CN102659950B - LHRH-bonded amphiphilic biodegradable polymer, preparation method and application - Google Patents
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- CN102659950B CN102659950B CN 201210116346 CN201210116346A CN102659950B CN 102659950 B CN102659950 B CN 102659950B CN 201210116346 CN201210116346 CN 201210116346 CN 201210116346 A CN201210116346 A CN 201210116346A CN 102659950 B CN102659950 B CN 102659950B
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- Medicinal Preparation (AREA)
Abstract
键合了LHRH的两亲性可生物降解聚合物、制备方法和用途，属于生物医药技术领域。解决现有技术的LHRH连接药物分子困难、LHRH在血液循环中的稳定性差和成本高的缺陷。该聚合物由LHRH多肽衍生物和两嵌段共聚物键合而成，或由LHRH多肽衍生物和三嵌段共聚物键合而成，LHRH多肽有两种，分别是10位端羧基化的LHRH-10-COOH和6位甘氨酸被右旋的赖氨酸置换的LHRH-6-NH2。本发明还提供键合了LHRH的两亲性可生物降解聚合物的制备方法，本发明的键合了LHRH的两亲性可生物降解聚合物可制备带有LHRH靶向功能的载药纳米粒，适合于多种药物，可发挥其对高表达LHRH受体的癌细胞的靶向功能。
The amphiphilic biodegradable polymer bonded with LHRH, its preparation method and application belong to the technical field of biomedicine. The invention solves the defects of difficulty in connecting drug molecules to LHRH, poor stability of LHRH in blood circulation and high cost in the prior art. The polymer is formed by bonding LHRH polypeptide derivatives and two-block copolymers, or by bonding LHRH polypeptide derivatives and three-block copolymers. There are two types of LHRH polypeptides, which are carboxylated at the 10-terminal LHRH-10-COOH and LHRH-6-NH 2 in which glycine at position 6 is replaced by D-lysine. The present invention also provides a preparation method of an LHRH-bonded amphiphilic biodegradable polymer, and the LHRH-bonded amphiphilic biodegradable polymer of the present invention can prepare drug-loaded nanoparticles with LHRH targeting function , suitable for a variety of drugs, and can exert its targeting function on cancer cells with high expression of LHRH receptors.
Description
Technical field
The invention belongs to the biological medicine technology field, amphiphilic biologically degradable polymkeric substance, preparation method and the purposes of LHRH that be specifically related to bonding.
Background technology
LHRH is the abbreviation of luteinising hormone-releasing hormo (luteinizing-hormone-releasing hormone), represents the following polypeptide of being made up of 10 amino acid, has following array structure:
U.S. scientist A.V.Schally 1971 separates from the pig hypothalamus and obtains LHRH (being also referred to as Gn-RH at that time), has illustrated its aminoacid sequence and has successfully carried out synthetic, has therefore obtained Nobel Prize in medicine in 1977.The function of LHRH, expressed the same of its name of erect image is the secretion that promotes progestin, the function of regulating human body sexual organ and reproductive system.People have studied the derivative of a lot of LHRH, and wherein a class and its function class are its agonists seemingly, and another kind of is its antagonist, weakens its effect, suppress the function of hypophysis and sexual gland.No matter discover, be agonist or antagonist, and the principle that plays a role all is the interaction with the LHRH acceptor.Research also finds, has the acceptor of LHRH except hypophysis and sexual gland in the human body, and other position does not all have basically, but has crossing of LHRH acceptor to express in some tumour.Statistic data shows, 52% mammary cancer, 80% ovarian cancer and carcinoma of endometrium, and 86% prostate cancer crosses expression LHRH acceptor, and most of healthy tissues is not expressed the LHRH acceptor.Therefore, LHRH or its agonist, antagonist might be realized targeted and targeted therapy to these tumours as the target primitive of medicine.A.V.Schally is connected to 14 hydroxyls of Zorubicin with multiple LHRH derivative by pentanedioic acid and forms the target anticancer anthracycline derivative among the Chinese patent CN 1202903A, than the Zorubicin medicine, this compound is to comprising mammary cancer, ovarian cancer, prostate cancer has better therapeutic in several interior cancers.World patent WO 2010033580A2 discloses LHRH and curcumine is connected to form key compound by pentanedioic acid, utilize the keying action of LHRH and cell surface receptor, improved the anticancer therapeutic of curcumine, to carcinoma of the pancreas, prostate cancer, ovarian cancer, several cancers of expressing the LHRH acceptor such as cervical cancer have better therapeutic.US Patent No. 20020183257A1 discloses the method for PEG bonding LHRH derivative.T.Minko has the PEG of taxol to an end with a kind of LHRH is analogue bonded, and is perhaps that LHRH is analogue bonded to the dendrimer PAMAM that is connected with drug molecule.Experiment shows that bonding has the medicine carrying macromole of LHRH to show the antitumor activity that is better than taxol in the body.Now, utilize LHRH that the specific effect of its acceptor is increased the approval that curative effect of medication progressively obtains scientist.In the derivative of these LHRH, the derivative that forms after the LHRH terminal carboxylization (hereinafter to be referred as LHRH-10-COOH) has the feature of endogenous LH RH:
10 peptides of following structure use as the target primitive, and synthetic being easier to and advantage such as stability is better arranged, and receive more concern:
The difference of it and aforementioned LHRH be 6 glycine by the displacement of the Methionin of dextrorotation, formed the side amino that activity is higher, hereinafter to be referred as LHRH-6-NH
2
Up to the present, the target of reporting that utilizes LHRH all is that it is connected on the drug molecule, perhaps is connected on the drug molecule by a water-soluble polymer segment.Though do certain effect like this, shortcoming is apparent: (1) drug molecule needs a LHRH, and cost is very high, has increased considerably the cost of product; (2) LHRH will be received on the drug molecule, and types of drugs is a lot, structure is different, may connect very difficultly, even does not connect at all, does not still have a kind of LHRH can have universality, and it is all very convenient to connect which kind of medicine; (3) the target primitive with other protein and polypeptide class is the same, LHRH is easy to be decomposed by the various enzymes in the human body, poor stability in blood circulation, all proteins that expose in appearance all might be destroyed by the immunity system of human body before not arriving target spot.
Summary of the invention
The objective of the invention is for the LHRH that solves prior art connects drug molecule difficulty, poor stability and the cost high defective of LHRH in blood circulation, and bonding is provided amphiphilic biologically degradable polymkeric substance, preparation method and the purposes of LHRH.
Bonding the amphiphilic biologically degradable polymkeric substance of LHRH, this polymkeric substance is formed by LHRH polypeptide derivative and di-block copolymer bonding, structure is suc as formula shown in a:
Among the formula a, di-block copolymer is made up of PEG and HPB, PEG is the polyoxyethylene glycol block that end group has functional group, number-average molecular weight is 2-10kg/mol, described functional group is hydroxyl, amino or carboxyl, HPB is hydrophobic biodegradable polymers block, comprises polyester, polycarbonate, polyamino acid, polyesteramide, polyestercarbonate or polyester polyamino acid, and number-average molecular weight is 1-20kg/mol.
