CN106913880A

CN106913880A - A kind of targeting drug delivery system containing RSPO1 and its preparation and application

Info

Publication number: CN106913880A
Application number: CN201510990488.1A
Authority: CN
Inventors: 徐宇虹; 曹婧; 安松柱
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2015-12-24
Filing date: 2015-12-24
Publication date: 2017-07-04
Anticipated expiration: 2035-12-24
Also published as: CN106913880B

Abstract

The invention belongs to field of pharmaceutical preparations, and in particular to a kind of targeting drug delivery system containing RSPO1 and its preparation and application.The targeting drug delivery system contains：RSPO1 coupling carriers molecule, medicine and pharmaceutical carrier particle, RSPO1 is in pharmaceutical carrier particle surface.The targeting drug delivery system, can significantly improve the targeting and oncotherapy effect of medicine on the basis of poisonous side effect of medicine is reduced.

Description

A kind of targeting drug delivery system containing RSPO1 and its preparation and application

Technical field

The invention belongs to field of pharmaceutical preparations, and in particular to a kind of targeting drug delivery system containing RSPO1 and its preparation and application.

Background technology

Traditional tumor pharmacother (chemotherapy) can also have killing while killing tumor cell to organism normal cell, because This toxic and side effect for producing not only influences the therapeutic effect even life of entail dangers to patient.Therefore, people endeavour always in recent years In improving chemotherapy to the specificity of tumour cell.In order to realize this purpose, scientists have attempted many methods, wherein wrapping Include the target that target administration is carried out to tumour using pharmaceutical carrier to realize raising curative effect, reduce toxic and side effect.

The composition of antineoplastic drug carrier is varied, have been directed to protein, lipid, inorganic material, macromolecular material and Inorganic and high molecular composite.The nano-carrier system of research and development includes polymer nanoparticle, liposome, tree-shaped poly- at present Compound, nano-emulsion, nm of gold or other metal nanoparticles etc..The targeting of pharmaceutical carrier includes passive target and active Targeting.For example, liposome is a kind of conventional pharmaceutical carrier, after the hydrophilic polyethylene glycol of its surface modification (PEG) Hidden liposome can be obtained, so as to dramatically increase internal circulation time, and by EPR effect passive target tumor tissues. Stealthy Evacet is cancer target liposome medicament most long of current most widely used, time.Compared with common adriamycin, Stealthy Evacet has longer Half-life in vivo and stronger drug effect.But played a role based on passive target, medicine Thing is by diffusing into tumour cell after cytoplasm is released, and the improvement to therapeutic effect is not obvious, and in clinic On generate new toxic and side effect, such as acra redness disease (palmar-plantar erythrodysesthesia, PPE) etc..Use at present Most active targeting carriers is antibody-mediated target medicine carrier.Existing multiple antibody coupling carrier enters clinical research rank It is clinical that the liposome for being loaded with DNA plasmid of section, such as transferrin antibodies fragment modification has been enter into the II phases.

Wnt signal transduction pathways participate in various biological process regulation and control, including embryo growth and morphological development, tissue Surely, the maintenance of the balance and stem cell of energetic supersession.The excessive activation of Wnt paths and kinds cancer (including colon cancer, Stomach cancer, breast cancer etc.) it is closely connected.Wnt signal paths can also promote the transfer of cancer cell, therefore recognized simultaneously To be the novel targets of oncotherapy.Tumour medicine currently for Wnt paths is mainly micromolecular compound and Wnt/Fzd Protein antibodies.FDA ratifies micromolecular inhibitor --- the Vismodegib (Erivedge) of Hedgehog signal paths within 2012 Listing, for treating pernicious basal-cell carcinoma.But suppression of the medicine to Wnt paths inevitably causes many side effects, Including muscle cramp, alopecia, dysgeusia, body weight reduction etc..

R-spondin1 (RSPO1) is newfound Wnt paths ligandin in recent years.RSPO1 albumen is by activating simultaneously Collaboration Wnt signal paths participate in the regulation and control of cell proliferation and differentiation.In the middle of prior art, it is badly in need of using the targeting of Wnt paths Part targets the targeting drug delivery system of Wnt path overacfivity tumour cells so as to specific efficient.

The content of the invention

In order to overcome the problems of in the prior art, can specific efficient targeting it is an object of the invention to provide one kind The targeting drug delivery system of Wnt path excessive activation tumour cells and its preparation and application.

To achieve these goals and other related purposes, the present invention is adopted the following technical scheme that：

The first aspect of the present invention provides a kind of RSPO1 coupling carriers molecule, and its structural formula is：RSPO1- carriers point Son, is connected between the RSPO1 and carrier molecule by covalent bond.

Preferably, connected by thioether bond between the RSPO1 and carrier molecule.

It is further preferred that carrier molecule is connected on the sulfydryl of cysteine of the 6th, the RSPO1N ends.

It is further preferred that the mol ratio between the RSPO1 and carrier molecule is 1：1.

It is further preferred that the structural formula of the RSPO1 coupling carriers molecule is：RSPO1-linker-R, the RSPO1 It is connected by covalent bond between linker, is connected by covalent bond between the linker and R.

Preferably, connected by thioether bond between the RSPO1 and linker.

Preferably, linker is connected on the sulfydryl of cysteine of the 6th, the RSPO1N ends.

Preferably, the mol ratio between described RSPO1, linker, R is 1：1：1.

Preferably, the R is selected from any one in lipid, polymeric material.

Preferably, the lipid is selected from any one in phosphatide, aliphatic acid, cholesterol.

It is further preferred that the phosphatide is selected from the phosphatide is selected from DSPE, two myristoyls Phosphatidyl-ethanolamine, DOPE, DPPE, DlinPE, dilauroyl phosphatide Acyl monoethanolamine, heptadecyl acyl phosphatidyl-ethanolamine, two mustard acylphosphatidyl ethanolamines, hydrogenated soya phosphatide acyl monoethanolamine, Any one in hydrogenation egg phosphatide acyl monoethanolamine, soybean phospholipid phosphatidyl monoethanolamine or egg phosphatide acyl monoethanolamine.

In a preferred embodiment of the invention, the phosphatide selects DSPE, two myristoyl phosphatide Acyl monoethanolamine.

It is further preferred that the aliphatic acid is selected from the misery acid of C8, C10 acid capric acid, C12 acid laurate, C14 acid nutmegs Acid, C16 acid palmitic acid, C18 acid stearic acid in any one.

In the preferred embodiment of the present invention, the aliphatic acid is from C12 acid laurate.

It is further preferred that the polymeric material be selected from block copolymer material, alternate copolymer material, random copolymer, Any one in graft copolymer material.

Preferably, the block copolymer material be selected from PLA, PLGA, PLG, Pluronic, PEO, PEI, PPO, Any one in PEG or two or more compound in them.

In the preferred embodiments of the present invention, R selects PLA.

Preferably, the linker is selected from any one in PEG, small peptide, oligonucleotides.

Small peptide, referred to as, English name Short chain polypeptide are made up of short-chain peptide the amino acid residue of 3-9 Short-chain peptide, is also oligopeptide sometimes.

Oligonucleotides, is that a class only has less than 20 general names of the short nucleotide of base, including DNA DNA Or the nucleotides in RNA.

It is further preferred that the molecular weight ranges of the polyethylene glycol are 400~8000Da.It is further preferred that described poly- The molecular weight ranges of ethylene glycol are 400~5000.It is further preferred that the molecular weight ranges of the polyethylene glycol are 2000~5000.

For example, the polyethylene glycol is selected from, but not limited to, PEG400, PEG550, PEG750, PEG1000, poly- second two Any one in alcohol 2000, polyethylene glycol 3400, Macrogol 4000, polyethylene glycol 5000.

In a preferred embodiment of the invention, the polyethylene glycol selects PEG2000, polyethylene glycol 5000.

