CN105999294A - CD147 targeting drug-delivery system and preparation and application thereof - Google Patents

CD147 targeting drug-delivery system and preparation and application thereof Download PDF

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Publication number
CN105999294A
CN105999294A CN201610316174.8A CN201610316174A CN105999294A CN 105999294 A CN105999294 A CN 105999294A CN 201610316174 A CN201610316174 A CN 201610316174A CN 105999294 A CN105999294 A CN 105999294A
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dspe
peg
tumor
medicine
pharmaceutical carrier
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王剑
杨�远
周伟平
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Shanghai Fortune Medical Equipment Co Ltd
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Shanghai Fortune Medical Equipment Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers

Abstract

The invention belongs to the field of pharmaceutic preparation research and development, and particularly relates to a CD147 targeting drug-delivery system and preparation and application thereof. The system contains Anti-CD147-polyethylene glycol-phospholipid, drug carrier particles and drugs, wherein the molar ratio of Anti-CD147 to polyethylene glycol-phospholipid in Anti-CD147-polyethylene glycol-phospholipid is 1:(1-3), the molar ratio of polyethylene glycol to phospholipid is 1:1, and Anti-CD147 and polyethylene glycol are connected through a thioether bond. In in-vivo and in-vitro anti-tumor practice, the CD147 targeting drug-delivery system has obvious advantages compared with a non-targeting drug-delivery system.

Description

CD147 targeting drug delivery system and preparation and application thereof
Technical field
The invention belongs to pharmaceutical preparation research and development field, be specifically related to a kind of CD147 targeting drug delivery system and preparation and application thereof.
Background technology
Primary hepatocarcinoma is one of modal malignant tumor in China and worldwide, although imaging diagnosis and surgery operating technology obtain significant progress, but in addition to early stage patient, primary hepatocarcinoma post-operative survival rates situation is the most not satisfactory, tumor single-shot > 5 years survival rates only 37% of patient of 5cm, tumor is multiple >=5 years survival rates only 26% of patient of 3.For there is no the patient of patient's such as BCLC C phase of surgical engine meeting, guide only recommends Sorafenib at present, but the tumor control rate of Sorafenib only 43%, middle position extends life span the most about 3 months, and the patient that maybe cannot tolerate for Suo Lafei Endodontic failure, the most still without effective two wires replacement therapy side.Therefore, the patient no matter postoperative patient that there is high-risk Recurrence or late period understood without surgical engine, guide does not the most recommend system chemotherapy, this tumor such as colorectal cancer, breast carcinoma with some with effective, perfect system chemotherapy regimen is compared, hence it is evident that constrain the effect of its clinical complex treatment.Therefore, find the effective chemotherapeutics to primary hepatocarcinoma and also become the difficult problem that many Medical Research Work persons make great efforts to solve.
For the current antitumor drug problem to primary liver cancer DeGrain, it is the task of top priority that development has the novel nano lipidosome drug carrier of immune active targeting.
Summary of the invention
In order to overcome the problem in the presence of prior art, it is an object of the invention to provide a kind of CD147 targeting drug delivery system and preparation and application thereof.
To achieve these goals and other relevant purposes, the present invention adopts the following technical scheme that
A first aspect of the present invention, it is provided that a kind of Anti-CD147-PEG-DSPE, the mol ratio between described Anti-CD147 and PEG-DSPE is 1:(1～3), the mol ratio between described Polyethylene Glycol and phospholipid is 1:1.
Preferably, all connected by covalent bond between described Anti-CD147, Polyethylene Glycol, phospholipid.
It is further preferred that be connected by thioether bond between described Anti-CD147 and Polyethylene Glycol.
It is further preferred that be connected by amido link between described Polyethylene Glycol and phospholipid.
Preferably, described Anti-CD147 selects CD147 monoclonal antibody.
In the preferred embodiments of the present invention, Anti-CD147 selects Anti-CD147F (ab) ' 2.
Preferably, described molecular weight polyethylene glycol scope is 200～6000Da.
In a preferred embodiment of the invention, PEG2000 selected by described Polyethylene Glycol.
Preferably, the molecular weight of described phospholipid and the molecular weight of described Polyethylene Glycol are quite or slightly smaller.
In the preferred embodiments of the present invention, described phospholipid selects DSPE (DSPE).
A second aspect of the present invention, it is provided that a kind of method preparing aforementioned Anti-CD147-PEG-DSPE, is comprised the following steps: by Anti-CD147 and PEG-DSPE, prepared by covalent bond coupling reaction.
Preferably, described method specifically includes following steps:
(1) by Anti-CD147 sulfhydrylation, the Anti-CD147 solution of sulfhydrylation is prepared；
(2) maleimide modified PEG-DSPE is dissolved in step (1) gained solution, reaction, it is thus achieved that Anti-CD147-PEG-DSPE.
Preferably, in step (1), use 2-iminothiolane hydrochlorate (2-IT, CAS 4781-83-3) by Anti-CD147 sulfhydrylation.
Preferably, in step (2), described reaction refers specifically at room temperature react 1～2 hour.
Third aspect present invention, it is provided that aforementioned Anti-CD147-PEG-DSPE purposes in preparing targetable drug carriers or targeting drug delivery system.
A fourth aspect of the present invention, it is provided that a kind of targetable drug carriers, containing aforementioned Anti-CD147-PEG-DSPE.
Further, described targetable drug carriers, containing Anti-CD147-PEG-DSPE and pharmaceutical carrier particle.
Preferably, described pharmaceutical carrier particle surface has Anti-CD147.
Preferably, each described pharmaceutical carrier particle surface has more than one Anti-CD147.
It is further preferred that Anti-CD147 is covalently attached to the surface of described pharmaceutical carrier particle.
Preferably, described pharmaceutical carrier particle is selected from, but not limited to, liposome.
nullPreferably，The constituent of described liposome is selected from egg phosphatide、HSPC、Hydrogenation Yolk lecithin、DLPC、Dimyristoyl phosphatidyl choline、Dipalmitoyl phosphatidyl choline、Distearoyl phosphatidylcholine、1-myristoyl-2-palmitoylphosphatidyl choline、1-palmityl-2-DSPC、1-stearoyl-2-palmitoylphosphatidyl choline、POPC、1-stearoyl-2-Asia oleoyl phosphatidylcholine、DOPC、Hydrogenation dipalmitoyl phosphatidyl choline、Distearoyl phosphatidylcholine、Two myristoyl phosphatidic acid、Two myristoyl phosphatidic acid、DPPA、DPPA、G 12S3P、DMPEA、DPPE、Cephalin acyl serine、Two myristoyl Phosphatidylserine、Two palmityl Phosphatidylserine、E-PG、PE、GLYCEROL,DIMYRISTOYL PHOSPHATIDYL、DPPG、DSPG、DOPG、Brain sphingomyelins、Two palmitoyl sphingomyelin or distearyl sphingomyelins、Cholesterol、Two oil epoxide hydroxypropyltrimonium chloride (DOTAP)、Dioleoyl chloropropyl chlorination trimethylammonium (DOTMA)、DDA (DDAB)、Dimethyl aminoethyl amido propiono-cholesterol (DC-Chol)、Spermine-5-carboxyamino acetic acid octacosyl amide (DOGS)、Dioleoyl succinyl glycerolcholine ester (DOSC)、Dioleoyl chlorine spermine Carboxylamide ethyl dimethyl propyl trifluoroacetic acid ammonium (DOSPA)、Two or more combination of any one or they in MVL5.
Further, described pharmaceutical carrier particle also can carry out other modifications.
A fifth aspect of the present invention, it is provided that the construction method of aforementioned targetable drug carriers, selected from following arbitrary:
Method one, comprise the following steps:
(2) using Anti-CD147-PEG-DSPE as one of the material building pharmaceutical carrier, directly mix for the material building pharmaceutical carrier with other and prepare targetable drug carriers；
Method two, rear insertion, comprise the following steps:
(3) pharmaceutical carrier is first built；
(4) Anti-CD147-PEG-DSPE is mixed with the pharmaceutical carrier built,.
Method three: rear connection method, comprises the following steps:
(2) use PEG-DSPE as one of drug carrier material, build the pharmaceutical carrier containing PEG-DSPE；
(2) pharmaceutical carrier containing PEG-DSPE built in Anti-CD147 and step (1) is passed through covalent bond coupling reaction, build described targetable drug carriers.
A sixth aspect of the present invention, it is provided that aforementioned targetable drug carriers is loaded in the purposes preparing in targeting drug delivery system.
A seventh aspect of the present invention, it is provided that a kind of targeting drug delivery system, containing aforementioned targeting drug delivery system and medicine.
Preferably, described medicine is selected from, but not limited to, antitumor drug.
