CN102166190B - Paclitaxel nano liposomes of dual targeting tumor and method for preparing paclitaxel nano liposomes - Google Patents
Paclitaxel nano liposomes of dual targeting tumor and method for preparing paclitaxel nano liposomes Download PDFInfo
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Abstract
The invention belongs to the field of biomedicine. In particular, the invention discloses paclitaxel nano liposomes of dual targeting tumor. The dual targeting paclitaxel nano liposomes consist of three parts: polypeptides of dual targeting tumor, lipid attachments and paclitaxel liposomes. The invention also discloses a method for preparing the paclitaxel nano liposomes of dual targeting tumor and application of paclitaxel nano liposomes to preparation of a tumor-inhibiting medicament.
Description
Technical field
The invention belongs to field of medicaments, more specifically, the invention discloses nanometer liposome of a kind of dual-target tumor and preparation method thereof.
Background technology
The M ﹠ M of malignant tumor is obvious ascendant trend in recent years, has become the principal disease that threatens human health and life.The report of international cancer research institution shows, between 1975 to 2000, global cases of cancer number has increased by one times, annual new cases approximately 1,200 ten thousand, and Died Patients surpasses 7,000,000.Cancer will leap to the primary cause of the death into the whole world in 2010, the year two thousand thirty the tumor patient number will be present three times, the new cases number will increase to 2000-2600 ten thousand.
Chemotherapy is one of Main Means in the Multimodality Therapy of Malignant Tumors, but the life span of rational chemotherapy strategy significant prolongation tumor patient is greatly improved patient's life quality.Paclitaxel is a kind of diterpene-kind compound, the natural product that from the Pacific yew bark, obtains the earliest, because it can stablize the microtubule of tumor cell, suppress mitosis and have unique active anticancer (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.), be widely used at present ovarian cancer (Thomas, H.and P.Rosenberg, Roleof weekly paclitaxel in the treatment of advanced ovarian cancer.CritRev Oncol Hematol, 2002.44Suppl:p.S43-51.), breast carcinoma (Saloustros, E., D.Mavroudis, and V.Georgoulias, Paclitaxel and docetaxel in thetreatment of breast cancer.Expert Opin Pharmacother, 2008.9 (15): p.2603-16.), incidence cancer (Aisner, J.and H.Cortes-Funes, Paclitaxel in headand neck and other cancers:future prospects.Semin Oncol, (1997.24 1Suppl 2): p.S2-113-S2-115.) and the treatment (Greco of nonsmall-cell lung cancer, F.A., Paclitaxel-based combination chemotherapy in advanced non-small celllung cancer.Lung Cancer, 2001.34Suppl 4:p.S53-6.).But paclitaxel is a kind of liposoluble substance, in aqueous solution and other many solvents, all be difficult to dissolving (Ceruti, M., et al., Preparation, characterization, cytotoxicity and pharmacokinetics ofliposomes containing water-soluble prodrugs of paclitaxel.J ControlRelease, 2000.63 (1-2): p.141-53.).Formulation for paclitaxel (the Taxol that uses clinically, commodity are called taxol) solvent that adopts is non-ionic surfactant polyoxyethylene Oleum Ricini (Cremophor EL, CrEL) and dehydrated alcohol (1: 1, V/V) mixture, but this dosage form is followed a lot of insoluble clinical problem (Hennenfent, K.L.and R.Govindan, Novel formulations of taxanes:areview.Old wine in a new bottle? Ann Oncol, 2006.17 (5): p.735-49.).The clinical practice of tradition formulation for paclitaxel can cause a series of anaphylaxis (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.), the lighter shows pruritus, erythra, severe one shows as angioedema, hypotension, (the Weiss such as respiratory failure, R.B., et al., Hypersensitivity reactions from taxol.J ClinOncol, 1990.8 (7): p.1263-8.Wiemik, P.H., et al., Phase I clinical andpharmacokinetic study of taxol.Cancer Res, 1987.47 (9): p.2486-93.).Patient's anaphylaxis more than 95% betide for the first time or for the second time formulation for paclitaxel administration during, 80% symptom betides front 10 minutes of infusion of drug, and often paclitaxel only infusion 1mg.Because but the CrEL induced animal produces similarly reaction (Wiernik, P.H., et al., Phase I clinical and pharmacokinetic study oftaxol.Cancer Res, 1987.47 (9): p.2486-93.), the existence of therefore thinking CrEL has caused the generation of above-mentioned untoward reaction.And the generation of untoward reaction (Lorenz relevant with infusion velocity, W., et al., Histamine release and hypotensive reactions in dogs by solubilizing agents and fattyacids:analysis of various components in cremophor El and development of acompound with reduced toxicity.Agents Actions, 1982.12 (1-2): p.64-80.), can not eliminate anaphylactoid appearance but prolong the infusion time.Researcher is attempted substituting CrEL with other solvents, and such as Polyethylene Glycol, but the existence of zoopery discovery Polyethylene Glycol can reduce the anti-tumor activity of paclitaxel.Therefore CrEL is still standard solvent (Wiernik, P.H., et al., Phase I clinical andpharmacokinetic study of taxol.Cancer Res, 1987.47 (9): p.2486-93.) of present paclitaxel.