Preferably, the small peptide is selected from any one in Gn, (GGS) n, (GGGS) n, (GGGGS) n, RGD.This In the preferred embodiment of invention, n=2~40.

Preferably, the molecular weight ranges of the oligonucleotides are 600-8000Da.

The second aspect of the present invention provides a kind of method for preparing foregoing RSPO1 coupling carriers molecule, comprises the following steps： RSPO1 and carrier molecule/linker-R is prepared by covalent bond coupling reaction.

Preferably, the RSPO1 needs to carry out the activation of free sulfhydryl groups.It is further preferred that can be lived using reducing agent TCEP Change the free sulfhydryl groups in R-Spondin1.

The preferred embodiments of the present invention, give the method for preparing RSPO1- PEG-DSPEs, methods described specifically include as Lower step：

(1) maleimide modified PEG-DSPE is taken by proportioning, is hydrated into micellar solution；

(2) R-Spondin1 that free sulfhydryl groups have been activated is added in step (1) gained micellar solution, is taken the photograph 4~25 Family name's degree, reacts 2~20 hours under nitrogen protective condition.

It is further preferred that the pH scopes of the aqua liquid used in step (1) are in 6.5-7.5.

It is further preferred that using the free sulfhydryl groups in reducing agent TCEP activation R-Spondin1 in step (2).

Third aspect present invention, there is provided foregoing RSPO1 coupling carriers molecule is preparing targetable drug carriers or target administration system Purposes in system.

A kind of the fourth aspect of the present invention, there is provided targetable drug carriers, contains foregoing RSPO1 coupling carriers molecule.

Further, the targetable drug carriers, contain RSPO1 coupling carriers molecule and pharmaceutical carrier particle.

Preferably, the pharmaceutical carrier particle surface has RSPO1.

It is further preferred that RSPO1 is by being covalently attached to the surface of the pharmaceutical carrier particle.

Preferably, each described pharmaceutical carrier particle surface has more than one RSPO1.It is further preferred that each described medicine There are 5~10000 RSPO1 on carrier particle surface.

In the embodiment of the present invention, there are 5~1000 R-Spondin1 on each described pharmaceutical carrier surface.

Preferably, RSPO1 coupling carriers molecule is with the molar ratio of pharmaceutical carrier particle：(0.01~100)：1000.

In the embodiment of the present invention, R-Spondin1- PEG-DSPEs are with the molar ratio of pharmaceutical carrier： (0.5~100)：1000.

Preferably, the particle size range of the pharmaceutical carrier particle is 10~1000nm.

Preferably, the pharmaceutical carrier particle is selected from, but not limited to, liposome, nanoparticle, polymer nanoparticle, cation lipid Any one in nanoparticle, polymer micelle, emulsion, micro-capsule, microballoon, nanocapsule, nanosphere.

Further, the pharmaceutical carrier can also carry out other modifications.

Preferably, the constituent of the liposome is selected from egg phosphatide, HSPC, hydrogenation egg phosphatide acyl courage Alkali, DLPC, dimyristoyl phosphatidyl choline, DPPC, distearyl phosphatide Phatidylcholine, 1- myristoyl -2- palmitoylphosphatidyl cholines, 1- palmityl -2- DSPCs, 1- stearoyls The sub- oleoyl phosphatidylcholine of -2- palmitoylphosphatidyl cholines, POPC, 1- stearoyls -2-, DOPC, hydrogenation DPPC, DSPC, two myristoyl phosphatidic acids, Two myristoyl phosphatidic acids, DPPA, DPPA, G 12S3P, two myristoyl phosphorus Acyl monoethanolamine, DPPE, cephalin acyl serine, two myristoyl phosphatidylserines, two palm fibres Palmitic acid acyl phosphatidylserine, E-PG, PE, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, two palm fibres Palmitic acid acyl phosphatidyl glycerol, DSPG, DOPG, brain sphingomyelins, two palmitoyl sphingomyelins Or distearyl sphingomyelins, cholesterol, two oily epoxides hydroxypropyltrimonium chloride (DOTAP), dioleoyl chloropropyl chlorination three First ammonium (DOTMA), DDA (DDAB), dimethyl aminoethyl amido propiono-cholesterol (DC-Chol), spermine -5- carboxyaminos acetic acid octacosyl acid amides (DOGS), dioleoyl succinyl glycerolcholine ester (DOSC), any in dioleoyl chlorine spermine Carboxylamide ethyl dimethyl propyl trifluoroacetic acid ammonium (DOSPA), MVL5 Plant or their two or more combination.

Preferably, the constituent of the polymer nanoparticle is：PLA、PLGA、PLG、Pluronic、PEO、PEI、 Any one in PPO, PEG or their two or more combination.

The fifth aspect of the present invention, additionally provides the construction method of foregoing targetable drug carriers, selected from following any：

Method one, comprise the following steps：

Using RSPO1 coupling carriers molecule as one of material for building pharmaceutical carrier, it is used to build pharmaceutical carrier with other Material directly mixes self assembly to prepare targetable drug carriers；

Method two, rear insertion, comprise the following steps：

(1) pharmaceutical carrier is first built；

(2) RSPO1 coupling carriers molecule is mixed with the pharmaceutical carrier for having built, you can；

Method three：Connection method, comprises the following steps afterwards：

(1) using carrier molecule as one of drug carrier material, the pharmaceutical carrier containing carrier molecule is built；

(2) pharmaceutical carrier containing carrier molecule that will be built in RSPO1 and step (1) is by covalent bond coupling reaction, Build the targetable drug carriers.

The sixth aspect of the present invention, there is provided purposes of the foregoing targetable drug carriers in targeting drug delivery system is prepared.

A kind of the seventh aspect of the present invention, there is provided targeting drug delivery system, contains foregoing targetable drug carriers and medicine.

Preferably, the medicine is selected from, but not limited to, antineoplastic；Antimetabolite；Cytotoxic drug；Antibiotic； Sensitising agent；Kinase inhibitor；Anti-inflammatory drug；Immunodepressant；It is anti-infective to use medicine；Antiviral drugs.

Preferably, the medicine is selected from, but not limited to, genes of interest, antisense gene, suicide gene, apoptosis gene, thin The combination of intracellular cytokine gene, siRNA, mRNA or said gene, or the carrier for expression of eukaryon DNA containing said gene.

For example, in one embodiment of the invention, also having selected siRNA or shRNA plasmid expressions to carry Body (pGPU6/GFP/Neo) is used as model drug.

It is further preferred that the medicine is antineoplastic.Antineoplastic of the present invention can be existing any number of The suppression growth of tumour cell of class, propagation, differentiation, the medicine of transfer, including but not limited to：Small-molecule chemical medicine, nucleic acid Molecule, carbohydrate, lipid, antibody medicine, polypeptide, albumen or interference slow virus.

In the preferred embodiments of the present invention, from adriamycin as model drug.

Preferably, the target cell that the targeting drug delivery system is directed to is tumour cell.

The targeting drug delivery system of the invention can carry antineoplastic and targeting is in tumour cell.

Preferably, the tumour is entity tumor or metastatic tumo(u)r.It is further preferred that the tumour is LGR5 expression high Tumour.

It is highly preferred that the tumour is selected from, but not limited to, the carcinoma of the rectum, stomach cancer, breast cancer, lung cancer, cancer of pancreas, liver cancer, food Pipe cancer or glioblast cancer.

