿瘤效果。The innovation of the present invention is to combine thermosensitive liposomes with active targeting technology to realize the cooperation of multiple targeting technologies to improve the targeting of thermosensitive liposomes and enhance the anti-tumor effect of thermosensitive liposomes Effect.

为此，本发明的一个目的是提供一种靶向热敏脂质体组合物，所述组合物包括表面修饰有肿瘤归巢肽CREKA或具有CREKA核心序列的肽段的热敏脂质体，所述热敏脂质体中包含有抗肿瘤药物。To this end, an object of the present invention is to provide a targeted thermosensitive liposome composition, which includes a thermosensitive liposome whose surface is modified with a tumor-homing peptide CREKA or a peptide segment having a CREKA core sequence, The thermosensitive liposome contains antitumor drugs.

本发明的另一个目的是提供所述靶向热敏脂质体组合物的制备方法。Another object of the present invention is to provide a preparation method of the targeted thermosensitive liposome composition.

本发明的另一个目的是提供所述靶向热敏脂质体组合物在制备用于治疗恶性肿瘤的药物中的用途。Another object of the present invention is to provide the use of the targeted thermosensitive liposome composition in the preparation of medicines for treating malignant tumors.

本发明的技术方案和要点将在下面作详细说明。The technical scheme and key points of the present invention will be described in detail below.

本发明还涉及以下技术方案：The present invention also relates to the following technical solutions:

1、一种靶向热敏脂质体组合物，其特征在于所述组合物包括表面修饰有肿瘤归巢肽CREKA或具有CREKA核心序列的肽段的热敏脂质体，所述热敏脂质体中包含有抗肿瘤药物。1. A targeted thermosensitive liposome composition, characterized in that the composition includes thermosensitive liposomes whose surface is modified with tumor-homing peptide CREKA or a peptide segment having a CREKA core sequence, and the thermosensitive liposome The plastid contains antineoplastic drugs.

2、如项1所述的靶向热敏脂质体组合物，其中所述抗肿瘤药物选自阿霉素、表阿霉素、柔红霉素、紫杉醇、多西紫杉醇、吡柔比星、阿柔比星、顺铂、卡铂、奥沙利铂和甲氨蝶呤中的一种或多种。2. The targeted thermosensitive liposome composition as described in item 1, wherein the antitumor drug is selected from the group consisting of adriamycin, epirubicin, daunorubicin, paclitaxel, docetaxel, pirarubicin One or more of , arubicin, cisplatin, carboplatin, oxaliplatin, and methotrexate.

3、如项2所述的靶向热敏脂质体组合物，其中抗肿瘤药物的药脂比(药物与脂材的质量比)为1∶10w/w～1∶50w/w，例如1∶10w/w、1∶20w/w、1∶30w/w、1∶40w/w、1∶50w/w。3. The targeted thermosensitive liposome composition as described in Item 2, wherein the drug-to-lipid ratio of the antineoplastic drug (the mass ratio of the drug to the lipid material) is 1:10w/w～1:50w/w, for example, 1 :10w/w, 1:20w/w, 1:30w/w, 1:40w/w, 1:50w/w.

4、如项1-3任一项所述的靶向热敏脂质体组合物，其中制成所述热敏脂质体的原料包括肿瘤归巢肽导向化合物DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段、热敏脂质体膜材、DSPE-PEG和抗肿瘤药物。4. The targeted thermosensitive liposome composition according to any one of items 1-3, wherein the raw materials for making the thermosensitive liposome include tumor homing peptide targeting compound DSPE-PEG-CREKA or DSPE- PEG-peptides with CREKA core sequence, thermosensitive liposome membrane material, DSPE-PEG and anti-tumor drugs.

5、如项4所述的靶向热敏脂质体组合物，其中所述肿瘤归巢肽导向化合物DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段是通过将肿瘤归巢肽CREKA或具有CREKA核心序列的肽段与DSPE-PEG-马来酰亚胺溶解于液体介质中，调节液体介质的pH值至6.0-8.0范围内，持续搅拌反应，纯化反应所得物，经冷冻干燥得到的肿瘤归巢肽导向化合物DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段。5. The targeted thermosensitive liposome composition as described in item 4, wherein the tumor homing peptide targeting compound DSPE-PEG-CREKA or DSPE-PEG-peptide segment having a CREKA core sequence is achieved by homing the tumor Dissolve the peptide CREKA or the peptide segment with the core sequence of CREKA and DSPE-PEG-maleimide in the liquid medium, adjust the pH value of the liquid medium to the range of 6.0-8.0, continue to stir the reaction, purify the reaction product, and freeze The dried tumor-homing peptide targeting compound DSPE-PEG-CREKA or DSPE-PEG-peptide segment having the core sequence of CREKA.

6、如项5所述的靶向热敏脂质体组合物，其中调节液体介质的pH值至6.5-7.5范围内，优选调节pH值至6.8-7.2的范围内，更优选调节pH值至6.9-7.1的范围，最优选调节至pH值为7.0。6. The targeted thermosensitive liposome composition as described initem 5, wherein the pH value of the liquid medium is adjusted to within the range of 6.5-7.5, preferably within the range of 6.8-7.2, more preferably adjusted to the pH value of The range of 6.9-7.1, most preferably adjusted to a pH value of 7.0.

7、如项5所述的靶向热敏脂质体组合物，其中所述液体介质为可将肿瘤归巢肽CREKA与DSPE-PEG-马来酰亚胺溶解于其中的任意种类的液体介质，例如水性溶剂和有机溶剂。7. The targeted thermosensitive liposome composition as described initem 5, wherein the liquid medium is any kind of liquid medium in which the tumor homing peptide CREKA and DSPE-PEG-maleimide can be dissolved , such as aqueous and organic solvents.

8、如项5所述的靶向热敏脂质体组合物，其中所述反应是在惰性气体的保护下进行的。8. The targeted thermosensitive liposome composition according toitem 5, wherein the reaction is carried out under the protection of an inert gas.

9、如项5所述的靶向热敏脂质体组合物，其中所述反应是在环境温度下进行的，优选在15℃-35℃的温度进行，更优选在20℃-30℃的温度进行，最优选在25℃的温度进行。9. The targeted thermosensitive liposome composition as described initem 5, wherein the reaction is carried out at ambient temperature, preferably at a temperature of 15°C-35°C, more preferably at a temperature of 20°C-30°C temperature, most preferably at a temperature of 25°C.

10、如项5所述的靶向热敏脂质体组合物，其中所述搅拌的时间为24小时以上。10. The targeted thermosensitive liposome composition according toitem 5, wherein the stirring time is more than 24 hours.

11、如项5所述的靶向热敏脂质体组合物，其中所述纯化反应所得物的步骤是通过对反应所得物进行透析的方法来实现的。11. The targeted thermosensitive liposome composition according toitem 5, wherein the step of purifying the reaction product is achieved by dialysis of the reaction product.

12、如项4所述的靶向热敏脂质体组合物，其中所述热敏脂质体膜材包括DPPC和DSPC。12. The targeted thermosensitive liposome composition according to item 4, wherein the thermosensitive liposome membrane material includes DPPC and DSPC.

13、如项4所述的靶向热敏脂质体组合物，其中所述热敏脂质体膜材包括DPPC、HSPC和CHOL。13. The targeted thermosensitive liposome composition according to item 4, wherein the thermosensitive liposome membrane material includes DPPC, HSPC and CHOL.

14、如项4所述的靶向热敏脂质体组合物，其中所述热敏脂质体膜材包括DPPC和MSPC。14. The targeted thermosensitive liposome composition according to item 4, wherein the thermosensitive liposome membrane material includes DPPC and MSPC.

15、如项4所述的靶向热敏脂质体组合物，其中所述热敏脂质体膜材包括DPPC、MSPC和DSPG。15. The targeted thermosensitive liposome composition according to item 4, wherein the thermosensitive liposome membrane material includes DPPC, MSPC and DSPG.

16、如项4所述的靶向热敏脂质体组合物，其中所述热敏脂质体膜材包括DPPC和MPPC。16. The targeted thermosensitive liposome composition according to item 4, wherein the thermosensitive liposome membrane material includes DPPC and MPPC.

17、如项1-3任一项所述的靶向热敏脂质体组合物，其中制成所述热敏脂质体的原料包括DPPC、DSPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段，其中DPPC、DSPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段之间的摩尔比为DPPC∶DSPC∶DSPE-PEG∶DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段＝60～100∶25～5∶0.2～10∶0.2～10，优选70～90∶20～10∶0.5～5∶0.5～5，例如80∶19∶0.5∶0.5、80∶18∶1∶1、80∶17∶1.5∶1.5、80∶16∶2∶2、80∶15∶2.5∶2.5、80∶14∶3∶3和80∶10∶5∶5。17. The targeted thermosensitive liposome composition according to any one of items 1-3, wherein the raw materials for making the thermosensitive liposome include DPPC, DSPC, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-peptide with CREKA core sequence, wherein the molar ratio between DPPC, DSPC, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-peptide with CREKA core sequence is DPPC: DSPC: DSPE- PEG: DSPE-PEG-CREKA or DSPE-PEG-peptide with CREKA core sequence = 60-100: 25-5: 0.2-10: 0.2-10, preferably 70-90: 20-10: 0.5-5: 0.5 ~5, such as 80:19:0.5:0.5, 80:18:1:1, 80:17:1.5:1.5, 80:16:2:2, 80:15:2.5:2.5, 80:14:3: 3 and 80:10:5:5.

18、如项1-3任一项所述的靶向热敏脂质体组合物，其中制成所述热敏脂质体的原料包括DPPC、HSPC、CHOL、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段，其中DPPC、HSPC、CHOL、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段之间的摩尔比为DPPC∶HSPC∶CHOL∶DSPE-PEG∶DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段＝90～110∶40～60∶20～40∶1～6∶1～6，例如95～105∶45～55∶25～35∶2～5∶2～5，优选100∶50∶30∶3∶3。18. The targeted thermosensitive liposome composition according to any one of items 1-3, wherein the raw materials for making the thermosensitive liposome include DPPC, HSPC, CHOL, DSPE-PEG, DSPE-PEG- CREKA or DSPE-PEG-the peptide segment with the CREKA core sequence, wherein the molar ratio between DPPC, HSPC, CHOL, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-the peptide segment with the CREKA core sequence is DPPC: HSPC:CHOL:DSPE-PEG:DSPE-PEG-CREKA or DSPE-PEG-peptide segment with CREKA core sequence=90～110:40～60:20～40:1～6:1～6, for example 95～105 :45-55:25-35:2-5:2-5, preferably 100:50:30:3:3.