Generally adopt clinically the conventional pretreatment before heavy dose of glucocorticoid and histamine receptor antagonists use as it, but its curative effect is still imprecise.The pharmacovigilance project Chinese medicine Adverse Event of Northwestern Univ USA's Feenberg medical college and report research (Research on Adverse Drug Events and Reports, RADAR) find, between 1997 to 2007 the U.S., Europe and Japan Report 171 examples anaphylactoid case occurs after using the polyoxyethylene castor oil paclitaxels, wherein death (34%) occurs in 58 routine patients, it is dead that 96 example reports have adopted among the patient of pretreating scheme 21 examples to occur, and mortality rate reaches 22%.Research (the Michaud of BoehnkeMichaud etc., L.B., V.Valero, and G.Hortobagyi, Risks and benefitsof taxanes in breast and ovarian cancer.Drug Saf, 2000.23 (5): p.401-28.) also show, the allergy of light disease can occur in 40% the patient of still having an appointment after pretreating scheme adopts, and patient's anaphylaxis of 3% can be fatal.The simultaneously execution of pretreating scheme has limited tumor and has merged the patient of ulcer, diabetes, hypertension etc. to the use of this medicine, and the use of hormone also may increase the relevant mortality rate for the treatment of in the pretreating scheme.
Adopt CrEL also can cause induced peripheral neurotoxicity: (Onetto, N., et al., Overview of Taxol safety.J Natl Cancer Inst Monogr, 1993 (15): p.131-9.) as solvent.Electrophysiologic study finds that the patient that peripheral neuropathy occurs after paclitaxel CrEL solution uses exists aixs cylinder degraded and demyelination.Peripheral neuropathy can occur in 25% the patient of having an appointment behind the cyclosporine CrEL solution intravenously administrable, oral administration then occurs without this untoward reaction, and CrEL and without gastrointestinal absorption (de Groen, P.C., et al., Central nervous system toxicity after liver transplantation.The role of cyclosporineand cholesterol.N Engl J Med, 1987.317 (14): p.861-6.).The CrEL plasma concentration that the paclitaxel of therapeutic dose or cyclosporine CrEL solution produce can cause the swelling of mice dorsal root ganglion neurons aixs cylinder, vacuolar degeneration and degraded (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.).Present studies show that, the epoxyethane derivative that CrEL produces is the topmost factor (Brat that causes nerve injury, D.J., A.J.Windebank, and S.Brimijoin, Emulsifier forintravenous cyclosporin inhibits neurite outgrowth, causes deficits in rapid axonaltransport and leads to structural abnormalities in differentiating N1E.115neuroblastoma.J Pharmacol Exp Ther, 1992.261 (2): p.803-10.).
Paclitaxel CrEL solution is inferior on every Wendesdays, each usage of 3 hours of infusion makes the plasma concentration of paclitaxel exceed body and removes the ability of metabolism, therefore the elimination in vivo of paclitaxel CrEL solution is nonlinear kinetics, also be called zero-order elimination kinetics (Sparreboom, A., et al., Cremophor EL-mediatedalteration of paclitaxel distribution in human blood:clinical pharmacokineticimplications.Cancer Res, 1999.59 (7): Tellingen p.1454-7.van, O., et al., CremophorEL causes (pseudo-) non-linear pharmacokinetics of paclitaxel in patients.Br JCancer, 1999.81 (2): p.330-5.).Zoopery confirms, paclitaxel nonlinear pharmacokinetics feature (Sparreboom relevant to CrEL, A., et al., Nonlinear pharmacokinetics of paclitaxel in miceresults from the pharmaceutical vehicle Cremophor EL.Cancer Res, 1996.56 (9): p.2112-5.), experiment in the human body has also obtained similar result (van Tellingen, O., et al., CremophorEL causes (pseudo-) non-linear pharmacokinetics of paclitaxel in patients.Br JCancer, 1999.81 (2): p.330-5.).The reduction of the clearance rate of paclitaxel causes body tissue to be exposed to the medicine time lengthening of high concentration, and the probability that produces serious whole body toxicity increases.CrEL also can form molecule in blood, parcel paclitaxel molecule, thereby affect distribution, metabolism and the excretion (Sparreboom of drug molecule in tumor tissues, A., et al., Cremophor EL-mediated alteration of paclitaxeldistribution in human blood:clinical pharmacokinetic implications.Cancer Res, 1999.59 (7): p.1454-7.).