The eighth aspect of the present invention, there is provided the construction method of foregoing targeting drug delivery system, selected from following any：

Method one, comprise the following steps：

Using RSPO1 coupling carriers molecule as one of material for building pharmaceutical carrier, it is used to build pharmaceutical carrier with other Material and medicine directly mix self assembly to prepare targeting drug delivery system；

Method two, rear insertion, comprise the following steps：

(1) pharmaceutical carrier for carrying medicine is first built；

(2) RSPO1 coupling carriers molecule is mixed with the pharmaceutical carrier of the carrying medicine for having built, you can；

Method three：Connection method, comprises the following steps afterwards：

(1) using carrier molecule as one of drug carrier material, the medicine for building the carrying medicine containing carrier molecule is carried Body；

(2) pharmaceutical carrier of the carrying medicine containing carrier molecule that will be built in RSPO1 and step (1) is by covalent Key coupling reaction, builds the targeting drug delivery system.

The ninth aspect of the present invention, there is provided foregoing targeting drug delivery system is preparing acoustic contrast agent, radiopaque contrast medium, nuclear medicine Purposes in contrast agent.

The tenth aspect of the present invention, additionally provides a kind of method for treating tumour, including step：Foregoing targeting drug delivery system is applied For patient.Dosage used, doctor can select according to actual conditions.

The patient can be tumor patient.The tumour is selected from, but not limited to, the carcinoma of the rectum, stomach cancer, breast cancer, lung cancer, pancreas Cancer, liver cancer, the cancer of the esophagus or glioblast cancer.

Compared with prior art, the present invention has the advantages that：

(1) present invention -- RSPO1 albumen coupling carrier molecules, can specifically target the active tumour cell of Wnt paths, Acted on by passive and active dual-target and not only improved the drug effect of chemotherapeutics but also its side effect can be reduced.It is coupled using RSPO1 Carrier molecule is modified pharmaceutical carrier, especially between the RSPO1 and carrier molecule by thioether bond connect and Mol ratio between the RSPO1 and carrier molecule is 1：The factor of 1 these two aspects, for RSPO1 coupling carrier molecules It is particularly critical as efficient " targeting head ".The present invention it is substantial amounts of research show, the targeting drug delivery system containing RSPO1, The RSPO1 on pharmaceutical carrier surface, can characteristic pharmaceutical carrier is efficiently directionally transported to the tumor group of Lgr5 expression high In knitting, the pharmaceutical carrier being distributed in target tissue can be combined with the target protein of tumor cell surface, and induced drug carrier The process such as endocytosis or insoluble drug release, so as to play drug effect.The targeting drug delivery system of RSPO1 modifications swells to Lgr5 expression high Oncocyte has stronger growth inhibition effect.Specifically, the targeting drug delivery system of RSPO1 modifications swells to Lgr5 expression high The inhibitory action of oncocyte is up to 5 times or so without RSPO1 modification delivery systems.

(2) present invention carries out R-Spondin1 modifications using liposome as pharmaceutical carrier model, by fluorescent traccer technique, examines The LoVo rectum cancer cells of LGR5 expression high are looked into respectively to the intake of RSPO1 modified liposomes and conventional liposome, is as a result proved RSPO1 can efficiently mediate liposome to enter the cell of LGR5 expression high in vitro.Specifically, LoVo cells are repaiied to RSPO1 The intake of the liposome of decorations is probably to more than 7 times of liposome intake without modification.

(3) by with adriamycin as model drug, liposome is pharmaceutical carrier model, investigated RSPO1 modified liposomes/ DOX to the growth in vitro inhibitory action and tumor growth inhibitory action of the LoVo rectum cancer cells of LGR5 expression high, in vitro The Evacet of RSPO1 modifications is probably unmodified liposome to the inhibitory action of carcinoma of the rectum LoVo cells, in vivo The Evacet of RSPO1 modifications is up to 59% to the inhibiting rate of carcinoma of the rectum tumour growth, than unmodified liposome to the carcinoma of the rectum The growth inhibition ratio of tumour is high by 25%.Show that RSPO1 modifications can dramatically increase Evacet thin to the cancer of LGR5 expression high Born of the same parents and the growth inhibition effect of tumor tissues.

(4) result of study prompting of the present invention, the targeting drug delivery system of R-Spondin1 modifications of the present invention, with passive and active Dual-target is acted on, the primary tumo(u)r and metastatic lesion that efficiently can be expressed the drug targetings such as antineoplastic to LGR5, Enable that medicine is accurately sent to inside tumor cells, realize real targeted therapy, greatly improve therapeutic effect.Through overtesting It was found that, compared with the targeting drug delivery system modified without R-Spondin1, the target administration system of R-Spondin1 modifications of the invention The effect for ruling treatment tumour improves more than four times.

Brief description of the drawings

Fig. 1 is the Polyacrylamide Gel Electrophoresis knot of the RSPO1-PEG2000-DSPE prepared by the embodiment of the present invention 1 Really.

Fig. 2 is the FITC mark liposome tumor cell in vitro targetings of the RSPO1 modifications prepared by the embodiment of the present invention 3 Result of study.

Fig. 3 is that the DiI mark liposome tumor cell in vitro targetings of the RSPO1 modifications prepared by the embodiment of the present invention 4 are ground Study carefully.

When Fig. 4 A are 24h, the Evacet of the RSPO1 modifications prepared by the embodiment of the present invention 5 is to LoVo rectum The growth in vitro inhibitory action of cancer cell.

When Fig. 4 B are 48h, the Evacet of the RSPO1 modifications prepared by the embodiment of the present invention 5 is to the LoVo carcinoma of the rectum The growth in vitro inhibitory action of cell.

Fig. 5 is the body of the Evacet to LoVo rectum cancer cells of the RSPO1 modifications prepared by the embodiment of the present invention 5 Interior growth inhibition effect.

When Fig. 6 A are 1h, the embodiment of the present invention 7 prepares the fluorescence labeling cation lipid gene composite Nano of RSPO1 modifications Particle is by LoVo cellular uptake experimental results.

Fig. 6 B be 24h when, the embodiment of the present invention 7 prepares RSPO1 modification fluorescence labeling cation lipid gene be combined receive Rice corpuscles is by LoVo cellular uptake experimental results.

Fig. 7 is that LoVo cells are much larger than not to the intake of the liposome of the RSPO1 modifications prepared in the embodiment of the present invention 8 There is the liposome of modification.

Specific embodiment

The present inventor synthesizes and there is provided one kind first in a preferred embodiment by in-depth study extensively R-Spondin1- PEG-DSPEs, the R-Spondin1, the mol ratio between polyethylene glycol, phosphatide are 1：1： 1。

The R-Spondin1- PEG-DSPEs are a kind of linear amphipathic nature block polymers, and hydrophilic section is polyethylene glycol, are dredged Water section is phosphatide.The macromolecule micelle being made up of hydrophily shell and lipophilicity kernel can be self-assembly of in water.

The R-Spondin1 is existing albumen, and its accession number in Genebank is NC_000001.11.Can be by commercially available way Footpath obtains.In the present invention, R-Spondin1 can be referred to as RSPO1.

The polyethylene glycol, also known as PEG, can be obtained by commercially available approach.The present invention is to the molecular weight of polyethylene glycol without special Limitation, molecular weight polyethylene glycol is preferably 400~8000.In a preferred embodiment of the invention, the polyethylene glycol is selected PEG2000, can also be using the polyethylene glycol of other molecular weight, however it is not limited to the poly- second two of the specific molecular weight cited by embodiment Alcohol.

The phosphatide can be obtained by commercially available approach.The present invention has no particular limits for phosphatide, can select short-chain phospholipid, point Son amount is suitable with the molecular weight of polyethylene glycol or slightly smaller.For example：The phosphatide may be selected from DSPE, two Myristoyl phosphatidyl-ethanolamine, dilauroyl phosphatidyl-ethanolamine, DPPE, dioleoyl phospholipid Acyl monoethanolamine, hydrogenated soya phosphatide acyl monoethanolamine, hydrogenation egg phosphatide acyl monoethanolamine, soybean phospholipid phosphatidyl monoethanolamine or egg phosphatide acyl second Any one in hydramine.In a preferred embodiment of the invention, the phosphatide selects DSPE, also Can select others phosphatide, however it is not limited to the specific phosphatide cited by embodiment.

