CN102166190B - Paclitaxel nano liposomes of dual targeting tumor and method for preparing paclitaxel nano liposomes - Google Patents

Abstract

The invention belongs to the field of biomedicine. More specifically, the invention discloses a double-targeted tumor paclitaxel nanoliposome. The double-targeted paclitaxel nanoliposome is connected by a double-targeted tumor polypeptide and lipid. It consists of three parts: drug and paclitaxel liposome. The invention also discloses the preparation method of the paclitaxel nano-liposome with double targeting tumor and its application as preparation of tumor suppressing drug.

Description

A kind of paclitaxel nano liposome with double targeting tumor and preparation method thereof

technical field

The invention belongs to the field of medicine, and more specifically, the invention discloses a nano-liposome with dual targeting tumors and a preparation method thereof.

Background technique

In recent years, the morbidity and mortality of malignant tumors have shown an obvious upward trend, and have become a major disease that threatens human health and life. According to a report by the International Agency for Research on Cancer, the number of cancer cases worldwide doubled between 1975 and 2000, with approximately 12 million new cases and more than 7 million deaths each year. In 2010, cancer will become the leading cause of death in the world. In 2030, the number of cancer patients will triple, and the number of new cases will increase to 20-26 million.

Chemotherapy is one of the main methods in the comprehensive treatment of malignant tumors. A reasonable chemotherapy strategy can significantly prolong the survival time of cancer patients and greatly improve the quality of life of patients. Paclitaxel is a diterpenoid compound, which was first obtained from the bark of the Pacific yew tree. It has unique anticancer activity because it can stabilize the microtubules of tumor cells and inhibit mitosis (Wani, M.C., et al., Plant antitumoragents.VI.The isolation and structure of taxol, a novel antileukemicand antitumor agent from Taxus brevifol ia.J Am Chem Soc, 1971.93(9):p.2325-7.), is currently widely used in ovarian cancer (Thomas, H. and P. Rosenberg, Role of weekly paclitaxel in the treatment of advanced ovarian cancer. CritRev Oncol Hematol, 2002.44 Suppl: p.S43-51.), breast cancer (Saloustros, E., D. Mavroudis, and V. Georgoulias, Paclitaxel and docetaxel in the treatment of breast cancer. Expert Opin Pharmacother, 2008.9(15): p.2603-16.), head and neck cancer (Aisner, J. and H. Cortes-Funes, Paclitaxel in head and neck and other cancers: future prospects.Semin Oncol, 1997.24 (1Suppl 2): p.S2-113-S2-115.) and treatment of non-small cell lung cancer (Greco, F.A., Paclitaxel-based combination chemotherapy in advanced non-small celllung cancer.Lung Cancer , 2001.34 Suppl 4: p.S53-6.). But paclitaxel is a fat-soluble substance, which is difficult to dissolve in aqueous solution and many other solvents (Ceruti, M., et al., Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing water-soluble prodrugs of paclitaxel.J Control Release, 2000.63( 1-2): p.141-53.). The solvent used in clinically used paclitaxel preparations (Taxol, trade name Taxol) is a mixture of non-ionic surfactant polyoxyethylene castor oil (Cremophor EL, CrEL) and absolute ethanol (1: 1, V/V), But this dosage form is accompanied by many difficult clinical problems (Hennenfent, K.L. and R.Govindan, Novel formulations of taxanes: review. Old wine in a new bottle? Ann Oncol, 2006.17 (5): p.735-49.). The clinical application of traditional paclitaxel preparations can lead to a series of allergic reactions (Hennenfent, K.L.and R.Govindan, Novel formulations of taxanes: a review. Old wine in a new bottle? AnnOncol, 2006.17(5): p.735-49.) , mild cases show itching, rash, severe cases show angioedema, hypotension, respiratory failure, etc. Wiemik, P.H., et al., Phase I clinical and pharmacokinetic study of taxol. Cancer Res, 1987.47(9): p.2486-93.). More than 95% of patients with allergic reactions occurred during the first or second administration of paclitaxel preparations, and 80% of the symptoms occurred in the first 10 minutes of drug infusion, and often only 1 mg of paclitaxel was infused. Since CrEL can induce similar reactions in animals (Wiernik, P.H., et al., Phase I clinical and pharmacokinetic study oftaxol. Cancer Res, 1987.47 (9): p.2486-93.), it is considered that the presence of CrEL has caused Occurrence of the above adverse reactions. And the occurrence of adverse reactions is related to the infusion rate (Lorenz, W., et al., Histamine release and hypotensive reactions in dogs by solubilizing agents and fatty acids: analysis of various components in cremophor El and development of a compound with reduced toxicity. Agents , 1982.12(1-2): p.64-80.), but prolonging the infusion time does not eliminate the occurrence of allergic reactions. Researchers tried to replace CrEL with other solvents, such as polyethylene glycol, but animal experiments found that the presence of polyethylene glycol can reduce the antitumor activity of paclitaxel. Therefore, CrEL is still the standard vehicle for paclitaxel (Wiernik, P.H., et al., Phase I clinical and pharmacokinetic study of taxol. Cancer Res, 1987.47 (9): p.2486-93.).