The resistance mechanism of paclitaxel is a lot, and it is one of important reason that the expression of P-glycoprotein raises.The P-glycoprotein is by a kind of transmembrane transporter of multidrug resistance gene coding, has atpase activity, chemotherapeutics can be pumped in cell, reduces intracellular active drug concentration and causes drug resistance.There is research to think, CrEL can be by regulating the P-glycoprotein, suppress the expression of multidrug resistance gene and strengthen the antitumor action of paclitaxel, but (Woodcock does not in vivo succeed in the experiment, D.M., et al., Reversal of the multidrugresistance phenotype with cremophor EL, a common vehicle for water-insolublevitamins and drugs.Cancer Res, 1990.50 (14): p.4199-203.Schuurhuis, G.J., et al., The polyoxyethylene castor oil Cremophor EL modifies multidrug resistance.Br JCancer, 1990.62 (4): p.591-4.Friche, E., et al., The solvents cremophor EL andTween 80 modulate daunorubicin resistance in the multidrug resistant Ehrlich ascitestumor.Cancer Commun, 1990.2 (9): p.297-303.).There is on the contrary research to find, CrEL can be by the retardance cell cycle, reduce tumor cell to the picked-up of paclitaxel and the cytotoxicity (Liebmann of antagonism paclitaxel, J., et al., The influence of Cremophor EL on the cell cycle effects ofpaclitaxel (Taxol) in human tumor cell lines.Cancer Chemother Pharmacol, 1994.33 (4): p.331-9.).
Studies show that the problem of paclitaxel CrEL solution existence and stability and the compatibility just easily precipitates once the dilution paclitaxel such as medicine; Can be from stripping plasticizer (divinyl hexyl phthalate) the clinically conventional PVC transfusion bag of using and the infusion pipeline, therefore whole use procedure must be adopted glass or non-PVC infusion device; With plastics or glass container non-specific adsorption etc. (Waugh is arranged, W.N., L.A.Trissel, andV.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 sum, the existence of CrEL has caused the appearance of a series of clinical toxicities in the existing dosage form of paclitaxel, CrEL also can produce complicated effect to curative effect and the pharmacokinetics of paclitaxel, therefore clinically in the urgent need to developing the paclitaxel novel form that does not contain CrEL, to overcome the defective of surfactant.
Numerous scholars are devoted to the research and development of paclitaxel novel form, and the novel form of having researched and developed successfully or having researched and developed at present comprises: albumin nano granular (Abraxame
TM) (Ibrahim, N.K., et al., Phase I andpharmacokinetic 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 withSolid Tumors.Jpn J Clin Oncol, 2010.), prodrugs of paclitaxel docosahexenoic acid paclitaxel (Taxoprexin) (Wolff, A.C., et al., Phase I study of docosahexaenoic acid-paclitaxel:a taxane-fatty acid conjugate with a unique pharmacology and toxicity profile.ClinCancer 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 epithelialovarian, fallopian tube, or primary peritoneal carcinoma.J Clin Oncol, 2004.22 (22): p.4523-31.), paclitaxel analogs (BMS-184476 (Rose, W.C., C.Fairchild, and F.Y. Lee, Preclinical antitumor activity of two novel taxanes.Cancer Chemother Pharmacol, 2001.47 (2): p.97-105.), DJ-927Ono, 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/IIstudy of a 3 weekly oral taxane (DJ-927) in patients with recurrent, advancednon-small cell lung cancer.J Thorac Oncol, 2008.3 (7): p.745-50.), BMS-275183 (Broker, L.E., et al., Effect of food on the pharmacokinetic behavior ofthe 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?withpromising?antitumor?activity.Clin?Caneer?Res，2006.12(6)：p.1760-7.)、OrtataxelCassinelli，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?tolerability?andtherapeutic?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?8to?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 solidtumors.J Clin Oncol, 2000.18 (17): p.3164-71.), paclitaxel micelle copolymer (Genexol-PM) (Kim, S.C., 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 microsphere (Paclimer) (Lissianskaya, A., et al., Paclitaxelinjectable emulsion:Phase 2a study of weekly administration in patients withplatinum-resistant ovarian cancer.Journal of Clinical Oncology, 2004.22 (14): p.460s-460s.Bogdanova, N., et al., Paclitaxel injectable emulsion:Phase 2a study ofweekly administration in patients with non-small cell lung cancer (NSCLC) .Journalof Clinical Oncology, 2004.22 (14): p.649s-649s.), Paclitaxel liposome (power is simple) etc.The part paclitaxel novel form (Abraxame that goes on the market wherein
TMGone on the market by FDA's approval in 2005; Taxusol-lipid muscle power is simple to be researched and developed successfully by Nanjing Cisco Pharmaceutical, at home listing of approval in 2003), most of paclitaxel novel form still is in the preclinical study stage, or in the I-III clinical trial phase.