19、如项1-3任一项所述的靶向热敏脂质体组合物，其中制成所述热敏脂质体的原料包括DPPC、MSPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段，其中DPPC、MSPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段之间的摩尔比为DPPC∶MSPC∶DSPE-PEG∶DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段＝80～120∶0.5～20∶0.2～5∶0.2～5，优选85～100∶1～10∶0.5～2.5∶0.5～2.5，更优选86∶10∶2∶2、90∶10∶2∶2、95∶5∶2∶2、97.5∶2.5∶2∶2、99∶1∶2∶2、89∶10∶2.5∶2.5、90∶10∶2∶2、91∶10∶1.5∶1.5、92∶10∶1∶1和93∶10∶0.5∶0.5。19. The targeted thermosensitive liposome composition according to any one of items 1-3, wherein the raw materials for making the thermosensitive liposome include DPPC, MSPC, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-peptide with CREKA core sequence, wherein the molar ratio between DPPC, MSPC, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-peptide with CREKA core sequence is DPPC: MSPC: DSPE- PEG: DSPE-PEG-CREKA or DSPE-PEG-peptide with CREKA core sequence = 80-120: 0.5-20: 0.2-5: 0.2-5, preferably 85-100: 1-10: 0.5-2.5: 0.5 ~2.5, more preferably 86:10:2:2, 90:10:2:2, 95:5:2:2, 97.5:2.5:2:2, 99:1:2:2, 89:10:2.5 :2.5, 90:10:2:2, 91:10:1.5:1.5, 92:10:1:1 and 93:10:0.5:0.5.

20、如项1-3任一项所述的靶向热敏脂质体组合物，其中制成所述热敏脂质体的原料包括DPPC、MSPC、DSPG、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段，其中DPPC、MSPC、DSPG、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段之间的摩尔比为DPPC∶MSPC∶DSPG∶DSPE-PEG∶DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段＝60～100∶2～20∶2～20∶0.5～5∶0.5～5，优选70～90∶6～10∶8～12∶1～3∶1～3，更优选82∶8∶10∶2∶2。20. The targeted thermosensitive liposome composition according to any one of items 1-3, wherein the raw materials for making the thermosensitive liposome include DPPC, MSPC, DSPG, DSPE-PEG, DSPE-PEG- CREKA or DSPE-PEG-the peptide segment with the CREKA core sequence, wherein the molar ratio between DPPC, MSPC, DSPG, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-the peptide segment with the CREKA core sequence is DPPC: MSPC: DSPG: DSPE-PEG: DSPE-PEG-CREKA or DSPE-PEG-peptide segment with CREKA core sequence = 60～100: 2～20: 2～20: 0.5～5: 0.5～5, preferably 70～90 :6-10:8-12:1-3:1-3, more preferably 82:8:10:2:2.

21、如项1-3任一项所述的靶向热敏脂质体组合物，其中制成所述热敏脂质体的原料包括DPPC、MPPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段，其中DPPC、MPPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段之间的摩尔比为DPPC∶MPPC∶DSPE-PEG∶DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段＝70～110∶2～20∶0.5～5∶0.5～5，优选80～100∶8～12∶1～3∶1～3，更优选90∶10∶2∶2。21. The targeted thermosensitive liposome composition according to any one of items 1-3, wherein the raw materials for making the thermosensitive liposome include DPPC, MPPC, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-peptide with CREKA core sequence, wherein the molar ratio between DPPC, MPPC, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-peptide with CREKA core sequence is DPPC:MPPC:DSPE- PEG: DSPE-PEG-CREKA or DSPE-PEG-peptide with CREKA core sequence = 70-110: 2-20: 0.5-5: 0.5-5, preferably 80-100: 8-12: 1-3:1 ~3, more preferably 90:10:2:2.

22、如项1-3任一项所述的靶向热敏脂质体组合物，其中所述的靶向热敏脂质体组合物被进一步制成药物剂型，例如注射剂、输液、冻干粉针剂或喷雾剂。22. The targeted thermosensitive liposome composition according to any one of items 1-3, wherein the targeted thermosensitive liposome composition is further made into pharmaceutical dosage forms, such as injection, infusion, freeze-dried Powder injection or spray.

23、如项1所述的靶向热敏脂质体组合物的制备方法，包括如下步骤：23. The method for preparing the targeted thermosensitive liposome composition as described in item 1, comprising the following steps:

以肿瘤归巢肽导向化合物DSPE-PEG-肿瘤归巢肽或DSPE-PEG-具有CREKA核心序列的肽段、热敏脂质体膜材、DSPE-PEG和抗肿瘤药物为原料，采用薄膜分散法或逆向蒸发法制备靶向热敏脂质体组合物。The tumor-homing peptide-guiding compound DSPE-PEG-tumor-homing peptide or DSPE-PEG-peptide segment with CREKA core sequence, thermosensitive liposome membrane material, DSPE-PEG and antitumor drugs are used as raw materials, and the film dispersion method is adopted Or reverse evaporation method to prepare targeted thermosensitive liposome composition.

24、如项23所述的靶向热敏脂质体组合物的制备方法，其中所述采用薄膜分散法制备靶向热敏脂质体组合物包括下述步骤：24. The method for preparing the targeted thermosensitive liposome composition as described in item 23, wherein the preparation of the targeted thermosensitive liposome composition by thin film dispersion method comprises the following steps:

将DSPE-PEG-肿瘤归巢肽或DSPE-PEG-具有CREKA核心序列的肽段、热敏脂质体膜材、DSPE-PEG和抗肿瘤药物溶解于有机溶剂中；Dissolve DSPE-PEG-tumor-homing peptide or DSPE-PEG-peptide segment with CREKA core sequence, thermosensitive liposome membrane material, DSPE-PEG and antitumor drug in an organic solvent;

除去有机溶剂，以在容器壁上形成脂质薄膜；removal of organic solvents to form a lipid film on the vessel walls;

加入任选含有抗肿瘤药物的缓冲液，进行水化处理，以形成粗脂质体混悬液；adding a buffer optionally containing an antineoplastic drug for hydration to form a crude liposome suspension;

对粗脂质体混悬液进行超声粉碎处理；Ultrasonic pulverization of the crude liposome suspension;

对超声粉碎处理后的脂质体混悬液进行纯化，从而得到靶向热敏脂质体组合物。Purify the liposome suspension after ultrasonic pulverization, so as to obtain the targeted thermosensitive liposome composition.

25、如项23所述的靶向热敏脂质体组合物的制备方法，其中所述采用薄膜分散法制备靶向热敏脂质体组合物包括下述步骤：25. The method for preparing the targeted thermosensitive liposome composition as described in item 23, wherein the preparation of the targeted thermosensitive liposome composition by thin film dispersion method comprises the following steps:

对超声粉碎处理后的脂质体混悬液进行纯化；Purify the liposome suspension after ultrasonic crushing;

将纯化后的脂质体混悬液与抗肿瘤药物的溶液进行恒温共孵育，并对共孵育后的脂质体进行纯化，从而得到靶向热敏脂质体组合物。The purified liposome suspension is co-incubated with the antitumor drug solution at a constant temperature, and the co-incubated liposome is purified to obtain a targeted thermosensitive liposome composition.

26、如项24或25所述的靶向热敏脂质体组合物的制备方法，其中所述除去有机溶剂是通过在一定温度的水浴条件下(例如30-60℃)减压旋转蒸发来实现的。26. The method for preparing the targeted thermosensitive liposome composition as described in item 24 or 25, wherein the organic solvent is removed by rotary evaporation under reduced pressure in a water bath at a certain temperature (for example, 30-60°C). Achieved.

27、如项24或25所述的靶向热敏脂质体组合物的制备方法，其中所述水化处理是通过对缓冲液进行涡旋震荡和/或超声处理来实现的。27. The method for preparing the targeted thermosensitive liposome composition according to item 24 or 25, wherein the hydration treatment is realized by vortexing and/or ultrasonic treatment of the buffer solution.

28、如项24或25所述的靶向热敏脂质体组合物的制备方法，其中所述任选含有抗肿瘤药物的缓冲液为任选含有抗肿瘤药物的硫酸铵缓冲液或pH＝4的柠檬酸缓冲液。28. The method for preparing the targeted thermosensitive liposome composition as described in item 24 or 25, wherein the buffer optionally containing antitumor drugs is ammonium sulfate buffer or pH= 4 citrate buffer.

29、如项24或25所述的靶向热敏脂质体组合物的制备方法，其中所述对粗脂质体混悬液进行超声粉碎处理是利用探头超声的超声波细胞粉碎机来实现的。29. The method for preparing the targeted thermosensitive liposome composition as described in item 24 or 25, wherein the ultrasonic pulverization of the crude liposome suspension is realized by using an ultrasonic cell pulverizer with ultrasonic probe .

30、如项24或25所述的靶向热敏脂质体组合物的制备方法，其中所述对超声粉碎处理后的脂质体混悬液进行纯化是通过将超声粉碎处理后的脂质体混悬液通过Sephadex G50柱，用磷酸盐缓冲溶液(PBS)或汉克平衡盐溶液(HBS)洗脱并收集脂质体部分来实现的。30. The method for preparing the targeted thermosensitive liposome composition as described in item 24 or 25, wherein said liposome suspension after ultrasonic pulverization is purified by The liposome suspension was passed through a Sephadex G50 column, eluted with phosphate buffered saline (PBS) or Hank's balanced salt solution (HBS) and the liposome fraction was collected.

31、如项24或25所述的靶向热敏脂质体组合物的制备方法，其中所述对超声粉碎处理后的脂质体混悬液进行纯化是通过将超声粉碎处理后的脂质体离心并收集上清部分来实现的。31. The method for preparing the targeted thermosensitive liposome composition as described in item 24 or 25, wherein said liposome suspension after ultrasonic pulverization is purified by This was achieved by centrifuging the body and collecting the supernatant.

32、如项25所述的靶向热敏脂质体组合物的制备方法，其中所述恒温共孵育是在37度恒温水浴震荡器中进行的。32. The method for preparing the targeted thermosensitive liposome composition as described in item 25, wherein the co-incubation at constant temperature is carried out in a 37-degree constant temperature water bath shaker.

33、如项25所述的靶向热敏脂质体组合物的制备方法，其中所述对共孵育后的脂质体进行纯化是通过将共孵育后的脂质体再次通过SephadexG50柱，除去未包封的抗肿瘤药物并收集脂质体部分来实现的。33. The method for preparing the targeted thermosensitive liposome composition as described in item 25, wherein the liposomes after co-incubation are purified by passing the co-incubated liposomes through a SephadexG50 column again to remove Unencapsulated antineoplastic drugs are achieved by collecting the liposomal fraction.