Clinically, large doses of glucocorticoids and histamine receptor antagonists are commonly used as routine pretreatment before use, but their efficacy is not yet clear. The Research on Adverse Drug Events and Reports (RADAR) of the Pharmacovigilance Project of the Northwestern University Feinberg School of Medicine found that from 1997 to 2007, 171 cases of polyoxygen were reported in the United States, Europe and Japan. Among the cases of anaphylaxis after ethylene castor oil paclitaxel, 58 patients died (34%), and 21 of 96 patients reported using the conditioning regimen died, with a mortality rate of 22%. The research of BoehnkeMichaud et al. (Michaud, L.B., V.Valero, and G.Hortobagyi, Risks and benefits of taxes in breast and ovarian cancer. Drug Saf, 2000.23(5): p.401-28.) also showed that the pretreatment program adopts Afterwards, about 40% of patients will still have mild allergies, and 3% of patients will have fatal anaphylaxis. At the same time, the implementation of the pretreatment regimen limits the use of the drug in patients with tumors complicated by ulcers, diabetes, hypertension, etc., and the use of hormones in the pretreatment regimen may also increase treatment-related mortality.

The use of CrEL as a solvent can also lead to peripheral nervous system toxicity (Onetto, N., et al., Overview of Taxol safety. J Natl Cancer Inst Monogr, 1993 (15): p.131-9.). Electrophysiological examination revealed axonal degradation and demyelination in patients with peripheral neuropathy after paclitaxel-CrEL solution administration. Peripheral neuropathy occurs in about 25% of patients after intravenous administration of cyclosporine CrEL solution, and no such adverse reactions occur in oral administration, and CrEL is not absorbed through the gastrointestinal tract (de Groen, P.C., et al. , Central nervous system toxicity after liver transplantation. The role of cyclosporine and cholesterol. N Engl J Med, 1987.317(14): p.861-6.). Therapeutic doses of paclitaxel or cyclosporine CrEL solutions produced CrEL plasma concentrations that resulted in axonal swelling, vacuolar degeneration, and degradation of dorsal root ganglion neurons in mice (Windebank, A.J., M.D. Blexrud, and P.C. de Groen, Potentialneurotoxicity of the solvent vehicle for cyclosporine. J Pharmacol Exp Ther, 1994.268(2): p.1051-6.). Current research shows that the ethylene oxide derivatives produced by CrEL are the most important factor leading to nerve damage (Brat, D.J., A.J. to structural abnormalities in differentiating N1E.115 neuroblastoma. J Pharmacol Exp Ther, 1992.261(2): p.803-10.).

The administration of paclitaxel CrEL solution three times a week, each infusion of 3 hours makes the plasma concentration of paclitaxel exceed the body's ability to eliminate metabolism, so the elimination of paclitaxel CrEL solution in the body exhibits nonlinear kinetics, also known as zero-order elimination kinetics (Sparreboom, A., et al., Cremophor EL-mediated alteration of paclitaxel distribution in human blood: clinical pharmacokinetic implications. Cancer Res, 1999.59 (7): p.1454-7. van Tellingen, O., et al., CremophorEL causes (pseudo-)non-linear pharmacokinetics of paclitaxel in patients. Br J Cancer, 1999.81(2): p.330-5.). Animal experiments have confirmed that the nonlinear pharmacokinetics of paclitaxel is related to CrEL (Sparreboom, A., et al., Nonlinear pharmacokinetics of paclitaxel in miceresults from the pharmaceutical vehicle Cremophor EL. Cancer Res, 1996.56(9): p.2112- 5.), similar results were obtained in experiments in humans (van Tellingen, O., et al., CremophorEL causes (pseudo-) non-linear pharmacokinetics of paclitaxel in patients. Br J Cancer, 1999.81 (2): p. 330-5.). The reduction of the paclitaxel clearance rate leads to prolonged exposure of body tissues to high concentrations of the drug, increasing the chance of serious systemic toxic side effects. CrEL can also form tiny particles in the blood to wrap paclitaxel molecules, thereby affecting the distribution, metabolism and excretion of drug molecules in tumor tissues (Sparreboom, A., et al., Cremophor EL-mediated alteration of paclitaxel distribution in human blood: Clinical pharmacokinetic implications. Cancer Res, 1999.59(7): p.1454-7.).