The common ground of above-mentioned paclitaxel novel form is to have avoided employing CrEL as solvent, therefore has unique advantage, such as the infusion time that can shorten medicine, the incidence rate that reduces the untoward reaction such as allergy, bone marrow depression and alopecia etc.The part dosage form be because therefore the change of structure and no longer be the effect substrate of P-glycoprotein can reduce the incidence rate of taxol resistance, part dosage form even can oral absorption.But it is on the knees of the gods that can paclitaxel novel form and paclitaxel conventional dosage forms compare the prognosis that improve tumor patient.
Summary of the invention
The present invention is intended to research and develop a kind of novel cancer target liposome drug-carrying nanometer particle, can improve physicochemical character and the pharmacokinetics behavior of paclitaxel after the envelope paclitaxel, improve dissolubility, thereby avoid every toxicity that CrEL causes in the conventional formulation, pass through simultaneously the tumor-targeting of this carrier, increase specifically paclitaxel in the concentration of tumor by local, when strengthening antitumor action, reduce the toxicity of medicine normal tissue organ.
The present invention is encapsulated in paclitaxel in the nanometer liposome of dual-target tumor, and two target molecules can be kept separately original biologic activity, produce cooperative effect in function.
The paclitaxel nano lipid body of dual-target tumor provided by the invention mainly is comprised of three parts, the polypeptide of dual-target tumor, lipid junctional complex and Paclitaxel liposome.
At first, the present invention adopts solid-phase synthesis to obtain the polypeptide of dual-target tumor, and the aminoacid sequence of described polypeptide is as follows: ARYC
RGDCFD
ATWLPPRARYC wherein
RGDCFDG: its core texture is the RGD tripeptides, and namely arginine-glycine-aspartic acid (Arg-Gly-Asp) sequence is integin αV family for target spot.α V integrin is one of important member of cell adhesion molecule family, activation and migration, the mediation endothelial cell proliferation by participating in endotheliocyte, suppress endothelial cell apoptosis, participate in angiogenesis that basic fibroblast growth factor and VEGF induce, genesis that the number of ways such as generation of inducing COX-2 promote neovascularization and tumor.α V integrin is low in static vascular endothelial cell and the normal structure organ under physiological status expresses, but high expressed is in tumor vascular endothelial cell and the tumor cell surface of activation.Pharmaceutical carrier connects the ability that can significantly strengthen its target tumor new vessels behind the small peptide contain the RGD sequence.The ATWLPPR sequence be for target spot be the neural endothelin-1 (Neuropilin-1, NRP-1) of co-receptor of VEGFR2.VEGFR-2 (Vascular Endothelial Growth Factor Receptor-2) is the VEGF/VEGFR family member, the low-molecular-weight VEGF of main identification plays an important role in migration of vascular endothelial cells, propagation, survival and vascular permeability are regulated.NRP-1 is-kind of non-tyrosine transmembrane glycoprotein, is the accessory receptor of VEGFR-2, and the coexpression of VEGFR-2 can significantly promote the combination of VEGF165 and VEGFR-2, the biological action of enhancing VEGF165 mediation, the propagation of promotion blood vessel endothelium.NRP-1 also high expressed is low the expression in tumor vascular endothelial cell and the tumor cell surface of activation in static vascular endothelial cell and the normal structure organ under physiological status.
The present invention adopts the Fmoc solid-phase synthesis to synthesize polypeptide ligand-LYS-GLY-glycine-Palmic acid (lysine-glycine-glycine, KGG-pal) connection thing, connects novel polypeptide and Paclitaxel liposome.