Further, antitumor drug of the present invention can be the medicine suppressing growth of tumour cell, breeding, break up, shift of existing any kind, includes but not limited to: small-molecule chemical medicine, nucleic acid molecules, carbohydrate, lipid, antibody medicine, polypeptide, albumen or interference slow virus.
In the preferred embodiments of the present invention, select amycin as model drug.
Preferably, described targeting drug delivery system for target cell be tumor cell.
The described targeting drug delivery system portability antitumor drug of the present invention targeting are in tumor cell.
Preferably, described tumor is entity tumor or metastatic tumo(u)r.It is further preferred that the tumor that described tumor is CD147 high expressed.
It is highly preferred that described tumor is selected from, but not limited to, hepatocarcinoma.
Further, described pharmaceutical carrier also can carry out other modifications.
A eighth aspect of the present invention, it is provided that the construction method of aforementioned targeting drug delivery system, selected from following arbitrary:
Method one: comprise the following steps:
(1) Anti-CD147-PEG-DSPE, medicine and being used for builds the material of pharmaceutical carrier directly mix and prepare targeting drug delivery system；
Method two, rear insertion, comprise the following steps:
(3) structure carries the pharmaceutical carrier of medicine；
(4) pharmaceutical carrier carrying medicine that Anti-CD147-PEG-DSPE builds with step (1) is mixed,；
Method three, rear connection method, comprise the following steps:
(3) use PEG-DSPE as one of drug carrier material, build the pharmaceutical carrier carrying medicine containing PEG-DSPE；
(4) pharmaceutical carrier carrying medicine containing PEG-DSPE built in Anti-CD147 and step (1) is passed through covalent bond coupling reaction, build described targeting drug delivery system.
A ninth aspect of the present invention, it is provided that aforementioned targeting drug delivery system purposes in preparing oncotherapy product.
Preferably, described tumor is selected from the tumor of CD147 high expressed.It is highly preferred that described tumor is selected from, but not limited to, hepatocarcinoma.
A tenth aspect of the present invention, it is provided that a kind of method treating tumor, including step: aforementioned targeting drug delivery system is applied to patient.Dosage used, doctor can select according to practical situation.
Described patient can be tumor patient.Described tumor is selected from, but not limited to, hepatocarcinoma.
Compared with prior art, there is advantages that
The present inventor is through above-mentioned extensively in-depth study discovery, use Anti-CD147 that pharmaceutical carrier is modified, it is connected by thioether bond between the most described Anti-CD147 and PEG-DSPE and mol ratio between described Anti-CD147 and PEG-DSPE is 1:(1～3) factor in terms of these, " targeting head " is become efficiently for Anti-CD147-PEG-DSPE particularly critical.The substantial amounts of research of the present invention shows, targeting drug delivery system containing Anti-CD147, the Anti-CD147 of pharmaceutical carrier particle surface, characteristic efficiently pharmaceutical carrier particle directionally can be transported in the tumor tissues of CD147 high expressed, the pharmaceutical carrier particle being distributed in target tissue can be combined with the target protein of tumor cell surface, and the process such as induced drug carrier endocytosis or drug release, thus play drug effect.The targeting drug delivery system that Anti-CD147 modifies has higher growth inhibited effect to the tumor of CD147 high expressed, and the targeting drug delivery system that Anti-CD147 modifies is that other modify more than 3～8 times of drug-supplying system without Anti-CD147 to the inhibitory action of the tumor of CD147 high expressed.Concrete, the targeting drug delivery system that Anti-CD147 modifies is up to 95.3% to the suppression ratio of the tumor of CD147 high expressed, and the drug-supplying system modified without Anti-CD147 only has 38.24% to the suppression ratio of tumor.
Accompanying drawing explanation
Fig. 1: synthesis CD147 targeting peptide-doxorubicin liposome schematic diagram.
CD147 and the CD133 expression of Fig. 2: hepatoma cell line；A, hepatoma cell line CD147 positive rate；B, hepatoma carcinoma cell CD147 average fluorescent strength；C, hepatoma carcinoma cell CD133 positive rate；D, hepatoma carcinoma cell CD133 average fluorescent strength.
Integrity and joint efficiency that Fig. 3: SDS-PAGE detection antibody connects detect；The black arrow of 4-6 swimming lane represent CD147 monoclonal antibody be combined with DSPE-PEG2000-MAL after band, band the highest expression molecular weight is the biggest, the most in conjunction with DSPE-PEG2000-MAL quantity.
Fig. 4: A, the particle size results of CD147 targeting peptide-doxorubicin liposome；The Zeta potential result of B, CD147 targeting peptide-doxorubicin liposome；C, the particle size results of non-targeted Evacet；D, the Zeta potential result of non-targeted Evacet.
The blood concentration-time curve of Fig. 5: three kinds of medicines.
Fig. 6: A, injection medicine is after 24 hours, amycin level in nude mouse tissue；B, injection medicine after 72 hours, amycin level in nude mouse tissue, * P < 0.05.
Fig. 7: Huh-7 to different pharmaceutical picked-up situation in vitro.
Fig. 8: Huh-7 cell endocytosis situation to targeting and non-targeted liposome in vitro；A, CD147 non-eluting of targeting peptide-doxorubicin liposome group (blue line) compare with (red line) after eluting；B, the non-eluting of non-targeted Evacet group (blue line) compare with (red line) after eluting；C, each comparison organizing average fluorescent strength.
Fig. 9: medicine inhibitory action to liver cancer cell growth in vitro；A, Huh-7 cell incubation 1 hour；B, Huh-7 cell incubation 24 hours；C, HepG2 cell incubation 1 hour；D, HepG2 cell incubation 24 hours；E, HCC 3736 cell incubation 1 hour；F, HCC 3736 cell incubation 24 hours.
The impact on Huh-7 cell dryness of Figure 10: the medicine；The impact that Huh-7 cell CD133 is expressed by A, medicine；Row plate clone test after B, drug treating；C, the Quantitative Comparison of plate clone quantity of formation；D, crystal violet eluting row OD570 light absorption value sxemiquantitative are compared.
After Figure 11: tumor bearing nude mice injection different pharmaceutical, in tumor cell, adriamycin positive rate compares, A, streaming result；B, positive rate compare, * P < 0.01.
The tumor suppression experiment of Figure 12: Huh-7 tumor bearing nude mice；A, tumor growth curve；*P<0.01；The schematic diagram of tumor, the most respectively normal saline group, free amycin group, non-targeted Evacet+free antibodies group, non-targeted Evacet group and CD147 targeting peptide-doxorubicin liposome group are respectively organized in B, experiment when terminating；C, nude mice body weight change curve；*P>0.05；D, the comparison of tumor weight, * P < 0.01.
Figure 13: A: the expression of CD147 in SABC detection PDX model LI-03-0005 tissue；B, tumor growth curve；* P < 0.05, * * P < 0.01；The schematic diagram of tumor, the most respectively normal saline group, non-targeted Evacet group and CD147 targeting peptide-doxorubicin liposome group are respectively organized in B, experiment when terminating；C, nude mice body weight change curve；*P>0.05；D, the comparison of tumor weight, * P < 0.05；E, the comparison of tumor weight.
Detailed description of the invention
Present invention firstly provides a kind of Anti-CD147-PEG-DSPE, the mol ratio between described Anti-CD147 and PEG-DSPE is 1:(1～3), the mol ratio between described Polyethylene Glycol and phospholipid is 1:1.All connected by covalent bond between described Anti-CD147, Polyethylene Glycol, phospholipid.Concrete, it is connected by thioether bond between described Anti-CD147 and Polyethylene Glycol.It is connected by amido link between described Polyethylene Glycol and phospholipid.
Anti-CD147-PEG-DSPE of the present invention is a kind of linear amphiphilic block copolymer, and hydrophilic section is Polyethylene Glycol, and hydrophobic section is phospholipid.Described Anti-CD147-PEG-DSPE can be self-assembly of the macromolecule micelle being made up of hydrophilic shell and lipotropy kernel in water.
Anti-CD147 of the present invention can be selected for CD147 monoclonal antibody, can obtain by being purchased approach.The present invention has no particular limits for CD147 monoclonal antibody, in a preferred embodiment of the invention, Anti-CD147 selects Anti-CD147F (ab) ' 2, also can use other CD147 monoclonal antibody, however it is not limited to the concrete monoclonal antibody cited by embodiment.
Polyethylene Glycol of the present invention, has another name called PEG, can obtain by being purchased approach.The molecular weight of Polyethylene Glycol is had no particular limits by the present invention, and molecular weight polyethylene glycol is preferably 200～6000Da.In a preferred embodiment of the invention, PEG2000 selected by described Polyethylene Glycol, also can use the Polyethylene Glycol of other molecular weight, however it is not limited to the Polyethylene Glycol of the concrete molecular weight cited by embodiment.