Described polyester is preferably PCL (poly-epsilon-caprolactone), PDLLA (poly-racemic lactic acid) or PLGA (polylactic acid-glycolic guanidine-acetic acid multipolymer); Polycarbonate is preferably PMCC (poly-2-methyl-2-carbonyl trimethylene-1, the ammediol carbonic ether), PDHP (poly-2,2-dihydroxymethyl trimethylene-1, the ammediol carbonic ether), PMBC (poly-2-methyl-2-carbobenzoxy-(Cbz)-1, the ammediol carbonic ether) or PMAC (poly-2-methyl-2-allyloxycarbonyl-1, ammediol carbonic ether); Polyamino acid is preferably PGA (polyglutamic acid) or PLL (polylysine); Polyesteramide is preferably PLGG (polylactide-poly-3s-propyloic morpholine-2, the 5-diketone) or PLGL (polylactide-poly-3s-ammonia butyl morpholine-2, the 5-diketone), polyestercarbonate is preferably P (LA-MCC) (polylactide-2-methyl-2-carbonyl trimethylene-1, the ammediol carbonate copolymer), P (CL-DHP) (poly-epsilon-caprolactone-2,2-dihydroxymethyl trimethylene-1, the ammediol carbonate copolymer) or P (LA-MAC) (polylactide-2-methyl-2-allyloxycarbonyl-1, ammediol carbonate copolymer); The polyester polyamino acid is preferably PLA-PGA (polylactide-polyglutamic acid) or PCL-PLL (poly-epsilon-caprolactone-polylysine).
Above-mentioned LHRH polypeptide derivative is the luteinising hormone-releasing hormo derivative, comprises LHRH-10-COOH (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-COOH) (structural formula I) and 6 LHRH-6-NH that glycine are replaced by the Methionin of dextrorotation of 10 end carboxylations
2(pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-GlyNH
2) (formula II);
Bonding the preparation method of amphiphilic biologically degradable polymkeric substance of LHRH, the polymkeric substance shown in the preparation formula a when the LHRH polypeptide derivative is LHRH-10-COOH, comprises the steps:
LHRH-10-COOH is dissolved in N, in dinethylformamide or the water, the 4-Dimethylamino pyridine (DMAP) that 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) that adding carboxyl mole number is 1.5-3 times and carboxyl mole number are 0.01 times, stirred 10-30 minute under the ice bath, add the carboxyl mole number 1-3 di-block copolymer that contains end amino or terminal hydroxy group doubly, keep ice bath and rise to room temperature naturally, reacted 12-24 hour, through dialysis lyophilize, the amphiphilic biologically degradable polymkeric substance of LHRH-10-COOH that obtained bonding.
Bonding the preparation method of amphiphilic biologically degradable polymkeric substance of LHRH, the polymkeric substance shown in the preparation formula a is when the LHRH polypeptide derivative is LHRH-6-NH
2The time, comprise the steps:
To contain and hold the di-block copolymer of carboxyl to be dissolved in N, in dinethylformamide or the water, the DMAP that the EDCHCl that adding carboxyl mole number is 1.5-3 times and carboxyl mole number are 0.01 times stirred 10-30 minute under the ice bath, added carboxyl mole number 0.3-1 LHRH-6-NH doubly
2, keep ice bath and rise to room temperature naturally, reacted 12-24 hour, through the lyophilize of dialysing, the LHRH-6-NH that obtained bonding
2The amphiphilic biologically degradable polymkeric substance.
Bonding the amphiphilic biologically degradable polymkeric substance of LHRH, this polymkeric substance is formed by LHRH polypeptide derivative and triblock copolymer bonding, structure is suc as formula shown in the b:
Among the formula b, triblock copolymer is made up of PEG1, PEG2 and HPB, and PEG1 is the polyoxyethylene glycol block, and the data molecular weight is 2-10kg/mol; PEG2 is the polyoxyethylene glycol block that side group contains functional group, and number-average molecular weight 1-5kg/mol, described functional group are hydroxyl, amino or carboxyl; HPB is hydrophobic biodegradable polymers block, comprises polyester, polycarbonate, polyamino acid, polyesteramide, polyestercarbonate or polyester polyamino acid, and number-average molecular weight is 1-20kg/mol.
Described polyester is preferably PCL (poly-epsilon-caprolactone), PDLLA (poly-racemic lactic acid) or PLGA (polylactic acid-glycolic guanidine-acetic acid multipolymer); Polycarbonate is preferably PMCC (poly-2-methyl-2-carbonyl trimethylene-1, the ammediol carbonic ether), PDHP (poly-2,2-dihydroxymethyl trimethylene-1, the ammediol carbonic ether), PMBC (poly-2-methyl-2-carbobenzoxy-(Cbz)-1, the ammediol carbonic ether) or PMAC (poly-2-methyl-2-allyloxycarbonyl-1, ammediol carbonic ether); Polyamino acid is preferably PGA (polyglutamic acid) or PLL (polylysine); Polyesteramide is preferably PLGG (polylactide-poly-3s-propyloic morpholine-2, the 5-diketone) or PLGL (polylactide-poly-3s-ammonia butyl morpholine-2, the 5-diketone), polyestercarbonate is preferably P (LA-MCC) (polylactide-2-methyl-2-carbonyl trimethylene-1, the ammediol carbonate copolymer), P (CL-DHP) (poly-epsilon-caprolactone-2,2-dihydroxymethyl trimethylene-1, the ammediol carbonate copolymer) or P (LA-MAC) (polylactide-2-methyl-2-allyloxycarbonyl-1, ammediol carbonate copolymer); The polyester polyamino acid is preferably PLA-PGA (polylactide-polyglutamic acid) or PCL-PLL (poly-epsilon-caprolactone-polylysine).
Above-mentioned LHRH polypeptide derivative is the luteinising hormone-releasing hormo derivative, comprises LHRH-10-COOH (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-COOH) (structural formula I) and 6 LHRH-6-NH that glycine are replaced by the Methionin of dextrorotation of 10 end carboxylations
2(pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-GlyNH
2) (formula II);
Bonding the preparation method of amphiphilic biologically degradable polymkeric substance of LHRH, the polymkeric substance shown in the preparation formula b when the LHRH polypeptide derivative is LHRH-10-COOH, comprises the steps:
LHRH-10-COOH is dissolved in N, in dinethylformamide or the water, the 4-Dimethylamino pyridine (DMAP) that 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) that adding carboxyl mole number is 1.5-3 times and carboxyl mole number are 0.01 times, stirred 10-30 minute under the ice bath, add the carboxyl mole number 1-3 triblock copolymer that contains side amino or pendant hydroxyl group doubly, keep ice bath and rise to room temperature naturally, reacted 12-24 hour, through dialysis lyophilize, the amphiphilic biologically degradable polymkeric substance of LHRH-10-COOH that obtained bonding.
Bonding the preparation method of amphiphilic biologically degradable polymkeric substance of LHRH, the polymkeric substance shown in the preparation formula b is when the LHRH polypeptide derivative is LHRH-6-NH
2The time, comprise the steps:
The triblock copolymer that will contain side carboxyl is dissolved in N, and in dinethylformamide or the water, the DMAP that the EDCHCl that adding carboxyl mole number is 1.5-3 times and carboxyl mole number are 0.01 times stirred 10-30 minute under the ice bath, added carboxyl mole number 0.3-1 LHRH-6-NH doubly
2, keep ice bath and rise to room temperature naturally, reacted 12-24 hour, through the lyophilize of dialysing, the LHRH-6-NH that obtained bonding
2The amphiphilic biologically degradable polymkeric substance.
Bonding the amphiphilic biologically degradable polymkeric substance of LHRH in the application of preparation in the drug-carrying nanometer particle.
Described drug-carrying nanometer particle the has been bonding amphiphilic biologically degradable polymkeric substance of LHRH and bonding the amphipathic nature block polymer self-assembly of drug molecule form, or by bonding the amphiphilic biologically degradable polymkeric substance of LHRH, amphipathic nature block polymer and drug molecule self-assembly form.Described drug molecule is the medicine that needs arbitrarily, as taxanes medicine, anthracene nucleus medicament, metal platinum medicine or gemcitabine.
