CN106913880B

CN106913880B - RSPO 1-containing targeted drug delivery system and preparation and application thereof

Info

Publication number: CN106913880B
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: 2021-02-12
Anticipated expiration: 2035-12-24
Also published as: CN106913880A

Abstract

The invention belongs to the field of pharmaceutical preparations, and particularly relates to a RSPO 1-containing targeted drug delivery system, and preparation and application thereof. The targeted drug delivery system comprises: RSPO1 is coupled with carrier molecule, medicine and medicine carrier particle, and RSPO1 is on the surface of the medicine carrier particle. The targeted drug delivery system can obviously improve the targeting property and the tumor treatment effect of the drug on the basis of reducing the toxic and side effects of the drug.

Description

RSPO 1-containing targeted drug delivery system and preparation and application thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations, and particularly relates to a RSPO 1-containing targeted drug delivery system, and preparation and application thereof.

Background

The traditional tumor drug therapy (chemotherapy) can kill tumor cells and also kill normal cells of the body at the same time, so the generated toxic and side effects not only influence the treatment effect, but also endanger the life of a patient. Therefore, in recent years, efforts have been made to improve the specificity of chemotherapy for tumor cells. To achieve this goal, scientists have tried a number of approaches, including the targeted delivery of drugs to tumors using drug carriers to achieve the goals of improved efficacy and reduced toxic side effects.

The components of the antitumor drug carrier are various, and protein, lipid, inorganic materials, high molecular materials and inorganic and high molecular composite materials are involved. The nano-carrier systems developed at present include polymer nanoparticles, liposomes, dendrimers, nano-emulsions, nano-gold or other metal nanoparticles, and the like. Targeting of drug carriers includes passive targeting and active targeting. For example, liposome is a common drug carrier, and a stealth liposome can be prepared by modifying hydrophilic polyethylene glycol (PEG) on the surface of the liposome, so that the circulation time in vivo is remarkably increased, and the liposome is passively targeted to tumor tissues through the EPR effect. The stealth adriamycin liposome is the tumor targeted liposome drug which is most widely applied and has the longest time at present. Compared with the common adriamycin, the stealth adriamycin liposome has longer in vivo half-life and stronger drug effect. However, the drug is released in the intercellular space and then enters the tumor cells by diffusion based on passive targeting, the improvement of the treatment effect is not obvious, and new toxic and side effects such as palmar-plantar erythrodysesthis (PPE) and the like are generated clinically. The most currently used active targeting vectors are antibody-mediated targeting drug carriers. A plurality of antibody coupling carriers enter the clinical research stage, for example, transferrin antibody fragment modified liposome carrying DNA plasmid enters the phase II clinic.

The Wnt signaling pathway is involved in the regulation of a variety of biological processes, including embryonic growth and morphological development, tissue stabilization, balance of energy metabolism, and maintenance of stem cells. Over-activation of the Wnt pathway is closely linked to the development of a variety of cancers, including colon, gastric, breast, etc. The Wnt signal pathway can promote the metastasis of cancer cells, so that the Wnt signal pathway is considered as a new target point for tumor treatment. The current tumor drugs aiming at the Wnt pathway are mainly small molecular compounds and Wnt/Fzd protein antibodies. The FDA approved vismodegib (eridge), a small molecule inhibitor of the Hedgehog signaling pathway in 2012 for the treatment of malignant basal cell carcinoma. However, inhibition of the Wnt pathway by drugs inevitably causes many side effects including muscle spasm, hair loss, taste disturbance, weight loss, etc.

R-spondin1(RSPO1) is a newly discovered Wnt pathway ligand protein in recent years. The RSPO1 protein is involved in the regulation of cell proliferation and differentiation through activating and coordinating with Wnt signal path. In the prior art, a targeting drug delivery system which utilizes a Wnt pathway targeting ligand to specifically and efficiently target tumor cells with over-active Wnt pathways is urgently needed.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide a targeting drug delivery system capable of specifically and efficiently targeting Wnt pathway over-activated tumor cells, and preparation and application thereof.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in a first aspect of the invention, there is provided a RSPO 1-coupled carrier molecule having the structural formula: RSPO 1-carrier molecule, said RSPO1 being linked to the carrier molecule by a covalent bond.

Preferably, the RSPO1 is linked to the carrier molecule via a thioether bond.

Further preferably, the carrier molecule is attached to the thiol group of the cysteine at position 6 at the end of said RSPO 1N.

Further preferably, the molar ratio between the RSPO1 and the carrier molecule is 1: 1.

further preferably, the RSPO1 coupled carrier molecule has the structural formula: RSPO1-linker-R, the RSPO1 is connected with the linker through a covalent bond, and the linker is connected with the R through a covalent bond.

Preferably, the RSPO1 is connected with the linker through a thioether bond.

Preferably, a linker is attached to the thiol group of the cysteine at position 6 at the end of the RSPO 1N.

Preferably, the molar ratio between the RSPO1, the linker and the R is 1: 1: 1.

preferably, the R is selected from any one of lipid, polymer material.

Preferably, the lipid is selected from any one of phospholipids, fatty acids, cholesterol.

Further preferably, the phospholipid is selected from any one of the phospholipids selected from distearoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, DlinPE, dilauroylphosphatidylethanolamine, heptadecylphosphatidylethanolamine, erucamoylphosphatidylethanolamine, hydrogenated soybean phosphatidylethanolamine, hydrogenated egg phosphatidylethanolamine, soybean phosphatidylethanolamine, or egg phosphatidylethanolamine.

In a preferred embodiment of the present invention, the phospholipid is distearoyl phosphatidyl ethanolamine or dimyristoyl phosphatidyl ethanolamine.

Further preferably, the fatty acid is selected from any one of caprylic acid C8, capric acid C10, lauric acid C12, myristic acid C14, palmitic acid C16 and stearic acid C18.

In the preferred embodiment of the invention, the fatty acid is lauric acid C12.

Further preferably, the polymer material is selected from any one of a block copolymer material, an alternating copolymer material, a random copolymer, and a graft copolymer material.