Phospholipid of the present invention can obtain by being purchased approach.The present invention has no particular limits for phospholipid, can be selected for short-chain phospholipid, and molecular weight and the molecular weight of Polyethylene Glycol are quite or slightly smaller.In a preferred embodiment of the invention, described phospholipid selects DSPE (DSPE), also can be selected for other phospholipid, however it is not limited to the concrete phospholipid cited by embodiment.
The present invention still further provides the preparation method of Anti-CD147-PEG-DSPE, and described preparation method is had no particular limits by the present invention, as long as this material can successfully be prepared.In a preferred embodiment of the invention, use covalent bond coupling method to prepare Anti-CD147-PEG-DSPE, also can use other preparation method, however it is not limited to concrete grammar cited in embodiment.
The present invention still further provides Anti-CD147-PEG-DSPE purposes in preparing targetable drug carriers or targeting drug delivery system.
Described targeting drug delivery system, containing Anti-CD147-PEG-DSPE.Further, described targeting drug delivery system contains: Anti-CD147-PEG-DSPE, medicine and pharmaceutical carrier.
Described medicine is had no particular limits by the present invention.Such as, described medicine can be antitumor drug.In the preferred embodiments of the present invention, select amycin as model drug, study the targeting ability of this antitumor drug sending out targeting drug delivery system conveying described.
Described pharmaceutical carrier refers to that pharmaceutical carrier refers to change medicine and enters the mode of human body and distribution in vivo, the rate of release of control medicine and conduct drugs to the system of target organs.The present invention has no particular limits for pharmaceutical carrier, as long as being capable of successfully carrying described medicine.Such as, any one during described pharmaceutical carrier can be selected for liposome, cation lipid nanoparticle, polymer micelle, Emulsion, microcapsule, microsphere, nanocapsule, nanosphere.In the preferred embodiments of the present invention, select liposome as model drug carrier, the most also can be selected for other suitable pharmaceutical carriers, however it is not limited to the concrete pharmaceutical carrier cited by embodiment.The construction method of pharmaceutical carrier is the content that those skilled in the art can be known.
The present invention does not has special restriction to the construction method of targeting drug delivery system.The structure of targeting drug delivery system can be carried out as required, for example, it is possible to first build the pharmaceutical carrier carrying medicine, be subsequently adding Anti-CD147-PEG-DSPE, mix.Can also, first build the pharmaceutical carrier carrying medicine containing PEG-DSPE, be subsequently adding Anti-CD147 and pass through covalent bond coupling reaction, build described targeting drug delivery system.Again for example, it is also possible to directly by Anti-CD147-PEG-DSPE, medicine and be used for the material mixing building pharmaceutical carrier to prepare targeting drug delivery system.
Further, described drug administration carrier also can through other modify, such as, also can modification carrying out single or multiple antibody interested, part polysaccharide etc. to described pharmaceutical carrier, be not limited in the present invention Anti-CD147 modify.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments rather than in order to limit the scope of the invention.The test method of unreceipted actual conditions in the following example, generally according to normal condition, or according to the condition proposed by each manufacturer.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, between two end points and two end points of each numerical range, any one numerical value all can be selected for.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology are generally understood that with those skilled in the art of the present technique.In addition to the concrete grammar used in embodiment, equipment, material, according to those skilled in the art's grasp to prior art and the record of the present invention, it is also possible to use any method, equipment and the material of the prior art similar or equivalent with the method described in the embodiment of the present invention, equipment, material to realize the present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all use conventional molecular biology, biochemistry, chromatin Structure and the analysis of the art, analytical chemistry, cell to cultivate, recombinant DNA technology and the routine techniques of association area.These technology have improved explanation in existing document, specifically can be found in the MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987 and periodic updates；The series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Term is explained:
Anti-CD147ILs-DOX CD147 targeting peptide-doxorubicin liposome
Ls-DOX Evacet
Free DOX dissociates amycin
The structure of embodiment 1CD147 targeting peptide-doxorubicin liposome and sign, pharmacokinetics, the research of tissue distribution
One, experimental procedure
1. flow cytomery hepatoma cell line CD147 and the expression of CD133
The good Huh-7 of cell state in cultivating, HepG2, PLC/PRF/5, Hep3B, 97L, 97H, LM3,7721 cells wash with PBS, add pancreas enzyme-EDTA and hatch in incubator 3 minutes, add DMEM culture medium and terminate digestion.Centrifugal collecting cell, washs three times with PBS, resuspended with streaming dye solution 100 microlitre, adds 1 microlitre CY5.5 labelling CD147 streaming antibody or 5 microlitre FITC labelling CD133 streaming antibody, hatches 15 minutes for 4 degrees Celsius.Washing three times with PBS after hatching, streaming dye solution 300 microlitre is resuspended, after 70 micrometer cell filter screens purchase filtration, gives flow cytomery.
2.CD147 the synthesis of targeting peptide-doxorubicin liposome (Anti-CD147ILs-DOX)
(1) prepare before experiment: dilution CD147 monoclonal antibody is standby to 2mg/ml, 5mg；Standby with PBS preparation 5mg/ml2-IT 1mL；Preparation PBS solution 2L containing 5mM EDTA, pH value 7.4, put into 4 degrees Celsius of refrigerator pre-coolings.
(2) CD147 monoclonal antibody sulfhydrylation: take 500 μ l 2mg/ml CD147 monoclonal antibody solution and 30 μ l 5mg/ml 2-IT (2-iminothiolane hydrochloride, 2-iminothiolane hydrochlorate, CAS 4781-83-3) mixing, low speed oscillations on shaking table, room temperature reaction 2 hours, makes CD147 monoclonal antibody sulfhydrylation.
(3) remove free residue 2-IT after sulfhydrylation: loaded by the liquid after sulfhydrylation in the dialyzer that molecular cut off is 1000D of an end closure, put into the PBS of the 5mM EDTA of 1L pre-cooling, 4 degrees Celsius of dialysis 8h.Change fresh dialysis fluid, 4 degrees Celsius of dialysed overnight, remove free 2-IT.
(4) synthesis DSPE-PEG2000-Mal-CD147 monoclonal antibody micelle: weigh DSPE-PEG2000-Mal2.5mg, it is dissolved in the CD147 monoclonal antibody solution of the sulfhydrylation that 1.7ml concentration is 2mg/ml, shaking table incubated at room 1-2 hour, make the sulfydryl in CD147 monoclonal antibody react the firm thioether bond of formation with maleimide, thus obtain DSPE-PEG2000-Mal-CD147 monoclonal antibody micelle.
(5) rear loading type method prepares CD147 targeting peptide-doxorubicin liposome (Anti-CD147ILs-DOX): by 1.7mlDSPE-PEG2000-Mal-CD147 monoclonal antibody micelle and Doxil injection (Evacet injection, ILs-DOX) 5ml (2mg/ml) mixing, put in 60 DEG C water baths, react 30 minutes, it is then placed into 5 minutes on ice, the DSPE end making DSPE-PEG2000-Mal-CD147 monoclonal antibody micelle is inserted in the phospholipid bilayer membrane structure of liposome, obtain CD147 targeting peptide-doxorubicin liposome solutions.
(6) free antibodies micelle is removed: put into by the CD147 targeting peptide-doxorubicin liposome solutions obtained in the dialyzer that molecular cut off is 300KD of an end closure, put into the HEPES buffer (pH value 7.4) that 1L concentration is 10mM, dialyse 8 hours, change fresh HEPES buffer, dialysed overnight, removes unnecessary free antibodies micelle.
This step experiment flow schematic diagram can be found in Fig. 1.
3.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) measures integrity and the joint efficiency of antibody
Owing to CD147 monoclonal antibody molecule amount is at about 110KD, therefore select 6%SDS-PAGE separation gel to carry out electrophoresis test, separation gel concrete configuration scheme is: 30% acrylamide solution 2ml, 1.5mol/L Tris (PH 8.8) 2.5ml, 10%SDS 0.1ml, TEMED 0.008ml, DD water is diluted to 10ml.Installing sandwich vertical slab electrophoresis groove, gap between the separation gel injection plate that will configure, isopropanol is closed.After glue to be separated solidifies completely, discard upper strata isopropanol, add and concentrate glue, and 15 hole stripping forks are installed.After glue to be concentrated solidifies completely, taking out stripping fork, inject DD water drive and catch up with bubble, install into electrophoresis tank, electrophoresis liquid there was not upper end.The leftmost side adds protein labeling, and with 2 μ g, 1 μ g, the antibody content of 0.5 μ g, untreated free CD147 monoclonal antibody is separately added into 1 to 3 swimming lanes；CD147 monoclonal antibody after sulfhydrylation is separately added into 4 to 6 swimming lanes with 2 μ g, 1 μ g, the antibody content of 0.5 μ g；Form CD147 target liposomes after inserting liposome, but be separately added into 7 to 9 swimming lanes without the sample of dialysis with about 2 μ g, 1 μ g, the antibody content of 0.5 μ g；The CD147 target liposomes inserting liposome removing Excess antibody of dialysing is separately added into 10 to 12 swimming lanes with about 2 μ g, 1 μ g, the antibody content of 0.5 μ g.The sample of all detections be with without DTT or mercaptoethanol Loading Buffer dilution, heating after loading.Open electrophoresis tank power supply, voltage is adjusted to 80V, after band enters separation gel, voltage is adjusted to 120V.Gel is taken out after terminating by electrophoresis, rejects and concentrates glue, puts in Coomassie brilliant blue and dyes, incubator overnight.Gel was carefully taken out in second day, put in Coomassie brilliant blue destaining solution and decolour, if destaining solution contaminates replaceable deeply.Thoroughly after decolouring, take pictures with slr camera, analyze.