The present invention has no particular limits for the preparation method of R-Spondin1- PEG-DSPEs, as long as can successfully make It is standby to obtain the copolymer.In the preferred embodiments of the present invention, R-Spondin1- is prepared using covalent bond coupling method and is gathered Ethylene glycol-phosphatide, can also use other preparation methods, however it is not limited to cited preparation method in embodiment.

Present invention also offers a kind of targeting drug delivery system, contain foregoing R-Spondin1- PEG-DSPEs.Further Ground, the targeting drug delivery system contains：R-Spondin1- PEG-DSPEs, medicine and pharmaceutical carrier.

The present invention has no particular limits to the medicine.For example, the medicine can be antineoplastic.It is of the invention In preferred embodiment, the antineoplastic of this hair targeting drug delivery system conveying is studied as model drug from adriamycin The targeting ability of thing.In another embodiment, also from siRNA or shRNA plasmid expression vectors (pGPU6/GFP/Neo) The targeting ability that the targetable drug carriers convey genomic medicine is studied as model drug.

The pharmaceutical carrier refers to that pharmaceutical carrier refers to that can change medicine into the mode of human body and distribution in vivo, control The rate of release of medicine simultaneously conducts drugs to the system of target organs.The present invention is not limited particularly for pharmaceutical carrier System, as long as can realize successfully carrying the medicine.For example, the pharmaceutical carrier can select liposome, cation Any one in lipid nano particle, polymer micelle, emulsion, micro-capsule, microballoon, nanocapsule, nanosphere.The present invention Preferred embodiment in, from liposome and cation lipid nanoparticle as model drug carrier, it is also can select certainly His suitable pharmaceutical carrier, however it is not limited to the specific pharmaceutical carrier cited by embodiment.The construction method of pharmaceutical carrier is this The content that the technical staff in field can be known.

The present invention does not have special limitation to the construction method of targeting drug delivery system.Targeting drug delivery system can as required be carried out Structure, for example, can first build carry medicine pharmaceutical carrier, be subsequently adding R-Spondin1- PEG-DSPEs, Mixing.Can also, the pharmaceutical carrier of the carrying medicine containing PEG-DSPE is first built, it is subsequently adding R-Spondin1 builds the targeting drug delivery system by covalent bond coupling reaction.Again for example, it is also possible to directly will R-Spondin1- PEG-DSPEs, medicine and mix for building the material of pharmaceutical carrier and prepare targeting drug delivery system.

Further, the drug administration carrier can also be modified through other, for example, single or multiple antibody interested, part polysaccharide Modification etc., is not limited in R-Spondin1 modifications of the invention.

Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to following spies Fixed specific embodiment；It is also understood that the term used in the embodiment of the present invention is to describe specific specific implementation Scheme, rather than in order to limit the scope of the invention.The test method of unreceipted actual conditions in the following example, leads to Often according to normal condition, or according to the condition proposed by each manufacturer.

When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two end points of each number range with And any one numerical value can select between two end points.Unless otherwise defined, all technologies for being used in the present invention and section are academic Language is identical with the meaning that those skilled in the art of the present technique are generally understood that.In addition to the specific method, equipment, the material that are used in embodiment, Grasp and record of the invention according to those skilled in the art to prior art, can also use and the embodiment of the present invention Described in method, any method in the similar or equivalent prior art of equipment, material, equipment and material realize the present invention.

Unless otherwise indicated, disclosed in this invention experimental technique, detection method, preparation method use the art Conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of association area.The perfect explanation in existing document of these technologies, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and 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..

The preparation of the RSPO1-PEG2000-DSPE of embodiment 1

(1) preparation of degasification HEPES solution：Ultra-pure water ebuillition of heated, fills N2 and is saturated to cooling, adds HEPES salt To concentration 50mM, NaOH regulation pH to 7.0, whole process N2 protections；

(2) DSPE-PEG2000-MAL powder is weighed, degasification HEPES salting liquids are added, hydration turns into micellar solution； During RSPO1 after TCEP is reduced under N2 protections adds the micellar solution；Reaction is 10 DEG C in temperature, and rotating speed is Constant temperature oscillation under the conditions of 500rpm, final product RSPO1-PEG2000-DSPE is obtained after reaction 24h.

Polyacrylamide Gel Electrophoresis result, referring to Fig. 1, such as Fig. 1, the RSPO1 after display connection DSPE-PEG2000 Shifting~3K on protein band, joint efficiency is calculated more than 95% through ImageJ, absolutely proves RSPO1-PEG2000-DSPE Synthesize successfully.

The preparation of the liposome of the RSPO1 of embodiment 2 modifications

Liposome, each raw material HSPC (hydrogenated soybeans are prepared with phosphatide, cholesterol, DSPE-PEG2000 as raw material Phosphatide)/Chol (cholesterol)/DSPE-PEG2000 (mPEG-DSPE) Mol ratio be 55：42：3.Specific method includes step：By proportioning, it is miscible in appropriate chloroform to take each raw material, 37 DEG C The rotary evaporation that depressurized under water-bath removes chloroform, forms uniform lipid film, adds 1ml PBS aquation 1h, water bath sonicator； 100nm and 80nm polycarbon resin films are pressed through successively, you can first prepare the liposome of PEG modifications.

Then the above-mentioned PEG for preparing of the present embodiment is added to repair the RSPO1-PEG2000-DSPE obtained in embodiment 1 In the liposome of decorations, at 37 DEG C, rotating speed is constant temperature oscillation under the conditions of 300rpm, and the fat of RSPO1 modifications is obtained after 24 hours Plastid.Nano particle diameter is determined using laser diffraction particle size analyzer (PCS), display particle diameter is 90 ± 10nm.

The preparation of the FITC mark liposomes of the RSPO1 of embodiment 3 modifications and tumor cell in vitro targeting Journal of Sex Research

(1) FITC of RSPO1 modifications marks the preparation of liposome

With phosphatide, cholesterol, DSPE-PEG2000, FITC-DHPE prepares liposome, each raw material HSPC for raw material (hydrogenated soya phosphatide)/Chol (cholesterol)/DSPE-PEG2000 (polyethylene glycol-distearoylphosphatidyl ethanol Amine compound)/FITC-DHPE mol ratio be 55：42：3：1.Specific method includes step：By proportioning, each original is taken Material is miscible in appropriate chloroform, and the rotary evaporation that depressurized under 37 DEG C of water-baths removes chloroform, forms uniform lipid film, adds 1ml PBS aquation 1h, water bath sonicator；100nm and 80nm polycarbon resin films are pressed through successively, obtain FITC modifications Liposome.

In the liposome for adding the present embodiment FITC to modify the RSPO1-PEG2000-DSPE obtained in embodiment 1, 37 DEG C, rotating speed is constant temperature oscillation under the conditions of 300rpm, and the FITC mark liposomes of RSPO1 modifications are obtained after 24 hours.Make Nano particle diameter is determined with laser diffraction particle size analyzer (PCS), display particle diameter is 95 ± 10nm.