Beneficial effect of the present invention:
1, bonding of the present invention the amphiphilic biologically degradable polymkeric substance of LHRH be a kind of polymkeric substance of complete biodegradable, reaction can be carried out in the aqueous solution, reacts simple and easy to control, LHRH derivative consumption is few, cost is low.
2, bonding of the present invention the amphiphilic biologically degradable polymkeric substance of LHRH; because polyoxyethylene glycol highly hydrophilic makes the periphery that it is in nanoparticle; polyoxyethylene glycol is to human immune system's formation " stealth " effect; make drug-carrying nanometer particle under polyoxyethylene glycol protection; in blood circulation the residence time longer and do not engulfed by immunocyte, have higher bioavailability and stability.
3; by bonding of the present invention the drug-carrying nanometer particle of amphiphilic biologically degradable polymer manufacture of LHRH; can be as required medicine by physics parcel or chemical bonding different varieties; has universality; drug distribution is in the inside of nanoparticle simultaneously; be subjected to duplicate protection; both avoided human immune system's attack; can reduce toxic side effects again; the LHRH molecule is in skin or the ectonexine near interface of nanoparticle; the targeting of taking the initiative; identified and combination by the LHRH acceptor easily, thereby make whole nanoparticle to the cell of LHRH expression of receptor or expression excessively targeting be arranged, preferentially medicine was delivered to the cell tissue of expressed receptor.
4, the targeting of the prepared drug-carrying nanometer particle of the present invention by experiment the result show: because the target effect of LHRH, nano particle is not only assembled in the SKOV3 surface of tumor cells, also faster by endocytosis in cell; Medicine presses down knurl curative effect aspect in vivo, mouse is carried out animal experiment method be: be that the subcutaneous injection of female nude mice left fore 0.1ml about 18-22g contains 106 SKOV3 tumour cell suspensions in weight at first, the tumour size is 50-100mm
3The time random packet, mark and administration for the first time, be administered three times altogether by the tail vein, be administered once every 3 days, each dosage is 20mg/kg (pressing taxol calculates), administration first day was the 0th day, every other day measure the knurl volume once, the knurl volume is calculated by following formula: knurl volume=major diameter * minor axis * minor axis/2, the result shows: prepared LHRH targeted medicament carrying nano grain has shown tumor-inhibiting action preferably: than the nanoparticle that is loaded with taxol that does not have target, administration is loaded with the nude mice gross tumor volume growth of the nanoparticle of taxol obviously slows down for the LHRH target, namely has and better presses down the knurl curative effect.
Description of drawings
Fig. 1 for bonding of the present invention the amphiphilic biologically degradable polymkeric substance ultraviolet-visible of LHRH absorb spectrogram;
The shape appearance figure of the target polypeptide mixing nanoparticle that is loaded with taxol drug that Fig. 2 obtains for the embodiment of the invention 12;
The size distribution figure of the target polypeptide mixing nanoparticle that is loaded with taxol drug that Fig. 3 obtains for the embodiment of the invention 12;
Fig. 4 is loaded with the polypeptide LHRH-6-NH of rhodamine B fluorescent molecular probe for embodiment 9 gained
2The burnt Photomicrograph of copolymerization that the mixing nanoparticle of target is engulfed human body ovarian cancer SKOV3 tumour cell;
The mixing nanoparticle of the bonding paclitaxel LHRH target polypeptide that Fig. 5 obtains for the embodiment of the invention 12 to lotus SKOV3 tumour nude mice time-the gross tumor volume curve;
Embodiment
In order further to understand the present invention, be described below in conjunction with the preferred embodiment of the invention of embodiment, but should be appreciated that these describe just in order to further specify the features and advantages of the present invention, rather than the present invention is weighed the restriction of requirement.
Bonding the amphiphilic biologically degradable polymkeric substance of LHRH, this polymkeric substance is formed by LHRH polypeptide derivative and di-block copolymer bonding, structure is suc as formula shown in a:
Among the formula a, di-block copolymer is made up of PEG and HPB, PEG is the polyoxyethylene glycol block that end group has functional group, number-average molecular weight is 2-10kg/mol, preferred 3-8kg/mol, more preferably 5-8kg/mol, 5kg/mol most preferably, described functional group is hydroxyl, amino or carboxyl, and HPB is hydrophobic biodegradable polymers block, comprises polyester, polycarbonate, polyamino acid, polyesteramide, polyestercarbonate or polyester polyamino acid, number-average molecular weight is 1-20kg/mol, preferred 2-15kg/mol, more preferably 5-10kg/mol, most preferably 10kg/mol.
Described polyester is preferably PCL, PDLLA or PLGA; Polycarbonate is preferably PMCC, PDHP, PMBC or PMAC; Polyamino acid is preferably PGA or PLL; Polyesteramide is preferably PLGG or PLGL, and polyestercarbonate is preferably P (LA-MCC), P (CL-DHP) or P (LA-MAC); The polyester polyamino acid is preferably PLA-PGA or PCL-PLL.
Above-mentioned LHRH polypeptide derivative is the luteinising hormone-releasing hormo derivative, comprises LHRH-10-COOH (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-COOH) (structural formula I) and 6 LHRH-6-NH that glycine are replaced by the Methionin of dextrorotation of 10 end carboxylations
2(pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-GlyNH
2) (formula II);
Bonding the preparation method of amphiphilic biologically degradable polymkeric substance of LHRH, the polymkeric substance shown in the preparation formula a when the LHRH polypeptide derivative is LHRH-10-COOH, comprises the steps:
LHRH-10-COOH is dissolved in N, make it dissolving in dinethylformamide or the water, the 4-Dimethylamino pyridine (DMAP) that 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) that adding carboxyl mole number is 1.5-3 times and carboxyl mole number are 0.01 times, stirred 10-30 minute under the ice bath, add carboxyl mole number 1-3 and doubly contain end amino or terminal hydroxy group di-block copolymer, keep ice bath and rise to room temperature naturally, stopped reaction after 12-24 hour, obtain reaction solution, it is that the dialysis tubing of 3500g/mol was with distill water dialysis 24-48 hour that reaction solution is placed molecular weight cut-off, changed water once in every 2-12 hour, dialyzate is through the lyophilize amphiphilic biologically degradable polymkeric substance of LHRH-10-COOH that obtained bonding.Reaction expression is as follows:
Bonding the preparation method of amphiphilic biologically degradable polymkeric substance of LHRH, the polymkeric substance shown in the preparation formula a is when the LHRH polypeptide derivative is LHRH-6-NH
2The time, comprise the steps:
To contain and hold the di-block copolymer of carboxyl to be dissolved in N, in dinethylformamide or the water, the DMAP that the EDCHCl that adding carboxyl mole number is 1.5-3 times and carboxyl mole number are 0.01 times stirred 10-30 minute under the ice bath, added 0.3-1 times of LHRH-6-NH of carboxyl mole number
2, keep ice bath and rise to room temperature naturally, reacted 12-24 hour, obtain reaction solution, with reaction solution place molecular weight cut-off be the dialysis tubing of 3500g/mol with distill water dialysis 24-48 hour, changed water once in every 2-12 hour, dialyzate is through the lyophilize LHRH-6-NH that obtained bonding
2The amphiphilic biologically degradable polymkeric substance.Reaction expression is as follows:
Bonding the amphiphilic biologically degradable polymkeric substance of LHRH, this polymkeric substance is formed by LHRH polypeptide derivative and triblock copolymer bonding, structure is suc as formula shown in the b:
Among the formula b, triblock copolymer is made up of PEG1, PEG2 and HPB, and PEG1 is the polyoxyethylene glycol block, and the data molecular weight is 2-10kg/mol, preferred 3-8kg/mol, more preferably 5-8kg/mol, most preferably 5kg/mol; PEG2 is the polyoxyethylene glycol block that side group contains functional group, number-average molecular weight 1-5kg/mol, and preferred 1-4kg/mol, more preferably 2-3kg/mol, 2kg/mol most preferably, described functional group is hydroxyl, amino or carboxyl; HPB is hydrophobic biodegradable polymers block, comprise polyester, polycarbonate, polyamino acid, polyesteramide, polyestercarbonate or polyester polyamino acid, number-average molecular weight is 1-20kg/mol, preferred 2-15kg/mol, more preferably 5-10kg/mol, most preferably 10kg/mol.