The present invention adopts membrane-sonic method to prepare Paclitaxel liposome, and press filtration method or high pressure homogenization method control liposome particle diameter are at 60-200nm.The nanoparticle of small particle diameter has its unique advantage as pharmaceutical carrier.The invasive growth of malignant tumor and shift the generation depend on blood vessel, although with respect to normal blood vessels, the tumor tissues medium vessels to the selectively penetrating power of medicine a little less than, maximum gauge still is no more than 400nm.The liposome of large particle diameter is more holding back of spleen, removes sooner from blood, and the liposome that arrives tumor tissues obviously reduces.Therefore granule path length circulation medicinal liposome more easily sees through the effect that the vascular space performance arrives tumor by local.
Cytology research confirms, the dual-target tumor Paclitaxel liposome that contains RGD and ATWLPPR sequence of the present invention, and because novel targeted polypeptide molecular weight is little, core medicine carrying grain diameter is controlled at 60-200nm, therefore easily the Penetrating Through Endothelial Cells barrier enters tumor tissues.Studies confirm that, RGD and ATWLPPR sequence connect the rear space conformation and are independent of each other, and can keep separately original biologic activity of sequence, thereby produce cooperative effect.Compared to the single targeting Paclitaxel liposome that only contains RGD or ATWLPPR sequence, the Paclitaxel liposome of the dual-target tumor of research and development has significantly and to be excellent and specific binding power new vessels endotheliocyte and tumor cell.With compare without the Paclitaxel liposome of targeting and single target tumor, the Paclitaxel liposome of dual-target tumor has stronger and new vessels endotheliocyte and tumor cell specific binding ability, and suppress the tumor growth ability, therefore can further be applied to the targeted therapy field of malignant tumor.
Description of drawings
Fig. 1: the Paclitaxel liposome scanning electron microscope (SEM) photograph of dual-target tumor
Fig. 2: the Paclitaxel liposome particle size distribution figure of dual-target tumor
Fig. 3: Taxol and each Paclitaxel liposome cellular uptake figure
Fig. 4: Taxol and each Paclitaxel liposome MTT detect figure
Fig. 5: Taxol and each Paclitaxel liposome tumor suppression experiment D60 tumor multigraph
The specific embodiment
Experiment material
1, reagent, medicine and cell
Ovum Gallus domesticus Flavus lecithin (egg phosphatide, egg PC), cholesterol (cholesterol, CHOL) are available from German Dong Shang company; MPEG2000-DSPE mPEG2000-DSPE is available from U.S. Avanti Polar Lipids company; The lipid polypeptide is synthetic by the biochemical (Shanghai) Co., Ltd. of gill; Taxol is available from Shi Guibao company; Paclitaxel is available from upper Hiroad standing grain medical sci-tech Development Co., Ltd; Sodium lauryl sulphate (Sodium dodecyl sulfonate, SDS), polyacrylamide, sucrose, glacial acetic acid, silver nitrate, glutaraldehyde, aprotinin etc. are all available from Shanghai branch company of traditional Chinese medicines group; HUVEC (Human umbilical vein endothelial cells), ATCC No.:CRL-2873; A549 cell (lung adenocarcinoma cell system), ATCC No.:CCL-185; DMEM (Dulbecco ' s Modified Eagle Media, high sugared cell culture medium), available from Invitrogen company; New-born calf serum is available from Austrian PAA company; Penicillin, streptomycin, trypsin etc. are available from magnificent company.
2, instrument and equipment and consumptive material
MillexTM FH pin type filter and Ultracel YM100 super filter tube (U.S. Millipore company); SK5210HP water-bath Ultrasound Instrument (Shanghai High Kudos Science Instrument Co., Ltd.); R502B Rotary Evaporators (Taikan, Xi'an instrument and equipment company limited); JEM-1200EXII type transmission electron microscope (Japanese JEOL company); Quanta 200FEG field launch environment scanning electron microscope (FEI Co.); MASTERSIZER 2000 laser particle analyzers (Britain Malvern company); Freezer dryer 6L (U.S. Labconco company); Remaining the same.
3, solution preparation
PBS liquid (0.01M, pH7.4): NaCl 8.0g; KCl 0.2g; Na2HP0412H20 1.44g; KH2PO4 0.24g; Ultra-pure water 800ml; Regulate pH value to 7.4, be settled to autoclaving behind the 1L, 4 ℃ of preservations.