34、如项23所述的靶向热敏脂质体组合物的制备方法，其中所述采用逆向蒸发法制备靶向热敏脂质体组合物包括下述步骤：34. The method for preparing the targeted thermosensitive liposome composition as described in item 23, wherein the preparation of the targeted thermosensitive liposome composition by reverse evaporation method comprises the following steps:

将DSPE-PEG-肿瘤归巢肽或DSPE-PEG-具有CREKA核心序列的肽段、热敏脂质体膜材和DSPE-PEG溶于有机溶剂(如氯仿、乙醚等)中，加入待包封的抗肿瘤药物的水溶液，其中抗肿瘤药物的水溶液与有机溶剂的体积比为抗肿瘤药物的水溶液∶有机溶剂＝1∶3～1∶6；Dissolve DSPE-PEG-tumor homing peptide or DSPE-PEG-peptide segment with CREKA core sequence, thermosensitive liposome membrane material and DSPE-PEG in an organic solvent (such as chloroform, ether, etc.), add The aqueous solution of the antineoplastic drug, wherein the volume ratio of the aqueous solution of the antineoplastic drug to the organic solvent is the aqueous solution of the antineoplastic drug:organic solvent=1:3～1:6;

对上述步骤形成的溶液进行超声，直至形成W/O型乳剂；The solution formed in the above steps is ultrasonicated until a W/O emulsion is formed;

减压蒸发溶剂至形成凝胶；The solvent was evaporated under reduced pressure to form a gel;

继续减压蒸发，从而得到靶向热敏脂质体组合物；或者在混匀器上机械振荡，使凝胶块破碎并转变成液体，减压蒸发挥去溶剂，从而得到靶向热敏脂质体组合物。Continue to evaporate under reduced pressure to obtain the targeted thermosensitive liposome composition; or mechanically vibrate on the mixer to break the gel block and turn it into a liquid, and evaporate under reduced pressure to remove the solvent, thereby obtaining the targeted thermosensitive liposome composition plastid composition.

35、如项1-3任一项所述的靶向热敏脂质体组合物在制备用于治疗恶性肿瘤的药物中的用途。35. Use of the targeted thermosensitive liposome composition according to any one of items 1-3 in the preparation of a medicament for treating malignant tumors.

36、如项35所述的用途，其中所述恶性肿瘤选自乳腺癌、前列腺癌、非小细胞肺癌和黑色素瘤。36. The use according to item 35, wherein the malignant tumor is selected from breast cancer, prostate cancer, non-small cell lung cancer and melanoma.

术语the term

“CREKA(cys-arg-glu-lys-ala)”表示一种由半胱氨酸、精氨酸、谷氨酸、赖氨酸和丙氨酸组成的肿瘤归巢肽。"CREKA (cys-arg-glu-lys-ala)" denotes a tumor-homing peptide composed of cysteine, arginine, glutamic acid, lysine and alanine.

“具有CREKA核心序列的肽段”表示核心序列为CREKA的肿瘤归巢肽。可提及的实例包括但不限于环状CREKA-CREKA(通过二硫键相连)。在本发明中，“CREKA”或“具有CREKA核心序列的肽段”用以修饰热敏脂质体，实现热敏脂质体的主动靶向。上述肽段是本领域的技术人员所容易设计和制备的。"Peptide segment with CREKA core sequence" means a tumor-homing peptide whose core sequence is CREKA. Examples that may be mentioned include, but are not limited to, cyclic CREKA-CREKA (linked by disulfide bonds). In the present invention, "CREKA" or "peptide segment having the core sequence of CREKA" is used to modify thermosensitive liposomes to realize active targeting of thermosensitive liposomes. The above peptides can be easily designed and prepared by those skilled in the art.

“DPPC”表示二棕榈酰磷脂酰胆碱。"DPPC" means dipalmitoylphosphatidylcholine.

“MSPC”表示1-肉豆蔻酰-2-硬脂酰磷脂酰胆碱。"MSPC" stands for 1-myristoyl-2-stearoylphosphatidylcholine.

“DSPG”表示二硬脂酰磷脂酰甘油。"DSPG" stands for distearoylphosphatidylglycerol.

“MPPC”表示肉豆蔻酰磷脂酰胆碱。"MPPC" means myristoylphosphatidylcholine.

“DSPC”表示二硬脂酰磷脂酰胆碱。"DSPC" means distearoylphosphatidylcholine.

“HSPC”表示氢化大豆卵磷脂。"HSPC" stands for hydrogenated soy lecithin.

“CHOL”表示胆固醇。"CHOL" means cholesterol.

“PBS”表示磷酸盐缓冲溶液。"PBS" means phosphate buffered saline.

“HBS”表示汉克平衡盐溶液。"HBS" means Hank's Balanced Salt Solution.

“DSPE-PEG-马来酰亚胺”表示1，2-硬脂酰基磷脂酰乙醇胺-聚乙二醇-马来酰亚胺。在本发明中，其用于与CREKA或具有CREKA核心序列的肽段反应构建肿瘤归巢肽导向化合物DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段。"DSPE-PEG-maleimide" means 1,2-stearylphosphatidylethanolamine-polyethylene glycol-maleimide. In the present invention, it is used to react with CREKA or a peptide having a CREKA core sequence to construct a tumor homing peptide targeting compound DSPE-PEG-CREKA or DSPE-PEG-peptide having a CREKA core sequence.

“DSPE-PEG”表示1，2-硬脂酰基磷脂酰乙醇胺-聚乙二醇，其是本领域用于对脂质体进行PEG化(即使脂质体表面修饰有亲水性的PEG分子从而实现脂质体的长循环效应)的常用物质。"DSPE-PEG" stands for 1,2-stearylphosphatidylethanolamine-polyethylene glycol, which is used in the art to PEGylate liposomes (i.e., liposomes are surface-modified with hydrophilic PEG molecules to thereby Commonly used substances to realize the long circulation effect of liposomes).

“母液”表示配制于适当溶剂中的抗肿瘤药物的溶液。"Mother solution" means a solution of an antineoplastic drug formulated in a suitable solvent.

“药脂比”表示：药物与脂材的质量比"Drug-to-lipid ratio" means: the mass ratio of drug to lipid material

“％”如无相反指示，“％”表示质量体积百分比。"%" If there is no contrary indication, "%" means mass volume percentage.

详细说明Detailed description

本发明的下列技术特征之间可以进行任意的组合，以形成不同的技术方案。The following technical features of the present invention can be combined arbitrarily to form different technical solutions.

本发明首先提供一种靶向热敏脂质体组合物，其特征在于所述组合物包括表面修饰有肿瘤归巢肽CREKA或具有CREKA核心序列的肽段的热敏脂质体，所述热敏脂质体中包含有抗肿瘤药物。The present invention firstly provides a targeted thermosensitive liposome composition, which is characterized in that the composition includes thermosensitive liposomes whose surface is modified with tumor-homing peptide CREKA or a peptide segment having a core sequence of CREKA, and the thermosensitive liposome Sensitive liposomes contain antineoplastic drugs.

在一个实施方案中，所述抗肿瘤药物选自阿霉素、表阿霉素、柔红霉素、紫杉醇、多西紫杉醇、吡柔比星、阿柔比星、顺铂、卡铂、奥沙利铂、甲氨蝶呤等中的一种或多种。In one embodiment, the antitumor drug is selected from the group consisting of adriamycin, epirubicin, daunorubicin, paclitaxel, docetaxel, pirarubicin, arubicin, cisplatin, carboplatin, One or more of saliplatin, methotrexate, etc.

在一个实施方案中，制成所述热敏脂质体的原料包括肿瘤归巢肽导向化合物DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段、热敏脂质体膜材、DSPE-PEG和抗肿瘤药物。In one embodiment, the raw materials for making the thermosensitive liposome include the tumor-homing peptide targeting compound DSPE-PEG-CREKA or DSPE-PEG-peptide segment with the core sequence of CREKA, thermosensitive liposome membrane material, DSPE-PEG and antineoplastic drugs.

在一个实施方案中，抗肿瘤药物的药脂比为1∶10w/w～1∶50w/w，例如1∶10w/w、1∶20w/w、1∶30w/w、1∶40w/w、1∶50w/w。In one embodiment, the drug-to-lipid ratio of the antineoplastic drug is 1:10w/w～1:50w/w, such as 1:10w/w, 1:20w/w, 1:30w/w, 1:40w/w , 1:50w/w.

在一个实施方案中，所述肿瘤归巢肽导向化合物DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段是通过将肿瘤归巢肽CREKA或具有CREKA核心序列的肽段与DSPE-PEG-马来酰亚胺溶解于液体介质中，调节液体介质的pH值至6.0-8.0范围内，持续搅拌反应，纯化反应所得物，经冷冻干燥得到的肿瘤归巢肽导向化合物DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段。In one embodiment, the tumor-homing peptide-targeting compound DSPE-PEG-CREKA or DSPE-PEG-peptide with a CREKA core sequence is obtained by combining the tumor-homing peptide CREKA or a peptide with a CREKA core sequence with DSPE- Dissolve PEG-maleimide in the liquid medium, adjust the pH value of the liquid medium to the range of 6.0-8.0, continue to stir the reaction, purify the reaction product, and freeze-dry the tumor-homing peptide-directing compound DSPE-PEG- CREKA or DSPE-PEG - Peptide with CREKA core sequence.

其中，优选调节液体介质的pH值至6.5-7.5范围内，更优选调节pH值至6.8-7.2的范围内，更优选调节pH值至6.9-7.1的范围，最优选调节至pH值为7.0。Among them, the pH value of the liquid medium is preferably adjusted to a range of 6.5-7.5, more preferably adjusted to a range of 6.8-7.2, more preferably adjusted to a range of 6.9-7.1, and most preferably adjusted to a pH value of 7.0.

所述液体介质为可将肿瘤归巢肽CREKA与DSPE-PEG-马来酰亚胺溶解于其中的任意种类的液体介质，例如水性溶剂和有机溶剂。适宜的溶剂是本领域的技术人员容易选择和确定的。The liquid medium is any kind of liquid medium in which the tumor-homing peptide CREKA and DSPE-PEG-maleimide can be dissolved, such as aqueous solvents and organic solvents. Suitable solvents are readily selected and determined by those skilled in the art.

所述反应优选是在惰性气体的保护下进行的。惰性气体的实例包括但不限于氮气、二氧化碳和惰性稀有气体，例如氖、氩、氪和氙。The reaction is preferably carried out under the protection of an inert gas. Examples of inert gases include, but are not limited to, nitrogen, carbon dioxide, and inert noble gases such as neon, argon, krypton, and xenon.

所述反应优选是在环境温度下进行的，更优选在15-35℃的温度进行，更优选在20-30℃的温度进行，最优选在25℃的温度进行。The reaction is preferably carried out at ambient temperature, more preferably at a temperature of 15-35°C, more preferably at a temperature of 20-30°C, most preferably at a temperature of 25°C.

所述搅拌的时间优选为24小时以上。The stirring time is preferably more than 24 hours.