Described polyester is preferably PCL, PDLLA or PLGA; Polycarbonate is preferably PMCC, PDHP, PMBC or PMAC; Polyamino acid is preferably PGA or PLL; Polyesteramide is preferably PLGG or PLGL, and polyestercarbonate is preferably P (LA-MCC), P (CL-DHP) or P (LA-MAC); The polyester polyamino acid is preferably PLA-PGA or PCL-PLL.
Above-mentioned LHRH polypeptide derivative is the luteinising hormone-releasing hormo derivative, comprises LHRH-10-COOH (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-COOH) (structural formula I) and 6 LHRH-6-NH that glycine are replaced by the Methionin of dextrorotation of 10 end carboxylations
2(pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-GlyNH
2) (formula II);
Bonding the preparation method of amphiphilic biologically degradable polymkeric substance of LHRH, the polymkeric substance shown in the preparation formula b when the LHRH polypeptide derivative is LHRH-10-COOH, comprises the steps:
LHRH-10-COOH is dissolved in N, make it dissolving in dinethylformamide or the water, the DMAP that the EDCHCl that adding carboxyl mole number is 1.5-3 times and carboxyl mole number are 0.01 times, stirred 10-30 minute under the ice bath, adding carboxyl mole number 1-3 doubly contains the triblock copolymer of side amino or pendant hydroxyl group, keep ice bath and rise to room temperature naturally, reacted 12-24 hour, obtain reaction solution, it is that the dialysis tubing of 3500g/mol was with distill water dialysis 24-48 hour that reaction solution is placed molecular weight cut-off, changed water once in every 2-12 hour, dialyzate is through the lyophilize amphiphilic biologically degradable polymkeric substance of LHRH-10-COOH that obtained bonding.Reaction expression is as follows:
Bonding the preparation method of amphiphilic biologically degradable polymkeric substance of LHRH, the polymkeric substance shown in the preparation formula b is when the LHRH polypeptide derivative is LHRH-6-NH
2The time, comprise the steps:
The triblock copolymer that will contain side carboxyl is dissolved in N, make it dissolving in dinethylformamide or the water, the DMAP that the EDCHCl that adding carboxyl mole number is 1.5-3 times and carboxyl mole number are 0.01 times stirred 10-30 minute under the ice bath, added 0.3-1 times of LHRH-6-NH of carboxyl mole number
2, keep ice bath and rise to room temperature naturally, reacted 12-24 hour, obtain reaction solution, with reaction solution place molecular weight cut-off be the dialysis tubing of 3500g/mol with distill water dialysis 24-48 hour, changed water once in every 2-12 hour, dialyzate through the lyophilize bonding LHRH-6-NH
2The amphiphilic biologically degradable polymkeric substance.Reaction expression is as follows:
Di-block copolymer described in the above-mentioned preparation method (end group contains functional group hydroxyl, amino or carboxyl) and triblock copolymer (side group contains functional group hydroxyl, amino or carboxyl) are known technology, referring to document Yue, J., X.Y.Li, et al. (2010). " Modular Functionalization of Amphiphilic Block Copolymers via Radical-Mediated Thiol-Ene Reaction. " Macromolecules43 (23): 9645-9654..
Bonding the amphiphilic biologically degradable polymkeric substance of LHRH in the application of preparation in the drug-carrying nanometer particle.
Described drug-carrying nanometer particle the has been bonding amphiphilic biologically degradable polymkeric substance of LHRH and bonding the amphipathic nature block polymer self-assembly of drug molecule form, or by bonding the amphiphilic biologically degradable polymkeric substance of LHRH, amphipathic nature block polymer and drug molecule self-assembly form.The preparation method of drug-carrying nanometer particle can obtain according to following four kinds of methods, and concrete steps are as follows:
First method: with bonding the amphiphilic biologically degradable polymkeric substance of LHRH, amphipathic nature block polymer and medicine are dissolved in the organic solvent, form mixing solutions, the amphiphilic biologically degradable polymkeric substance of the milliliter number of the organic solvent volume LHRH that is bonding and the 4-100 of amphipathic nature block polymer total mass grams are doubly, the drug molecule quality the has been bonding amphiphilic biologically degradable polymkeric substance of LHRH and the 1-20% of amphipathic nature block polymer total mass, under agitation condition, to above-mentioned mixed solution and dripping distilled water, form and mix the drug-carrying nanometer particle aqueous solution, the volume of described distilled water is 1-5 times of organic solvent volume; Remove the organic solvent in the mixing solutions, with dialysate filter and lyophilize, obtain having the nano-granule freeze-dried powder of the polymer packaging medicine of LHRH target function.
Described bonding amphiphilic biologically degradable polymkeric substance and the amphipathic nature block polymer mol ratio of LHRH be 1: 4-1: 19.
Second method: with bonding the amphiphilic biologically degradable polymkeric substance of LHRH and bonding the amphipathic nature block polymer of drug molecule be dissolved in the organic solvent, form mixing solutions, the amphiphilic biologically degradable polymkeric substance of the milliliter number of the organic solvent volume LHRH that is bonding and bonding drug molecule amphipathic nature block polymer total mass grams 4-100 doubly, under agitation condition, to above-mentioned mixed solution and dripping distilled water, form and mix the drug-carrying nanometer particle aqueous solution, the volume of described distilled water is 1-5 times of organic solvent volume; Remove the organic solvent in the mixing solutions, with dialysate filter and lyophilize, obtain having the nano-granule freeze-dried powder of the polymer packaging medicine of LHRH target function.
Described bonding the amphiphilic biologically degradable polymkeric substance of LHRH and bonding the amphipathic nature block polymer mol ratio of drug molecule be 1: 4 to 1: 19.
The third method: will be with the amphiphilic biologically degradable polymkeric substance of function end group or side group, amphipathic nature block polymer and drug molecule are dissolved in the organic solvent, form mixing solutions, the milliliter number of organic solvent volume is the amphiphilic biologically degradable polymkeric substance of band function end group or side group and 4-100 times of amphipathic nature block polymer total mass grams, the drug molecule quality is band function end group or the amphiphilic biologically degradable polymkeric substance of side group and the 1-20% of amphipathic nature block polymer total mass, under agitation condition, to above-mentioned mixed solution and dripping distilled water, form and mix the drug-carrying nanometer particle aqueous solution, the volume of described distilled water is 1-5 times of organic solvent volume; Remove the organic solvent in the mixing solutions, doubly LHRH derivative of the amphiphilic biologically degradable polymkeric substance mole number 0.3-1 that in the nanoparticle aqueous solution, adds band function end group or side group, 1.5-3 EDCHCl and 0.01 times DMAP stirring at room 24 hours doubly, obtain reaction product, with reaction product diafiltration and lyophilize, obtain having the nano-granule freeze-dried powder of the polymer packaging medicine of LHRH target function.
The amphiphilic biologically degradable polymkeric substance of described band function end group or side group and amphipathic nature block polymer mol ratio are 1: 4 to 1: 19.