Measure 4.CD147 targeting peptide-doxorubicin liposome characterizes
Non-targeted Evacet (ILs-DOX) before the CD147 targeting peptide-doxorubicin liposome ((Anti-CD147ILs-DOX)) synthesized and synthesis is carried out sample detection on Malvern particle instrument (Zetasizer Nano S), draws particle diameter, polydispersity index (PDI) and Zeta potential.
5. pharmacokinetic trial
Maturation SD rat is randomly divided into three groups by us, often group three, injection CD147 targeting peptide-doxorubicin liposome (Anti-CD147ILs-DOX) respectively, non-targeted Evacet (ILs-DOX) and three kinds of medicines of free amycin (DOX).Within 1 minute after injection, 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, carry out tail venous blood sampling respectively, the blood gathered gives loading by triple quadrupole mass spectrometer LCMS-8030, detection doxorubicin concentration, it is depicted as blood concentration-time curve, and according to this curve by Phoenix WinNonlin computed in software half-life (t1/2), area (AUC), apparent volume of distribution (V under pharmaceutical concentration-time curved), Cl (CL), mean residence time (MRT).
6. drug entities distribution detection
We will be in the Huh-7 cells rinsed with PBS of exponential phase, add pancreas enzyme-EDTA and hatch in incubator 3 minutes, add DMEM culture medium and terminate digestion.Centrifugal collecting cell, resuspended with PBS, cell counting under microscope, by 5 × 106Individual Huh-7 cell infusion is to the 5 right dorsal sc of week old nude mice.After nude mice dorsal tumors transplantation model is successfully established, tumor size is monitored, gross tumor volume computing formula: volume=(major diameter × minor axis2)/2.When tumor length to 500mm3Time, select that nude mice body weight, gross tumor volume are the most homogeneous 18, are randomly divided into 3 groups, often group 6, respectively through tail vein injection CD147 targeting peptide-doxorubicin liposome, non-targeted Evacet and three kinds of medicines of free amycin, dosage is 5mg amycin/kg body weight.After 24 hours, often group is after random choose 3 only gives euthanasia, and excision nude mouse tumor, liver, heart, spleen, lung and kidney fully wash in PBS, wrap up with masking foil after absorbent paper exhaustion liquid, puts in liquid nitrogen 30 minutes.-80 degrees Celsius of Refrigerator stores are put in rear taking-up.By triple quadrupole mass spectrometer LCMS-8030, the doxorubicin concentration in tissue is measured.
Two, experimental result
1. hepatoma cell line CD147 and the expression of CD133
Pass through flow cytometer detection, it has been found that the CD147 of the most conventional hepatoma cell line presents obvious high expressed (Fig. 2 A), and the cell of all cells system above 90% is CD147 positive cell.And in the detection of CD133, the expression of Huh-7 and Hep3B cell is higher (Fig. 2 C), being respectively 85.8 ± 0.32% and 97.6 ± 0.57%, the average fluorescent strength of these two groups of cells is the strongest (Fig. 2 D) simultaneously, respectively 1420.3 ± 17.6 and 3657.7 ± 57.1.Then we are in the double positive cell line of only two CD147, CD133, choose CD147 average fluorescent strength higher (27033 ± 251.7vs 11800 ± 360.6) and the lower slightly Hun-7 cell line of CD133 expression, in order to the impact on cell CD133 ratio of the Part II drugs.Therefore the cell line selection of remaining experiment is essentially Huh-7 cell line in this research.
2.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) measures integrity and the joint efficiency of antibody
As it is shown on figure 3, free CD147 monoclonal antibody is positioned at the position of 110KD band on 1 to 3 swimming lanes, and the band less than 110KD does not occur on swimming lane afterwards, therefore antibody integrity is preferable, situation about being hydrolyzed does not occurs.nullThree higher bands of molecular weight (black arrow) are had above 110KD band on 4 to 6 swimming lanes，Represent the CD147 monoclonal antibody being combined with DSPE-PEG2000-MAL，Position is high one section，Above 110KD band, band pointed by first black arrow represents an Anti-CD147 monoclonal antibody and combines 1 DSPE-PEG-MAL，Above 110KD band, band pointed by second black arrow represents an Anti-CD147 monoclonal antibody and combines 2 DSPE-PEG-MAL，Above 110KD band, the 3rd band pointed by black arrow represents an Anti-CD147 monoclonal antibody and combines 3 DSPE-PEG-MAL，White arrow represents unconjugated free CD147 monoclonal antibody.On 7 to 9 swimming lanes, black arrow represents the DSPE-PEG2000-MAL-CD147 monoclonal antibody being inserted into surface of liposome, and white arrow represents unconjugated free CD147 monoclonal antibody.And on 10-12 swimming lane, 110KD position shown in white arrow is without detailed band before, representing that free antibodies has been dialyzed clear, band shown in upper black arrow is the CD147 monoclonal antibody being attached on CD147 targeting peptide-doxorubicin liposome.
Calculating gray value through Image J software, the linkage rate of antibody (black arrow band gray value sum/all band gray value sums) of 4 to 9 swimming lanes is followed successively by 83.5%, 77.9%, 61.5%, 87.5%, 84.9%, 80.5% (Fig. 3 B).
3. the sign of liposome measures
As shown in Fig. 4, table 1-1, pass through the CD147 targeting peptide-doxorubicin liposome of synthesis compared with non-targeted Evacet, its particle diameter less (90.97 ± 0.91vs, 109.4 ± 2.34nm), and Zeta potential absolute value is bigger (-17.1 ± 0.35vs-8.92 ± 0.04mv), the ratio that PDI controls is relatively low (0.098 ± 0.007vs 0.078 ± 0.002), ideal.
Table 1-1CD147 targeting peptide-doxorubicin liposome and the characterization parameter of non-targeted Evacet.Table 1-1:Characterization of liposomes.Data are expressed as mean ± SD (n=3) from three independent samples.
4. pharmacokinetics detection
Can find out that the distribution phase curvature of CD147 targeting peptide-doxorubicin liposome is bigger intuitively by drawing blood concentration-time curve (Fig. 5), and eliminate the mildest, presenting two Room distributed models of typical intravascular administration, this prompting internal there may be targeting cumulative action.And compared with two kinds of liposome medicaments, the blood drug level of free amycin is almost negligible.
Calculate as shown in table 1-2 according to blood concentration-time curve, CD147 targeting peptide-doxorubicin liposome and non-targeted Evacet are basically identical in half-life, mean residence time the two parameter, CD147 targeting peptide-doxorubicin liposome apparent volume of distribution (374.1 ± 60.3vs, 157.3 ± 5.7ml/kg) and Cl (16.5 ± 1.8vs, 7.4 ± 0.6ml/h/kg) are higher, and area significant lower (240963.7 ± 25056.4vs, 569292.9 ± 34558.3ng h/ml) under pharmaceutical concentration-time curve.
The pharmacokinetic parameter of 1-2: three kinds of medicines of table
5. tissue distribution detection
Tissue distribution results is explicitly shown (Fig. 6), little constantly at injection medicine 24, CD147 targeting peptide-doxorubicin liposome the most topmost accumulation position is at liver (14562.73 ± 5895.6ng/g), next to that spleen (3898.7 ± 1569.5ng/g) and tumor (3148 ± 596.2ng/g), it is all remarkably higher than non-targeted group (P < 0.05) in its doxorubicin concentration of these three position；Concrete, during tail vein injection 24h, CD147 targeting peptide-doxorubicin liposome group is general more than 1.96 times that the mean concentration of intra-tumor is non-targeted group.Although after injection CD147 targeting peptide-doxorubicin liposome 72 hours, intra-tumor amycin level has declined (1113.26 ± 542.9ng/g) relatively before, but it is still significantly higher than non-targeted group (P < 0.05) in tumor, liver, the level of spleen；Concrete, during tail vein injection 72h, CD147 targeting peptide-doxorubicin liposome group is general more than 2.94 times that the mean concentration of intra-tumor is non-targeted group.