Preferably, the block copolymer material is selected from any one of PLA, PLGA, PLG, Pluronic, PEO, PEI, PPO, PEG or a composite of two or more thereof.

In the preferred embodiment of the invention, PLA is selected as R.

Preferably, the linker is selected from any one of PEG, short peptide, oligonucleotide.

Short peptides, Short chain peptides for Short, Short chain polypeptide, Short chain peptides consisting of 3-9 amino acid residues, sometimes called oligopeptides.

Oligonucleotides, which are a generic term for short strands of nucleotides having only 20 or fewer bases, include nucleotides within deoxyribonucleic acid DNA or ribonucleic acid RNA.

Further preferably, the molecular weight range of the polyethylene glycol is 400-8000 Da. Further preferably, the molecular weight of the polyethylene glycol is 400-5000. Further preferably, the molecular weight of the polyethylene glycol is 2000-5000.

For example, the polyethylene glycol is selected from any one of, but not limited to, PEG400, PEG550, PEG750, PEG1000, polyethylene glycol 2000, polyethylene glycol 3400, polyethylene glycol 4000, and polyethylene glycol 5000.

In the preferred embodiment of the present invention, the polyethylene glycol is selected from PEG2000 and polyethylene glycol 5000.

Preferably, the short peptide is selected from any one of Gn, (GGS) n, (GGGS) n, (GGGGS) n, RGD. In a preferred embodiment of the present invention, n is 2 to 40.

Preferably, the molecular weight range of the oligonucleotide is 600-8000 Da.

A second aspect of the invention provides a method for preparing the aforementioned RSPO 1-coupled carrier molecule, comprising the steps of: the RSPO1 and the carrier molecule/linker-R are prepared through covalent bond coupling reaction.

Preferably, the RSPO1 requires activation of the free thiol group. Further preferably, the free thiol group in R-Spondin1 can be activated with a reducing agent TCEP.

In a preferred embodiment of the present invention, a method for preparing RSPO 1-polyethylene glycol-phospholipid is provided, the method comprising the steps of:

(1) taking maleimide modified polyethylene glycol-phospholipid according to the proportion, and hydrating to form micelle solution;

(2) and (2) adding the R-Spondin1 activated by the free sulfydryl into the micelle solution obtained in the step (1), and reacting for 2-20 hours at 4-25 ℃ under the protection of nitrogen.

Further preferably, the pH of the hydration liquid used in step (1) is in the range of 6.5-7.5.

Further preferably, the free thiol group in R-Spondin1 is activated in step (2) with a reducing agent TCEP.

In a third aspect of the invention, the use of the RSPO1 conjugated carrier molecule in the preparation of a targeted drug delivery carrier or a targeted drug delivery system is provided.

In a fourth aspect of the invention, a targeting drug delivery carrier is provided, which comprises the RSPO1 coupling carrier molecule.

Further, the targeted drug delivery carrier contains RSPO1 coupled carrier molecules and drug carrier particles.

Preferably, the drug carrier particle has RSPO1 on the surface.

Further preferably, RSPO1 is covalently attached to the surface of the drug carrier particle.

Preferably, there is more than one RSPO1 on the surface of each drug carrier particle. Further preferably, 5-10000 RSPO1 are arranged on the surface of each drug carrier particle.

In the embodiment of the invention, 5-1000R-Spondin 1 are arranged on the surface of each drug carrier.

Preferably, the molar ratio of RSPO1 coupled carrier molecule to drug carrier particle is in the range of: (0.01-100): 1000.

in the embodiment of the invention, the molar ratio range of the R-Spondin 1-polyethylene glycol-phospholipid to the drug carrier is as follows: (0.5-100): 1000.

preferably, the particle size range of the drug carrier particles is 10-1000 nm.

Preferably, the drug carrier particle is selected from any one of, but not limited to, liposome, nanoparticle, polymer nanoparticle, cationic lipid nanoparticle, polymer micelle, emulsion, microcapsule, microsphere, nanocapsule, and nanosphere.

Further, the drug carrier may be further modified.

Preferably, the liposome is composed of a member selected from the group consisting of egg phospholipids, hydrogenated soybean phosphatidylcholine, hydrogenated egg phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1-stearoyl-2-linoleoyl phosphatidylcholine, dioleoyl phosphatidylcholine, hydrogenated dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dimyristoyl phosphatidic acid, di-palmitoyl phosphatidylcholine, di-stearoyl phosphatidylcholine, Distearoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, cephalylphosphatidylserine, dimyristoylphosphatidylserine, egg phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, sphingomyelin, dipalmitoyl sphingomyelin or distearoylsphingomyelin, cholesterol, dioleoyloxypropyltrimethylammonium chloride (DOTAP), dioleoylchlorotrimethylammonium chloride (DOTMA), dioctadecyldimethylammonium bromide (DDAB), dimethylaminoethylpropionyl-cholesterol (DC-Chol), spermine-5-carboxyaminoacetic acid octacosylamide (DOGS), Dioleoylsuccinylcholinesteramide (DOSC), dioleoylceramidoethyldimethylpropyltrimethylammonium trifluoroacetate (DOSPA), Any one of MVLs 5 or a combination of two or more thereof.

Preferably, the composition of the polymer nanoparticles is as follows: any one of PLA, PLGA, PLG, Pluronic, PEO, PEI, PPO, PEG, or a combination of two or more thereof.

In the fifth aspect of the present invention, a method for constructing the aforementioned targeting drug delivery vector is further provided, which is selected from any one of the following:

the method comprises the following steps:

the RSPO1 coupled carrier molecule is used as one of the materials for constructing the drug carrier, and is directly mixed with other materials for constructing the drug carrier to self-assemble to prepare the targeted drug delivery carrier;

the second method, post-insertion method, includes the following steps:

(1) firstly, constructing a drug carrier;

(2) mixing RSPO1 coupled carrier molecule with the constructed drug carrier;

the third method comprises the following steps: the post-joining method comprises the following steps:

(1) adopting carrier molecules as one of the drug carrier materials to construct a drug carrier containing the carrier molecules;

(2) and (3) carrying out covalent bond coupling reaction on RSPO1 and the drug carrier containing the carrier molecule constructed in the step (1) to construct the targeted drug delivery carrier.