The 4th kind of method: will with the amphiphilic biologically degradable polymkeric substance of function end group or side group and bonding the amphipathic nature block polymer of drug molecule be dissolved in the organic solvent, form mixing solutions, the amphiphilic biologically degradable polymkeric substance of milliliter number band function end group or the side group of organic solvent volume and bonding drug molecule amphipathic nature block polymer total mass grams 4-100 doubly, under agitation condition, to above-mentioned mixed solution and dripping distilled water, form and mix the drug-carrying nanometer particle aqueous solution, the volume of described distilled water is 1-5 times of organic solvent volume; Remove the organic solvent in the mixing solutions, doubly LHRH derivative of the amphiphilic biologically degradable polymkeric substance mole number 0.3-1 that in the nanoparticle aqueous solution, adds band function end group or side group, 1.5-3 EDCHCl and 0.01 times DMAP stirring at room 24 hours doubly, obtain reaction product, with reaction product diafiltration and lyophilize, obtain having the nano-granule freeze-dried powder of the polymer packaging medicine of LHRH target function.
The amphiphilic biologically degradable polymkeric substance of described band function end group or side group and bonding the amphipathic nature block polymer mol ratio of drug molecule be 1: 4-1: 19.
Used organic solvent is acetone, tetrahydrofuran (THF), N in above-mentioned four kinds of methods, the mixed solvent of dinethylformamide, dimethyl sulfoxide (DMSO) or any two or three in them.
Among above-mentioned four kinds of preparation methods, the method of removing solvent in the mixing solutions is the method for this area routine, can be methods such as dialysis method, rotary evaporation or room temperature volatilization, dialysis method is: will mix the capable dialysis of the nanoparticle aqueous solution, remove the organic solvent in the solution, the dialysis membrane molecular weight cut-off is 3500g/mol, and the dialysis medium is distilled water, changed once, dialysed altogether 24-48 hour in 2-12 hour; The time of rotary evaporation is 1-4 hour; It is 2-8 hour that the time of room temperature volatilization is looked room temperature; After the desolventizing, with the membrane filtration postlyophilization of the nanoparticle aqueous solution through 0.45um, or be concentrated to by solvent evaporation or ultrafiltrationmembrane process and refilter behind the mass concentration 0.5-5% and lyophilize.
Among above-mentioned four kinds of preparation methods, in order to increase the solvability of the nanoparticle after the freeze-drying, can be before freeze-drying, in the nanoparticle aqueous solution, add solubilizing agent such as lactose, seminose, gelatin hydrolysate, sodium-chlor, glucose or their mixture, mix, described solubilizing agent consumption is the 50-200% of high molecular polymerization amount in the nanoparticle aqueous solution.
Among above-mentioned four kinds of preparation methods, the amphiphilic biologically degradable polymkeric substance of described band function end group or side group is that (end group contains the functional group hydroxyl to di-block copolymer, amino or carboxyl) or triblock copolymer (side group contains the functional group hydroxyl, amino or carboxyl), described amphipathic nature block polymer is the similar multipolymer of skeleton structure with the amphiphilic biologically degradable polymkeric substance of band function end group or side group, amphipathic nature block polymer comprises hydrophilic section and hydrophobic section, hydrophilic section is polyoxyethylene glycol, in the amphiphilic biologically degradable polymkeric substance of length and band function end group or side group the difference of the number-average molecular weight of hydrophilic section be no more than ± 10%; Hydrophobic section is biodegradable polymers, comprise polyester, polycarbonate, polyamino acid, polyesteramide, polyestercarbonate or polyester polyamino acid, in the amphiphilic biologically degradable polymkeric substance of length and band function end group or side group the difference of the number-average molecular weight of hydrophobic section be no more than ± 10%; Described polyester is preferably PCL, PDLLA or PLGA; Polycarbonate is preferably PMCC, PDHP, PMBC or PMAC; Polyamino acid is preferably PGA or PLL; Polyesteramide is preferably PLGG or PLGL, and polyestercarbonate is preferably P (LA-MCC), P (CL-DHP) or P (LA-MAC); The polyester polyamino acid is preferably PLA-PGA or PCL-PLL.
Described drug molecule comprises the medicine of any needs, as taxanes medicine, anthracene nucleus medicament, metal platinum medicine or gemcitabine etc.Described taxanes medicine is preferably taxol or Docetaxel; Anthracene nucleus medicament is preferably Zorubicin, epirubicin or complies with the gentle star that compares; The metal platinum medicine is preferably cis-platinum, oxaliplatin or carboplatin.
In above-mentioned four kinds of methods, amphipathic nature block polymer and bonding the amphipathic nature block polymer of drug molecule be known technology, referring to corresponding document, (Hu, X.L.and X.B.Jing, (2009). " Biodegradable amphiphilic polymer-drug conjugate micelles. " Expert Opinion on Drug Delivery6, (10): 1079-1090.) and patent, (the far refined grade of scape has the high molecule adriamycin bonding medicine nano capsule of target function and preparation method thereof Chinese patent application number: 200810050408.4 applyings date: 2008-02-28)
Embodiment 1: polypeptide target is to LHRH-6-NHCO-PEG
5K-PLA
2KThe preparation of segmented copolymer
0.5mmol is held carboxyl di-block copolymer COOH-PEG
5K-PLA
2K(index number in the molecular formula represents the number-average molecular weight of this block, down together), 0.61mg DMAP and 0.75mmol EDCHCl place dry peace bottle, add 10ml distilled water under the ice bath and stir 30 minutes, add 0.25mmolLHRH-6-NH
2, keep ice bath and rise to room temperature naturally, stir in the dialysis tubing that was placed on molecular weight cut-off 3500g/mol in 12 hours and use distill water dialysis, changed water every 6 hours, freeze-drying gets white polypeptide target to segmented copolymer LHRH-6-NHCO-PEG after 24 hours
5K-PLA
2KLyophilized powder.Fig. 1 is that the ultraviolet-visible of the segmented copolymer of bonding LHRH derivative absorbs spectrogram, and the curve 2 among the figure is the LHRH-6-NHCO-PEG of embodiment 1
5K-PLA
2K, the absorption peak that the target segmented copolymer occurs about 270nm in the collection of illustrative plates namely derives from the LHRH polypeptide, the target segmented copolymer that proved successful preparation.
Embodiment 2: polypeptide target is to LHRH-6-PEG
10K-PGA
20KThe preparation of segmented copolymer
1mmol is held carboxyl block polymer COOH-PEG
10K-PGA
20K, 1.22mg DMAP and 3mmol EDCHCl place dry peace bottle, add 10ml N under the ice bath, and the dinethylformamide dissolving was stirred 30 minutes, added 1mmol LHRH-6-NH
2, keep ice bath and rise to room temperature naturally, stir in the dialysis tubing that was placed on molecular weight cut-off 3500g/mol in 24 hours and use distill water dialysis, changed water every 12 hours, freeze-drying gets white polypeptide target to segmented copolymer LHRH-6-PEG after 48 hours
10K-PGA
20KLyophilized powder.
Embodiment 3: polypeptide target is to LHRH-10-CONH-PEG
2K-PMBC
5KThe preparation of segmented copolymer
With 0.1mmol LHRH-10-COOH, 0.12mg DMAP and 0.15mmol EDCHCl place dry peace bottle, add the 10ml dissolved in distilled water under the ice bath and stir after 10 minutes, add 0.15mmol and hold amino di-block copolymer NH
2-PEG
2K-PMBC
5KTemperature rose to room temperature naturally, stirs in the dialysis tubing be placed on molecular weight cut-off 3500g/mol in 12 hours and uses distill water dialysis, changed water in initial 6 hours every 2 hours, changed water once every 6 hours after 6 hours, freeze-drying gets white polypeptide target to segmented copolymer LHRH-10-CONH-PEG after 24 hours
2K-PMBC
5KLyophilized powder.