所述纯化反应所得物的步骤优选是通过对反应所得物进行透析的方法来实现的。The step of purifying the reaction product is preferably achieved by dialysis of the reaction product.

在一个实施方案中，所述热敏脂质体膜材包括DPPC(二棕榈酰磷脂酰胆碱)和DSPC(二硬脂酰磷脂酰胆碱)。In one embodiment, the thermosensitive liposome membrane material includes DPPC (dipalmitoylphosphatidylcholine) and DSPC (distearoylphosphatidylcholine).

在一个实施方案中，所述热敏脂质体膜材包括DPPC、HSPC(氢化大豆卵磷脂)和CHOL(胆固醇)。In one embodiment, the thermosensitive liposome membrane material includes DPPC, HSPC (hydrogenated soybean lecithin) and CHOL (cholesterol).

在一个实施方案中，所述热敏脂质体膜材包括DPPC及MSPC(1-肉豆蔻酰-2-硬脂酰磷脂酰胆碱)。In one embodiment, the thermosensitive liposome membrane material includes DPPC and MSPC (1-myristoyl-2-stearoylphosphatidylcholine).

在一个实施方案中，所述热敏脂质体膜材包括DPPC、MSPC和DSPG(二硬脂酰磷脂酰甘油)。In one embodiment, the thermosensitive liposome membrane material includes DPPC, MSPC and DSPG (distearoylphosphatidylglycerol).

在一个实施方案中，所述热敏脂质体膜材包括DPPC和MPPC(肉豆蔻酰磷脂酰胆碱)。In one embodiment, the thermosensitive liposome membrane material includes DPPC and MPPC (myristoylphosphatidylcholine).

在一个实施方案中，制成所述热敏脂质体的原料包括DPPC、DSPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段，其中DPPC、DSPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段之间的摩尔比为DPPC∶DSPC∶DSPE-PEG∶DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段＝60～100∶25～5∶0.2～10∶0.2～10，优选70～90∶20～10∶0.5～5∶0.5～5，例如80∶19∶0.5∶0.5、80∶18∶1∶1、80∶17∶1.5∶1.5、80∶16∶2∶2、80∶15∶2.5∶2.5、80∶14∶3∶3和80∶10∶5∶5。In one embodiment, the raw materials for making the thermosensitive liposomes include DPPC, DSPC, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-peptide segment with CREKA core sequence, wherein DPPC, DSPC, DSPE - PEG, DSPE-PEG-CREKA or DSPE-PEG - the molar ratio between the peptides with the CREKA core sequence is DPPC: DSPC: DSPE-PEG: DSPE-PEG-CREKA or DSPE-PEG - the peptide with the CREKA core sequence Segment = 60-100: 25-5: 0.2-10: 0.2-10, preferably 70-90: 20-10: 0.5-5: 0.5-5, for example 80: 19: 0.5: 0.5, 80: 18: 1: 1. 80:17:1.5:1.5, 80:16:2:2, 80:15:2.5:2.5, 80:14:3:3 and 80:10:5:5.

在一个实施方案中，制成所述热敏脂质体的原料包括DPPC、HSPC、CHOL、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段，其中DPPC、HSPC、CHOL、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段之间的摩尔比为DPPC∶HSPC∶CHOL∶DSPE-PEG∶DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段＝90～110∶40～60∶20～40∶1～6∶1～6，例如95～105∶45～55∶25～35∶2～5∶2～5，优选100∶50∶30∶3∶3。In one embodiment, the raw materials for making the thermosensitive liposomes include DPPC, HSPC, CHOL, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-peptide segment with CREKA core sequence, wherein DPPC, HSPC , CHOL, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-the molar ratio between peptides with CREKA core sequence is DPPC:HSPC:CHOL:DSPE-PEG:DSPE-PEG-CREKA or DSPE-PEG- Peptide segment with CREKA core sequence = 90-110: 40-60: 20-40: 1-6: 1-6, such as 95-105: 45-55: 25-35: 2-5: 2-5, preferably 100:50:30:3:3.

在一个实施方案中，制成所述热敏脂质体的原料包括DPPC、MSPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段，其中DPPC、MSPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段之间的摩尔比为DPPC∶MSPC∶DSPE-PEG∶DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段＝80～120∶0.5～20∶0.2～5∶0.2～5，优选85～100∶1～10∶0.5～2.5∶0.5～2.5，更优选86∶10∶2∶2、90∶10∶2∶2、95∶5∶2∶2、97.5∶2.5∶2∶2、99∶1∶2∶2、89∶10∶2.5∶2.5、90∶10∶2∶2、91∶10∶1.5∶1.5、92∶10∶1∶1和93∶10∶0.5∶0.5。In one embodiment, the raw materials for making the thermosensitive liposomes include DPPC, MSPC, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-peptide segment with CREKA core sequence, wherein DPPC, MSPC, DSPE - PEG, DSPE-PEG-CREKA or DSPE-PEG - the molar ratio between the peptides with the CREKA core sequence is DPPC: MSPC: DSPE-PEG: DSPE-PEG-CREKA or DSPE-PEG - the peptide with the CREKA core sequence Segment=80~120: 0.5~20: 0.2~5: 0.2~5, preferably 85~100: 1~10: 0.5~2.5: 0.5~2.5, more preferably 86:10:2:2, 90:10:2 :2, 95:5:2:2, 97.5:2.5:2:2, 99:1:2:2, 89:10:2.5:2.5, 90:10:2:2, 91:10:1.5:1.5 , 92:10:1:1 and 93:10:0.5:0.5.

在一个实施方案中，制成所述热敏脂质体的原料包括DPPC、MSPC、DSPG、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段，其中DPPC、MSPC、DSPG、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段之间的摩尔比为DPPC∶MSPC∶DSPG∶DSPE-PEG∶DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段＝60～100∶2～20∶2～20∶0.5～5∶0.5～5，优选70～90∶6～10∶8～12∶1～3∶1～3，更优选82∶8∶10∶2∶2。In one embodiment, the raw materials for making the thermosensitive liposomes include DPPC, MSPC, DSPG, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-peptide segment with CREKA core sequence, wherein DPPC, MSPC , DSPG, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-the molar ratio between peptides with CREKA core sequence is DPPC: MSPC: DSPG: DSPE-PEG: DSPE-PEG-CREKA or DSPE-PEG- Peptide segment with CREKA core sequence = 60-100: 2-20: 2-20: 0.5-5: 0.5-5, preferably 70-90: 6-10: 8-12: 1-3: 1-3, more Preferably 82:8:10:2:2.

在一个实施方案中，制成所述热敏脂质体的原料包括DPPC、MPPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段，其中DPPC、MPPC、DSPE-PEG、DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段之间的摩尔比为DPPC∶MPPC∶DSPE-PEG∶DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段＝70～110∶2～20∶0.5～5∶0.5～5，优选80～100∶8～12∶1～3∶1～3，更优选90∶10∶2∶2。In one embodiment, the raw materials for making the thermosensitive liposomes include DPPC, MPPC, DSPE-PEG, DSPE-PEG-CREKA or DSPE-PEG-peptide segment with CREKA core sequence, wherein DPPC, MPPC, DSPE - PEG, DSPE-PEG-CREKA or DSPE-PEG - the molar ratio between peptides with CREKA core sequence is DPPC:MPPC:DSPE-PEG:DSPE-PEG-CREKA or DSPE-PEG-peptide with CREKA core sequence Segment=70-110:2-20:0.5-5:0.5-5, preferably 80-100:8-12:1-3:1-3, more preferably 90:10:2:2.

所述的靶向热敏脂质体组合物可以进一步制成药物剂型，例如注射剂、输液、冻干粉针剂或喷雾剂。The targeted thermosensitive liposome composition can be further made into pharmaceutical dosage forms, such as injection, infusion, freeze-dried powder injection or spray.

本发明的另一个方面提供所述靶向热敏脂质体组合物的制备方法，包括如下步骤：Another aspect of the present invention provides a method for preparing the targeted thermosensitive liposome composition, comprising the steps of:

在一个实施方案中，所述采用薄膜分散法制备靶向热敏脂质体组合物包括下述步骤：In one embodiment, the preparation of targeted thermosensitive liposome composition by thin film dispersion method comprises the following steps:

其中，所述除去有机溶剂优选是通过在一定温度的水浴条件下(例如30-60℃)减压旋转蒸发来实现的。Wherein, the removal of the organic solvent is preferably achieved by rotary evaporation under reduced pressure in a water bath at a certain temperature (for example, 30-60° C.).

所述水化处理优选是通过对缓冲液进行涡旋震荡和/或超声处理来实现的。The hydration treatment is preferably achieved by vortexing and/or sonicating the buffer solution.

所述任选含有抗肿瘤药物的缓冲液优选为任选含有抗肿瘤药物的硫酸铵缓冲液或pH＝4的柠檬酸缓冲液。The buffer optionally containing anti-tumor drugs is preferably ammonium sulfate buffer or pH=4 citrate buffer optionally containing anti-tumor drugs.

所述对粗脂质体混悬液进行超声粉碎处理优选是利用探头超声的超声波细胞粉碎机来实现的。The ultrasonic pulverization treatment of the coarse liposome suspension is preferably realized by using an ultrasonic cell pulverizer with ultrasonic probe.

所述对超声粉碎处理后的脂质体混悬液进行纯化优选是通过将超声粉碎处理后的脂质体混悬液通过Sephadex G50柱，用磷酸盐缓冲溶液(PBS)或汉克平衡盐溶液(HBS)洗脱并收集脂质体部分来实现的。Purifying the liposome suspension after ultrasonic crushing is preferably by passing the liposome suspension after ultrasonic crushing through Sephadex G50 column, using phosphate buffered saline (PBS) or Hank's balanced salt solution (HBS) was eluted and the liposome fraction was collected.

所述对超声粉碎处理后的脂质体混悬液进行纯化优选是通过将超声粉碎处理后的脂质体离心并收集上清部分来实现的。The purification of the liposome suspension after ultrasonic crushing is preferably achieved by centrifuging the liposomes after ultrasonic crushing and collecting the supernatant.

所述恒温共孵育优选是在37度恒温水浴震荡器中进行的。The constant temperature co-incubation is preferably carried out in a 37 degree constant temperature water bath shaker.

所述对共孵育后的脂质体进行纯化优选是通过将共孵育后的脂质体再次通过Sephadex G50柱，除去未包封的抗肿瘤药物并收集脂质体部分来实现的。Purifying the co-incubated liposomes is preferably achieved by passing the co-incubated liposomes through a Sephadex G50 column again to remove unencapsulated antitumor drugs and collecting liposomes.