In a sixth aspect of the invention, the use of the targeted drug delivery carrier in the preparation of a targeted drug delivery system is provided.

In a seventh aspect of the present invention, a targeted drug delivery system is provided, which comprises the aforementioned targeted drug delivery carrier and a drug.

Preferably, the drug is selected from, but not limited to, antineoplastic drugs; an antimetabolite; a cytotoxic drug; (ii) an antibiotic; a photosensitizer; a kinase inhibitor; anti-inflammatory agents; an immunosuppressant; anti-infective agents; an antiviral drug.

Preferably, the drug is selected from, but not limited to, a target gene, an antisense gene, a suicide gene, an apoptosis gene, a cytokine gene, siRNA, mRNA, or a combination of the above genes, or eukaryotic expression vector DNA containing the above genes.

For example, in one embodiment of the present invention, siRNA or shRNA plasmid expression vector (pGPU6/GFP/Neo) is also selected as a model drug.

Further preferably, the drug is an anti-tumor drug. The anti-tumor drug can be any existing drug for inhibiting the growth, proliferation, differentiation and metastasis of tumor cells, and comprises but is not limited to: small molecule chemical drugs, nucleic acid molecules, carbohydrates, lipids, antibody drugs, polypeptides, proteins, or interfering lentiviruses.

In a preferred embodiment of the invention, doxorubicin is selected as the model drug.

Preferably, the target cell targeted by the targeted drug delivery system is a tumor cell.

The targeted drug delivery system can carry anti-tumor drugs and can be targeted to tumor cells.

Preferably, the tumor is a solid tumor or a metastatic tumor. Further preferably, the tumor is a tumor with high expression of LGR 5.

More preferably, the tumor is selected from, but not limited to, rectal cancer, gastric cancer, breast cancer, lung cancer, pancreatic cancer, liver cancer, esophageal cancer, or glioblastomas.

In an eighth aspect of the present invention, there is provided a method for constructing the targeted drug delivery system, which is selected from any one of the following:

the method comprises the following steps:

RSPO1 coupled carrier molecules are used as one of materials for constructing a drug carrier, and are directly mixed with other materials for constructing the drug carrier and drugs to be self-assembled to prepare a targeted drug delivery system;

the second method, post-insertion method, includes the following steps:

(1) firstly, constructing a drug carrier carrying drugs;

(2) mixing RSPO1 coupled carrier molecule with the constructed drug carrier carrying the drug;

(1) adopting carrier molecules as one of the drug carrier materials to construct a drug carrier containing the carrier molecules and carrying drugs;

(2) and (3) carrying out covalent bond coupling reaction on the RSPO1 and the drug carrier which is constructed in the step (1) and contains the carrier molecule and carries the drug to construct the targeted drug delivery system.

In a ninth aspect of the invention, the use of the targeted drug delivery system in the preparation of ultrasound contrast agents, radiocontrast agents, nuclear medicine contrast agents is provided.

In a tenth aspect of the present invention, there is also provided a method for treating a tumor, comprising the steps of: the aforementioned targeted drug delivery system is administered to a patient. The dosage used can be selected by the physician according to the actual circumstances.

The patient may be a tumor patient. The tumor is selected from but not limited to rectal cancer, gastric cancer, breast cancer, lung cancer, pancreatic cancer, liver cancer, esophageal cancer or glioblastomas.

Compared with the prior art, the invention has the following beneficial effects:

(1) the-RSPO 1 protein coupled carrier molecule of the invention can specifically target tumor cells with active Wnt pathway, and can improve the drug effect of chemotherapeutic drugs and reduce the side effect thereof through passive and active dual targeting effects. The drug carrier is modified by coupling RSPO1 with carrier molecules, particularly, the RSPO1 and the carrier molecules are connected through thioether bonds, and the molar ratio of the RSPO1 to the carrier molecules is 1:1, it is particularly critical for RSPO1 to become an efficient "targeting head" for coupling to a carrier molecule. A large number of researches show that the RSPO1 on the surface of the drug carrier and the target drug delivery system containing the RSPO1 can directionally transport the drug carrier to Lgr5 high-expression tumor tissues with characteristics and high efficiency, the drug carrier distributed in the target tissues can be combined with target proteins on the surface of tumor cells, and the processes of endocytosis or drug release of the drug carrier and the like are induced, so that the drug effect is exerted. The RSPO1 modified targeting drug delivery system has stronger growth inhibition effect on Lgr5 high-expression tumor cells. Specifically, the RSPO1 modified targeting drug delivery system has about 5 times of the inhibition effect on Lgr5 high-expression tumor cells without the RSPO1 modified drug delivery system.

(2) The invention takes liposome as a drug carrier model to carry out R-Spondin1 modification, and examines the uptake of RSPO1 modified liposome and common liposome by LoVo rectal cancer cells with high expression of LGR5 through a fluorescence tracing method, and the result proves that RSPO1 can efficiently mediate liposome to enter cells with high expression of LGR5 in vitro. Specifically, LoVo cells uptake RSPO1 modified liposomes was probably 7 times more than unmodified liposomes.

(3) By taking adriamycin as a model drug and liposome as a drug carrier model, the in vitro growth inhibition effect and the in vivo growth inhibition effect of RSPO1 modified liposome/DOX on LoVo rectal cancer cells highly expressed by LGR5 are studied, the inhibition effect of the in vitro RSPO1 modified adriamycin liposome on the colorectal cancer LoVo cells is probably that of unmodified liposome, the inhibition rate of the in vivo RSPO1 modified adriamycin liposome on the growth of the colorectal cancer tumors is up to 59 percent, and the inhibition rate of the in vivo RSPO1 modified adriamycin liposome on the growth of the colorectal cancer tumors is 25 percent higher than that of the unmodified liposome. The RSPO1 modification can obviously increase the growth inhibition effect of the adriamycin liposome on cancer cells and tumor tissues with high expression of LGR 5.