Embodiment 4: polypeptide target is to LHRH-10-CONH-PEG
5K-PLA
2KThe preparation of segmented copolymer
With 0.1mmol LHRH-10-COOH, 0.12mg DMAP and 0.15mmol EDCHCl place dry peace bottle, add the 10ml dissolved in distilled water under the ice bath and stir after 10 minutes, add 0.15mmol and hold amino di-block copolymer NH
2-PEG
5K-PLA
2KTemperature rose to room temperature naturally, stirs in the dialysis tubing be placed on molecular weight cut-off 3500g/mol in 12 hours and uses distill water dialysis, changed water in initial 6 hours every 2 hours, changed water once every 6 hours after 6 hours, freeze-drying gets white polypeptide target to segmented copolymer LHRH-10-CONH-PEG after 24 hours
5K-PLA
2KLyophilized powder.The ultraviolet-visible of the segmented copolymer of the LHRH derivative of embodiment of the invention preparation absorbs spectrogram shown in the curve 1 among Fig. 1, and curve 3 is COOH-PEG
5K-PLA
2K, curve 4 is NH
2-PEG
5K-PLA
2K, as we know from the figure, the absorption peak that curve 1 occurs about 270nm namely derives from the LHRH polypeptide, the target segmented copolymer that proved successful preparation.
Embodiment 5: polypeptide target is to LHRH-10-COO-PEG
5K-PLA
15KThe preparation of segmented copolymer
With 0.2mmol LHRH-10-COOH, 0.24mg DMAP and 0.4mmol EDCHCl place dry peace bottle, add 10ml N under the ice bath, and the dinethylformamide dissolving was stirred after 10 minutes, add 0.4mmol terminal hydroxy group di-block copolymer OH-PEG
5K-PLA
15K, temperature rose to room temperature naturally, stirs in the dialysis tubing that was placed on molecular weight cut-off 3500g/mol in 18 hours and uses distill water dialysis, changed water every 6 hours, and freeze-drying gets white polypeptide target to segmented copolymer LHRH-10-COO-PEG after 36 hours
5K-PLA
15KLyophilized powder, accompanying drawing Fig. 3 are its UV spectrograms.
Embodiment 6: polypeptide target is to PEG
5K-PEG
1K(CONH-6-LHRH)
1-P (CL-DHP)
15KThe preparation of segmented copolymer
0.2mmol is contained side carboxyl triblock copolymer PEG
5K-PEG
1K(COOH)
3-P (CL-DHP)
15K, 0.24mg DMAP and 0.4mmol EDCHCl place dry peace bottle, add 10ml water under the ice bath and stir 10 minutes, add 0.08mmol LHRH-6-NH
2Keep ice bath and rise to room temperature naturally, stirring was placed in the dialysis tubing of molecular weight cut-off 3500g/mol and uses distill water dialysis in 18 hours, began to change every 2 hours in 6 hours water, changed water once every 4 hours after 6 hours, freeze-drying gets white polypeptide target to segmented copolymer PEG after 24 hours
5K-PEG
1K(CONH-6-LHRH)
1-P (CL-DHP)
15KLyophilized powder.
Embodiment 7: polypeptide target is to PEG
5K-PEG
1K(NHCO-10-LHRH)
2-P (LA-MCC)
3KThe preparation of segmented copolymer
With 0.05mmol LHRH-10-COOH, 0.06mg DMAP and 0.15mmol EDCHCl place dry peace bottle, add 25mL water under the ice bath and stir 30 minutes, add 0.15mmol and contain the amino triblock copolymer PEG of side
5K-PEG
1K(NH
2)
4-P (LA-MCC)
3K, temperature rose to room temperature naturally, stirs in the dialysis tubing that was placed on molecular weight cut-off 3500g/mol in 24 hours and uses distill water dialysis, changed water every 12 hours, and freeze-drying gets white polypeptide target to segmented copolymer PEG after 48 hours
5K-PEG
1K(NHCO-10-LHRH)
2-P (LA-MCC)
3KLyophilized powder.
Embodiment 8: polypeptide target is to PEG
10K-PEG
5K(OOC-10-LHRH)
3-P (LA-PPA)
20KThe preparation of segmented copolymer
With 0.15mmol LHRH-10-COOH, 1.83mg DMAP and 0.3mmol EDCHCl place dry peace bottle, add 25ml N under the ice bath, and the dinethylformamide dissolving was stirred 30 minutes, add 0.15mmol and contain amino triblock copolymer PEG
10K-PEG
5K(OH)
10-P (LA-PPA)
20K, temperature rose to room temperature naturally, stirs in the dialysis tubing that was placed on molecular weight cut-off 3500g/mol in 24 hours and uses distill water dialysis, changed water every 8 hours, and freeze-drying gets white polypeptide target to segmented copolymer PEG after 36 hours
10K-PEG
5K(OOC-10-LHRH)
3-P (LA-PPA)
20KLyophilized powder.
Embodiment 9: preparation MPEG
5K-P (LA-DHP-RhB)
15KAnd PEG
5K-PEG
1K(CONH-6-LHRH)
1.5-P (CL-DHP)
15KThe mixing nanoparticle
Get 0.1mmol block polymer MPEG
5K-P (LA-DHP-RhB)
15KWith the polypeptide target of 0.025mmol embodiment 6 preparation to polymer P EG
5K-PEG
1K(CONH-6-LHRH)
3-P (CL-DHP)
15KBe dissolved in 30ml acetone, form mixing solutions, in mixing solutions, add 90ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, stirring at room was removed organic solvent in 4 hours, nanoparticle solution 0.45um membrane filtration, add 1g glucose dissolving postlyophilization in the filtrate, obtain having the nano-granule freeze-dried powder that is loaded with the rhodamine B fluorescent molecular probe of LHRH target.
The rhodamine fluorescent molecular probe is used for aids drug, the nano-granule freeze-dried powder solution that is loaded with the rhodamine B fluorescent molecular probe with LHRH target that above-described embodiment 9 obtains is used for human body ovarian cancer SKOV3 tumour cell and engulfs experiment, the result as shown in Figure 4, redness is the fluorescence of aids drug rhodamine, the targeting of LHRH targeted nano granule can be tested preliminary identification by cell in vitro, because the target effect of LHRH, nano particle is not only assembled in the SKOV3 surface of tumor cells, also by endocytosis in cell.
Embodiment 10: preparation MPEG
5K-P (CL-MAC)
3KAnd PEG
5K-PEG
1K(NHCO-10-LHRH)
2-P (LA-MCC)
3KThe mixing nanoparticle of parcel Zorubicin
Get 0.05mmol block polymer MPEG
5K-P (CL-MAC)
3K, the polypeptide target of 0.05g Zorubicin and 0.005mmol embodiment 7 preparations is to polymer P EG
5K-PEG
1K(NHCO-10-LHRH)
2-P (LA-MCC)
3KBe dissolved in the 10ml dimethyl sulfoxide (DMSO), form mixing solutions, in mixing solutions, add 20ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, stir in the dialysis tubing that was placed on molecular weight cut-off 3500g/mol in 60 minutes and use distill water dialysis, changed water every 6 hours, use the 0.45um membrane filtration after 36 hours, add 0.5g seminose dissolving postlyophilization in the filtrate, obtain having the nano-granule freeze-dried powder that is loaded with the Zorubicin medicine of LHRH target.
Embodiment 11: preparation MPEG
12K-PLGA
20KAnd LHRH-6-PEG
10K-PGA
20KThe mixing nanoparticle of parcel cis-platinum
Get 0.05mmol block polymer MPEG
12K-PLGA
20K, the polypeptide target polymer LHRH-6-PEG of 0.05g cis-platinum and 0.003mmol embodiment 2 preparations
10K-PGA
20KBe dissolved in the 20ml tetrahydrofuran (THF), form mixing solutions, add 100ml distilled water in mixing solutions, form and mix the drug-carrying nanometer particle aqueous solution, rotary evaporation is after 2 hours, place the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, change water every 12 hours, use 0.45um membrane filtration, filter freezing drying to obtain having the nano-granule freeze-dried powder that is loaded with cisplatin medicine of LHRH target after 48 hours.