在一个实施方案中，所述采用逆向蒸发法制备靶向热敏脂质体组合物包括下述步骤：In one embodiment, the preparation of targeted thermosensitive liposome composition by reverse evaporation method comprises the following steps:

优选的，本发明提供一种所述靶向热敏脂质体组合物的制备方法，所述热敏脂质体中包含水溶性药物(例如阿霉素)，所述方法包括如下步骤：Preferably, the present invention provides a method for preparing the targeted thermosensitive liposome composition, wherein the thermosensitive liposome contains water-soluble drugs (such as doxorubicin), and the method comprises the following steps:

将肿瘤归巢肽导向化合物DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段、热敏脂质体膜材和DSPE-PEG置于茄形瓶中，加入有机溶剂将其溶解，在45℃水浴条件下减压旋转蒸发，形成均匀的透明薄膜；向茄形瓶中加入硫酸铵溶液或pH＝4的柠檬酸缓冲液，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈现蓝色乳光；将所得脂质体过Sephadex G50柱，用PBS或用pH＝7的HBS，收集脂质体部分，得空白脂质体；将水溶性抗肿瘤药物(例如阿霉素)溶于少量蒸馏水中，与空白脂质体在37度恒温水浴震荡器中共孵育，再次过Sephadex G50柱，除去未包裹的水溶性抗肿瘤药物(例如阿霉素)，收集脂质体部分，即得靶向热敏脂质体组合物。Put the tumor-homing peptide-directing compound DSPE-PEG-CREKA or DSPE-PEG-peptide segment with CREKA core sequence, thermosensitive liposome membrane material and DSPE-PEG in an eggplant-shaped bottle, add an organic solvent to dissolve it, Under reduced pressure and rotary evaporation in a water bath at 45°C, a uniform transparent film is formed; add ammonium sulfate solution or citric acid buffer solution with pH = 4 to the eggplant-shaped bottle, vortex, and ultrasonically until the lipid film is completely peeled off and dissolved. Ultrasonic cell pulverizer with ultrasonic probe sonicates the sample until it presents blue opalescence; passes the obtained liposomes through a Sephadex G50 column, collects the liposome part with PBS or HBS with pH=7, and obtains blank liposomes; Water-soluble antitumor drugs (such as doxorubicin) were dissolved in a small amount of distilled water, co-incubated with blank liposomes in a 37-degree constant temperature water bath shaker, and passed through a Sephadex G50 column again to remove unencapsulated water-soluble antitumor drugs (such as adriamycin). Mycin), the liposome part is collected, and the targeted thermosensitive liposome composition is obtained.

还优选的，本发明提供一种所述靶向热敏脂质体组合物的制备方法，所述热敏脂质体中包含脂溶性药物，所述方法包括如下步骤：Also preferably, the present invention provides a method for preparing the targeted thermosensitive liposome composition, wherein the thermosensitive liposome contains fat-soluble drugs, and the method comprises the following steps:

将肿瘤归巢肽导向化合物DSPE-PEG-CREKA或DSPE-PEG-具有CREKA核心序列的肽段、热敏脂质体膜材和DSPE-PEG置于茄形瓶中，加入有机溶剂将其溶解，再加入脂溶性抗肿瘤药物母液，在45℃水浴条件下减压旋转蒸发，形成均匀的透明薄膜；向茄形瓶中加入预热(45℃)的PBS或pH＝7的HBS溶液，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈蓝色乳光；将所得脂质体离心，收集上清部分，即得靶向热敏脂质体组合物。Put the tumor-homing peptide-directing compound DSPE-PEG-CREKA or DSPE-PEG-peptide segment with CREKA core sequence, thermosensitive liposome membrane material and DSPE-PEG in an eggplant-shaped bottle, add an organic solvent to dissolve it, Then add the fat-soluble antineoplastic drug mother solution, and evaporate under reduced pressure in a water bath at 45°C to form a uniform transparent film; add preheated (45°C) PBS or HBS solution with pH=7 to the eggplant-shaped bottle, and vortex Vibrate and sonicate until the lipid membrane is completely detached and dissolved, sonicate the sample with an ultrasonic cell pulverizer with a probe sonication until it turns blue opalescent; centrifuge the obtained liposomes and collect the supernatant to obtain targeted thermosensitive liposomes combination.

本发明的另一个方面提供所述靶向热敏脂质体组合物在制备用于治疗恶性肿瘤的药物中的用途。Another aspect of the present invention provides the use of the targeted thermosensitive liposome composition in the preparation of medicines for treating malignant tumors.

在一个实施方案中，所述恶性肿瘤选自乳腺癌、前列腺癌、非小细胞肺癌和黑色素瘤。In one embodiment, the malignancy is selected from breast cancer, prostate cancer, non-small cell lung cancer and melanoma.

本发明的优点和/或能够产生的效果(特别是预料不到的技术效果)在于：The advantages of the present invention and/or the effect that can produce (especially unexpected technical effect) are:

将热敏脂质体在局部加热条件下大量释放所载药物所产生的物理化学靶向与肿瘤归巢肽CREKA的主动靶向相结合，起到协同抗肿瘤作用，对肿瘤的杀伤力更强，对正常组织的毒副作用更低。Combining the physicochemical targeting produced by the mass release of the loaded drug by the thermosensitive liposome under local heating conditions and the active targeting of the tumor-homing peptide CREKA, it has a synergistic anti-tumor effect and is more lethal to tumors , less toxic and side effects on normal tissues.

附图说明：Description of drawings:

图1为阿霉素摄取(流式细胞仪)实验结果Figure 1 is the experimental results of doxorubicin uptake (flow cytometry)

图2为阿霉素摄取(激光共聚焦)实验结果Figure 2 shows the experimental results of doxorubicin uptake (laser confocal)

图3为药效实验结果Figure 3 is the result of drug efficacy experiment

具体实施方式：Detailed ways:

实施例1Example 1

肿瘤归巢肽CREKA与DSPE-PEG2000-马来酰亚胺的连接Linkage of tumor-homing peptide CREKA to DSPE-PEG2000-maleimide

称取CREKA(1.1mg)、DSPE-PEG2000-马来酰亚胺(5mg)，即CREKA与DSPE-PEG2000-马来酰亚胺的摩尔比为2∶1，溶解于3ml PBS中(即反应物浓度为2mg/ml)，调节pH为7，在氮气保护下，室温搅拌24小时。取上述反应所得物料置于截留分子量为2000的透析袋中透析48小时，经冷冻干燥得到DSPE-PEG2000-CREKA。Weigh CREKA (1.1mg), DSPE-PEG2000-maleimide (5mg), that is, the molar ratio of CREKA and DSPE-PEG2000-maleimide is 2: 1, dissolve in 3ml PBS (i.e. reactant concentration of 2 mg/ml), adjust the pH to 7, and stir at room temperature for 24 hours under the protection of nitrogen. The material obtained from the above reaction was placed in a dialysis bag with a molecular weight cut off of 2000 for dialysis for 48 hours, and then freeze-dried to obtain DSPE-PEG2000-CREKA.

实施例2Example 2

称取CREKA(1.1mg)、DSPE-PEG2000-马来酰亚胺(10mg)，即CREKA与DSPE-PEG2000-马来酰亚胺的摩尔比为1∶1，溶解于5.55ml PBS中(即反应物浓度为2mg/ml)，调节pH为7，在氮气保护下，室温搅拌24小时。取上述反应所得物料置于截留分子量为2000的透析袋中透析48小时，经冷冻干燥得到DSPE-PEG2000-CREKA。Weigh CREKA (1.1mg), DSPE-PEG2000-maleimide (10mg), that is, the molar ratio of CREKA and DSPE-PEG2000-maleimide is 1: 1, dissolve in 5.55ml PBS (i.e. the reaction concentration of 2 mg/ml), adjust the pH to 7, and stir at room temperature for 24 hours under the protection of nitrogen. The material obtained from the above reaction was placed in a dialysis bag with a molecular weight cut off of 2000 for dialysis for 48 hours, and then freeze-dried to obtain DSPE-PEG2000-CREKA.

实施例3Example 3

称取CREKA(1.1mg)、DSPE-PEG2000-马来酰亚胺(5mg)，即CREKA与DSPE-PEG2000-马来酰亚胺的摩尔比为2∶1，溶解于6.1ml PBS中(即反应物浓度为1mg/ml)，调节pH为7，在氮气保护下，室温搅拌24小时。取上述反应所得物料置于截留分子量为2000的透析袋中透析48小时，经冷冻干燥得到DSPE-PEG2000-CREKA。Weigh CREKA (1.1mg), DSPE-PEG2000-maleimide (5mg), that is, the molar ratio of CREKA and DSPE-PEG2000-maleimide is 2:1, dissolve in 6.1ml of PBS (i.e. the reaction concentration of 1 mg/ml), adjust the pH to 7, and stir at room temperature for 24 hours under the protection of nitrogen. The material obtained from the above reaction was placed in a dialysis bag with a molecular weight cut off of 2000 for dialysis for 48 hours, and then freeze-dried to obtain DSPE-PEG2000-CREKA.

实施例4Example 4

称取CREKA(1.1mg)、DSPE-PEG2000-马来酰亚胺(5mg)，即CREKA与DSPE-PEG2000-马来酰亚胺的摩尔比为2∶1，溶解于2ml PBS中(即反应物浓度为3mg/ml)，调节pH为7，在氮气保护下，室温搅拌24小时。取上述反应所得物料置于截留分子量为2000的透析袋中透析48小时，经冷冻干燥得到DSPE-PEG2000-CREKA。Weigh CREKA (1.1mg), DSPE-PEG2000-maleimide (5mg), that is, the molar ratio of CREKA and DSPE-PEG2000-maleimide is 2: 1, dissolve in 2ml of PBS (i.e. reactant The concentration is 3 mg/ml), adjust the pH to 7, and stir at room temperature for 24 hours under the protection of nitrogen. The material obtained from the above reaction was placed in a dialysis bag with a molecular weight cut off of 2000 for dialysis for 48 hours, and then freeze-dried to obtain DSPE-PEG2000-CREKA.

实施例5Example 5

称取CREKA(1.1mg)、DSPE-PEG2000-马来酰亚胺(5mg)，即CREKA与DSPE-PEG2000-马来酰亚胺的摩尔比为2∶1，溶解于1.22ml PBS中(即反应物浓度为5mg/ml)，调节pH为7，在氮气保护下，室温搅拌24小时。取上述反应所得物料置于截留分子量为2000的透析袋中透析48小时，经冷冻干燥得到DSPE-PEG2000-CREKA。Weigh CREKA (1.1mg), DSPE-PEG2000-maleimide (5mg), that is, the molar ratio of CREKA and DSPE-PEG2000-maleimide is 2: 1, dissolve in 1.22ml PBS (i.e. the reaction concentration of 5 mg/ml), adjust the pH to 7, and stir at room temperature for 24 hours under the protection of nitrogen. The material obtained from the above reaction was placed in a dialysis bag with a molecular weight cut off of 2000 for dialysis for 48 hours, and then freeze-dried to obtain DSPE-PEG2000-CREKA.