There are many drug-resistant mechanisms of paclitaxel, and the increased expression of P-glycoprotein is one of the important reasons. P-glycoprotein is a transmembrane transport protein encoded by the multidrug resistance gene. It has ATPase activity, can pump chemotherapy drugs out of cells, and reduce the effective drug concentration in cells to cause drug resistance. Studies have suggested that CrEL can enhance the antitumor effect of paclitaxel by regulating P-glycoprotein and inhibiting the expression of multidrug resistance genes, but in vivo experiments have not been successful (Woodcock, D.M., et al., Reversal of the multidrugresistance phenotype with cremophor EL, a common vehicle for water-insoluble vitamins and drugs. Cancer Res, 1990.50(14): p.4199-203. Schuurhuis, G.J., et al., The polyoxyethylene castor oil Cremophor EL modifies multidBrrugJ resistance. 1990.62(4): p.591-4. Friche, E., et al., The solvents cremophor EL and Tween 80 modulate daunorubicin resistance in the multidrug resistant Ehrlich ascitestumor. Cancer Commun, 1990.2(9): p.297-303. ). On the contrary, studies have found that CrEL can antagonize the cytotoxic effect of paclitaxel by blocking the cell cycle and reducing the uptake of paclitaxel by tumor cells (Liebmann, J., et al., The influence of Cremophor EL on the cell cycle effects of paclitaxel (Taxol ) in human tumor cell lines. Cancer Chemother Pharmacol, 1994.33(4): p.331-9.).

Studies have shown that paclitaxel CrEL solution has stability and compatibility problems. For example, once the drug is diluted, paclitaxel is prone to precipitation; plasticizers (divinylhexyl o- Phthalates), so glass or non-PVC infusion sets must be used throughout the use process; non-specific adsorption with plastic or glass containers, etc. (Waugh, W.N., L.A. Trissel, and V.J. Stella, Stability, compatibility, and plasticizer extraction of taxol (NSC-125973) injection diluted in infusion solutions and stored in various containers. Am J HospPharm, 1991.48 (7): p.1520-4. Song, D., L.F.Hsu, and J.L.Au, Binding of taxol toplastic and glass containers and protein under in vitro conditions. J Pharm Sci, 1996.85(1): p.29-31.).

In summary, the existence of CrEL in the existing dosage forms of paclitaxel has led to the emergence of a series of clinical toxic and side effects, and CrEL can also have complex effects on the curative effect and pharmacokinetics of paclitaxel. A new formulation of paclitaxel in CrEL to overcome the defects of surfactants.

Many scholars are committed to the research and development of new formulations of paclitaxel. The new formulations that have been successfully developed or are currently being developed include: albumin nanoparticles (Abraxame ^TM ) (Ibrahim, NK, et al., Phase I and pharmacokinetic study of ABI-007, a Cremophor -free, protein-stabilized, nanoparticle formulation of paclitaxel. Clin Cancer Res, 2002.8(5): p.1038-44.

Yamada, K., et al., Phase I and Pharmacokinetic Study of ABI-007, Albumin-bound Paclitaxel, Administered Every 3Weeks in Japanese Patients with Solid Tumors.Jpn J Clin Oncol, 2010.), paclitaxel prodrug docosahexa Taxoprexin (Taxoprexin) (Wolff, AC, et al., Phase I study of docosahexaenoic acid-paclitaxel: a taxane-fatty acid conjugate with a unique pharmacology and toxicity profile. Clin Cancer Res, 2003.9 (10Pt 1): p.3589 -97.); polyglutamic acid paclitaxel (Xyotax ^TM ) (Sabbatini, P., et al., Phase II study of CT-2103 in patients with recurrent epithelianovarian, fallopian tube, or primary peritoneal carcinoma.J Clin Oncol, 2004.22(22): p.4523-31.), paclitaxel analogs (BMS-184476 (Rose, WC, C. Fairchild, and FY Lee, Preclinical antitumor activity of two novel taxanes. Cancer Chemother Pharmacol, 2001.47(2): p.97-105.), DJ-927 Ono, C., A. Takao, and R. Atsumi, Absorption, distribution, and excretion of DJ-927, a novel orally effective taxane, in mice, dogs, and monkeys. Biol Pharm Bull, 2004.27(3): p.345-51. Baas, P., et al., Phase I/II study of a 3 weekly oral taxane (DJ-927) in patients with recurrent, advanced non-small cell lung cancer. J Thorac Oncol, 2008.3 (7): p.745-50.), BMS-275183 (Broker, LE, et al., Effect of food on the pharmacokinetic behavior of the potent oral taxane BMS-275183 .Clin Caneer Res, 2008.14(13): p.4186-91.