Embodiment 2CD147 targeting peptide-doxorubicin liposome cellular uptake in vitro, cell killing and the research on the impact of tumor dryness
One, experimental procedure
The foundation of 1.PDC cell and the expression of its CD147 of flow cytomery
(1) male's Patients with Primary of one 56 years old taken from by PDC model.This patient has carried out right liver tumor excision, and postoperative pathological is diagnosed as hepatocellular carcinoma.In operation in patients during tumor resection, cut tumor specimen in an aseptic environment, cut the fresh not downright bad tumor tissues of fritter with knife blade, put in HTK Organ Preservation Solution, transport on ice.To super-clean bench, being poured into by tissue in culture dish, be cut into small pieces shape with sterile scissors, pour in the Digestive system containing IV Collagenase Type (0.1%w/v) and DNA enzymatic I (0.003%), 37 degrees Celsius of incubators digest 1 hour.Rear collection Digestive system after 70 micrometer cell strainer filterings, centrifugal collecting cell, add 1ml erythrocyte cracked liquid, softly blow and beat, after standing 5 minutes, centrifugal, collect cell, be put in overnight incubation in routine DMEM culture fluid.Second day supernatant discarded, changes fresh medium, waits cell attachment, growth.The named HCC3736 of the cell line passed on obtained.HCC3736 used in experiment is the cell after original cuiture less than 5 generations.Patient tumors is drawn materials and is implemented after patient's written consent.
(2) HCC 3736 cell good for growth conditions is washed with PBS, add pancreas enzyme-EDTA and hatch in incubator 3 minutes, add DMEM culture medium and terminate digestion.Centrifugal (1000rpm, 5 minutes) collects cell, manages with EP so that 100 μ l stainingbuffer are resuspended, and often pipe adds 1 microlitre CY5.5 labelling CD147 streaming antibody, hatches 15 minutes for 4 degrees Celsius.Washing three times with PBS after hatching, streaming dye solution 300 microlitre is resuspended, after 70 micrometer cell filter screens purchase filtration, gives flow cytomery.
2. hepatoma cell line picked-up to CD147 targeting peptide-doxorubicin liposome in vitro
Huh-7 cell good for growth conditions is washed with PBS, adds pancreas enzyme-EDTA and hatch in incubator 3 minutes, add DMEM culture medium and terminate digestion.Centrifugal (1000rpm, 5 minutes) collects cell, and resuspended with PBS, cell counting under microscope, by 2 × 105Individual Huh-7 plating cells in 30mm glass bottom copolymerization Jiao's special culture dish, adherent overnight.Second day supernatant discarded, CD147 targeting peptide-doxorubicin liposome, non-targeted Evacet, non-targeted Evacet+free CD147 monoclonal antibody, free amycin are hatched 1 hour with 37 degrees Celsius of incubators of concentration (calculating with amycin) of 200 μ g/ml, rear supernatant discarded, after washing three times with PBS, add 4% paraformaldehyde and fix 15 minutes.Wash with PBS again and add DAPI after three times and dye three minutes, after wash three times with PBS, use confocal laser scanning microscope.
3. hepatoma cell line endocytosis to CD147 targeting peptide-doxorubicin liposome in vitro
Cell has combination and endocytosis two kinds to the picked-up of nano drug-carrying, and the former is only combined in surface of cell membrane, and the latter is the effect that endocytosis such as endochylema or entrance nucleus, the most only the latter could really play tumor cytotoxicity.Therefore, we report according to document before, with Pronase as the eluant of liposome.
Concrete steps: the Huh-7 cell that the most just growth conditions is good spreads incubated overnight in six orifice plates.Second day supernatant discarded, CD147 targeting peptide-doxorubicin liposome, non-targeted Evacet are hatched 1 hour with 37 degrees Celsius of incubators of concentration (calculating with amycin) of 200 μ g/ml, rear supernatant discarded, PBS washs, add pancreas enzyme-EDTA to hatch in incubator 3 minutes, add DMEM culture medium and terminate digestion.Centrifugal (1000rpm, 5 minutes) collects cell.Now the cell of two process groups is respectively divided into two parts, a hatches 30 minutes with 2mg/ml Pronase 4 degrees Celsius, a with resuspended 30 minutes of PBS.Be centrifuged respectively, wash each group of cell, with staining buffer resuspended after, cross 70 micrometer cell filter screens, flow cytometer detection doxorubicin fluorescence intensity.
The 4.CD147 targeting peptide-doxorubicin liposome killing effect in vitro to hepatoma cell line
(1) by Huh-7 plating cells in good condition in 2 piece of 96 orifice plate, every hole about 3 × 103Individual cell, adherent overnight.
Within (2) second days, discard culture medium, add the most from top to down 300 μ g/ml, 200 μ g/ml, 100 μ g/ml, 33 μ g/ml, 11 μ g/ml, 3.7 μ g/ml, 1.2 μ g/ml concentration CD147 targeting peptide-doxorubicin liposome (with doxorubicin content calculate concentration, dilute with culture medium), each concentration does three secondary orifices, and same method adds the non-targeted Evacet of respective concentration, non-targeted Evacet+free CD147 monoclonal antibody, free amycin in same 96 orifice plate.37 degrees Celsius of incubators are hatched 1 hour, supernatant discarded, after every hole is washed with PBS, add fresh DMEM medium and continue to cultivate 47 hours.Another block 96 orifice plate in kind drug incubation 24 hours.Add fresh DMEM medium after PBS washing, continue to cultivate 24 hours.
(3) every piece of 96 orifice plates the most additionally arrange positive control: three holes of the cell being left intact, and Background control: without three secondary orifices of any cell in plate hole.
(4) after incubation time terminates, discard culture medium, add the CCK-8 reagent of 1% concentration, continue 37 degrees Celsius of incubators and hatch 1.5 hours.
(5) fully react two pieces of 96 orifice plates are put into Anthos Zenyth 3100 microplate reader, at 450nm, measure absorbance.Finally calculating cell viability value formula is: (ODX-ODBackground)/(ODPositive-ODBackground) %.OD in formulaXAbsorbance for certain hole；ODBackgroundFor being not added with cell, but add the Background control group mean light absorbency of CCK-8 reagent；ODPositiveThe mean light absorbency of the cell gained of drug treating normal growth it is not added with for positive controls.
(6) the IC50 value of medicine is often organized with Graphpad Prism computed in software.
(7) identical experimental program repeated authentication on HepG2 and HCC 3736 cell.
The most in vitro under environment, the medicine impact on hepatoma cell line dryness
(1) cultivation will be laid in six orifice plates after Huh-7 cell dissociation good for growth conditions, adherent overnight after, discarding culture medium, be separately added into the CD147 targeting peptide-doxorubicin liposome of 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 37 degrees Celsius of incubators hatch 36 hours.Rear supernatant discarded, PBS washs, and adds pancreas enzyme-EDTA and hatches in incubator 3 minutes, adds DMEM culture medium and terminates digestion.Centrifugal (1000rpm, 5 minutes) collects cell, manages with EP so that 100 μ l staining buffer are resuspended, and often pipe adds 5 microlitre FITC labelling CD133 streaming antibody, hatches 15 minutes for 4 degrees Celsius.Washing three times with PBS after hatching, streaming dye solution 300 microlitre is resuspended, after 70 micrometer cell filter screens purchase filtration, gives flow cytomery.The impact that Huh-7 cell line CD133 is expressed by the same method non-targeted Evacet of detection, non-targeted Evacet+free CD147 monoclonal antibody, free amycin.
(2) during Huh-7 cell is incubated at the CD147 targeting peptide-doxorubicin liposome of 50 μ g/ml concentration, non-targeted Evacet, non-targeted Evacet+free CD147 monoclonal antibody, free amycin by respectively, hatch 36 hours, rear supernatant discarded, PBS washs, add pancreas enzyme-EDTA to hatch in incubator 3 minutes, add DMEM culture medium and terminate digestion.Centrifugal (1000rpm, 5 minutes) collects cell, takes on 10 μ l samples and cell counting count board after PBS is resuspended, and Trypan Blue calculates viable cell density in cell suspension.Taking 6 orifice plates, 1000 living cells processed through different pharmaceutical, conventional DMEM culture medium culturing 14 days are put in every hole.Supernatant discarded, PBS washs 3 times, adds 4% paraformaldehyde and fix 15 minutes.Add 0.2% crystal violet solution room temperature after washing three times with PBS again to hatch 30 minutes, more fully wash with PBS, dry rear slr camera and take pictures and count.
(3) configuration adds 1 milliliter containing 50% ethanol, the destaining solution of 0.1% acetic acid, every hole, on shaking table after abundant dissolving crystallized purple, puts into Anthos Zenyth 3100 microplate reader, obtain light absorption value at 570nm.
Two, experimental result
The CD147 of 1.HCC 3736 cell expresses
By flow cytomery, our newly-established PDC model, HCC 3736 cell is also the same with common hepatoma cell line, is CD147 high expressed (positive rate: 99.9 ± 0.1%, MFI:11366.7 ± 57.7).Result shows at Part I Fig. 2 A, B.