实施例6Example 6

称取CREKA(1.1mg)、DSPE-PEG2000-马来酰亚胺(5mg)，即CREKA与DSPE-PEG2000-马来酰亚胺的摩尔比为2∶1，溶解于3ml DMF中(即反应物浓度为2mg/ml)，用三乙胺调节pH为7，在氮气保护下，室温搅拌24小时。取上述反应所得物料置于截留分子量为2000的透析袋中透析48小时，经冷冻干燥得到DSPE-PEG2000-CREKA。Take CREKA (1.1mg), DSPE-PEG2000-maleimide (5mg), that is, the molar ratio of CREKA and DSPE-PEG2000-maleimide is 2: 1, dissolve in 3ml DMF (i.e. reactant The concentration is 2 mg/ml), the pH is adjusted to 7 with triethylamine, and stirred at room temperature for 24 hours under the protection of nitrogen. The material obtained from the above reaction was placed in a dialysis bag with a molecular weight cut off of 2000 for dialysis for 48 hours, and then freeze-dried to obtain DSPE-PEG2000-CREKA.

实施例7Example 7

CREKA靶向的阿霉素热敏脂质体的构建Construction of Doxorubicin Thermosensitive Liposomes Targeted by CREKA

称量DPPC、MSPC、DSPE-PEG和DSPE-PEG-CREKA，置于茄形瓶中，加入异丙醇溶解，45度水浴条件下减压旋转蒸发，成均匀的透明薄膜。向茄形瓶中加入pH＝4的柠檬酸缓冲液，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈蓝色乳光。将所得脂质体过Sephadex G50柱，用HBS(pH＝7)洗脱，收集脂质体部分，得空白脂质体，向空白脂质体中加入2mg/ml的阿霉素母液0.5ml，使药脂比为1∶20m/m，37度恒温水浴震荡器中共孵育，再次过Sephadex G50柱，除去未包裹的游离阿霉素，收集脂质体部分，即得最终阿霉素靶向脂质体。Weigh DPPC, MSPC, DSPE-PEG and DSPE-PEG-CREKA, place them in an eggplant-shaped bottle, add isopropanol to dissolve, and evaporate under reduced pressure in a 45-degree water bath to form a uniform transparent film. Add pH=4 citric acid buffer solution to the eggplant-shaped flask, vortex, and sonicate until the lipid film is completely detached and dissolved, and sonicate the sample until it turns blue opalescent using an ultrasonic cell pulverizer with a probe sonication. Gained liposome is passed through Sephadex G50 column, elutes with HBS (pH=7), collects liposome part, gets blank liposome, adds the doxorubicin mother liquor 0.5ml of 2mg/ml in blank liposome, Make the drug-lipid ratio 1:20m/m, co-incubate with a 37-degree constant temperature water bath shaker, pass through the Sephadex G50 column again, remove unencapsulated free doxorubicin, collect the liposome part, and obtain the final doxorubicin-targeted lipid plastid.

实施例8Example 8

称量DPPC、MSPC、DSPE-PEG和DSPE-PEG-CREKA，置于茄形瓶中，加入二氯甲烷溶解，45度水浴条件下减压旋转蒸发，成均匀的透明薄膜。向茄形瓶中加入pH＝4的柠檬酸缓冲液，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈蓝色乳光。将所得脂质体过Sephadex G50柱，用HBS(pH＝7)洗脱，收集脂质体部分，得空白脂质体，向空白脂质体中加入2mg/ml的阿霉素母液0.5ml，使药脂比为1∶20m/m，37度恒温水浴震荡器中共孵育，再次过Sephadex G50柱，除去未包裹的游离阿霉素，收集脂质体部分，即得最终阿霉素靶向脂质体。Weigh DPPC, MSPC, DSPE-PEG and DSPE-PEG-CREKA, put them in an eggplant-shaped bottle, add dichloromethane to dissolve, and evaporate under reduced pressure in a 45-degree water bath to form a uniform transparent film. Add pH=4 citric acid buffer solution to the eggplant-shaped flask, vortex, and sonicate until the lipid film is completely detached and dissolved, and sonicate the sample until it turns blue opalescent using an ultrasonic cell pulverizer with a probe sonication. Gained liposome is passed through Sephadex G50 column, elutes with HBS (pH=7), collects liposome part, gets blank liposome, adds the doxorubicin mother liquor 0.5ml of 2mg/ml in blank liposome, Make the drug-lipid ratio 1:20m/m, co-incubate with a 37-degree constant temperature water bath shaker, pass through the Sephadex G50 column again, remove unencapsulated free doxorubicin, collect the liposome part, and obtain the final doxorubicin-targeted lipid plastid.

实施例9Example 9

称量DPPC、MSPC、DSPE-PEG和DSPE-PEG-CREKA，置于茄形瓶中，加入氯仿溶解，45度水浴条件下减压旋转蒸发，成均匀的透明薄膜。向茄形瓶中加入pH＝4的柠檬酸缓冲液，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈蓝色乳光。将所得脂质体过Sephadex G50柱，用HBS(pH＝7)洗脱，收集脂质体部分，得空白脂质体，向空白脂质体中加入2mg/ml的阿霉素母液0.5ml，使药脂比为1∶20m/m，37度恒温水浴震荡器中共孵育，再次过Sephadex G50柱，除去未包裹的游离阿霉素，收集脂质体部分，即得最终阿霉素靶向脂质体。Weigh DPPC, MSPC, DSPE-PEG and DSPE-PEG-CREKA, place them in an eggplant-shaped bottle, add chloroform to dissolve, and evaporate under reduced pressure in a 45-degree water bath to form a uniform transparent film. Add pH=4 citric acid buffer solution to the eggplant-shaped flask, vortex, and sonicate until the lipid film is completely detached and dissolved, and sonicate the sample until it turns blue opalescent using an ultrasonic cell pulverizer with a probe sonication. Gained liposome is passed through Sephadex G50 column, elutes with HBS (pH=7), collects liposome part, gets blank liposome, adds the doxorubicin mother liquor 0.5ml of 2mg/ml in blank liposome, Make the drug-lipid ratio 1:20m/m, co-incubate with a 37-degree constant temperature water bath shaker, pass through the Sephadex G50 column again, remove unencapsulated free doxorubicin, collect the liposome part, and obtain the final doxorubicin-targeted lipid plastid.

实施例10Example 10

称量DPPC、MSPC、DSPE-PEG和DSPE-PEG-CREKA，置于茄形瓶中，加入异丙醇溶解，45度水浴条件下减压旋转蒸发，成均匀的透明薄膜。向茄形瓶中加入pH＝4的柠檬酸缓冲液，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈蓝色乳光。将所得脂质体过Sephadex G50柱，用HBS(pH＝7)洗脱，收集脂质体部分，得空白脂质体，向空白脂质体中加入2mg/ml的阿霉素母液1ml，使药脂比为1∶10m/m，37度恒温水浴震荡器中共孵育，再次过Sephadex G50柱，除去未包裹的游离阿霉素，收集脂质体部分，即得最终阿霉素靶向脂质体。Weigh DPPC, MSPC, DSPE-PEG and DSPE-PEG-CREKA, place them in an eggplant-shaped bottle, add isopropanol to dissolve, and evaporate under reduced pressure in a 45-degree water bath to form a uniform transparent film. Add pH=4 citric acid buffer solution to the eggplant-shaped flask, vortex, and sonicate until the lipid film is completely detached and dissolved, and sonicate the sample until it turns blue opalescent using an ultrasonic cell pulverizer with a probe sonication. Gained liposomes are passed through Sephadex G50 column, elute with HBS (pH=7), collect liposome part, get blank liposome, add 1ml of doxorubicin mother solution of 2mg/ml in blank liposome, make The drug-lipid ratio is 1:10m/m, co-incubated in a 37-degree constant temperature water bath shaker, and passed through a Sephadex G50 column again to remove unencapsulated free doxorubicin, and collect the liposome part to obtain the final doxorubicin-targeted lipid body.

实施例11Example 11

称量DPPC、MSPC、DSPE-PEG和DSPE-PEG-CREKA，置于茄形瓶中，加入异丙醇溶解，45度水浴条件下减压旋转蒸发，成均匀的透明薄膜。向茄形瓶中加入pH＝4的柠檬酸缓冲液，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈蓝色乳光。将所得脂质体过Sephadex G50柱，用HBS(pH＝7)洗脱，收集脂质体部分，得空白脂质体，向空白脂质体中加入2mg/ml的阿霉素母液0.25ml，使药脂比为1∶40m/m，37度恒温水浴震荡器中共孵育，再次过Sephadex G50柱，除去未包裹的游离阿霉素，收集脂质体部分，即得最终阿霉素靶向脂质体。Weigh DPPC, MSPC, DSPE-PEG and DSPE-PEG-CREKA, place them in an eggplant-shaped bottle, add isopropanol to dissolve, and evaporate under reduced pressure in a 45-degree water bath to form a uniform transparent film. Add pH=4 citric acid buffer solution to the eggplant-shaped flask, vortex, and sonicate until the lipid film is completely detached and dissolved, and sonicate the sample until it turns blue opalescent using an ultrasonic cell pulverizer with a probe sonication. Gained liposome is passed through Sephadex G50 column, elutes with HBS (pH=7), collects liposome part, obtains blank liposome, adds the doxorubicin mother liquor 0.25ml of 2mg/ml in blank liposome, Make the drug-lipid ratio 1:40m/m, co-incubate with a 37-degree constant temperature water bath shaker, pass through the Sephadex G50 column again, remove unencapsulated free doxorubicin, collect the liposome part, and obtain the final doxorubicin-targeted lipid plastid.

实施例12Example 12

称量DPPC、MSPC、DSPE-PEG和DSPE-PEG-CREKA，置于茄形瓶中，加入异丙醇溶解，45度水浴条件下减压旋转蒸发，成均匀的透明薄膜。向茄形瓶中加入硫酸铵溶液(123mM，pH5.4)，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈蓝色乳光。将所得脂质体过Sephadex G50柱，用PBS洗脱，收集脂质体部分，得空白脂质体，向空白脂质体中加入2mg/ml的阿霉素母液0.5ml，使药脂比为1∶20m/m，37度恒温水浴震荡器中共孵育，再次过Sephadex G50柱，除去未包裹的游离阿霉素，收集脂质体部分，即得最终阿霉素靶向脂质体。Weigh DPPC, MSPC, DSPE-PEG and DSPE-PEG-CREKA, place them in an eggplant-shaped bottle, add isopropanol to dissolve, and evaporate under reduced pressure in a 45-degree water bath to form a uniform transparent film. Add ammonium sulfate solution (123mM, pH 5.4) into the eggplant-shaped bottle, vortex, and sonicate until the lipid film is completely detached and dissolved, and sonicate the sample until it turns blue opalescent using an ultrasonic cell pulverizer with a probe sonication. Gained liposome is crossed Sephadex G50 column, elutes with PBS, collects liposome part, obtains blank liposome, adds the doxorubicin mother liquor 0.5ml of 2mg/ml in blank liposome, makes medicine lipid ratio be 1:20m/m, co-incubated in a 37-degree constant temperature water bath shaker, passed through a Sephadex G50 column again to remove unencapsulated free doxorubicin, and collected the liposome part to obtain the final doxorubicin-targeted liposome.