Broker, L.E., et al., Phase I trial with BMS-275183, a novel oral taxane with promising antitumor activity. Clin Caneer Res, 2006.12(6): p.1760-7.), Ortataxel Cassinelli, G., et al., Cellular bases of the antitumor activity of the novel taxane IDN5109(BAY59-8862) on hormone-refractory prostate cancer. Clin Caneer Res, 2002.8(8): p.2647-54. Polizzi, D., et al., A novel taxane with improved tolerance and therapeutic activity in a panel of human tumor xenografts. Cancer Res, 1999.59 (5): p.1036-40.), RPRl09881A (Gelmon, K.A., et al., Phase I dose-finding study of a newtaxane, RPR 109881A, administered as a one-hour intravenous infusion days 1 and 8 to patients with advanced solid tumors. J Clin Oncol, 2000.18(24): p.4098-108.

Kurata, T., et al., Phase I and pharmacokinetic study of a new taxoid, RPR10988lA, given as a 1-hour intravenous infusion in patients with advanced solid tumors. J Clin Oncol, 2000.18(17): p.3164-71. ), paclitaxel micellar copolymer (Genexol-PM) (Kim, SC, et al., In vivo evaluation of polymeric micellar paclitaxel formulation: toxicity and efficacy.J Control Release, 2001.72(1-3): p.191-202 .), paclitaxel vitamin E emulsion (TOCOSOL), paclitaxel microspheres (Paclimer) (Lissianskaya, A., et al., Paclitaxelinjectable emulsion: Phase 2a study of weekly administration in patients with platinum-resistant ovarian cancer. Journal of Clinical Oncology, 2004.22 (14): p.460s-460s. Bogdanova, N., et al., Paclitaxel injectable emulsion: Phase 2a study of weekly administration in patients with non-small cell lung cancer (NSCLC). Journal of Clinical Oncology, 2004.22(14): p.649s-649s.), paclitaxel liposome (Liposu), etc. Some of the new dosage forms of paclitaxel have been listed (Abraxame ^TM was approved by the US Food and Drug Administration in 2005; Paclitaxel Liposupplement was successfully developed by Nanjing Cisco Pharmaceuticals and approved for domestic marketing in 2003), and most of the new dosage forms of paclitaxel are still available. In preclinical research stage, or in phase I-III clinical trials.

The common point of the above-mentioned new dosage forms of paclitaxel is that it avoids the use of CrEL as a solvent, so it has unique advantages, such as shortening the infusion time of the drug, and reducing the incidence of adverse reactions such as allergies, bone marrow suppression, and hair loss. Due to structural changes, some dosage forms are no longer the substrates of P-glycoprotein, so the incidence of paclitaxel resistance can be reduced, and some dosage forms can even be absorbed orally. However, whether the new dosage form of paclitaxel can improve the prognosis of cancer patients compared with the traditional dosage form of paclitaxel is still unknown.

Contents of the invention

The present invention aims to develop a new type of tumor-targeted liposome drug-loaded nanoparticles, which can improve the physical and chemical properties and pharmacokinetic behavior of paclitaxel after encapsulating paclitaxel, and improve solubility, thereby avoiding various problems caused by CrEL in traditional preparations. At the same time, through the active tumor targeting of the carrier, the local concentration of paclitaxel in the tumor can be specifically increased, and the toxicity of the drug to normal tissues and organs can be reduced while enhancing the anti-tumor effect.

In the present invention, paclitaxel is encapsulated in nano-liposomes with dual targeting tumors, and the two target molecules can maintain their respective original biological activities and produce synergistic effects in function.

The paclitaxel nano-liposome with double targeting tumor mainly consists of three parts, the polypeptide with double targeting tumor, lipid linker and paclitaxel liposome.

First, the present invention adopts the solid-phase synthesis method to obtain a dual-targeting tumor polypeptide, and the amino acid sequence of the polypeptide is as follows: ARYC RGD CFD ATWLPPR . Among them, ARYC RGD CFDG: its core structure is the RGD tripeptide, that is, the sequence of arginine-glycine-aspartic acid (Arg-Gly-Asp), and the target is the integrin αV family. αV integrin is one of the important members of the cell adhesion molecule family. It participates in the activation and migration of endothelial cells, mediates endothelial cell proliferation, inhibits endothelial cell apoptosis, participates in angiogenesis induced by basic fibroblast growth factor and VEGF, Induction of cyclooxygenase 2 and other pathways promote neovascularization and tumor development. αV integrin is lowly expressed in quiescent vascular endothelial cells and normal tissues and organs under physiological conditions, but highly expressed on the surface of activated tumor vascular endothelial cells and tumor cells. After the drug carrier is linked with a short peptide containing RGD sequence, it can significantly enhance its ability to target tumor neovascularization. The ATWLPPR sequence is aimed at the co-receptor neuroendothelin-1 (Neuropilin-1, NRP-1) of VEGFR2. VEGFR-2 (Vascular Endothelial Growth Factor Receptor-2) is a member of the VEGF/VEGFR family, mainly recognizes low molecular weight VEGF, and plays an important role in the migration, proliferation, survival and regulation of vascular permeability of vascular endothelial cells. NRP-1 is a non-tyrosine transmembrane glycoprotein, and it is a co-receptor of VEGFR-2. The co-expression of NRP-1 and VEGFR-2 can significantly promote the combination of VEGF165 and VEGFR-2, and enhance the biological effects mediated by VEGF165 , promote the proliferation of vascular endothelium. NRP-1 is also highly expressed on the surface of activated tumor vascular endothelial cells and tumor cells, and is lowly expressed in quiescent vascular endothelial cells and normal tissues and organs under physiological conditions.