(2) the FITC mark liposome tumor cell in vitro targeting Journal of Sex Research of RSPO1 modifications

Take the logarithm the monolayer cultivation LoVo cells in growth period, single-layer culturing cell digested with 0.25% trypsase/EDTA, Single cell suspension is made into the DMEM/F12 culture mediums containing 20% hyclone, with every hole 4X10⁵Individual cell is inoculated in 12 In porocyte culture plates, per pore volume 2ml, during culture plate moved into CO2gas incubator, 37 degrees Celsius, 5% titanium dioxide Cultivated under the conditions of carbon and saturated humidity, make cell attachment.The next day, configure FITC with the culture medium without hyclone and mark fat Plastid and the FITC mark liposomes of RSPO1 modifications.Culture medium in culture plate is suctioned out, FITC mark liposomes are added And the FITC mark liposomes of RSPO1 modifications, 37 degrees Celsius of incubation 1h, inhale and abandon supernatant.With the PBS solution of precooling Board-washing twice, 0.25% trypsase/EDTA vitellophags, with the DMEM/F12 culture medium ends containing 20% hyclone Only digest, cell adds paraformaldehyde to fix 10 minutes after blowing and beating into single cell suspension.Cell is collected by centrifugation, supernatant is abandoned in suction, Add PBS resuspended, be repeated twice.Finally use cell internalizing situation in flow cytomery single cell suspension.Referring to figure 2, as a result show, the intake of the liposome that LoVo cells are modified RSPO1 is much larger than the liposome without modification.Specifically Ground, the intake of the liposome that LoVo cells are modified RSPO1 is probably to more than 7 times of liposome intake without modification.

The preparation and targeting research of the DiI mark liposomes of the RSPO1 of embodiment 4 modifications

(1) DiI of RSPO1 modifications marks the preparation of liposome

Liposome is prepared with phosphatide, cholesterol, DSPE-PEG2000, DiI as raw material.Each raw material HSPC (hydrogenations Soybean lecithin) (PEG2000-DSPE is combined/Chol (cholesterol)/DSPE-PEG2000 Thing)/DiI mol ratio be 55：42：3：0.8.Specific method includes step：By proportioning, each raw material are taken miscible in suitable The rotary evaporation that depressurized in amount chloroform, under 37 DEG C of water-baths removes chloroform, forms uniform lipid film, adds 1ml PBS water Change 1h, water bath sonicator；100nm and 80nm polycarbon resin films are pressed through successively, obtain the liposome of DiI modifications.

The RSPO1-PEG2000-DSPE that will be obtained in embodiment 1 adds the liposome of DiI modifications manufactured in the present embodiment In, at 37 DEG C, rotating speed is constant temperature oscillation under the conditions of 300rpm, and the DiI mark lipids of RSPO1 modifications are obtained after 24 hours Body.Nano particle diameter is determined using laser diffraction particle size analyzer (PCS), display particle diameter is 95 ± 10nm.

(2) the DiI mark liposome tumor cell in vitro targeting Journal of Sex Research of RSPO1 modifications

Take the logarithm the monolayer cultivation LoVo cells in growth period, single-layer culturing cell digested with 0.25% trypsase/EDTA, Single cell suspension is made into the DMEM/F12 culture mediums containing 20% hyclone, with every hole 2X10⁵Individual cell is inoculated in 24 On 14mm cover glasses in orifice plate, per pore volume 1ml, during culture plate moved into CO2gas incubator, 37 degrees Celsius, Cultivated under the conditions of 5% carbon dioxide and saturated humidity, make cell attachment.The next day, configure DiI with the culture medium without hyclone Mark liposome and the DiI mark liposomes of RSPO1 modifications.Culture medium in culture plate is suctioned out, DiI mark fat is added Plastid and the DiI mark liposomes of RSPO1 modifications, 37 degrees Celsius are incubated 15min, 30min, and after 1h, supernatant is abandoned in suction. Cleaned with the PBS solution of precooling three times, add paraformaldehyde to fix 10 minutes, PBS takes out cover glass and is placed in load after washing twice Piece fluid-tight piece is sealed up on slide, the observation of cell internalization situation under fluorescence microscope.Referring to Fig. 3, the DiI of RSPO1 modifications Mark liposome and DiI mark liposomes in 37 degrees Celsius respectively with LoVo cytosiies 15 minutes, 30 minutes, it is one small When after fluorescence micrograph.Result shows that DiI mark liposomes substantially can not be by LoVo cellular uptakes, and RSPO1 The liposome of modification then by huge uptake, illustrates under the mediation of RSPO1 that RSPO1 modified liposomes are thin to the carcinoma of the rectum Born of the same parents have good targeting.

The preparation and tumor-inhibiting action research of the liposome for containing water-soluble ADMh of the RSPO1 of embodiment 5 modifications

(1) preparation of the liposome for containing water-soluble ADMh of RSPO1 modifications

Liposome is prepared with phosphatide, cholesterol, DSPE-PEG2000 as raw material.Each raw material HSPC (hydrogenated soybeans Phosphatide)/Chol (cholesterol)/DSPE-PEG2000 (mPEG-DSPE) Mol ratio be 55：42：3.Specific method includes step：By proportioning, it is miscible in appropriate chloroform to take each raw material, 37 DEG C The rotary evaporation that depressurized under water-bath removes chloroform, forms uniform lipid film, adds the ammonium sulfate aquation 1h of certain volume, water Bath is ultrasonic to obtain liposome turbid liquor.In 60 degree of water-baths, using micro extruder, 400nm, 200nm are pressed through successively, 100nm and 80nm polycarbon resin films, obtain blank liposome；After changing outer water phase by dialysing, 1 is compared according to medicine fat:10(w/w) Add the adriamycin aqueous solution, 60 degree of water-baths 20 minutes；Dialysis removes free drug, obtains Evacet.

During the RSPO1-PEG2000-DSPE obtained in embodiment 1 added into the Evacet prepared by the present embodiment, At 37 DEG C, rotating speed is constant temperature oscillation under the conditions of 300rpm, and the Evacet of RSPO1 modifications is obtained after 24 hours.Make Nano particle diameter is determined with laser diffraction particle size analyzer (PCS), display particle diameter is 95 ± 10nm.

(2) growth in vitro inhibitory action of the Evacet of RSPO1 modifications to LoVo rectum cancer cells

The Evacet modified using CCK-8 kit measurements RSPO1 is pressed down to the growth in vitro of LoVo rectum cancer cells Make and use.The monolayer cultivation LoVo cells in exponential phase are taken, is digested with 0.25% trypsase/EDTA, with containing The DMEM/F12 culture mediums of 20% hyclone are made into single cell suspension, cell count, with every hole 1X10⁴Individual cell connects Kind in 96 orifice plates, per pore volume 100ul, during culture plate moved into CO2gas incubator, 37 degrees Celsius, 5% titanium dioxide Cultivated under the conditions of carbon and saturated humidity, make cell attachment.The next day, the adriamycin lipid for being modified RSPO1 with cell culture medium Body and Evacet are diluted to doxorubicin concentration 30ug/ml, 15ug/ml, 7.5ug/ml, suck the training of 96 orifice plate inner cells Nutrient solution, each hole add the liposome liquid of each concentration of 100ul changed into after being incubated 4 hours complete medium continue to cultivate 24 hours, 48 hours.Each concentration is all provided with three multiple holes, reserves three holes and only adds nutrient solution as control wells.10 microlitres are added per hole CCK-8 solution, continues to be incubated 3 hours in cell culture incubator, and ELIASA detects 450nm extinctions after concussion is mixed 2 minutes Degree, and 600nm absorbances are referred to as background value.Result referring to shown in Fig. 4 A and 4B, Ah mould of RSPO1 modifications Plain liposome has stronger growth inhibition effect to carcinoma of the rectum LoVo cells.Specifically, the adriamycin fat of RSPO1 modifications Plastid to the inhibitory action of carcinoma of the rectum LoVo cells is probably unmodified liposome 5 times.