实施例13Example 13

CREKA靶向的紫杉醇热敏脂质体的构建Construction of paclitaxel thermosensitive liposomes targeted by CREKA

称量DPPC、MSPC、DSPE-PEG和DSPE-PEG-CREKA，置于茄形瓶中，加入氯仿溶解，再加入2mg/ml乙腈溶解的紫杉醇母液0.5ml，使药脂比为1∶20m/m，45度水浴条件下减压旋转蒸发，成均匀的透明薄膜。向茄形瓶中加入2ml 45度预热的5％葡萄糖溶液，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈蓝色乳光。将所得脂质体离心，12000rpm，5min，收集上清部分，即得最终紫杉醇靶向热敏脂质体。Weigh DPPC, MSPC, DSPE-PEG and DSPE-PEG-CREKA, put them in an eggplant-shaped bottle, add chloroform to dissolve, then add 0.5ml of paclitaxel mother solution dissolved in 2mg/ml acetonitrile, so that the ratio of drug to lipid is 1:20m/m , Rotary evaporation under reduced pressure under the condition of 45 degree water bath to form a uniform transparent film. Add 2ml of 5% glucose solution preheated at 45°C to the eggplant-shaped bottle, vortex, and sonicate until the lipid film is completely detached and dissolved. Use an ultrasonic cell pulverizer with a probe sonicator to sonicate the sample until it turns blue opalescent. The obtained liposome was centrifuged at 12000 rpm for 5 min, and the supernatant was collected to obtain the final paclitaxel-targeted thermosensitive liposome.

实施例14Example 14

称量DPPC、MSPC、DSPE-PEG和DSPE-PEG-CREKA，置于茄形瓶中，加入氯仿溶解，再加入2mg/ml乙腈溶解的紫杉醇母液0.75ml，使药脂比为1∶30m/m，45度水浴条件下减压旋转蒸发，成均匀的透明薄膜。向茄形瓶中加入2ml 45度预热的5％葡萄糖溶液，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈蓝色乳光。将所得脂质体离心，12000rpm，5min，收集上清部分，即得最终紫杉醇靶向热敏脂质体。Weigh DPPC, MSPC, DSPE-PEG and DSPE-PEG-CREKA, put them in an eggplant-shaped bottle, add chloroform to dissolve, then add 0.75ml of paclitaxel mother solution dissolved in 2mg/ml acetonitrile, so that the drug-to-lipid ratio is 1:30m/m , Rotary evaporation under reduced pressure under the condition of 45 degree water bath to form a uniform transparent film. Add 2ml of 5% glucose solution preheated at 45°C to the eggplant-shaped bottle, vortex, and sonicate until the lipid film is completely detached and dissolved. Use an ultrasonic cell pulverizer with a probe sonicator to sonicate the sample until it turns blue opalescent. The obtained liposome was centrifuged at 12000 rpm for 5 min, and the supernatant was collected to obtain the final paclitaxel-targeted thermosensitive liposome.

实施例15Example 15

称量DPPC、MSPC、DSPE-PEG和DSPE-PEG-CREKA，置于茄形瓶中，加入氯仿溶解，再加入2mg/ml的乙腈溶解的紫杉醇母液1ml，使药脂比为1∶40m/m，45度水浴条件下减压旋转蒸发，成均匀的透明薄膜。向茄形瓶中加入2ml 45度预热的5％葡萄糖溶液，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈蓝色乳光。将所得脂质体离心，12000rpm，5min，收集上清部分，即得最终紫杉醇靶向热敏脂质体。Weigh DPPC, MSPC, DSPE-PEG and DSPE-PEG-CREKA, place them in an eggplant-shaped bottle, add chloroform to dissolve, then add 1ml of paclitaxel mother solution dissolved in 2mg/ml acetonitrile, so that the ratio of drug to fat is 1:40m/m , Rotary evaporation under reduced pressure under the condition of 45 degree water bath to form a uniform transparent film. Add 2ml of 5% glucose solution preheated at 45°C to the eggplant-shaped bottle, vortex, and sonicate until the lipid film is completely detached and dissolved. Use an ultrasonic cell pulverizer with a probe sonicator to sonicate the sample until it turns blue opalescent. The obtained liposome was centrifuged at 12000 rpm for 5 min, and the supernatant was collected to obtain the final paclitaxel-targeted thermosensitive liposome.

实施例16Example 16

CREKA靶向的多西紫杉醇热敏脂质体的构建Construction of docetaxel thermosensitive liposomes targeted by CREKA

称量DPPC、MSPC、DSPE-PEG和DSPE-PEG-CREKA，置于茄形瓶中，加入氯仿溶解，再加入2mg/ml乙腈溶解的DTX母液0.5ml，使药脂比为1∶20m/m，45度水浴条件下减压旋转蒸发，成均匀的透明薄膜。向茄形瓶中加入2ml 45度预热的5％葡萄糖溶液，涡旋震荡，超声，至脂膜完全脱落溶解，使用探头超声的超声波细胞粉碎机将样品超声至呈蓝色乳光。将所得脂质体离心，12000rpm，5min，收集上清部分，即得最终多西紫杉醇靶向热敏脂质体。Weigh DPPC, MSPC, DSPE-PEG and DSPE-PEG-CREKA, put them in an eggplant-shaped bottle, add chloroform to dissolve, then add 0.5ml of DTX mother solution dissolved in 2mg/ml acetonitrile, so that the ratio of drug to lipid is 1:20m/m , Rotary evaporation under reduced pressure under the condition of 45 degree water bath to form a uniform transparent film. Add 2ml of 5% glucose solution preheated at 45°C to the eggplant-shaped bottle, vortex, and sonicate until the lipid film is completely detached and dissolved. Use an ultrasonic cell pulverizer with a probe sonicator to sonicate the sample until it turns blue opalescent. The obtained liposome was centrifuged at 12000 rpm for 5 min, and the supernatant was collected to obtain the final docetaxel-targeted thermosensitive liposome.

实施例17Example 17

为检验本发明的靶向热敏脂质体的抗肿瘤效果，我们以实施例7的阿霉素靶向热敏脂质体为例，进行了以下方面的考察：In order to test the antitumor effect of the targeted thermosensitive liposome of the present invention, we took the doxorubicin targeted thermosensitive liposome of Example 7 as an example, and carried out the following investigations:

1、脂质体性质的表征1. Characterization of liposome properties

采用荧光分光光度法对所制备的阿霉素靶向热敏脂质体进行包封率的考察，结果表明包封率在98％以上，采用动态光散射法(dynamic lightscattering，DLS)对其进行粒径和zeta电位的测定，结果见表1。表明本发明中所述阿霉素靶向热敏脂质体的制备方法优良、可行。The encapsulation efficiency of the prepared doxorubicin-targeted thermosensitive liposomes was investigated by fluorescence spectrophotometry. The results showed that the encapsulation efficiency was above 98%, and it was carried out by dynamic light scattering (dynamic lightscattering, DLS). The determination of particle size and zeta potential, the results are shown in Table 1. It shows that the preparation method of the doxorubicin-targeted thermosensitive liposome in the present invention is excellent and feasible.

表1Table 1

2、体外释放实验2. In vitro release experiment

在37、39、41、43和45℃下进行体外释放，于不同时间点(2.5、5、10、20和30min)取样，测定荧光强度(Ex/Em＝494/590nm)。在最后一个时间点用10％triton破坏脂质体，作为最大释放。以公式：释放百分率％＝(It-I0)/(I∞-I0)*100％计算释放百分率。结果表明在43℃和45℃时，靶向热敏脂质体的阿霉素释放量已经达到85％，说明本发明的阿霉素靶向热敏脂质体能在加热的条件下迅速释放所载药物，具有优良的热敏效果。In vitro release was carried out at 37, 39, 41, 43 and 45°C, samples were taken at different time points (2.5, 5, 10, 20 and 30 min), and the fluorescence intensity (Ex/Em=494/590nm) was measured. Liposomes were disrupted with 10% triton at the last time point as maximal release. The release percentage was calculated by the formula: release percentage %=(It-I0)/(I∞-I0)*100%. The results showed that at 43°C and 45°C, the release amount of doxorubicin targeting thermosensitive liposomes had reached 85%, indicating that the doxorubicin-targeting thermosensitive liposomes of the present invention can release rapidly under heating conditions. Loaded with drugs, it has excellent thermosensitivity effect.

3、细胞毒性实验3. Cytotoxicity experiment

阿霉素游离药母液配置：将20mg盐酸阿霉素溶于10ml容量瓶中配成2mg/ml的阿霉素游离药母液Doxorubicin free drug mother solution configuration: Dissolve 20mg doxorubicin hydrochloride in a 10ml volumetric flask to make 2mg/ml doxorubicin free drug mother solution

阿霉素靶向普通脂质体：以EPC(蛋黄卵磷脂)∶CHOL(胆固醇)∶DSPE-PEG∶DSPE-PEG-CREKA＝20∶10∶1∶1采用薄膜分散法制备空白脂质体，pH梯度法包载阿霉素制成阿霉素靶向普通脂质体Doxorubicin targeting common liposomes: prepare blank liposomes by film dispersion method with EPC (egg yolk lecithin): CHOL (cholesterol): DSPE-PEG: DSPE-PEG-CREKA=20: 10: 1: 1, Preparation of doxorubicin-targeted ordinary liposomes by encapsulating doxorubicin by pH gradient method

采用硫氰酸铵B(sulforhodamineB)(SRB)测定方法，在第0天将MCF-7/ADR细胞以6000个/孔的浓度接种于96孔板中，在第1天，将从阿霉素游离药母液、阿霉素靶向普通脂质体和阿霉素靶向热敏脂质体原液中制备一系列药物的稀释液(100μg/ml，50μg/ml，10μg/ml，5μg/ml，1μg/ml，0.5μg/ml，0.1μg/ml，0.05μg/ml)，加入到肿瘤细胞中，每种样品6孔(200μl)，立即45℃加热5min，然后孵育24小时，弃去培养基，用10％三氯乙酸在4℃固定细胞1小时，用自来水冲洗后，晾干并用0.4％SRB在4℃染色10分钟，取出用0.1％乙酸冲洗，晾干后加入10μM的Tris溶液，置摇床震荡30分钟后，用酶标仪在540nm处测定吸光值。Ammonium thiocyanate B (sulforhodamineB) (SRB) assay method was used to inoculate MCF-7/ADR cells in a 96-well plate at a concentration of 6000 cells/well on the 0th day. Prepare a series of drug dilutions (100 μg/ml, 50 μg/ml, 10 μg/ml, 5 μg/ml, 1μg/ml, 0.5μg/ml, 0.1μg/ml, 0.05μg/ml), added to tumor cells, 6 wells (200μl) of each sample, immediately heated at 45°C for 5min, then incubated for 24 hours, discarded the medium , fix the cells with 10% trichloroacetic acid at 4°C for 1 hour, rinse with tap water, dry and stain with 0.4% SRB at 4°C for 10 minutes, remove and rinse with 0.1% acetic acid, add 10 μM Tris solution after drying, set After shaking on a shaker for 30 minutes, the absorbance was measured at 540 nm with a microplate reader.