The invention adopts the Fmoc solid-phase synthesis method to synthesize a polypeptide ligand-lysine-glycine-glycine-palmitic acid (lysine-glycine-glycine, KGG-pal) conjugate, and connects the novel polypeptide and paclitaxel liposome.

The invention adopts a thin-film ultrasonic dispersion method to prepare paclitaxel liposomes, and controls the particle size of the liposomes to be 60-200nm by an extrusion filtration method or a high-pressure homogenization method. Nanoparticles with small particle size have unique advantages as drug carriers. The invasive growth and metastasis of malignant tumors depend on angiogenesis. Compared with normal blood vessels, blood vessels in tumor tissue have weaker selective permeability to drugs, but the maximum diameter still does not exceed 400nm. Liposomes with large particle size have more entrapment in the spleen, are cleared from the blood faster, and the liposomes reaching the tumor tissue are significantly reduced. Therefore, liposomes with small particle size and long circulation are more likely to reach the local tumor through the vascular space.

Cytological studies have confirmed that the dual-targeting tumor paclitaxel liposome containing RGD and ATWLPPR sequences of the present invention can easily penetrate the endothelial cell barrier because of the small molecular weight of the new targeting polypeptide and the particle size of the core drug-loaded particles is controlled at 60-200nm into the tumor tissue. Studies have confirmed that the spatial conformation of the RGD and ATWLPPR sequences after linkage does not affect each other, and can maintain the original biological activities of the sequences, thereby producing a synergistic effect. Compared with single-targeting paclitaxel liposomes containing only RGD or ATWLPPR sequences, the developed dual-targeting tumor paclitaxel liposomes have significantly better specific binding ability to neovascular endothelial cells and tumor cells. Compared with paclitaxel liposomes without targeting and single-targeting tumors, paclitaxel liposomes dual-targeting tumors have stronger specific binding ability to neovascular endothelial cells and tumor cells, and the ability to inhibit tumor growth. It can be further applied to the field of targeted therapy of malignant tumors.

Description of drawings

Figure 1: Scanning electron micrographs of paclitaxel liposomes dual targeting tumors

Figure 2: Particle size distribution of paclitaxel liposomes for dual targeting tumors

Figure 3: Cellular uptake of Taxol and various paclitaxel liposomes

Figure 4: MTT detection chart of Taxol and various paclitaxel liposomes

Figure 5: D60 tumor weight map of Taxol and various paclitaxel liposome inhibition experiments

Detailed ways

Experimental Materials

1. Reagents, drugs and cells

Egg phosphatide (egg PC) and cholesterol (cholesterol, CHOL) were purchased from Dongshang Company in Germany; monomethoxypolyethylene glycol 2000 distearoylphosphatidylethanolamine mPEG2000-DSPE was purchased from Avanti Polar Lipids in the United States ; Lipid peptides were synthesized by Jill Biochemical (Shanghai) Co., Ltd.; Taxol was purchased from Bristol-Myers Squibb Company; Paclitaxel was purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd.; Sodium dodecyl sulfonate (SDS), polyacrylamide , sucrose, glacial acetic acid, silver nitrate, glutaraldehyde, aprotinin, etc. were purchased from Sinopharm Shanghai Branch; HUVEC (human umbilical vein endothelial cells), ATCC No.: CRL-2873; A549 cells (lung adenocarcinoma cells Department), ATCC No.: CCL-185; DMEM (Dulbecco's Modified Eagle Media, high-glucose cell culture medium), purchased from Invitrogen Company; newborn bovine serum was purchased from Austria PAA Company; penicillin, streptomycin, trypsin, etc. were purchased from Huamei Company.

2. Instruments and consumables

MillexTM FH needle filter and Ultracel YM100 ultrafiltration tube (Millipore, USA); SK5210HP water bath ultrasonic instrument (Shanghai Kedao Ultrasonic Instrument Co., Ltd.); R502B rotary evaporator (Xi’an Taikang Instrument Equipment Co., Ltd.); JEM-1200EXII type transmission Electron microscope (Japan JEOL company); Quanta 200FEG field emission environment scanning electron microscope (FEI company); MASTERSIZER 2000 laser particle size analyzer (Malvern company, UK); Freeze dryer 6L (Labconco company, USA);

3. Solution preparation

PBS solution (0.01M, pH7.4): NaCl 8.0g; KCl 0.2g; Na2HP04·12H20 1.44g; KH2PO4 0.24g; ultrapure water 800ml; Store at ℃.