2. hepatoma cell line picked-up situation to medicine in vitro
As shown in Figure 7, after hatching 1 hour by 200 μ g/ml, different pharmaceutical imaging under Laser Scanning Confocal Microscope is compared, we have found that cell that CD147 targeting peptide-doxorubicin liposome-treated crosses at intracellular red fluorescence (Doxorubicin autofluorescence is redness) compared with non-targeted Evacet group, hence it is evident that stronger.And the result of amycin group of dissociating shows, amycin enters nucleus with the overwhelming majority, overlapping with DAPI dyeing part, and fluorescence intensity is stronger.
3. hepatoma cell line situation to medicine endocytosis in vitro
After being illustrated in figure 8 two groups of medicines of targeting and non-targeted liposome process Huh-7 cell the most in the same manner, through Pronase and without the flow cytometer detection result after Pronase eluting.Black line is the blank group of non-drug treating, and A figure blue line represents the targeting group place fluorescence area of non-eluting, targeting group place fluorescence area after the eluting that red line represents, and contrast blue line slightly moves to left.B figure blue line represents non-targeted group of fluorescence area of non-eluting, and red line represents the targeting group place fluorescence area after eluting, and relatively blue line has and significantly moves to left.The targeting group MFI value of non-eluting is: 713 ± 29.9, it is significantly higher than non-targeted group of MFI value of non-eluting: 647.7 ± 21.5 (P=0.008), being also significantly greater than the targeting group MFI value after eluting: 602 ± 23.9 (P < 0.001), non-targeted group of MFI value of non-eluting is also significantly greater than non-targeted group of MFI value after eluting: 325.67 ± 11.1 (P < 0.001).Visible non-targeted Evacet is easier to by Pronase eluting, therefore CD147 targeting peptide-doxorubicin liposome is easier to mediated adriablastina by cell endocytic.
4.CCK-8 detects each medicine killing effect in vitro to hepatoma carcinoma cell
Fig. 9 is variable concentrations CD147 targeting peptide-doxorubicin liposome, non-targeted Evacet, non-targeted Evacet+free antibodies, free amycin be respectively acting on Huh-7, HepG2, HCC3736 cell 1 hour, the cell viability curve chart of 24 hours；Each medicine is calculated to the different cell line effects IC50 of 1,24 hours (table 2-1) according to this curve；We can see that CD147 targeting peptide-doxorubicin liposome most has superiority at the IC50 of 1 hour, in Huh-7 cell, the IC50 of target liposomes and two groups of non-targeted liposomees differ 4.2 times and 5.6 times respectively, in HepG2 cell, then reach 8 times and 11.4 times, in HCC 3736, be respectively 7.2 and 8.9 times；But target liposomes does not the most have advantage at the IC50 of 24 hours compared with non-targeted liposome；Being worth one, the effect of the amycin that dissociates in vitro suppression liver cancer cell growth is the most obvious, puts the IC50 value to any cell at any time the most minimum.
Table 2-1 medicine is to Huh-7, HepG2 and HCC 3736 cytotoxic effect of cell
5. medicine impact on hepatoma cell line dryness in vitro
After hatching 36 hours with the CD147 targeting peptide-doxorubicin liposome of variable concentrations, non-targeted Evacet, non-targeted Evacet+free antibodies, free amycin, the CD133 detection of expression of Huh-7 cell line is found that CD147 targeting peptide-doxorubicin liposome has the most significantly down regulation trend (Figure 10 A) for the expression of Huh-7 cell CD133, there were significant differences compared with two groups of non-targeted liposome-treated (P=0.035,0.041), and free amycin has slight rise trend for the expression of Huh-7 cell CD133.
The cell row plate clone test that drug treating is crossed, find that CD147 targeting peptide-doxorubicin liposome is the most obvious (Figure 10 B) to the suppression of Huh-7 cell clonal formation, the plastidogenetic clone's quantity being processed is significantly less than non-targeted Evacet (P=0.033) and non-targeted Evacet+free antibodies group (P=0.039), and clone's quantity of free amycin group is with untreated control group (NEG) substantially quite (Figure 10 C).Crystal violet eluting after plate clone is formed the conclusion of semiquantitative determination OD570 light absorption value gained and plate clone basically identical (Figure 10 D), light absorption value (the P=0.026 that the OD570 light absorption value of targeting peptide-doxorubicin group is non-targeted significantly less than two groups, 0.016) OD570 of amycin group of, dissociating is basically identical with matched group.
The internal targeting of embodiment 3CD147 targeting peptide-doxorubicin liposome and antitumous effect research
One, experimental procedure
1. the detection in-vivo tumour picked-up situation to medicine
(1) we will be in the Huh-7 cells rinsed with PBS of exponential phase, add pancreas enzyme-EDTA and hatch in incubator 3 minutes, add DMEM culture medium and terminate digestion.Centrifugal collecting cell, resuspended with PBS, cell counting under microscope, by 5 × 106Individual Huh-7 cell infusion is to the 5 right dorsal sc of week old nude mice.After nude mice dorsal tumors transplantation model is successfully established, tumor size is monitored, gross tumor volume computing formula: volume=(major diameter × minor axis2)/2.When tumor length to 500mm3Time, select that nude mice body weight, gross tumor volume are the most homogeneous 9, it is randomly divided into 3 groups, often group 3, respectively through tail vein injection CD147 targeting peptide-doxorubicin liposome, non-targeted Evacet and free amycin, dosage is 5mg amycin/kg body weight, separately sets a mice with tumor injecting normal saline and does blank.
After (2) 24 hours, nude mice gives euthanasia, excises nude mouse tumor.To super-clean bench, being put into by tumor tissues in culture dish, be cut into small pieces shape with aseptic operation shears, pour in the Digestive system containing IV Collagenase Type (0.1%w/v) and DNA enzymatic I (0.003%), 37 degrees Celsius of incubators digest 1 hour.Rear collection Digestive system after 70 micrometer cell strainer filterings, centrifugal collecting cell, add 1ml erythrocyte cracked liquid, soft piping and druming, after standing 5 minutes, centrifugal collecting cell, with 300 μ lstainning buffer resuspended after, cross 70 micrometer cell filter screens, flow cytometer detection doxorubicin fluorescence.
2. in-vivo tumour Inhibition test
(1) we will be in the Huh-7 cells rinsed with PBS of exponential phase, add pancreas enzyme-EDTA and hatch in incubator 3 minutes, add DMEM culture medium and terminate digestion.Centrifugal collecting cell, resuspended with PBS, cell counting under microscope, by 5 × 106Individual Huh-7 cell infusion is to the 5 right dorsal sc of week old nude mice.Tumor size is monitored, gross tumor volume computing formula: volume=(major diameter × minor axis2)/2。
(2) after nude mice dorsal tumors transplantation model is successfully established, gross tumor volume is selected at 100-200mm3And 50 that nude mice body weight is the most homogeneous, it is randomly divided into 5 groups, often group 10, respectively through tail vein injection CD147 targeting peptide-doxorubicin liposome, non-targeted Evacet, non-targeted Evacet+free antibodies and four kinds of medicines of free amycin, dosage is 5mg amycin/kg body weight, and another group injection same volume normal saline is matched group.Medicine injection in every 7 days once, is injected four times altogether.
(3) after medication, every three days gross tumor volumes of record and nude mice body weight.Until treatment group occurs that nude mice is dead, terminate experiment.After nude mice euthanasia, excise Subcutaneous tumor, Taking Pictures recording.Weigh often organizing Subcutaneous tumor record.
The foundation of 3.PDX model
(1) male's Patients with Primary of one 56 years old taken from by PDX model.This patient has carried out right liver tumor excision+cholecystectomy, and postoperative pathological is diagnosed as hepatocellular carcinoma.In operation in patients during tumor resection, cut tumor specimen in an aseptic environment, cut the fresh not downright bad tumor tissues of fritter with knife blade, put in HTK Organ Preservation Solution, transport on ice.
(2) in super-clean bench, take out tissue, be placed in the culture dish of 10cm, be quickly transferred to containing in dual anti-PBS, and clean 2 times, reject slough, the degree of necrosis of record organization.Guarantee that downright bad part is not used in inoculation as far as possible.Tissue is proceeded to containing in the dual anti-DMEM culture medium of 1% Pen .-Strep (serum-free), tissue is cut into small pieces (30-50mm with Aseptic sterilisation operating scissors3).Matrigel infiltrates, at syringe needle, fill in No. 18 trocars.The skin on the left of nude mice is caught to make skin-tightening on the right side of it with left hand, it is easy to puncture, the skin of back of nude mice is carried out disinfection by the ethanol with 75%, and it is coated with local anaesthetics lignocaine (0.5% solution) at position to be punctured, then skin is penetrated with No. 18 puncture needles from the centre position of right dorsal part, upwards inserting needle is at scapula, a folliculus is formed subcutaneous stripping with trocar front end, tumor being released plants in herein, if tumor mass is withdrawn from syringe needle and shift, available trocar root is resetted, 1 point of every animal inoculation.