0.25% trypsinization liquid (1L): pancreatin 2.5g; NaCl 8g; KCl 0.4g; Na2HP0412H20 0.06g; KH2PO4 0.06g; NaHCO3 0.35g; Phenol red 0.02g; The pressure filtration degerming is preserved DMEM culture medium (1L) for-20 ℃ after the packing: 2 bags in DMEM powder; NaHCO37.4g; Penicillin 0.12g; Streptomycin 0.2g; HCl, NaOH regulate pH value to 7.2-7.4, pressure filtration degerming, 4 ℃ of preservations; Cells frozen storing liquid: DMEM culture medium 70%; Dimethyl sulfoxine (DMSO) 10%; New-born calf serum 20%;-20 ℃ of preservations after the packing; Cell pyrolysis liquid: Tris 10mmol/L; Nacl 100mmol/L; EDTA 1mmol/L; EGTA 1mmol/L; NaF 1mmol/L; Na4P2O710H2O 20mmol/L; Vanadic acid sodium 2mmol/L; Triton X-1001%; Glycerol 10%; Sodium lauryl sulphate 0.1%; Dexycholate 0.5%; PMSF 100 μ g/ μ L.
Further specify the present invention by following examples, but not as restriction of the present invention.
Embodiment 1: the synthesis step of ARYCRGDCFDATWLPPR
Adopt 9-fluorenylmethyloxycarbonyl (fluorenylmethyloxycarbonyl, FMOC) polypeptide of the synthetic dual-target tumor of solid-phase synthesis, adopt high performance liquid chromatography (high performance liquidchromatography, HPLC) method purification, adopt Mass Spectrometric Identification.Concrete synthesis step is as follows:
1) resin swelling: the FMOC-AA-Wang-Resin resin is put into reaction tube, add (15ml/g) 30min of dimethyl formamide (N, N-dimethylformamide, DMF);
Deprotection: inhale and abandon DMF, add 20% piperidines DMF solution (15ml/g) 5min, add again 20% piperidines DMF solution (15ml/g) 15min after suction is abandoned;
2) detect: take out piperidine solution, get tens of resins, wash three times with ethanol, add 1,2,3-indantrione monohydrate, potassium cyanide, each one of phenol solution, 105 ℃-110 ℃ heating 5min deepen blue positive reaction.
3) washing: DMF (10ml/g) twice, methanol (10ml/g) twice, twice of DMF (10ml/g);
4) condensation: three times of protected amino acids (FOMC-Asp-OH) are excessive, two three times of the dimethylamine carboxyl benzotriazole tetrafluoride boron salt of 1-oxygen-3-are excessive, all with lack the DMF dissolving as far as possible, add reaction tube, add at once ten times of N-methylmorpholines excessive, reaction 30min;
5) washing: DMF (10ml/g) once, methanol (10ml/g) twice, twice of DMF (10ml/g);
6) repetitive operation step 2)--6), connect successively 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) last washing: DMF (10ml/g) twice, methanol (10ml/g) twice, DMF (10ml/g) twice, twice of DCM (10ml/g);
8) cracking: lysate (10ml/g) (contains TFA 94.5%; Water 2.5%; EDT 2.5%; TIS 1%) 120min;
9) dry up washing: nitrogen dries up lysate as far as possible, ether washing six times, and room temperature volatilizes;
10) sealing ,-20 degree are preserved.
Gained is the white powder material, HPLC purification, purity＞95%;
Embodiment 2: the preparation of the Paclitaxel liposome of non-targeted, single targeting and dual-target tumor
Adopt membrane-sonic method to prepare each Paclitaxel liposome
Non-active targeting Paclitaxel liposome prescription: PC: CHOL: mPEG-DSPE: PTX 9: 1: 0.5: 0.33, initiatively the targeting Paclitaxel liposome adds different lipid polypeptide (ARYC in above-mentioned prescription
RGDCFDG-KGG, ARYC
RGDCFD
ATWLPPR-KGG) (be 1: 542 with total fat mol ratio).Because in patent 1 embodiment, polypeptide A TWLPPR and ATWLPPR-fluorescent lipid body all do not demonstrate and are better than polypeptide A RYC
RGDCFDG, ARYC
RGDCFDG-fluorescent lipid body and polypeptide A RYC
RGDCFD
ATWLPPR, ARYC
RGDCFD
ATWLPPR-fluorescent lipid body with the cell-specific affinity, therefore synthetic, and in follow-on test, also do not give application.