0.25% trypsin digestion solution (1L): trypsin 2.5g; NaCl 8g; KCl 0.4g; Na2HP04 12H20 0.06g; KH2PO4 0.06g; NaHCO3 0.35g; Store DMEM medium (1L) at -20°C: 2 bags of DMEM powder; 37.4g NaHCO; 0.12g penicillin; 0.2g streptomycin; ;Cell cryopreservation medium: DMEM medium 70%; Dimethyl sulfoxide (DMSO) 10%; Newborn bovine serum 20%; Store at -20°C after aliquoting; Cell lysate: Tris 10mmol/L; Nacl 100mmol/L; EDTA 1mmol/L; EGTA 1mmol/L; NaF 1mmol/L; Na4P2O7·10H2O 20mmol/L; Sodium vanadate 2mmol/L; Triton X-1001%; Glycerin 10%; Sodium lauryl sulfate 0.1%; Salt 0.5%; PMSF 100μg/μL.

The present invention is further illustrated by the following examples, but not as a limitation of the present invention.

Example 1: Synthesis steps of dual targeting tumor polypeptides

The dual-targeting tumor peptide was synthesized by 9-fluorenylmethyloxycarbonyl (FMOC) solid-phase synthesis method, purified by high performance liquid chromatography (HPLC), and identified by mass spectrometry. Concrete synthetic steps are as follows:

1) Resin swelling: put FMOC-AA-Wang-Resin resin into the reaction tube, add dimethylformamide (N, N-dimethylformamide, DMF) (15ml/g) for 30min;

Deprotection: discard DMF, add 20% piperidine DMF solution (15ml/g) for 5min, then add 20% piperidine DMF solution (15ml/g) for 15min;

2) Detection: Take out the piperidine solution, take more than a dozen resins, wash them three times with ethanol, add ninhydrin, potassium cyanide, and phenol solution one drop each, heat at 105°C-110°C for 5 minutes, and turn dark blue as a positive reaction.

3) Washing: DMF (10ml/g) twice, methanol (10ml/g) twice, DMF (10ml/g) twice;

4) Condensation: three times excess of protected amino acid (FOMC-Asp-OH), three times excess of 1-oxo-3-bisdimethylamine carboxybenzotriazole boron tetrafluoride salt, dissolved with as little DMF as possible, and added To the reaction tube, immediately add N-methylmorpholine in a ten-fold excess, and react for 30 minutes;

5) Washing: DMF (10ml/g) once, methanol (10ml/g) twice, DMF (10ml/g) twice;

6) Repeat steps 2)--6) to connect FMOC-Tyr-OH, FMOC-Thr-OH, FMOC-Met-OH, FMOC-Asn-OH, FMOC-Pro-OH, FMOC-Arg-OH, FMOC-Lys-OH, FMOC-Leu-OH, FMOC-Phe-OH;

7) The last washing: DMF (10ml/g) twice, methanol (10ml/g) twice, DMF (10ml/g) twice, DCM (10ml/g) twice;

8) Lysis: lysate (10ml/g) (containing TFA 94.5%; water 2.5%; EDT 2.5%; TIS 1%) 120min;

9) Drying and washing: dry the lysate with nitrogen as much as possible, wash with ether six times, and evaporate to dry at room temperature;

10) Sealed and stored at -20°C.

The resulting white powdery substance was purified by HPLC with a purity > 95%;

Example 2: Preparation of non-targeting, single-targeting and dual-targeting tumor paclitaxel liposomes

Preparation of Paclitaxel Liposomes by Thin Film Ultrasonic Dispersion

Non-active targeting paclitaxel liposome formula: PC: CHOL: mPEG-DSPE: PTX 9: 1: 0.5: 0.33, active targeting paclitaxel liposomes added different lipid polypeptides (ARYC RGD CFDG-KGG , ARYC RGD CFD ATWLPPR -KGG) (the molar ratio to the total fat is 1:542). Because in the patent 1 embodiment, the polypeptide ATWLPPR and ATWLPPR-fluorescent liposomes are not shown to be superior to the polypeptide ARYC RGD CFDG, ARYC RGD CFDG-fluorescent liposome and the polypeptide ARYC RGD CFD ATWLPPR , ARYC RGD CFD ATWLPPR -fluorescent liposome Plastids have specific cell-specific affinity, so they were not synthesized and were not used in subsequent experiments.