(3) tissue frozen, pass on: P0-P3 generation can according to tumor with or without growth sign, select growth sign weekly (to growing tumor faster) or every 2 weeks (to growing slower tumor) measures 1 tumor size, the tumor reaching a certain size is passed in time, after inoculating 120 days, if tumor is without growth sign, then by nude mice euthanasia.P0-P3 to 500-800mm3, can take off tumor mass for tumor growth, selects without downright bad position, and a part is frozen with Bambanker frozen stock solution-80 degrees Celsius, and a part is continued to be inoculated in other nude mices by above-mentioned flow process.The named LI-03-0005 of PDX model that we are set up.
Patient tumors is drawn materials and is implemented after patient's written consent.
4. SABC checking LI-03-0005 organizes CD147 to express
(1) wax stone makes: the nude mice selecting tumor size about 500mm3 gives euthanasia, completely takes off tumor 4% paraformaldehyde and fixes 48 hours.Put into 75% ethanol overnight, after be respectively put into 85% ethanol 2 hours, 95% ethanol 2 times each 1.5 hours, dehydrated alcohol 3 times each 15 minutes, be fully dehydrated.After to put into dimethylbenzene twice each 10 minutes, put in paraffin wax embedding liquid paraffin 2 hours, rear paraffin embedding, after cooling, make wax stone.
(2) tissue slice: after wax stone-20 C overnight, microtome, drags for tissue to microscope slide, dries 60 degrees Celsius of sheet machine and toast 1 hour.
(3) dewaxing, aquation: each immersion of dimethylbenzene three bottles 10 minutes, dehydrated alcohol, 85% ethanol, 75% ethanol respectively soak 5 minutes.DD water soaking 5 minutes.
(4) cell-permeant, closing endogenous peroxydase: configure 3% hydrogen peroxide-methanol solution, soak 20 minutes.DD washes three times.
(5) antigen retrieval exposes antigenic determinant: configuration sodium citrate antigen retrieval buffers (containing trisodium citrate 1.8g+ citric acid 1.05g) 1500ml is positioned over pressure cooker and boils, put into section, lid pot continues 2 minutes after steaming, and is vented steam, natural cooling of uncapping.
(6) closing nonspecific proteins: PBS to soak 5 minutes, 1%BSA room temperature is closed 30 minutes.
(7) one resist overnight: Anti-CD147 antibody [MEM-M6/1] is diluted to 1:500, is covered in tissue, put into wet box, 4 C overnight.
(8) two anti-hatch: by one anti-fall fall and wash 5 minutes with PBS, be repeated 3 times.With filter paper, the water around circle is sucked, add 1 two diluted anti-after put in 37 degrees Celsius of constant temperature roasters 30 minutes.Wash 5 minutes with PBS afterwards, be repeated 3 times.
(9) colour developing: add DAB nitrite ion, developing time control 1 point about 40 seconds, by Microscopic observation color control the time.Getting rid of DAB, putting into can color development stopping in water.DD water soaking 5 minutes, washes twice.
(10) redye: microscope slide is put in the haematoxylin crossed with filter paper filtering, redye 10 minutes, tap water cleaning down.
(11) differentiation: microscope slide is put in the hydrochloride alcohol (5ml hydrochloric acid is diluted to 400ml with 75% ethanol) configured, break up 2 times.DD water rinses, anti-blue.
(12) dehydration: be sequentially placed into 75% ethanol, 85% ethanol, each 30 seconds of dehydrated alcohol.
(13) mounting: drip a small amount of resinene on microscope slide, thoroughly clean coverslip, cover resin gently.
Tumor killing effect in 5.PDX modelling verification medicine body
One is vaccinated with LI-03-0005 tissue and successfully one-tenth tumor to 800mm3Nude mice row euthanasia, Subcutaneous tumor is divested, puts into and fill in the culture dish containing dual anti-PBS, reject slough, be transferred to, containing in dual anti-DMEM culture medium, cut, with aseptic operation, the (30-50mm that is cut into small pieces by tissue3), matrigel is sent into nude mice by subcutaneous by No. 18 trocars after infiltration.After close observation about 30 days, nude mice by subcutaneous initially forms tumor, selects tumor size the most homogeneous, about 50-150mm3Between nude mice 15, be randomly divided into three groups, often group 5, respectively injection CD147 targeting peptide-doxorubicin liposome and non-targeted Evacet, dosage is 5mg amycin/kg body weight, and another group injection same volume normal saline is matched group.Medicine injection in every 7 days once, is injected four times altogether.After medication, every three days gross tumor volumes of record and nude mice body weight.Terminate experiment, after nude mice euthanasia, excise Subcutaneous tumor, Taking Pictures recording.Weigh often organizing Subcutaneous tumor record.
Two, experimental result
1. the tumor picked-up situation to medicine under internal milieu
Pass through flow cytometer detection, it has been found that the positive rate of the tumor tissue cell's picked-up amycin after injection CD147 targeting peptide-doxorubicin liposome is higher than non-targeted group, free amycin group minimum (Figure 11 A).The average positive rate 30.77 ± 4.4% (Figure 11 B) of targeting group, is significantly higher than non-targeted group 7.54% ± 2 (P < 0.01).And free amycin group positive rate only 1 ± 0.32%.
2.Huh-7 tumor bearing nude mice tumor inhibition
Through four drug injections, the nude mouse tumor growth of Anti-CD147ILs-DOX treatment group is substantially suppressed, its effect is considerably better than Ls-DOX and Ls-DOX+Anti-CD147 treatment group (P < 0.01), and the curative effect of Ls-DOX and Ls-DOX+Anti-CD147 treatment group is better than free amycin group (Figure 12 A, B).After terminating experiment, the tumor of nude mice by subcutaneous is taken out by we, precise weighing, finds that the tumor weight after CD147 targeting peptide-doxorubicin liposome therapeutic is minimum (0.156 ± 0.09g), significant difference (P < 0.01, Figure 12 D) is all had compared with other each group.Concrete, last time point, the average external volume of the nude mouse tumor of Anti-CD147ILs-DOX treatment group is: 217.33 ± 67.67mm3；The average external volume of the nude mouse tumor of Ls-DOX treatment group is: 903.13 ± 246mm3；The average external volume of the nude mouse tumor of Ls-DOX+Anti-CD147 treatment group is: 834.69 ± 223.81mm3；The average external volume of the nude mouse tumor of free DOX treatment group is: 1867.54 ± 473.21mm3。
As can be seen here, the average external volume of the nude mouse tumor of Anti-CD147ILs-DOX treatment group is minimum, and the average external volume of nude mouse tumor is 3.84 times of Anti-CD147ILs-DOX treatment group after Ls-DOX+Anti-CD147 treatment, after Ls-DOX treatment, the average external volume of nude mouse tumor is 4.15 times of Anti-CD147ILs-DOX treatment group, and after Free DOX treatment, nude mouse tumor average external volume is 8.59 times of Anti-CD147ILs-DOX treatment group.
The body weight of nude mice is also monitored by we simultaneously, after finding medication, although the body weight of targeting group is higher (Figure 12 C), but the statistical testing results shows no significant difference between each group (P > 0.05).
In 3.PDX model tissue, the expression of CD147 measures
Figure 13 A is the expression of CD147 in the PDX model LI-03-0005 tissue that we set up.Even if we have found that when employing anti-concentration (1:500) lower than description concentration, we have still obtained the SABC photo of a strong positive, and by group it is clear that discovery CD147 be expressed in the cell membrane of tumor cell.
The tumor inhibition of 4.PDX model LI-03-0005
By the dosage regimen identical with Huh-7 nude mice lotus tumor model, on PDX model, we have obtained the most similar result.Figure 13 B is the tumor growth curve of LI-03-0005 model, we can see that CD147 targeting peptide-doxorubicin liposome has good inhibition for PDX model transplantations tumor, Anti-CD147ILs-DOX treatment group is up to 95.3% to the suppression ratio of tumor, non-targeted liposome effect is taken second place, and suppression ratio only has 38.24%.Through statistical test, the nude mouse tumor of injection CD147 targeting peptide-doxorubicin liposome is significantly less than non-targeted group (P=0.047).At the end of experiment, by nude mice euthanasia, take out tumor and take pictures (Figure 13 C) and claim to obtain weight.The exemplary embodiment lock of CD147 targeting peptide-doxorubicin liposome therapeutic is 0.056 ± 0.01g, significantly less than non-targeted Evacet group (0.244 ± 0.19g, P=0.044).
The body weight of nude mice during experiment is monitored (Figure 12 C), between discovery group, is not significantly different from (P > 0.05).