(3) tumor growth inhibitory action of the Evacet of RSPO1 modifications to carcinoma of the rectum tumor tissues

The monolayer cultivation LoVo cells in exponential phase are taken, is digested with 0.25% trypsase/EDTA, PBS washings, Cell count, is scattered in PBS, with 1X10⁶Individual cell (100ul) inoculated with subcutaneous injections underarm region on the right side of nude mice, Raised under SPF environment.LoVo hypodermic tumours (50~100mm of gross tumor volume has been formed by 21³) nude mice divide at random It is 3 groups (every group 7) to be set to PBS, the Evacet group of Evacet and RSPO1 modifications.From Divide cage daystart by the corresponding preparation tail vein injections of 100ul to nude mouse, inject once within every three days, each adriamycin Dosage is 0.5mg/kg, a co-injection 8 times, and accumulated dose is 4mg/kg.Hypodermic tumour tissue size, tumor tissues are measured weekly

Volume computing formula is as follows：

V(mm³)=(d²X D)/2

Wherein d and D are respectively the minor axis and major diameter of tumor tissues, and unit is mm.Such as Fig. 5, as a result show and Evacet Compare, the Evacet of RSPO1 modifications is significantly increased to the inhibitory action of tumour growth.Specifically, RSPO1 is modified Evacet 59% is up to the inhibiting rate of carcinoma of the rectum tumour growth, and unmodified liposome is to carcinoma of the rectum tumour growth Inhibiting rate there was only 34%.

The preparation of the cation lipid gene composite nanoparticle of the RSPO1 of embodiment 6 modifications

DSPC, cholesterol, DLin-MC2-MPZ and DSPE-PEG2000 are dissolved in the ratio of 20/38.5/40/1.5 In a certain amount of ethanol, inject under the conditions of at the uniform velocity in the pH4.0 buffer solutions of certain volume, ethanol is with the volume ratio of buffer solution 2:8.By equal alcohol water than preparing the solution of corresponding siRNA, and it is well mixed in equal volume with lipid soln, 37 degree of bars It is incubated 50 minutes under part；Under the conditions of 4 degree, the final resulting solution of step 2 was first dialysed through 4 hours pH4.0 buffer solutions, to the greatest extent The ethanol in lipidic gene composite nanoparticle may be removed；Dialysed 8 hours to 12 hours by pH7.4 buffer solutions again, make fat Matter gene composite nanoparticle keeps its electroneutral under the conditions of pH7.4.

Then the above-mentioned PEG for preparing of the present embodiment is added to repair the RSPO1-PEG2000-DSPE obtained in embodiment 1 In the lipidic gene composite nanoparticle of decorations, at 37 DEG C, rotating speed is constant temperature oscillation under the conditions of 300rpm, after 20-24 hours To the lipidic gene composite nanoparticle of RSPO1 modifications.Nano-particle is determined using laser diffraction particle size analyzer (PCS) Particle diameter, display particle diameter is about 170nm, and zeta potentials are about -15.8mV, and agarose gel electrophoresis surveys its siRNA envelop rate It is 80%~90%.

The preparation of the fluorescence labeling cation lipid gene composite nanoparticle of the RSPO1 of embodiment 7 modifications and tumor cell in vitro target To Journal of Sex Research

(1) preparation of the fluorescence labeling cation lipid gene composite nanoparticle of RSPO1 modifications

DSPC, cholesterol, DLin-MC2-MPZ and DSPE-PEG2000, DiI are pressed the ratio of 20/38.5/40/1.5/1 Example is dissolved in a certain amount of ethanol, is injected under the conditions of at the uniform velocity in the pH4.0 buffer solutions of certain volume, the volume of ethanol and buffer solution Than being 2:8.By equal alcohol water than preparing the solution of corresponding Cy5-Luciferase siRNA, and by it with lipid soln etc. body Product is well mixed, is incubated 50 minutes under the conditions of 37 degree；Under the conditions of 4 degree, by the final resulting solution of step 2 first through 4 hours pH4.0 Buffer solution is dialysed, and the ethanol in lipidic gene composite nanoparticle is removed as far as possible；Again 8 hours are dialysed by pH7.4 buffer solutions extremely 12 hours, make lipidic gene composite nanoparticle that its electroneutral is kept under the conditions of pH7.4.Then will be obtained in embodiment 1 RSPO1-PEG2000-DSPE add the present embodiment it is above-mentioned prepare PEG modification fluorescent lipid gene composite Nano In particle, at 37 DEG C, rotating speed is constant temperature oscillation under the conditions of 300rpm, and the fluorescence mark of RSPO1 modifications is obtained after 20-24 hours Note lipidic gene composite nanoparticle.

(2) the tumor cell in vitro targeting Journal of Sex Research of the fluorescence labeling cation lipid gene composite nanoparticle of RSPO1 modifications

Take the logarithm the monolayer cultivation LoVo cells in growth period, single-layer culturing cell digested with 0.25% trypsase/EDTA, Single cell suspension is made into the DMEM/F12 culture mediums containing 20% hyclone, with every hole 2X10⁵Individual cell is inoculated in 24 On 14mm cover glasses in orifice plate, per pore volume 1ml, during culture plate moved into CO2gas incubator, 37 degrees Celsius, Cultivated under the conditions of 5% carbon dioxide and saturated humidity, make cell attachment.The next day, the culture medium in culture plate is suctioned out, add The fluorescence labeling that the fluorescence labeled fatty acid gene composite nanoparticle and RSPO1 configured with the culture medium without hyclone are modified Lipidic gene composite nanoparticle, after 37 degrees Celsius are incubated 1h and 24h, supernatant is abandoned in suction.PBS solution with precooling is clear Wash three times, add paraformaldehyde to fix 10 minutes, PBS takes out cover glass and is placed on slide and seals up piece fluid-tight piece after washing twice, The observation of cell internalization situation under fluorescence microscope.Referring to Fig. 7, the fluorescence labeled fatty acid gene composite Nano of RSPO1 modifications Particle and fluorescence labeled fatty acid gene composite nanoparticle in 37 degrees Celsius respectively with LoVo cytosiies 1 hour and 24 hours Fluorescence micrograph afterwards.Result shows that fluorescence labeled fatty acid gene composite nanoparticle can only be taken the photograph by LoVo cells on a small quantity Take, and the fluorescence labeled fatty acid gene composite nanoparticle of RSPO1 modifications is then by huge uptake.Simultaneously it is observed that nothing is repaiied The siRNA that the nanoparticle group of decorations is hardly visible fluorescent decoration enters cell, and a large amount of glimmering in the nanoparticle group of RSPO1 modifications The siRNA of light modification enters into intracellular.Illustrate under the mediation of RSPO1, RSPO1 modification lipidic genes are combined Nano-particle has good targeting to rectum cancer cell.

The surface connection method of embodiment 8 prepares the FITC mark liposomes and its tumor cell in vitro targeting Journal of Sex Research of RSPO1 modifications

(1) surface connection method prepares the FITC mark liposomes of RSPO1 modifications

The preparation of degasification HEPES solution：Ultra-pure water ebuillition of heated, fills N2 and is saturated to cooling, adds HEPES salt to concentration 50mM, NaOH adjust pH to 7.0, whole process N2 protections；

With phosphatide, cholesterol, DSPE-PEG5000, DPPE-PEG5000-Maleimide, FITC-DHPE as raw material Prepare liposome.Each raw material HSPC (hydrogenated soya phosphatide)/Chol (cholesterol)/DSPE-PEG5000 (poly- second Glycol-DSPE compound)/DPPE-PEG5000-Maleimide/FITC-DHPE mol ratio It is 55：42：3：1：1.Specific method includes step：By proportioning, it is miscible in appropriate chloroform to take each raw material, 37 DEG C of water The lower decompression rotary evaporation of bath removes chloroform, forms uniform lipid film, adds 1ml degasification HEPES solution aquation 1h, and water-bath surpasses Sound；100nm and 80nm polycarbon resin films are pressed through successively, obtain marking liposome with the amine-modified FITC of maleimide. During RSPO1 albumen after TCEP is reduced under N2 protections adds the liposome for preparing, reaction is 10 DEG C in temperature, Rotating speed is constant temperature oscillation under the conditions of 500rpm, and the FITC mark liposomes of final product RSPO1 modifications are obtained after reaction 24h. Nano particle diameter is determined using laser diffraction particle size analyzer (PCS), display particle diameter is 120 ± 10nm.