Above-mentioned prescription is dissolved in chloroform, and 0.2 μ m plate filter filters, and puts the follow-up continuous vacuum draw of Rotary Evaporators 37 ℃ of rotations film forming and further removes chloroform in 1 hour; 9% sucrose solution that adds an amount of filtration sterilization, fully being swelling to thin film comes off, the gained suspension divides to be filled to cillin bottle after Mini-extruder extrudes (1 μ m, 0.4 μ m, 0.2 μ m, 0.1 μ m each 10 times), puts in the vacuum freeze drier,-38 ℃ * 2h,-35 ℃ * 18h ,-16 ℃ * 2h, 20 ℃ * 2h, the capping of jumping a queue after the drying ,-20 ℃ of Refrigerator stores are stand-by.Freeze-dried lipidosome PBS redissolution liquid is all adopted in every detection.
Scanning electron microscope is seen the rounded or similar round of two targeting Paclitaxel liposomes, and granular size is more consistent, particle diameter＜100nm (seeing Fig. 1).
Embodiment 3: the Paclitaxel liposome extracorporeal releasing experiment
Paclitaxel liposome and each 3ml of taxol (Taxol) of non-active targeting Paclitaxel liposome, single targeting and the dual-target tumor of redissolving put in the molecular weight 14KD bag filter, bag adds the aquesterilisa simulated in vivo environment that 20ml contains the 45g/L bovine serum albumin, constant temperature blender with magnetic force stirs (300rpm), take out respectively dialysis solution 80 μ L in 30min, 1h, 2h, 4h, 6h, 8h, 24h for subsequent use, the outer add-back 80 μ L of bag filter contain the aquesterilisa of 45g/L bovine serum albumin simultaneously.The dialysis solution that takes out adds the chromatographic grade methanol solution protein precipitation of 320 μ L, and high speed centrifugation 13000g * 10min after whirlpool mixes gets the capable HPLC of supernatant and detects content of taxol.Detect simultaneously the concentration of paclitaxel in each Paclitaxel liposome of redissolving before the dialysis and the Taxol solution, with (in the dialysis solution in paclitaxel concentration/stock solution paclitaxel concentration) * 100% index as the measurement slow-release capability.
Found that (seeing Fig. 2), paclitaxel discharges the fastest from the CrEL adjuvant, and the 24h cumulative release reaches 15.96%.After paclitaxel adopts liposome as pharmaceutical carrier, external rate of release obviously reduces, the Paclitaxel liposome 24h cumulative release of Paclitaxel liposome, single targeting Paclitaxel liposome, dual-target tumor is respectively 4.40%, 4.55%, 3.6%, and slow releasing function is remarkable.Also point out simultaneously the lipid polypeptide chain to be connected to the structural intergrity of not destroying liposome behind the surface of liposome, the sustained release performance of enhancing can further improve paclitaxel pharmacokinetics behavior in vivo.
Embodiment 4: the cell endocytic experiment
HUVEC, A549 cell are inoculated in 24 orifice plates, 1 * 10
6/ hole, put incubator 24h and make cell attachment, every hole adds respectively Taxol, Paclitaxel liposome, single targeting Paclitaxel liposome, two targeting Paclitaxel liposome (in paclitaxel 5 μ g), 4 in multiple hole, hatch 4h for 37 ℃, ice PBS liquid washing 2 times adds lysis liquid 100 μ L/ holes, collects lysate, add 100 μ L methanol solution protein precipitations, whirlpool mixing 5min, the centrifugal 30min of 10000g gets the capable HPLC of supernatant and detects intracellular content of taxol.
After Taxol and each Paclitaxel liposome and cell were hatched, the cellular uptake situation was seen Fig. 3.As seen from the figure, A549 cell and HUVEC are extremely low to the intake of traditional paclitaxel solution (Taxol), and content of taxol reaches HPLC and detects minimum (being respectively 5ng/ hole and 25ng/ hole).After paclitaxel is encapsulated in liposome, because liposome can have an effect by the number of ways such as contact release, absorption, endocytosis, fusion, lipid exchange and cell, therefore increased the picked-up ability of cell to paclitaxel.A549 cell and HUVEC reach respectively 145ng/ hole, 157ng/ hole to the intake of non-targeted Paclitaxel liposome.The Paclitaxel liposome that connects tumor specific target polypeptide has further strengthened the picked-up ability of cell to paclitaxel, wherein A549 cell and HUVEC are respectively 212ng/ hole and 232ng/ hole to the intake of single target spot Paclitaxel liposome, and the intake of the Paclitaxel liposome of dual-target tumor is respectively 346ng/ hole and 319ng/ hole.Above-mentioned data are also pointed out simultaneously, and the ability of the Paclitaxel liposome of cellular uptake dual-target tumor is stronger, confirm that the novel polypeptide of dual-target tumor has and tumor cell, the better specific adhesion of vascular endothelial cell.