The above formula was dissolved in chloroform, filtered through a 0.2 μm plate filter, placed in a rotary evaporator at 37°C to form a film, and then vacuumed for 1 hour to further remove chloroform; add an appropriate amount of filter-sterilized 9% sucrose solution, fully swell until the film falls off, and the obtained After the suspension was extruded by Mini-extruder (1 μm, 0.4 μm, 0.2 μm, 0.1 μm each 10 times), it was divided into vials and placed in a vacuum freeze dryer at -38°C×2h, -35°C×18h , -16°C x 2h, 20°C x 2h, dry and seal with a stopper, store in a -20°C refrigerator until use. All tests were performed using freeze-dried liposome PBS reconstituted solution.

Scanning electron microscopy showed that the dual-targeted paclitaxel liposomes were round or nearly round, and the particle size was relatively consistent, with a particle diameter of <100nm (see Figure 1).

Embodiment 3: Paclitaxel liposome release experiment in vitro

3ml of reconstituted non-active targeting paclitaxel liposomes, paclitaxel liposomes with single and dual targeting tumors, and Taxol were placed in a dialysis bag with a molecular weight of 14KD, and 20ml containing 45g/L bovine serum white was added to the bag Protein sterilized water simulates the internal environment, stirred with a constant temperature magnetic stirrer (300rpm), and 80μL of dialysate was taken out at 30min, 1h, 2h, 4h, 6h, 8h, and 24h for use, and at the same time, 80μL containing 45g/L cattle was added back to the dialysis bag. Serum albumin in sterile water. Add 320 μL of chromatographic grade methanol solution to the dialysate taken out to precipitate protein, vortex and mix, then centrifuge at 13000g×10min at high speed, and take the supernatant to detect paclitaxel content by HPLC. At the same time, the concentration of paclitaxel in each paclitaxel liposome and Taxol solution reconstituted before dialysis was detected, and (concentration of paclitaxel in the dialysate/concentration of paclitaxel in the stock solution)×100% was used as an index to measure the sustained release ability.

It was found (see Figure 2) that paclitaxel was released fastest from the CrEL adjuvant, and the cumulative release reached 15.96% in 24 hours. After using liposomes as drug carriers for paclitaxel, the release rate in vitro was significantly reduced. The 24-hour cumulative release of paclitaxel liposomes, single-targeted paclitaxel liposomes, and dual-targeted tumor paclitaxel liposomes was 4.40%, 4.55%, and 4.5%, respectively. 3.6%, the slow-release effect is remarkable. At the same time, it is also suggested that the structural integrity of the liposome is not destroyed after the lipopolypeptide is linked to the surface of the liposome, and the enhanced sustained-release performance can further improve the pharmacokinetic behavior of paclitaxel in vivo.

Example 4: Cell Endocytosis Experiment

HUVEC and A549 cells were seeded in 24-well plates, 1×10 ⁶ /well, placed in an incubator for 24 hours to allow the cells to adhere to the wall, and Taxol, paclitaxel liposomes, single-targeted paclitaxel liposomes, and double-targeted paclitaxel lipids were added to each well Plastid (5 μg as paclitaxel), 4 duplicate wells, incubate at 37°C for 4 hours, wash twice with ice PBS, add 100 μL/well of cell lysate, collect the lysate, add 100 μL methanol solution to precipitate protein, vortex for 5 min, 10000 g After centrifugation for 30 min, the supernatant was taken for HPLC to detect the paclitaxel content in the cells.

After Taxol and various paclitaxel liposomes were incubated with cells, the uptake by cells is shown in Figure 3. It can be seen from the figure that the uptake of traditional paclitaxel solution (Taxol) by A549 cells and HUVEC is extremely low, and the paclitaxel content reaches the minimum limit of HPLC detection (5 ng/well and 25 ng/well, respectively). After paclitaxel is encapsulated in liposomes, since liposomes can interact with cells through contact release, adsorption, endocytosis, fusion, lipid exchange and other ways, the uptake of paclitaxel by cells is increased. The uptake of non-targeted paclitaxel liposomes by A549 cells and HUVECs was 145ng/well and 157ng/well, respectively. Paclitaxel liposomes linked to tumor-specific targeting polypeptides further enhanced the uptake of paclitaxel by cells. The uptake of single-target paclitaxel liposomes by A549 cells and HUVECs was 212ng/well and 232ng/well, respectively. The uptake of tumor-targeted paclitaxel liposomes was 346ng/well and 319ng/well, respectively. At the same time, the above data also suggested that the ability of cells to take up paclitaxel liposomes with dual targeting tumors was stronger, confirming that the novel polypeptides with dual targeting tumors had better specific binding force with tumor cells and vascular endothelial cells.