(4) Research results of the invention indicate that the R-Spondin1 modified targeted drug delivery system has passive and active dual targeting effects, and can efficiently target drugs such as antitumor drugs to primary tumors and metastasis lesions expressed by LGR5, so that the drugs can be accurately delivered to the inside of tumor cells, real targeted therapy is realized, and the therapeutic effect is greatly improved. Experiments show that compared with a targeted drug delivery system which is not modified by the R-Spondin1, the effect of the R-Spondin1 modified targeted drug delivery system on treating tumors is improved by more than four times.

Drawings

FIG. 1 shows the result of polyacrylamide gel electrophoresis analysis of RSPO1-PEG2000-DSPE prepared in example 1 of the present invention.

FIG. 2 shows the results of in vitro tumor cell targeting studies of RSPO 1-modified FITC-labeled liposome prepared in example 3 of the present invention.

FIG. 3 is the in vitro tumor cell targeting study of RSPO1 modified DiI-labeled liposome prepared in example 4 of the present invention.

FIG. 4A shows the in vitro growth inhibitory effect of RSPO1 modified doxorubicin liposome prepared in example 5 of the present invention on LoVo rectal cancer cells at 24 h.

FIG. 4B shows the in vitro growth inhibitory effect of RSPO1 modified doxorubicin liposome prepared in example 5 of the present invention on LoVo rectal cancer cells at 48 h.

FIG. 5 shows the in vivo growth inhibitory effect of RSPO1 modified adriamycin liposome on LoVo rectal cancer cells, which is prepared in example 5 of the present invention.

FIG. 6A shows that the RSPO1 modified fluorescent-labeled cationic lipid gene composite nanoparticle prepared in example 7 of the present invention is taken up by LoVo cells at 1 h.

FIG. 6B shows the result of the experiment of the fluorescent-labeled cationic lipid gene composite nanoparticle modified by RSPO1, which is prepared in example 7 of the present invention, taken up by LoVo cells after 24 h.

FIG. 7 shows that the uptake of RSPO1 modified liposomes prepared in example 8 of the invention by LoVo cells is much greater than that of unmodified liposomes.

Detailed Description

The inventors of the present invention, after extensive and intensive studies, synthesized and provided for the first time in a preferred embodiment R-Spondin 1-polyethylene glycol-phospholipid, wherein the molar ratio of R-Spondin1, polyethylene glycol, phospholipid is 1: 1: 1.

the R-Spondin 1-polyethylene glycol-phospholipid is a linear amphiphilic block copolymer, the hydrophilic segment is polyethylene glycol, and the hydrophobic segment is phospholipid. Can self-assemble in water to form a high molecular micelle consisting of a hydrophilic shell and a lipophilic inner core.

The R-Spondin1 is an existing protein with accession number NC-000001.11 in Genebank. Are available from commercial sources. In the present invention, R-Spondin1 may be abbreviated as RSPO 1.

The polyethylene glycol, also known as PEG, is commercially available. The molecular weight of the polyethylene glycol is not particularly limited, and the molecular weight of the polyethylene glycol is preferably 400 to 8000. In the preferred embodiment of the present invention, the polyethylene glycol is PEG2000, and polyethylene glycols with other molecular weights can be used, and are not limited to the specific molecular weights listed in the examples.

The phospholipids are commercially available. The phospholipid used in the present invention is not particularly limited, and a short-chain phospholipid having a molecular weight equivalent to or slightly smaller than that of polyethylene glycol can be used. For example: the phospholipid can be selected from one of distearoyl phosphatidyl ethanolamine, dimyristoyl phosphatidyl ethanolamine, dilauroyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine, hydrogenated soybean phosphatidyl ethanolamine, hydrogenated egg phosphatidyl ethanolamine, soybean phosphatidyl ethanolamine or egg phosphatidyl ethanolamine. In the preferred embodiment of the present invention, the phospholipid is distearoyl phosphatidyl ethanolamine, and other phospholipids can be selected, and are not limited to the specific phospholipids listed in the examples.

The present invention is not particularly limited with respect to the method for preparing R-Spondin 1-polyethylene glycol-phospholipid, as long as the copolymer can be successfully prepared. In the preferred embodiment of the present invention, the R-Spondin 1-polyethylene glycol-phospholipid is prepared by covalent bond coupling, but other preparation methods can be used, and the preparation method is not limited to the preparation methods listed in the examples.

The invention also provides a targeted drug delivery system, which contains the R-Spondin 1-polyethylene glycol-phospholipid. Further, the targeted drug delivery system comprises: R-Spondin 1-polyethylene glycol-phospholipid, a drug, and a pharmaceutical carrier.

The present invention is not particularly limited to the drug. For example, the drug may be an anti-tumor drug. In the preferred embodiment of the invention, adriamycin is selected as a model drug to study the targeting ability of the antitumor drug delivered by the targeted drug delivery system. In another embodiment, siRNA or shRNA plasmid expression vectors (pGPU6/GFP/Neo) are also selected as model drugs to study the targeting ability of the targeted drug delivery vectors for delivering gene drugs.

The drug carrier is a system which can change the mode of entering the human body and the distribution of the drug in the human body, control the release speed of the drug and deliver the drug to a target organ. The present invention is not particularly limited to drug carriers as long as successful carrying of the drug can be achieved. For example, the drug carrier can be any one of liposome, cationic lipid nanoparticle, polymer micelle, emulsion, microcapsule, microsphere, nanocapsule and nanosphere. In the preferred embodiment of the present invention, liposomes and cationic lipid nanoparticles are used as model drug carriers, but other suitable drug carriers can be used, and the invention is not limited to the specific drug carriers listed in the examples. Methods of constructing pharmaceutical carriers are known to those skilled in the art.