Embodiment 12: preparation MPEG
5K-P (LA-MCC-PTX)
2KAnd LHRH-6-NHCO-PEG
5K-PLA
2KThe mixing nanoparticle
Get 0.02mmol paclitaxel bonded drug MPEG
5K-P (LA-MCC-PTX)
2KPolypeptide target polymer LHRH-6-NHCO-PEG with 0.005mmol embodiment 4 preparations
5K-PLA
2KBe dissolved in the 10ml tetrahydrofuran (THF), form mixing solutions, in mixing solutions, add 30ml distilled water, form to mix the drug-carrying nanometer particle aqueous solution, stirs after 10 minutes rotary evaporation and remove tetrahydrofuran (THF), interpolation solubilizing agent lactose 0.15g in the nanoparticle aqueous solution that obtains, dissolving mixes, and lyophilize obtains having the polymer paclitaxel bonded drug nano-granule freeze-dried powder of LHRH target.The size distribution figure of the target polypeptide mixing nanoparticle that typically is loaded with taxol drug that embodiment 12 obtains and shape appearance figure as shown in Figures 2 and 3, they are respectively by light scattering method and transmissioning electric mirror determining.As can be seen from the figure, it is spherical mixing nanoparticle, and particle diameter is about 80nm, and uniform particle diameter meets " EPR effect " fully to the requirement of nanoparticle particle diameter and pattern.
Fig. 5 for bonding paclitaxel mixing nanoparticle to lotus SKOV3 tumour nude mice time-the gross tumor volume curve, the curve 1 among the figure is MPEG
2K-P (LA-MCC-PTX)
2K, the MPEG that curve 2 obtains for embodiment 12
2K-P (LA-MCC-PTX)
2K/ LHRH-6-NHCO-PEG
5K-PLA
2K, curve 3 is MPEG
2K-P (LA-MCC-PTX)
2K/ LHRH-10-COO-PEG
5K-PLA
2K, as can be seen from the figure, than the paclitaxel nano grain that does not have target, but this LHRH targeted medicament carrying nano grain has the nude mice of SKOV3 tumour cell to show better the knurl curative effect to the subcutaneous lotus of left fore.
Embodiment 13: preparation MPEG
2K-P (CL-DHP-CO-NHNH
2-DOX)
5KAnd LHRH-10-CONH-PEG
2K-PMBC
5KThe mixing nanoparticle
Get 0.01mmol adriamycin bonding medicine MPEG
2K-P (CL-DHP-CO-NHNH
2-DOX)
5KPolypeptide target polymer LHRH-10-CONH-PEG with 0.0025mmol embodiment 3 preparations
2K-PMBC
5KBe dissolved in the 10ml acetone, form mixing solutions, in mixing solutions, add 50ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, stirring at room was removed organic solvent in 4 hours, and the nanoparticle aqueous solution lyophilize that obtains obtains having the high molecule adriamycin bonding medicine nano grain lyophilized powder of LHRH target.
Embodiment 14: preparation MPEG
15K-P (LA-MPC/Pt)
20KAnd PEG
10K-PEG
5K(OOC-10-LHRH)
3-P (LA-PPA)
20KThe mixing nanoparticle
Get 0.005mmol bonding metal cis-platinum MPEG
15K-P (LA-co-MPC/Pt)
20KWith the polypeptide target of 0.0003mmol embodiment 8 preparation to polymer P EG
10K-PEG
5K(OOC-10-LHRH)
3-P (LA-PPA)
20KBe dissolved in 15ml N, in the dinethylformamide, form mixing solutions, in mixing solutions, add 15ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, stir in the dialysis tubing that was placed on molecular weight cut-off 3500g/mol in 10 minutes and use distill water dialysis, changed water every 8 hours, 48 hours postlyophilizations obtain having the polymer platinum bonding medicine nano grain lyophilized powder of LHRH target.
Embodiment 15: the LHRH polypeptide target of preparation parcel epirubicin medicine to the mixing nanoparticle
Get 0.05mmol amphipathic nature block polymer MPEG
2K-PDLLA
1K, 0.03g epirubicin and 0.0125mmol hold amino di-block copolymer NH
2-PEG
2K-PDLLA
1KBe dissolved in the 10ml tetrahydrofuran (THF), form mixing solutions, in mixing solutions, add 20ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, stir 20 minutes rotary evaporations and removed tetrahydrofuran (THF) in 2 hours, place the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, in the nanoparticle aqueous solution that is loaded with the epirubicin medicine that obtains, add 0.01mmol LHRH-10-COOH, 0.02mg DMAP and 0.035mmol EDCHCl, stirring at room after 24 hours places reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water every 6 hours, 36 hours postlyophilizations obtain having the nano-granule freeze-dried powder that is loaded with the epirubicin medicine of LHRH target.
Embodiment 16: the LHRH polypeptide target of preparation parcel Docetaxel medicine to the mixing nanoparticle
Get 0.02mmol amphipathic nature block polymer MPEG
5K-PLGA
5k, 0.04g Docetaxel and 0.0015mmol terminal hydroxy group di-block copolymer OH-PEG
5K-PLGA
5KBe dissolved in the 5ml acetone, form mixing solutions, in mixing solutions, add 20ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, stirring at room was removed acetone in 4 hours, the nanoparticle solution 0.45um membrane filtration that obtains, in the nanoparticle filtrate that is loaded with the Docetaxel medicine that obtains, add 0.00075mmol LHRH-10-COOH, 0.002mgDMAP and 0.003mmol EDCHCl, stirring at room after 12 hours placed reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water every 4 hours, and 24 hours postlyophilizations obtain having the nano-granule freeze-dried powder that is loaded with the Docetaxel medicine of LHRH target.
Embodiment 17: the LHRH polypeptide target of preparation parcel oxaliplatin medicine to the mixing nanoparticle
Get 0.03mmol amphipathic nature block polymer MPEG
5K-PLA
15K, the amino triblock copolymer PEG of 0.05g oxaliplatin and 0.003mmol side
5K-PEG
1K(NH
2)
3-PLA
15KBe dissolved in 5ml N, in the dinethylformamide, form mixing solutions, in mixing solutions, add 10ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, place the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, in the nanoparticle aqueous solution that is loaded with the oxaliplatin medicine that obtains, add 0.001mmol LHRH-10-COOH, 0.004mg DMAP and 0.005mmol EDCHCl, stirring at room after 24 hours places reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water every 6 hours, use the 0.45um membrane filtration after 48 hours, the filtrate lyophilize obtains having the nano-granule freeze-dried powder that is loaded with the oxaliplatin medicine of LHRH target.
Embodiment 18: the LHRH polypeptide target of preparation parcel carboplatin medicine to the mixing nanoparticle
Get 0.01mmol amphipathic nature block polymer MPEG
10K-PPA
20K, 0.07g carboplatin and 0.001mmol pendant hydroxyl group triblock copolymer PEG
10K-PEG
2K(OH)
4-PPA
20KBe dissolved in the 10ml dimethyl sulfoxide (DMSO), form mixing solutions, in mixing solutions, add 10ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, place the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, in the nanoparticle aqueous solution that is loaded with the carboplatin medicine that obtains, add 0.0005mmol LHRH-10-COOH, 0.001mg DMAP and 0.003mmol EDCHCl, stirring at room after 12 hours placed reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water every 8 hours, used the 0.45um membrane filtration after 24 hours, the filtrate lyophilize obtains having the nano-granule freeze-dried powder that is loaded with the carboplatin medicine of LHRH target.