Embodiment 5: cytotoxicity test

A549 and HUVEC cells were diluted to 4×10 ⁴ /mL, added to a 96-well plate, 100 μL per well, and placed in an incubator for 24 hours to allow the cells to adhere to the wall; Taxol, paclitaxel liposome, single-targeted paclitaxel Liposomes, paclitaxel liposomes with dual targeting tumors are diluted to the final concentration of 0.9375mg/L, 1.875mg/L, 3.75mg/L, 7.5mg/L, 15mg/L working solution, add working solution to each well Add 20 μl/well of 5 mg/mL MTT solution, incubate in the incubator for 4 hours, centrifuge at 1500 rpm for 10 minutes, discard the culture medium, add 200 μL of DMSO to each well, and mix on a decolorizing shaker for 30 minutes On a microplate reader, select a wavelength of 530nm to detect the absorbance value, and calculate IC50, the formula is as follows:

It can be seen from Fig. 4 that the IC50 values of Taxol to A549 cells and HUVEC are 6.027mg/L and 6.56mg/L respectively; The IC50 values of paclitaxel liposomes and dual-targeted tumor paclitaxel liposomes were 1.46mg/L, 1.16mg/L and 3.40mg/L, 3.25mg/L, respectively. The results suggested that the surface of paclitaxel liposomes linked with vascular targeting polypeptides enhanced the toxicity to tumor cells and vascular endothelial cells, and the dual-targeting tumor paclitaxel liposomes had the strongest cytotoxicity.

Example 6: In vivo tumor suppression evaluation

A549 lung adenocarcinoma cells were routinely cultured to the logarithmic growth phase, digested with 0.25% trypsin to form a cell suspension, and then inoculated 1×10 ⁷ /0.1mL under the right armpit of 4-week-old (18-20g) female Balb/c nude mice , the tumor body grows to 50mm ³ -80mm ³ and begins to administer. The nude mice were divided into 5 groups according to different administrations, with 5 mice in each group. They were given normal saline, Taxol, paclitaxel liposome, single-targeted paclitaxel liposome and dual-targeted tumor-targeted paclitaxel liposome on d1, d4, and d8 respectively. plastid. Among them, the normal saline group was given 0.5 mL of normal saline, and the dosage of other groups was 7.5 mg/kg based on paclitaxel. After administration, the body weight (twice/week) and tumor size (twice/week) of the nude mice were monitored, and all nude mice were sacrificed on d60, the tumors were stripped off and weighed, and the tumor inhibition rate was calculated.

Tumor size calculation formula: tumor volume V=ab ² /2, where a is the longest diameter of the tumor and b is the shortest diameter of the tumor, and the calculation formula of tumor inhibition rate: (1-tumor weight of the experimental group)/tumor weight of the control group×100% .

The results showed (see FIG. 5 ) that the transplanted tumors in the control group given normal saline grew rapidly, with a d60 of 1925mm ³ and an average tumor weight of (1.62±0.09g). The growth of tumor volume in nude mice given Taxol and various paclitaxel liposome groups was relatively slow ^. The average volume of d60 transplanted tumors in Taxol group was 1104mm ³ , and the tumor inhibition rate was 46.9%. The tumor weight was (0.61±0.10g), and the tumor inhibition rate was 62.3%. The average volume of tumors in the single-targeted paclitaxel liposome group and the dual-targeted tumor-targeted paclitaxel liposome group was further reduced to 590 mm ³ and 455 mm ³ , respectively. The average tumor weights were (0.44±0.07g) and (0.30±0.04g), and the tumor inhibition rates were 72.8% and 81.5%. It was indirectly confirmed that the dual-targeting tumor peptides were effectively linked to the surface of paclitaxel liposomes, which promoted the uptake of the drug carrier by tumor tissue, increased the local effective concentration of the drug, and enhanced the anti-tumor ability of the drug.

Claims (3)

1. the paclitaxel nano lipid body of a dual-target tumor is characterized in that it mainly by the polypeptide of dual-target tumor, and lipid junctional complex and Paclitaxel liposome three parts form; The structure of described polypeptide and lipid junctional complex is ARYCRGDCFDATWLPPR-KGG; The prescription of Paclitaxel liposome is that PC:CHOL:mPEG-DSPE:PTX is 9:1:0.5:0.33.

2. the preparation method of the paclitaxel nano lipid body of dual-target tumor claimed in claim 1, it is characterized in that at first synthesizing the ARYCRGDCFDATWLPPR polypeptide of dual-target tumor, then synthesize polypeptide junctional complex KGG, prepare at last the paclitaxel nano lipid body of dual-target tumor; The prescription of Paclitaxel liposome is that PC:CHOL:mPEG-DSPE:PTX is 9:1:0.5:0.33.

3. the application of the paclitaxel nano lipid body of dual-target tumor claimed in claim 1 in preparation inhibition tumour medicine.

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