The construction method of the targeted drug delivery system is not particularly limited in the present invention. The targeted drug delivery system can be constructed as required, for example, a drug carrier carrying the drug can be constructed firstly, then the R-Spondin 1-polyethylene glycol-phospholipid is added, and the mixture is mixed. Alternatively, a drug carrier carrying the drug and containing polyethylene glycol-phospholipid is constructed, and then R-Spondin1 is added to carry out coupling reaction through covalent bonds, so as to construct the targeted drug delivery system. For another example, R-Spondin 1-polyethylene glycol-phospholipid, a drug, and materials used to construct a drug carrier can also be mixed directly to make a targeted drug delivery system.

Further, the administration vehicle may be modified with other modifications, e.g., single or multiple antibodies of interest, modification of the ligand polysaccharide, etc., and is not limited to the R-Spondin1 modification of the invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, 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; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

EXAMPLE 1 preparation of RSPO1-PEG2000-DSPE

(1) Preparation of degassed HEPES solution: boiling with ultrapure water, filling N2 to saturate and cool, adding HEPES salt to the concentration of 50mM, adjusting pH to 7.0 with NaOH, and protecting with N2 in the whole process;

(2) weighing DSPE-PEG2000-MAL powder, adding degassing HEPES salt solution, and hydrating to obtain micelle solution; adding TCEP-reduced RSPO1 into the micellar solution under the protection of N2; the reaction is carried out under the conditions that the temperature is 10 ℃ and the rotating speed is 500rpm and the constant temperature oscillation is carried out, and the final product RSPO1-PEG2000-DSPE is obtained after 24 hours of reaction.

The result of polyacrylamide gel electrophoresis analysis, as shown in FIG. 1, shows that the RSPO1 protein band after DSPE-PEG2000 is connected is shifted to 3K, and the connection efficiency calculated by ImageJ is above 95%, which fully indicates the success of RSPO1-PEG2000-DSPE synthesis.

Example 2 preparation of RSPO1 modified liposomes

Preparing liposome by taking phospholipid, cholesterol and DSPE-PEG2000 as raw materials, wherein the molar ratio of each raw material HSPC (hydrogenated soybean phospholipid)/Chol (cholesterol)/DSPE-PEG 2000 (polyethylene glycol-distearoyl phosphatidyl ethanolamine compound) is 55: 42: 3. the method comprises the following steps: mixing the raw materials in proper amount of chloroform, performing rotary evaporation under reduced pressure in water bath at 37 ℃ to remove chloroform to form uniform lipid membrane, adding 1ml of PBS buffer solution, hydrating for 1h, and performing ultrasonic treatment in water bath; sequentially extruding the mixture to pass through 100nm and 80nm polycarbonate membranes to obtain the PEG modified liposome.

Then, RSPO1-PEG2000-DSPE obtained in example 1 was added to the PEG-modified liposome obtained in the above-mentioned preparation of this example, and the mixture was shaken at a constant temperature of 37 ℃ and a rotation speed of 300rpm for 24 hours to obtain RSPO 1-modified liposome. The particle size of the nanoparticles was measured using a laser scattering particle size analyzer (PCS), showing a particle size of 90. + -.10 nm.

Example 3 preparation of FITC-labeled liposome modified by RSPO1 and in vitro tumor cell targeting study

(1) Preparation of RSPO1 modified FITC labeled liposome

Preparing liposome by taking phospholipid, cholesterol, DSPE-PEG2000 and FITC-DHPE as raw materials, wherein the molar ratio of each raw material HSPC (hydrogenated soybean phospholipid)/Chol (cholesterol)/DSPE-PEG 2000 (polyethylene glycol-distearoyl phosphatidyl ethanolamine compound)/FITC-DHPE is 55: 42: 3: 1. the method comprises the following steps: mixing the raw materials in proper amount of chloroform, performing rotary evaporation under reduced pressure in water bath at 37 ℃ to remove chloroform to form uniform lipid membrane, adding 1ml of PBS buffer solution, hydrating for 1h, and performing ultrasonic treatment in water bath; sequentially extruding the mixture to pass through 100nm and 80nm polycarbonate membranes to obtain the FITC modified liposome.

RSPO1-PEG2000-DSPE obtained in example 1 was added to the FITC-modified liposome of this example, and the mixture was shaken at constant temperature at 37 ℃ and 300rpm for 24 hours to obtain RSPO 1-modified FITC-labeled liposome. The particle size of the nanoparticles was measured using a laser scattering particle size analyzer (PCS), showing a particle size of 95. + -.10 nm.

(2) RSPO1 modified FITC labeled liposome in-vitro tumor cell targeting research

Collecting LoVo cell cultured in logarithmic phase, digesting the LoVo cell with 0.25% trypsin/EDTA, preparing single cell suspension with DMEM/F12 medium containing 20% fetal calf serum, and culturing at 4X10 per well⁵And inoculating each cell in a 12-hole cell culture plate with 2ml of each hole, transferring the culture plate into a carbon dioxide incubator, and culturing at 37 ℃, 5% carbon dioxide and saturated humidity to allow the cells to adhere to the wall. On alternate days, FITC-labeled liposomes and RSPO 1-modified FITC-labeled liposomes were prepared in a medium without fetal bovine serum. The culture medium in the culture plate is sucked out, FITC labeled liposome and RSPO1 modified FITC labeled liposome are added, incubation is carried out for 1h at 37 ℃, and the supernatant is sucked away. Washing the plate twice with precooled PBS solution, digesting the cells with 0.25% trypsin/EDTA, stopping digestion with DMEM/F12 medium containing 20% fetal calf serum, blowing the cells into single cell suspension, and then adding paraformaldehyde for fixing for 10 minutes. Cells were harvested by centrifugation, the supernatant was aspirated off, resuspended in PBS and repeated twice. And finally detecting the cell internalization condition in the single cell suspension by using a flow cytometer. Referring to fig. 2, the results show that the uptake of RSPO1 modified liposomes by LoVo cells is much greater than that of unmodified liposomes. Specifically, LoVo cells uptake RSPO1 modified liposomes was probably 7 times more than unmodified liposomes.