Take the logarithm the monolayer cultivation LoVo cells in growth period, single-layer culturing cell digested with 0.25% trypsase/EDTA, Single cell suspension is made into the DMEM/F12 culture mediums containing 20% hyclone, with every hole 4X10⁵Individual cell is inoculated in 12 In porocyte culture plates, per pore volume 2ml, during culture plate moved into CO2gas incubator, 37 degrees Celsius, 5% titanium dioxide Cultivated under the conditions of carbon and saturated humidity, make cell attachment.The next day, configure FITC with the culture medium without hyclone and mark fat Plastid and the FITC mark liposomes of RSPO1 modifications.Culture medium in culture plate is suctioned out, FITC mark liposomes are added And the FITC mark liposomes of RSPO1 modifications, 37 degrees Celsius of incubation 1h, inhale and abandon supernatant.With the PBS solution of precooling Board-washing twice, 0.25% trypsase/EDTA vitellophags, with the DMEM/F12 culture medium ends containing 20% hyclone Only digest, cell adds paraformaldehyde to fix 10 minutes after blowing and beating into single cell suspension.Cell is collected by centrifugation, supernatant is abandoned in suction, Add PBS resuspended, be repeated twice.Finally use cell internalizing situation in flow cytomery single cell suspension.Referring to figure 7, as a result show, the intake of the liposome that LoVo cells are modified RSPO1 is much larger than the liposome without modification.

The preparation of the RSPO1-PEG-PLA of embodiment 9 and tumour cell targeting Journal of Sex Research

The preparation of RSPO1-PEG-PLA：

(2) weigh PLA-PEG-MAL to be dissolved in acetonitrile, rotary evaporation film forming, add degasification HEPES salting liquids, water Synthesize micellar solution；During RSPO1 after TCEP is reduced under N2 protections adds the micellar solution；React and be in temperature 10 DEG C, rotating speed is constant temperature oscillation under the conditions of 500rpm, and final product RSPO1-PEG-PLA is obtained after reaction 24h.

The preparation of the liposome of RSPO1 modifications and targeting Journal of Sex Research：

The Evacet of RSPO1 modifications is prepared with reference to the method in embodiment 5, and carries out tumor cell in vitro target To Journal of Sex Research.Result shows that there is the Evacet of RSPO1 modifications stronger growth to press down to carcinoma of the rectum LoVo cells Make and use.Specifically, the Evacet of RSPO1 modifications is probably not repair to the inhibitory action of carcinoma of the rectum LoVo cells 5 times of decorations liposome.

The preparation of embodiment 10 RSPO1- (GGGGS) n-Pluronic and tumour cell targeting Journal of Sex Research

The preparation of RSPO1- (GGGGS) n-Pluronic：

(1) the Pluronic copolymers for taking carboxylated are dissolved in acetonitrile, and rotary evaporation film forming is hydrated as micella after adding pure water Solution；1- ethyls -3_ (3- dimethyl aminopropyls)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS) are added, 200rpm Stirrings 15min adds (GGGGS) n polypeptides, 200rpm Stirring 2h, with hydrogen with activated carboxyl Sodium oxide molybdena adjusts pH value to 7, obtains (GGGGS) n-Pluronic micellas；

(2) 1- ethyls -3_ (3- dimethyl aminopropyls)-carbodiimides is added in (GGGGS) n-Pluronic micellas And N- [maleimidocaproic acid] hydrazides trifluoroacetic acid (EMCH), 200rpm Stirrings 15min are activating (EDC) Carboxyl, during the RSPO1 after TCEP is reduced under N2 protections adds the micellar solution；Obtained after normal temperature oscillating reactions 2h Final product RSPO1- (GGGGS) n-Pluronic.

The Evacet of RSPO1 modifications is prepared with reference to the method in embodiment 5, and carries out tumor cell in vitro targeting Journal of Sex Research.Result shows that there is the Evacet of RSPO1 modifications stronger growth inhibition to make to carcinoma of the rectum LoVo cells With.Specifically, the Evacet of RSPO1 modifications is up to unmodified liposome to the inhibitory action of carcinoma of the rectum LoVo cells 5 times.

The lauric preparations of the RSPO1-CpG ODN- of embodiment 11 and tumour cell targeting Journal of Sex Research

The lauric preparations of RSPO1-CpG ODN-：

(1) alkynyl-modified CpG ODN, copper sulphate and sodium ascorbate powder are dissolved in ultra-pure water respectively, and concussion is well mixed.

(2) nitrine laurate and TBTA powder are dissolved in DMF respectively, and concussion is well mixed.

(3) copper-bath, TBTA solution, triethylamine acetate buffer solution, CpG ODN are sequentially added in centrifuge tube molten Liquid, nitrine bay acid solution and sodium ascorbate solution, shake often plus after a kind of material and mix, after all adding Closed, room temperature concussion reaction overnight obtains CpG ODN- laurate.

(4) added after CpG ODN- laurate is mixed with PMPI (N- [p- maleimides benzene] isocyanates) solution RSPO1 after TCEP reduction；Rotating speed is normal temperature vibration under the conditions of 500rpm, obtains final after reaction 2h Product RSPO1-CpG ODN- laurate.

The present inventor is carried using RSPO1 coupling carriers molecule by above-mentioned in-depth study discovery extensively to medicine Body is modified, and is especially connected and the RSPO1 and load by thioether bond between the RSPO1 and carrier molecule Mol ratio between body molecule is 1：The factor of 1 these two aspects, efficient " target is turned into for RSPO1 coupling carriers molecule To head " it is particularly critical.The present invention it is substantial amounts of research show, the targeting drug delivery system containing RSPO1, pharmaceutical carrier table The RSPO1 in face, can characteristic efficiently pharmaceutical carrier is directionally transported in the tumor tissues of Lgr5 expression high, point Pharmaceutical carrier in cloth to target tissue can be combined with the target protein of tumor cell surface, and induced drug carrier endocytosis or medicine The processes such as thing release, so as to play drug effect.Tumour cell tool of the targeting drug delivery system of RSPO1 modifications to Lgr5 expression high There is stronger growth inhibition effect.Specifically, the targeting drug delivery system of RSPO1 modifications is to the tumour cell of Lgr5 expression high Inhibitory action is up to 5 times or so without RSPO1 modification delivery systems.And for its in RSPO1 coupling carrier molecules Other drugs carrier and other drugs in his carrier molecule, targeting drug delivery system, the present invention are no longer repeated one by one.

The above, only presently preferred embodiments of the present invention, it is not any to the present invention in form and substantial limitation, should Point out, for those skilled in the art, on the premise of the inventive method is not departed from, can also make some Improve and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art, not In the case of departing from the spirit and scope of the present invention, when a little change made using disclosed above technology contents, repair Decorations and the equivalent variations for developing, are Equivalent embodiments of the invention；Meanwhile, it is all according to substantial technological of the invention to above-mentioned reality The variation, modification and evolution of any equivalent variations that example is made are applied, is still fallen within the range of technical scheme.

Claims

1. a kind of RSPO1 coupling carriers molecule, its structural formula is：RSPO1- carrier molecules, the RSPO1 and carrier molecule Between by covalent bond connect.

2. RSPO1 coupling carriers molecule according to claim 1, it is characterised in that the RSPO1 and carrier molecule it Between by thioether bond connect.

3. RSPO1 coupling carriers molecule according to claim 1, it is characterised in that carrier molecule is connected to the RSPO1 On the 6th sulfydryl of cysteine of N-terminal.

4. RSPO1 coupling carriers molecule according to claim 1, it is characterised in that the RSPO1 and carrier molecule it Between mol ratio be 1：1.

5. RSPO1 coupling carriers molecule according to claim 1, it is characterised in that the RSPO1 coupling carriers molecule Structural formula be：RSPO1-linker-R, is connected, the linker between the RSPO1 and linker by covalent bond It is connected by covalent bond between R.