结果表明，阿霉素靶向热敏脂质体具有同阿霉素游离药相同的抑制肿瘤细胞生长的作用，说明本发明的阿霉素靶向热敏脂质体具有优良的热敏抗肿瘤效果。The results show that the doxorubicin-targeted thermosensitive liposome has the same tumor cell growth inhibitory effect as the free drug of doxorubicin, indicating that the doxorubicin-targeted thermosensitive liposome of the present invention has excellent thermosensitive anti-tumor Effect.

4、细胞摄取实验(流式细胞仪)4. Cell uptake experiment (flow cytometry)

将细胞分三组(MCF-7/ADR细胞以2×10⁵的浓度)接种于6孔板中，24小时后，分别给予阿霉素靶向热敏脂质体、阿霉素靶向普通脂质体(制备方法同细胞毒性实验)和游离阿霉素(配置方法同细胞毒性实验)三种药物(40μg/ml)，给药后一组直接孵育两个小时；另一组立即45℃加热5分钟，再37℃孵育两个小时；第三组先37℃孵育2小时，再吸尽药液，换成培养基，45℃加热5min。三组都用PBS清洗三次，用胰酶消化细胞，离心弃去上清，用500μl PBS重悬细胞，流式细胞仪测定。实验结果见图1，靶向热敏脂质体先加热时阿霉素摄取进入细胞的量是不加热和后加热的2.1和1.09倍。说明本发明的阿霉素靶向热敏脂质体在加热的条件下具有明显的增加体外肿瘤细胞药物摄取作用。The cells were divided into three groups (MCF-7/ADR cells at a concentration of 2×¹⁰⁵ ) and seeded in 6-well plates. After 24 hours, adriamycin-targeted thermosensitive liposomes and adriamycin-targeted common Liposome (preparation method is the same as the cytotoxicity test) and free doxorubicin (the preparation method is the same as the cytotoxicity test) three drugs (40μg/ml), one group is directly incubated for two hours after administration; the other group is immediately incubated at 45°C Heat for 5 minutes, and then incubate at 37°C for two hours; for the third group, incubate at 37°C for 2 hours, then absorb the drug solution, replace with medium, and heat at 45°C for 5 minutes. The three groups were washed three times with PBS, the cells were digested with trypsin, the supernatant was discarded by centrifugation, the cells were resuspended in 500 μl PBS, and flow cytometry was performed. The experimental results are shown in Figure 1. The amount of doxorubicin uptake into cells when the targeted thermosensitive liposome is heated first is 2.1 and 1.09 times that of no heating and post-heating. It shows that the doxorubicin-targeted thermosensitive liposome of the present invention can significantly increase drug uptake by tumor cells in vitro under heating conditions.

5、细胞摄取实验(激光共聚焦)5. Cell uptake experiment (laser confocal)

将细胞分三组(MCF-7/ADR细胞以2×10⁵的浓度)接种于6孔板中，24小时后，分别给予阿霉素靶向热敏脂质体、阿霉素靶向普通脂质体(制备方法同细胞毒性实验)和游离阿霉素(配置方法同细胞毒性实验)三种药物(40μg/ml)，给药后一组直接孵育两个小时；另一组立即45℃加热5分钟，再37℃孵育两个小时；第三组先37℃孵育2小时，再吸尽药液，换成培养基，45℃加热5min。三组都用PBS清洗三次，激光共聚焦显微镜下观察。实验结果见图2，靶向热敏脂质体先加热时阿霉素摄取进入细胞的量是不加热和后加热的2.28和1.96倍。说明本发明的阿霉素靶向热敏脂质体在加热的条件下具有明显的增加体外肿瘤细胞对药物的摄取作用。The cells were divided into three groups (MCF-7/ADR cells at a concentration of 2×¹⁰⁵ ) and seeded in 6-well plates. After 24 hours, adriamycin-targeted thermosensitive liposomes and adriamycin-targeted common Liposome (preparation method is the same as the cytotoxicity test) and free doxorubicin (the preparation method is the same as the cytotoxicity test) three drugs (40μg/ml), one group is directly incubated for two hours after administration; the other group is immediately incubated at 45°C Heat for 5 minutes, and then incubate at 37°C for two hours; for the third group, incubate at 37°C for 2 hours, then absorb the drug solution, replace with medium, and heat at 45°C for 5 minutes. All three groups were washed three times with PBS, and observed under a laser confocal microscope. The experimental results are shown in Figure 2. The amount of doxorubicin uptake into cells when the targeted thermosensitive liposome is heated first is 2.28 and 1.96 times that of no heating and post-heating. It shows that the doxorubicin-targeted thermosensitive liposome of the present invention can significantly increase the drug uptake effect of tumor cells in vitro under heating conditions.

6、靶向性实验6. Targeted experiments

荷瘤鼠(原位接种MCF-7/ADR细胞1×10⁷个/只鼠)尾静脉分别注射生理盐水和包载荧光素CY7的靶向热敏脂质体(薄膜分散法)，给药后6小时于活体成像仪下观察。结果表明，靶向热敏脂质体组的荧光主要集中在肿瘤部位，说明本发明的靶向热敏脂质体具有很好的靶向肿瘤的作用。Tumor-bearing mice (orthotopic inoculation of MCF-7/ADR cells 1×¹⁰⁷ /mouse) were injected with normal saline and targeted thermosensitive liposomes loaded with fluorescein CY7 (thin-film dispersion method) in the tail vein respectively. After 6 hours, it was observed under an in vivo imager. The results show that the fluorescence of the targeted thermosensitive liposome group is mainly concentrated in the tumor site, indicating that the targeted thermosensitive liposome of the present invention has a good tumor-targeting effect.

7、药效试验7. Drug efficacy test

将荷瘤鼠(原位接种MCF-7/ADR细胞1×10⁷个/只鼠)分为对照组和靶向热敏脂质体组(每组6只)，分别于第0、2、4天给予生理盐水或7mg/kg的阿霉素靶向热敏脂质体，并且实验组给药前肿瘤部位局部用45℃水浴预热30分钟，给药后再持续加热1小时。实验结果见图3，阿霉素靶向热敏脂质体能更好地抑制肿瘤的生长，具有很好的抗肿瘤效果。The tumor-bearing mice (orthotopically inoculated with MCF-7/ADR cells 1×¹⁰⁷ /mouse) were divided into the control group and the targeted thermosensitive liposome group (6 mice in each group). Physiological saline or 7 mg/kg doxorubicin-targeted thermosensitive liposomes were administered for 4 days, and the tumor site in the experimental group was preheated in a 45°C water bath for 30 minutes before administration, and continued heating for 1 hour after administration. The experimental results are shown in Figure 3. Doxorubicin-targeted thermosensitive liposomes can better inhibit the growth of tumors and have a good anti-tumor effect.

Claims

1. a targeting thermal sensitive liposome compositions is characterized in that said compositions comprises the thermal sensitive liposome that finishing has tumor-homing peptide CREKA or has the peptide section of CREKA core sequence, includes antitumor drug in the said thermal sensitive liposome.

2. targeting thermal sensitive liposome compositions as claimed in claim 1, wherein said antitumor drug is selected from one or more in amycin, epirubicin, daunorubicin, paclitaxel, Docetaxel, pirarubicin, aclarubicin, cisplatin, carboplatin, oxaliplatin and the methotrexate.

3. targeting thermal sensitive liposome compositions as claimed in claim 2, wherein the medicine fat of antitumor drug is 1 than (mass ratio of medicine and fat material): 10w/w～1: 50w/w, for example 1: 10w/w, 1: 20w/w, 1: 30w/w, 1: 40w/w, 1: 50w/w.

4. like each described targeting thermal sensitive liposome compositions of claim 1-3, the raw material of wherein processing said thermal sensitive liposome comprises that tumor-homing peptide lead compound DSPE-PEG-CREKA or DSPE-PEG-have the peptide section of CREKA core sequence, thermal sensitive liposome film material, DSPE-PEG and antitumor drug.

5. targeting thermal sensitive liposome compositions as claimed in claim 4; The peptide section that wherein said tumor-homing peptide lead compound DSPE-PEG-CREKA or DSPE-PEG-have a CREKA core sequence is through tumor-homing peptide CREKA or peptide section and DSPE-PEG-maleimide with CREKA core sequence are dissolved in the liquid medium; The pH value of regulator solution body medium is to the 6.0-8.0 scope; Continue stirring reaction; The purification reaction gains, the peptide section that tumor-homing peptide lead compound DSPE-PEG-CREKA that obtains through lyophilization or DSPE-PEG-have the CREKA core sequence.

6. targeting thermal sensitive liposome compositions as claimed in claim 5; Wherein the pH value of regulator solution body medium is to the 6.5-7.5 scope; The preferred pH value of regulating is more preferably regulated the scope of pH value to 6.9-7.1 to the scope of 6.8-7.2, most preferably being adjusted to pH value is 7.0.

7. targeting thermal sensitive liposome compositions as claimed in claim 5, wherein said liquid medium is for being dissolved in tumor-homing peptide CREKA and DSPE-PEG-maleimide the liquid medium of any kind wherein, for example aqueous solvent and organic solvent.

8. targeting thermal sensitive liposome compositions as claimed in claim 5, wherein said reaction is under the protection of noble gas, to carry out.

9. targeting thermal sensitive liposome compositions as claimed in claim 5, wherein said reaction is carried out at ambient temperature, preferably carries out 15 ℃-35 ℃ temperature, more preferably carries out 20 ℃-30 ℃ temperature, most preferably carries out 25 ℃ temperature.

10. targeting thermal sensitive liposome compositions as claimed in claim 5, the time of wherein said stirring is more than 24 hours.