Example 4 preparation and targeting study of RSPO 1-modified DiI-labeled liposomes

(1) Preparation of RSPO1 modified DiI-labeled liposome

The liposome is prepared by taking phospholipid, cholesterol, DSPE-PEG2000 and DiI as raw materials. The molar ratio of each raw material HSPC (hydrogenated soybean phospholipid)/Chol (cholesterol)/DSPE-PEG 2000 (polyethylene glycol-distearoyl phosphatidyl ethanolamine complex)/DiI is 55: 42: 3: 0.8. the method comprises the following steps: mixing the raw materials in proper amount of chloroform, performing rotary evaporation under reduced pressure in water bath at 37 ℃ to remove chloroform to form uniform lipid membrane, adding 1ml of PBS buffer solution, hydrating for 1h, and performing ultrasonic treatment in water bath; sequentially extruding the mixture to pass through 100nm and 80nm polycarbonate membranes to obtain the DiI modified liposome.

RSPO1-PEG2000-DSPE obtained in example 1 is added into the DiI modified liposome prepared in the example, and the mixture is shaken at constant temperature at 37 ℃ and the rotating speed of 300rpm for 24 hours to obtain the RSPO1 modified DiI labeled liposome. The particle size of the nanoparticles was measured using a laser scattering particle size analyzer (PCS), showing a particle size of 95. + -.10 nm.

(2) RSPO1 modified DiI labeled liposome in-vitro tumor cell targeting research

Collecting LoVo cell cultured in logarithmic growth phase, digesting the LoVo cell with 0.25% trypsin/EDTA, preparing single cell suspension with DMEM/F12 medium containing 20% fetal calf serum, and culturing at 2X10 per well⁵The cells are inoculated on a 14mm cover glass in a 24-well plate, each pore volume is 1ml, the culture plate is moved into a carbon dioxide incubator and cultured under the conditions of 37 ℃, 5% carbon dioxide and saturated humidity, so that the cells are attached to the wall. Every other day, DiI-labeled liposomes and RSPO 1-modified DiI-labeled liposomes were prepared in a medium without fetal bovine serum. Sucking out the culture medium from the culture plate, adding the DiI-labeled liposome and the RSPO1 modified DiI-labeled liposome, incubating at 37 ℃ for 15min, incubating for 30min, and sucking out the supernatant after 1 h. And (3) washing with precooled PBS (phosphate buffer solution) for three times, adding paraformaldehyde for fixing for 10 minutes, washing with PBS for two times, taking out the cover glass, placing the cover glass on a glass slide, adding a mounting solution for mounting, and observing the cell internalization condition under a fluorescence microscope. Referring to fig. 3, fluorescence micrographs of RSPO 1-modified DiI-labeled liposomes and DiI-labeled liposomes after 15 minutes, 30 minutes, and one hour exposure, respectively, to LoVo cells at 37 ℃. The result shows that the DiI labeled liposome can not be taken up by LoVo cells basically, while the RSPO1 modified liposome is taken up in large quantity, which indicates that the RSPO1 modified liposome has good in vitro targeting property to the colorectal cancer cells under the mediation effect of RSPO 1.

Example 5 preparation of RSPO 1-modified Water-soluble Doxorubicin hydrochloride-entrapped liposomes and study of antitumor Effect

(1) Preparation of RSPO1 modified liposome entrapping water-soluble adriamycin hydrochloride

The liposome is prepared by taking phospholipid, cholesterol and DSPE-PEG2000 as raw materials. The molar ratio of each raw material HSPC (hydrogenated soybean phospholipid)/Chol (cholesterol)/DSPE-PEG 2000 (polyethylene glycol-distearoyl phosphatidyl ethanolamine complex) was 55: 42: 3. the method comprises the following steps: proportionally mixing the raw materials, dissolving in chloroform, performing rotary evaporation under reduced pressure in water bath at 37 ℃ to remove chloroform to form a uniform lipid membrane, adding ammonium sulfate solution with a certain volume, hydrating for 1h, and performing ultrasonic treatment in water bath to obtain liposome suspension. Sequentially extruding the mixture through 400nm, 200nm, 100nm and 80nm polycarbonate membranes by using a micro extruder in a water bath at 60 ℃ to obtain blank liposomes; after replacing the external water phase by dialysis, adding adriamycin aqueous solution according to the drug-lipid ratio of 1:10(w/w), and carrying out water bath at 60 ℃ for 20 minutes; dialyzing to remove free drug to obtain adriamycin liposome.

RSPO1-PEG2000-DSPE obtained in example 1 was added to the doxorubicin liposome prepared in this example, and the mixture was shaken at a constant temperature of 37 ℃ and a rotation speed of 300rpm for 24 hours to obtain RSPO 1-modified doxorubicin liposome. The particle size of the nanoparticles was measured using a laser scattering particle size analyzer (PCS), showing a particle size of 95. + -.10 nm.

(2) In-vitro growth inhibition effect of RSPO1 modified adriamycin liposome on LoVo rectal cancer cells

The in vitro growth inhibition effect of the RSPO1 modified adriamycin liposome on LoVo rectal cancer cells is determined by adopting a CCK-8 kit. Collecting single-layer cultured LoVo cells in logarithmic growth phase, digesting with 0.25% trypsin/EDTA, preparing into single-cell suspension with DMEM/F12 medium containing 20% fetal calf serum, counting cells, and adding 1X10 per well⁴The cells are inoculated in a 96-well plate, each pore volume is 100ul, the culture plate is transferred into a carbon dioxide incubator and cultured under the conditions of 37 ℃, 5% carbon dioxide and saturated humidity, so that the cells adhere to the wall. Every other day, the RSPO1 modified adriamycin liposome and adriamycin liposome are diluted to adriamycin concentration of 30ug/ml, 15ug/ml and 7.5ug/ml by using cell culture medium, the cell culture solution in a 96-well plate is sucked out, 100ul liposome medicinal solution with each concentration is added into each well, and after incubation for 4 hours, the wells are changed into complete culture medium to be continuously cultured for 24 hours and 48 hours. Three duplicate wells were set for each concentration, leaving three wells to which culture medium was added only as control wells. Adding 10 microliter of CCK-8 solution into each hole, and placing in a cell culture boxAnd continuously incubating for 3 hours, shaking and uniformly mixing for 2 minutes, detecting the absorbance at 450nm by using an enzyme-labeling instrument, and taking the absorbance at 600nm as a background value reference. The results are shown in fig. 4A and 4B, and the RSPO 1-modified doxorubicin liposome has a stronger growth inhibition effect on colorectal cancer LoVo cells. Specifically, the inhibitory effect of the RSPO1 modified adriamycin liposome on colorectal cancer LoVo cells is about 5 times that of the unmodified liposome.