In sum, the present invention selects amycin as drug model, Anti-CD147, as pharmaceutical carrier model, is connected on pharmaceutical carrier by Evacet by Anti-CD147-PEG-DSPE, constructs the targetable drug carriers that can target CD147 high expression tumour cell.First we are successful to the synthesis of CD147 targeting peptide-doxorubicin liposome, are demonstrated the integrity of antibody by SDS-PAGE, and tentatively detect its joint efficiency.This is also the research having target liposomes in hepatocarcinoma for the first time simultaneously.Pharmacokinetics prompting CD147 targeting peptide-doxorubicin liposome has obvious two Room characteristic distributions, has longer half-life, apparent volume of distribution and clearance rate.Tissue distribution display medicine has accumulation at liver, spleen and tumor locus.Experiment in vitro display hepatoma cell line is significantly stronger than non-targeted liposome to the external picked-up of CD147 targeting peptide-doxorubicin liposome, and non-targeted group is had a clear superiority in by its IC50 when short incubation period.Endocytosis experiment proves that CD147 targeting peptide-doxorubicin liposome is easier to the mediated cell endocytosis to chemotherapeutics.Furthermore it has been found that CD147 targeting peptide-doxorubicin liposome can substantially reduce the expression of the dryness mark CD133 of hepatoma carcinoma cell in vitro, and affect its dryness.Experiment in vivo display CD147 targeting peptide-doxorubicin liposome has higher inhibition to nude mice lotus tumor, and tumor is also the highest to the picked-up of CD147 targeting peptide-doxorubicin liposome in vivo.During injection, there are not the untoward reaction such as obvious weight loss in nude mice.At present on the market of oncotherapy, only Evacet goes through to list, and is only approved in the oncotherapys such as Kaposi sarcoma in China.Meanwhile, our medicine, on the characterization parameter such as particle diameter, Zeta potential, is compared than the how U.S. element (Doxil injection) listed and is had certain advantage.And our research proves that the Evacet of CD147 targeting is respectively provided with clear superiority than non-targeted Evacet in vivo with in extracorporeal anti-tumor further.
As can be seen here, use Anti-CD147 that pharmaceutical carrier is modified, it is connected by thioether bond between the most described Anti-CD147 and PEG-DSPE and mol ratio between described Anti-CD147 and PEG-DSPE is 1:(1～3) factor in terms of these, " targeting head " is become efficiently for Anti-CD147-PEG-DSPE particularly critical.The substantial amounts of research of the present invention shows, targeting drug delivery system containing Anti-CD147, the Anti-CD147 of pharmaceutical carrier particle surface, characteristic efficiently pharmaceutical carrier particle directionally can be transported in the tumor tissues of CD147 high expressed, the pharmaceutical carrier particle being distributed in target tissue can be combined with the target protein of tumor cell surface, and the process such as induced drug carrier endocytosis or drug release, thus play drug effect.The targeting drug delivery system that Anti-CD147 modifies has higher growth inhibited effect to the tumor of CD147 high expressed, and the targeting drug delivery system that Anti-CD147 modifies is that other modify more than 3～8 times of drug-supplying system without Anti-CD147 to the inhibitory action of the tumor of CD147 high expressed.Concrete, the targeting drug delivery system that Anti-CD147 modifies is up to 95.3% to the suppression ratio of tumor, and the drug-supplying system modified without Anti-CD147 only has 38.24% to the suppression ratio of tumor.And for other phospholipid in Anti-CD147-PEG-DSPE, the other drug carrier particle in targeting drug delivery system and other drug, the present invention repeats the most one by one.
The above; it is only presently preferred embodiments of the present invention; not any formal and substantial to present invention restriction; should be understood that; for those skilled in the art; on the premise of without departing from the inventive method, also can make some improvement and supplement, these improve and supplement and also should be regarded as protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, a little change of making when available disclosed above technology contents, the equivalent variations modified and develop, it is the Equivalent embodiments of the present invention；Meanwhile, the change of any equivalent variations that above-described embodiment is made by all substantial technological according to the present invention, modify and develop, all still fall within the range of technical scheme.

Claims (22)

1. an Anti-CD147-PEG-DSPE, the mol ratio between described Anti-CD147 and PEG-DSPE For 1:(1～3), the mol ratio between described Polyethylene Glycol and phospholipid is 1:1.
Anti-CD147-PEG-DSPE the most according to claim 1, it is characterised in that described Anti-CD147, All connected by covalent bond between Polyethylene Glycol, phospholipid.
Anti-CD147-PEG-DSPE the most according to claim 1, it is characterised in that described Anti-CD147, It is connected by thioether bond with between Polyethylene Glycol.
Anti-CD147-PEG-DSPE the most according to claim 1, it is characterised in that described Polyethylene Glycol with Connected by amido link between phospholipid.
Anti-CD147-PEG-DSPE the most according to claim 1, it is characterised in that described Anti-CD147 selects Use CD147 monoclonal antibody.
Anti-CD147-PEG-DSPE the most according to claim 1, it is characterised in that described molecular weight polyethylene glycol Scope is 200～6000Da.
Anti-CD147-PEG-DSPE the most according to claim 1, it is characterised in that described phospholipid selects distearyl Acyl PHOSPHATIDYL ETHANOLAMINE.
8. prepare a method for Anti-CD147-PEG-DSPE as described in claim 1～7 any claim, including Following steps: by Anti-CD147 and PEG-DSPE, prepared by covalent bond coupling reaction.
Method the most according to claim 8, it is characterised in that described method specifically includes following steps:
(1) by Anti-CD147 sulfhydrylation, the Anti-CD147 solution of sulfhydrylation is prepared；
(2) maleimide modified PEG-DSPE is dissolved in step (1) gained solution, reaction, it is thus achieved that Anti-CD147-PEG-DSPE.
Method the most according to claim 9, it is characterised in that in step (1), employing 2-iminothiolane hydrochlorate will Anti-CD147 sulfhydrylation.
11. methods according to claim 9, it is characterised in that in step (2), described reaction refers specifically at room temperature React 1～2 hour.
12. as described in claim 1～7 any claim Anti-CD147-PEG-DSPE prepare targetable drug carriers or Purposes in targeting drug delivery system.
13. 1 kinds of targetable drug carriers, containing as described in claim 1～7 any claim Anti-CD147-Polyethylene Glycol- Phospholipid.
14. targetable drug carriers according to claim 13, it is characterised in that described targetable drug carriers, contain Anti-CD147-PEG-DSPE and pharmaceutical carrier particle.
15. targetable drug carriers according to claim 14, it is characterised in that each described pharmaceutical carrier particle surface has More than one Anti-CD147.
16. targetable drug carriers according to claim 14, it is characterised in that described pharmaceutical carrier particle is selected from liposome.
17. as described in claim 13～16 any claim the construction method of targetable drug carriers, selected from following arbitrary:
Method one, comprise the following steps:
(1) using Anti-CD147-PEG-DSPE as one of material building pharmaceutical carrier, it is used for building medicine with other The material of thing carrier directly mixes to prepare targetable drug carriers；
Method two, rear insertion, comprise the following steps:
(1) pharmaceutical carrier is first built；
(2) Anti-CD147-PEG-DSPE is mixed with the pharmaceutical carrier built,.
Method three: rear connection method, comprises the following steps:
(1) use PEG-DSPE as one of drug carrier material, build the medicine containing PEG-DSPE and carry Body；
(2) pharmaceutical carrier containing PEG-DSPE built in Anti-CD147 and step (1) is passed through covalency Key coupling reaction, builds described targetable drug carriers.
18. targetable drug carriers as described in claim 13～16 any claim is loaded in the purposes preparing in targeting drug delivery system.
19. 1 kinds of targeting drug delivery systems, containing targetable drug carriers and medicine as described in claim 13～16 any claim.
20. targeting drug delivery systems according to claim 19, described medicine is selected from antitumor drug.
21. as described in claim 19～20 any claim the construction method of targeting drug delivery system, selected from following arbitrary:
Method one: comprise the following steps:
(1) Anti-CD147-PEG-DSPE, medicine and being used for builds the material of pharmaceutical carrier directly mix and make Standby targeting drug delivery system；
Method two, rear insertion, comprise the following steps:
(1) structure carries the pharmaceutical carrier of medicine；
(2) pharmaceutical carrier carrying medicine that Anti-CD147-PEG-DSPE builds with step (1) is mixed, ?；
Method three, rear connection method, comprise the following steps:
(1) PEG-DSPE is used to carry medicine as one of drug carrier material, structure containing PEG-DSPE The pharmaceutical carrier of thing；
(2) medicine carrying medicine containing PEG-DSPE built in Anti-CD147 and step (1) is carried Body passes through covalent bond coupling reaction, builds described targeting drug delivery system.
22. as described in claim 19～20 any claim targeting drug delivery system purposes in preparing oncotherapy product.
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