6. RSPO1 coupling carriers molecule according to claim 5, it is characterised in that between the RSPO1 and linker Connected by thioether bond.

7. RSPO1 coupling carriers molecule according to claim 5, it is characterised in that linker is connected to the RSPO1N Hold on the 6th sulfydryl of cysteine.

8. RSPO1 coupling carriers molecule according to claim 5, it is characterised in that described RSPO1, linker, R it Between mol ratio be 1：1：1.

9. RSPO1 coupling carriers molecule according to claim 5, it is characterised in that the R is selected from lipid, polymer Any one in material.

10. RSPO1 coupling carriers molecule according to claim 9, it is characterised in that the lipid is selected from phosphatide, fat Acid, any one in cholesterol.

11. RSPO1 coupling carriers molecules according to claim 10, it is characterised in that the phosphatide is selected from distearyl Phosphatidyl-ethanolamine, two myristoyl phosphatidyl-ethanolamines, DOPE, two palmityl phosphatidyls Monoethanolamine, DlinPE, dilauroyl phosphatidyl-ethanolamine, heptadecyl acyl phosphatidyl-ethanolamine, two mustard acyl group phosphorus Acyl monoethanolamine, hydrogenated soya phosphatide acyl monoethanolamine, hydrogenation egg phosphatide acyl monoethanolamine, soybean phospholipid phosphatidyl monoethanolamine or egg It is any in phosphatidyl-ethanolamine.

12. RSPO1 coupling carriers molecules according to claim 10, it is characterised in that it is misery that the aliphatic acid is selected from C8 Acid, C10 acid capric acid, C12 acid laurate, C14 acid myristic acid, C16 acid palmitic acid, C18 acid stearic acid appoint One.

13. RSPO1 coupling carriers molecules according to claim 9, it is characterised in that the polymeric material is selected from block Any one in copolymer material, alternate copolymer material, random copolymer, graft copolymer material.

14. RSPO1 coupling carriers molecules according to claim 13, it is characterised in that the block copolymer material is selected from Two kinds or two in any one or they in PLA, PLGA, PLG, Pluronic, PEO, PEI, PPO, PEG Plant the compound of the above.

15. RSPO1 coupling carriers molecules according to claim 5, it is characterised in that the linker be selected from polyethylene glycol, Any one in small peptide, oligonucleotides.

16. RSPO1 coupling carriers molecules according to claim 15, it is characterised in that the molecular weight of the polyethylene glycol Scope is 400~8000Da.

17. RSPO1 coupling carriers molecules according to claim 15, it is characterised in that the small peptide is selected from Gn, (GGS) Any one in n, (GGGS) n, (GGGGS) n, RGD.

18. RSPO1 coupling carriers molecules according to claim 15, it is characterised in that the molecular weight model of the oligonucleotides It is 600-8000Da to enclose.

19. as described in claim 1~18 any claim RSPO1 coupling carriers molecule preparation method, comprise the following steps： RSPO1 and carrier molecule/linker-R is prepared by covalent bond coupling reaction.

20. as described in claim 1~18 any claim RSPO1 coupling carriers molecule prepare targetable drug carriers or target to Purposes in medicine system.

A kind of 21. targetable drug carriers, contain the RSPO1 coupling carriers molecule as described in claim 1~18 any claim.

22. targetable drug carriers according to claim 21, it is characterised in that the targeting drug delivery system contains：RSPO1 Coupling carrier molecule and pharmaceutical carrier particle.

23. targetable drug carriers according to claim 22, it is characterised in that the pharmaceutical carrier particle surface has RSPO1.

24. targetable drug carriers according to claim 22, it is characterised in that each described pharmaceutical carrier particle surface has 5~10000 RSPO1.

25. targetable drug carriers according to claim 22, it is characterised in that the particle size range of the pharmaceutical carrier particle It is 10~1000nm.

26. targetable drug carriers according to claim 22, it is characterised in that the pharmaceutical carrier is selected from liposome, lipid Any one in nanoparticle, polymer nanoparticle.

27. targetable drug carriers according to claim 26, it is characterised in that the constituent of the liposome is selected from egg phosphorus Fat, HSPC, hydrogenation Yolk lecithin, DLPC, two myristoyl phosphorus Phosphatidylcholine, DPPC, DSPC, 1- myristoyl -2- palmityl phosphatide Phatidylcholine, 1- palmityl -2- DSPCs, 1- stearoyl -2- palmitoylphosphatidyl cholines, 1- palm fibres Palmitic acid acyl -2- oleoyl phosphatidylcholines, 1- stearoyls -2- Asias oleoyl phosphatidylcholine, DOPC, hydrogen Change DPPC, DSPC, two myristoyl phosphatidic acids, two myristoyl phosphatide Acid, DPPA, DPPA, G 12S3P, DMPEA, DPPE, cephalin acyl serine, two myristoyl phosphatidylserines, two palmityl phosphatide Acyl serine, E-PG, PE, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, two palmityls Phosphatidyl glycerol, DSPG, DOPG, brain sphingomyelins, two palmitoyl sphingomyelins Or distearyl sphingomyelins, cholesterol, two oily epoxides hydroxypropyltrimonium chloride (DOTAP), dioleoyl chloropropyl chlorination Trimethylammonium (DOTMA), DDA (DDAB), dimethyl aminoethyl amido propiono-courage are solid Alcohol (DC-Chol), spermine -5- carboxyaminos acetic acid octacosyl acid amides (DOGS), dioleoyl succinyl glycerine courage In alkali ester (DOSC), dioleoyl chlorine spermine Carboxylamide ethyl dimethyl propyl trifluoroacetic acid ammonium (DOSPA), MVL5 Any one or their two or more combination.

28. targetable drug carriers according to claim 26, it is characterised in that the constituent of the polymer nanoparticle is： Any one in PLA, PLGA, PLG, Pluronic, PEO, PEI, PPO, PEG or their two kinds or two Plant the combination of the above.

29. as described in claim 21~28 any claim targetable drug carriers construction method, selected from following any： Method one, comprise the following steps：

Using RSPO1 coupling carriers molecule as one of material for building pharmaceutical carrier, it is used to build pharmaceutical carrier with other Material directly mixes self assembly to prepare targetable drug carriers； Method two, rear insertion, comprise the following steps：

(1) pharmaceutical carrier is first built；

Method three：Connection method, comprises the following steps afterwards：

Purposes of the targetable drug carriers in targeting drug delivery system is prepared as described in 30. such as claim 21~28 any claims.

A kind of 31. targeting drug delivery systems, contain the targetable drug carriers as described in claim 21~28 any claim and medicine point Son.

32. targeting drug delivery systems according to claim 29, it is characterised in that the medicine is selected from antineoplastic；Anti- generation Thank to medicine；Cytotoxic drug；Antibiotic；Sensitising agent；Kinase inhibitor；Anti-inflammatory drug；Immunodepressant；It is anti- Infection medicine；Antiviral drugs.

33. targeting drug delivery systems according to claim 29, it is characterised in that the medicine is selected from genes of interest, antisense base Cause, suicide gene, apoptosis gene, cytokine gene, the combination of siRNA, mRNA or said gene, Or the carrier for expression of eukaryon DNA containing said gene.

34. as described in claim 29~31 any claim targeting drug delivery system construction method, selection is following any：

Method one, comprise the following steps：

Method two, rear insertion, comprise the following steps：

(1) pharmaceutical carrier for carrying medicine is first built；

Method three：Connection method, comprises the following steps afterwards：

35. targeting drug delivery systems as described in claim 29~31 any claim are preparing acoustic contrast agent, radiopaque contrast medium, core Purposes in medical science contrast agent.

A kind of 36. methods for treating tumour, including step：Will as described in claim 29~31 any claim targeting drug delivery system It is applied to patient.