(3) In vivo growth inhibition effect of RSPO1 modified adriamycin liposome on colorectal cancer tumor tissue

Monolayer cultured LoVo cells in logarithmic growth phase were taken, digested with 0.25% trypsin/EDTA, washed with PBS, counted, dispersed in PBS at 1X10⁶The cells (100ul) were inoculated subcutaneously to the right underarm of nude mice and fed under SPF environment. 21 subcutaneous tumors of LoVo (tumor volume 50-100 mm)³) The nude mice of (a) were randomly divided into 3 groups (7 per group), which were set as PBS, doxorubicin liposome and RSPO 1-modified doxorubicin liposome groups, respectively. 100ul of the corresponding preparation was injected into nude mice from the first day of cage separation, every three days, each doxorubicin dose was 0.5mg/kg, 8 injections were performed in total, and the total dose was 4 mg/kg. Weekly measurements of subcutaneous tumor tissue size, tumor tissue

The volume calculation formula is as follows:

V(mm³)＝(d²X D)/2

wherein D and D are the minor and major diameters of the tumor tissue, respectively, in mm. As shown in fig. 5, the results showed that the inhibitory effect of RSPO 1-modified doxorubicin liposomes on tumor growth was significantly enhanced compared to doxorubicin liposomes. Specifically, the rate of inhibiting the growth of the colorectal cancer tumor by RSPO1 modified adriamycin liposome is up to 59%, while the rate of inhibiting the growth of the colorectal cancer tumor by unmodified liposome is only 34%.

Example 6 preparation of RSPO1 modified cationic lipid Gene composite nanoparticles

Dissolving DSPC, cholesterol, DLin-MC2-MPZ and DSPE-PEG2000 in a certain amount of ethanol at a ratio of 20/38.5/40/1.5, and injecting a certain volume of buffer solution with pH of 4.0 at a constant speed, wherein the volume ratio of the ethanol to the buffer solution is 2: 8. Preparing corresponding siRNA solution according to the same alcohol-water ratio, mixing the siRNA solution with the lipid solution in equal volume uniformly, and incubating for 50 minutes at 37 ℃; under the condition of 4 ℃, the solution finally obtained in the step 2 is dialyzed by a buffer solution with pH4.0 for 4 hours, and the ethanol in the lipid gene composite nanoparticles is removed as much as possible; then dialyzing by pH7.4 buffer solution for 8-12 hr to make the lipid gene composite nano particle keep its electric neutrality under the condition of pH7.4.

Then, the RSPO1-PEG2000-DSPE obtained in the example 1 was added to the PEG modified lipid gene composite nanoparticle obtained in the above preparation of this example, and the mixture was shaken at a constant temperature of 37 ℃ and a rotation speed of 300rpm for 20 to 24 hours to obtain the RSPO1 modified lipid gene composite nanoparticle. The particle size of the nanoparticles was measured using a laser scattering particle size analyzer (PCS), which showed a particle size of about 170nm, a zeta potential of about-15.8 mV, and a siRNA encapsulation efficiency of 80% -90% as measured by agarose gel electrophoresis.

Example 7 preparation of fluorescence labeling cationic lipid gene composite nanoparticle modified by RSPO1 and in vitro tumor cell targeting study

(1) Preparation of RSPO1 modified fluorescence labeling cationic lipid gene composite nano particle

Dissolving DSPC, cholesterol, DLin-MC2-MPZ, DSPE-PEG2000 and DiI in a certain amount of ethanol according to the ratio of 20/38.5/40/1.5/1, and injecting a certain volume of buffer solution with pH value of 4.0 under the condition of uniform speed, wherein the volume ratio of the ethanol to the buffer solution is 2: 8. Preparing corresponding Cy5-Luciferase siRNA solution according to the same alcohol-water ratio, uniformly mixing the solution with a lipid solution in the same volume, and incubating for 50 minutes at 37 ℃; under the condition of 4 ℃, the solution finally obtained in the step 2 is dialyzed by a buffer solution with pH4.0 for 4 hours, and the ethanol in the lipid gene composite nanoparticles is removed as much as possible; then dialyzing by pH7.4 buffer solution for 8-12 hr to make the lipid gene composite nano particle keep its electric neutrality under the condition of pH7.4. Then, the RSPO1-PEG2000-DSPE obtained in the example 1 is added into the PEG modified fluorescent lipid gene composite nanoparticle obtained in the embodiment, and the mixture is oscillated at constant temperature of 37 ℃ and at the rotating speed of 300rpm for 20 to 24 hours to obtain the RSPO1 modified fluorescent labeled lipid gene composite nanoparticle.

(2) In-vitro tumor cell targeting research of RSPO1 modified fluorescence-labeled cationic lipid gene composite nanoparticle