Embodiment 5: cell toxicity test
A549 and HUVEC cell dilution to 4 * 10
4/ mL adds 96 orifice plates, and every hole 100 μ L put incubator 24h and make cell attachment; The DMEM culture fluid of employing serum-free is the working solution of final concentration 0.9375mg/L, 1.875mg/L, 3.75mg/L, 7.5mg/L, 15mg/L with the Paclitaxel liposome doubling dilution of Taxol, Paclitaxel liposome, single targeting Paclitaxel liposome, dual-target tumor, every hole adds working solution 100 μ L, puts cell culture incubator again and hatches 72h; Add the MTT liquid 20 μ l/ holes of 5mg/mL, hatch 4h in the incubator, the centrifugal 10min of 1500rpm, discard culture fluid, each hole adds respectively DMSO200 μ L, upper microplate reader behind the decolorization swinging table mixing 30min, select wavelength 530nm to detect light absorption value, calculate IC50, formula is as follows:
Fig. 4 as seen, Taxol is respectively 6.027mg/L, 6.56mg/L to the IC50 value among A549 cell, the HUVEC; Paclitaxel liposome is respectively 3.37mg/L, 4.56mg/L to the IC50 value of two kinds of cells; The Paclitaxel liposome of single targeting Paclitaxel liposome, dual-target tumor is respectively 1.46mg/L, 1.16mg/L and 3.40mg/L, 3.25mg/L to the IC50 value of two kinds of cells.Strengthened the toxicity to tumor cell and vascular endothelial cell behind the results suggest taxusol-lipid surface connection blood-vessels target polypeptide, wherein the Paclitaxel liposome of dual-target tumor has the strongest cytotoxicity.
Embodiment 6: the in vivo antitumor effect assessment
A549 lung adenocarcinoma cell cellar culture is to exponential phase, and 0.25% trypsinization gets 1 * 10 after becoming cell suspension
7It is subcutaneous that/0.1mL is inoculated in the right axil of 4 week (18-20g) female Balb/c nude mices in age, and the tumor body grows to 50mm
3-80mm
3Play the beginning administration.Nude mice is divided into 5 groups by the administration difference, 5 every group, gives respectively the Paclitaxel liposome of normal saline, Taxol, Paclitaxel liposome, single targeting Paclitaxel liposome and dual-target tumor in d1, d4, d8 days.Wherein the normal saline group gives normal saline 0.5mL, and the remaining dosage of respectively organizing is counted 7.5mg/kg by paclitaxel.Monitoring nude mice body weight (2 times/week), tumorous size (2 times/week) after the administration put to death all nude mices to d60, peels off the tumor body and weighs, and calculates tumor control rate.
Tumor size computing formula: gross tumor volume V=ab
2/ 2, wherein a is that the major diameter of tumor and b are the minor axis of tumor, tumor control rate computing formula: (1-experimental group tumor is heavy)/matched group tumor heavy * 100%.
The result shows (seeing Fig. 5), and the nude mice of control group growth of xenografted that gives normal saline is rapid, and d60 reaches 1925mm
3, average tumor heavily is (1.62 ± 0.09g).Give Taxol and each Paclitaxel liposome group nude mice tumor volume growth is relatively slow, Taxol group d60 transplanted tumor average external volume is 1104mm
3, tumour inhibiting rate is 46.9%; Paclitaxel liposome group d60 transplanted tumor average external volume is 912mm
3, average tumor heavily is that (0.61 ± 0.10g), tumour inhibiting rate is 62.3%; The Paclitaxel liposome group tumor average volume of single targeting Paclitaxel liposome group and dual-target tumor further dwindles, and is respectively 590mm
3And 455mm
3, average tumor heavily is respectively (0.44 ± 0.07g) and (0.30 ± 0.04g), tumour inhibiting rate is respectively 72.8% and 81.5%.The polypeptide that indirectly confirms the dual-target tumor effectively is connected in the taxusol-lipid surface, has promoted the picked-up of tumor tissues to pharmaceutical carrier, has improved the valid density of medicine in the part, has strengthened the anti-tumor capacity of medicine.
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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