Embodiment 19: preparation parcel according to gentle LHRH polypeptide target than star medicine to the mixing nanoparticle
Get 0.01mmol amphipathic nature block polymer MPEG
15K-PPA
20K, 0.01g is according to softening than star and 0.001mmol side carboxyl triblock copolymer PEG
10K-PEG
5K(COOH)
5-PPA
20KBe dissolved in 10ml acetone, form mixing solutions, in mixing solutions, add 50ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, stirring at room was removed acetone in 8 hours, placed the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, to being loaded with according to adding 0.001mmolLHRH-6-NH in the gentle nanoparticle aqueous solution than star medicine of obtaining
20.001mg DMAP and 0.003mmol EDCHCl, stirring at room after 24 hours places reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water every 8 hours, use the 0.45um membrane filtration after 48 hours, the filtrate lyophilize obtains having being loaded with of LHRH target and complies with gentle nano-granule freeze-dried powder than star medicine.
Embodiment 20: the LHRH polypeptide target of preparation parcel gemcitabine medicine to the mixing nanoparticle
Get 0.01mmol amphipathic nature block polymer MPEG
5K-PLGA
10K, 0.02g gemcitabine and 0.0006mmol end carboxyl di-block copolymer COOH-PEG
5K-PLGA
10KBe dissolved in the 10ml tetrahydrofuran (THF), form mixing solutions, in mixing solutions, add 50ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, rotary evaporation was removed tetrahydrofuran (THF) in 4 hours, placed the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, added 0.0003mmolLHRH-6-NH in the nanoparticle aqueous solution that is loaded with the gemcitabine medicine that obtains
20.0007mg DMAP and 0.0015mmol EDCHCl, stirring at room after 24 hours places reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water every 5 hours, use the 0.45um membrane filtration after 36 hours, the filtrate lyophilize obtains having the nano-granule freeze-dried powder that is loaded with the gemcitabine medicine of LHRH target.
Embodiment 21: the LHRH polypeptide target of preparation bonding paclitaxel medicine to the mixing nanoparticle
Get 0.1mmol paclitaxel bonded drug MPEG
2K-P (LA-MCC-PTX)
2KWith 0.01mmol end carboxyl di-block copolymer COOH-PEG
2K-P (LA-MCC)
2K, be dissolved in the 10ml acetone, form mixing solutions, in mixing solutions, add 20ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, rotary evaporation was removed acetone in 1 hour.In the nanoparticle aqueous solution of the bonding paclitaxel medicine that obtains, add 0.01mmolLHRH-6-NH
20.02mg DMAP and 0.02mmol EDCHCl, stirring at room after 24 hours placed reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water every 6 hours, and 24 hours postlyophilizations obtain having the nano-granule freeze-dried powder of the bonding paclitaxel medicine of LHRH target.
Embodiment 22: the LHRH polypeptide target of preparation bonding Docetaxel medicine to the mixing nanoparticle
Get 0.05mmol docetaxel bonded drug MPEG
5K-P (LA-MAC-MPA-DTX)
5KHold amino di-block copolymer NH with 0.01mmol
2-PEG
5K-P (LA-MAC)
4K, be dissolved in the 10ml tetrahydrofuran (THF), form mixing solutions, in mixing solutions, add 50ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, stirring at room was removed tetrahydrofuran (THF) in 8 hours.In the nanoparticle aqueous solution of the bonding Docetaxel medicine that obtains, add 0.01mmol LHRH-10-COOH, 0.02mg DMAP and 0.03mmol EDCHCl, stirring at room after 12 hours places reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water every 8 hours, 48 hours postlyophilizations obtain having the nano-granule freeze-dried powder of the bonding Docetaxel medicine of LHRH target.
Embodiment 23: the preparation adriamycin bonded drug the LHRH polypeptide target to the mixing nanoparticle
Get 0.1mmol adriamycin bonding medicine MPEG
2K-P (LA-DHP-DOX)
2KWith 0.01mmol terminal hydroxy group di-block copolymer OH-PEG
2K-P (LA-MCC)
1KBe dissolved in 5ml N, in the dinethylformamide, form mixing solutions, in mixing solutions, add 20ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, place the dialysis tubing of molecular weight cut-off 3500g/mol to use distill water dialysis 24 hours, changed water every 4 hours, in the nanoparticle aqueous solution of the adriamycin bonded drug that obtains, add 0.005mmol LHRH-10-COOH, 0.02mg DMAP and 0.015mmol EDCHCl, stirring at room after 24 hours places reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water every 8 hours, 24 hours postlyophilizations obtain having the nano-granule freeze-dried powder of the adriamycin bonded drug of LHRH target.
Embodiment 24: the LHRH polypeptide target of preparation bonding metal platinum medicine to the mixing nanoparticle
Get 0.05mmol tetravalence cis-platinum bonding medicine MPEG
5K-P (LA-MCC-OH-Pt)
10KWith 0.01mmol side carboxyl triblock copolymer PEG
2K-PEG
2K(COOH)
n-P (LA-MCC)
10K, be dissolved in the 5ml acetone, form mixing solutions, in mixing solutions, add 20ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, rotary evaporation was removed acetone in 1 hour, added 0.01mmol LHRH-6-NH in the nanoparticle aqueous solution of the bonding tetravalence cisplatin medicine that obtains
20.02mg DMAP and 0.02mmol EDCHCl, stirring at room after 24 hours placed reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water every 6 hours, and 24 hours postlyophilizations obtain having the nano-granule freeze-dried powder of the bonding metal platinum medicine of LHRH target.
Embodiment 25: the LHRH polypeptide target of preparation bonding gemcitabine medicine to the mixing nanoparticle
Get 0.1mmol gemcitabine bonding medicine MPEG
10K-P (LL-SA-Gemcitabine)
15KWith the amino triblock copolymer PEG of 0.01mmol side
5K-PEG
2K(NH
2)
n-PLL
15KBe dissolved in the 10ml dimethyl sulfoxide (DMSO), form mixing solutions, in mixing solutions, add 10ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, place the dialysis tubing of molecular weight cut-off 3500g/mol to use distill water dialysis 24 hours, in the nanoparticle aqueous solution of the bonding gemcitabine medicine that obtains, add 0.005mmol LHRH-10-COOH, 0.02mg DMAP and 0.03mmol EDCHCl, stirring at room after 24 hours places reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water in initial 6 hours every 2 hours, changed water once every 6 hours after 6 hours, add 5g sodium-chlor after 36 hours in the dialyzate, the stirring and evenly mixing postlyophilization obtains having the nano-granule freeze-dried powder of the bonding gemcitabine medicine of LHRH target.
Embodiment 26: the LHRH polypeptide target of preparation bonding metal platinum medicine to the mixing nanoparticle
Get 0.1mmol bonding medicine MPEG
10K-b-P (LA-co-MCC/Asp-DACHPt)
20KWith 0.01mmol side carboxyl triblock copolymer PEG
5K-PEG
3K(COOH)
4-P (LA-MCC)
20K, be dissolved in the 10ml acetone, form mixing solutions, in mixing solutions, add 20ml distilled water, form and mix the drug-carrying nanometer particle aqueous solution, rotary evaporation was removed acetone in 1 hour, added 0.003mmolLHRH-6-NH in the bonding medicine nano grain aqueous solution that obtains
20.02mg DMAP and 0.025mmol EDCHCl, stirring at room after 24 hours placed reaction solution the dialysis tubing distill water dialysis of molecular weight cut-off 3500g/mol, changed water every 6 hours, and 24 hours postlyophilizations obtain having the nano-granule freeze-dried powder of the bonding metal platinum medicine of LHRH target.
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