Collecting LoVo cell cultured in logarithmic growth phase, digesting the LoVo cell with 0.25% trypsin/EDTA, preparing single cell suspension with DMEM/F12 medium containing 20% fetal calf serum, and culturing at 2X10 per well⁵The cells are inoculated on a 14mm cover glass in a 24-well plate, each pore volume is 1ml, the culture plate is moved into a carbon dioxide incubator and cultured under the conditions of 37 ℃, 5% carbon dioxide and saturated humidity, so that the cells are attached to the wall. On the next day, the culture medium in the culture plate is sucked out, the fluorescence-labeled lipid gene composite nanoparticles prepared by the culture medium without fetal calf serum and the fluorescence-labeled lipid gene composite nanoparticles modified by RSPO1 are added, and after incubation for 1h and 24h at 37 ℃, the supernatant is sucked away. And (3) washing with precooled PBS (phosphate buffer solution) for three times, adding paraformaldehyde for fixing for 10 minutes, washing with PBS for two times, taking out the cover glass, placing the cover glass on a glass slide, adding a mounting solution for mounting, and observing the cell internalization condition under a fluorescence microscope. Referring to fig. 7, fluorescence micrographs of RSPO 1-modified fluorescently labeled lipid gene-complexed nanoparticles and fluorescently labeled lipid gene-complexed nanoparticles after 1 hour and 24 hours of interaction with LoVo cells at 37 ℃. The results show that the fluorescent-labeled lipid gene composite nanoparticles can be only taken up by LoVo cells in a small amount, while the fluorescent-labeled lipid gene composite nanoparticles modified by RSPO1 are taken up in a large amount. Meanwhile, it can be observed that the fluorescence modified siRNA can hardly enter the cell in the unmodified nanoparticle group, and a large amount of fluorescence modified siRNA in the RSPO1 modified nanoparticle group enters the cell. The RSPO1 modified lipid gene composite nanoparticle has good in-vitro targeting property on the rectal cancer cells under the mediation effect of the RSPO 1.

Example 8 preparation of RSPO1 modified FITC labeled liposome by surface attachment method and in vitro tumor cell targeting study thereof

(1) Preparation of RSPO1 modified FITC labeled liposome by surface attachment method

Preparation of degassed HEPES solution: boiling with ultrapure water, filling N2 to saturate and cool, adding HEPES salt to the concentration of 50mM, adjusting pH to 7.0 with NaOH, and protecting with N2 in the whole process;

the liposome is prepared by taking phospholipid, cholesterol, DSPE-PEG 5000, DPPE-PEG5000-Maleimide and FITC-DHPE as raw materials. The molar ratio of each raw material HSPC (hydrogenated soybean phospholipid)/Chol (cholesterol)/DSPE-PEG 5000 (polyethylene glycol-distearoyl phosphatidyl ethanolamine complex)/DPPE-PEG 5000-Maleimide/FITC-DHPE is 55: 42: 3: 1: 1. the method comprises the following steps: mixing the raw materials in proper amount of chloroform, performing rotary evaporation at 37 deg.C under reduced pressure in water bath to remove chloroform to obtain uniform lipid membrane, adding 1ml degassing HEPES solution, hydrating for 1 hr, and performing ultrasonic treatment in water bath; sequentially extruding the mixture to pass through 100nm and 80nm polycarbonate membranes to obtain the FITC labeled liposome modified by maleimide. Adding RSPO1 protein subjected to TCEP reduction into the prepared liposome under the protection of N2, reacting at 10 ℃, oscillating at constant temperature under the condition of 500rpm, and reacting for 24 hours to obtain the final product RSPO1 modified FITC labeled liposome. The particle size of the nanoparticles was measured using a laser scattering particle size analyzer (PCS), showing a particle size of 120. + -.10 nm.

(2) RSPO1 modified FITC labeled liposome in-vitro tumor cell targeting research

Collecting LoVo cell cultured in logarithmic phase, digesting the LoVo cell with 0.25% trypsin/EDTA, preparing single cell suspension with DMEM/F12 medium containing 20% fetal calf serum, and culturing at 4X10 per well⁵And inoculating each cell in a 12-hole cell culture plate with 2ml of each hole, transferring the culture plate into a carbon dioxide incubator, and culturing at 37 ℃, 5% carbon dioxide and saturated humidity to allow the cells to adhere to the wall. On alternate days, FITC-labeled liposomes and RSPO 1-modified FITC-labeled liposomes were prepared in a medium without fetal bovine serum. The culture medium in the culture plate is sucked out, FITC labeled liposome and RSPO1 modified FITC labeled liposome are added, incubation is carried out for 1h at 37 ℃, and the supernatant is sucked away. Washing the plate twice with precooled PBS solution, digesting the cells with 0.25% trypsin/EDTA, stopping digestion with DMEM/F12 medium containing 20% fetal calf serum, blowing the cells into single cell suspension, and then adding paraformaldehyde for fixing for 10 minutes. Cells were harvested by centrifugation, the supernatant was aspirated off, resuspended in PBS and repeated twice. Finally detecting with flow cytometerCell internalization in single cell suspensions. Referring to fig. 7, the results show that the uptake of RSPO1 modified liposomes by LoVo cells is much greater than that of unmodified liposomes.

Example 9 preparation of RSPO1-PEG-PLA and tumor cell targeting study

Preparation of RSPO 1-PEG-PLA:

(2) weighing PLA-PEG-MAL, dissolving in acetonitrile, rotary evaporating to form film, adding degassing HEPES salt solution, and hydrating to obtain micelle solution; adding TCEP-reduced RSPO1 into the micellar solution under the protection of N2; the reaction is carried out under the conditions that the temperature is 10 ℃ and the rotating speed is 500rpm and the constant temperature oscillation is carried out, and the final product RSPO1-PEG-PLA is obtained after 24 hours of reaction.

Preparation and in vitro targeting study of RSPO1 modified liposome:

RSPO1 modified doxorubicin liposomes were prepared and subjected to in vitro tumor cell targeting studies, according to the method of example 5. The result shows that the RSPO1 modified adriamycin liposome has stronger growth inhibition effect on colorectal cancer LoVo cells. Specifically, the inhibitory effect of the RSPO1 modified adriamycin liposome on colorectal cancer LoVo cells is about 5 times that of the unmodified liposome.

Example 10 preparation of RSPO1- (GGGGS) n-Pluronic and tumor cell targeting study

Preparation of RSPO1- (GGGGS) n-Pluronic:

(1) dissolving a carboxylated Pluronic copolymer in acetonitrile, performing rotary evaporation to form a film, and adding pure water to hydrate the film into a micelle solution; adding 1-ethyl-3 \ (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS), rotationally stirring at 200rpm for 15min to activate carboxyl, adding (GGGGS) N polypeptide, rotationally stirring at 200rpm for 2h, and adjusting the pH value to 7 by using sodium hydroxide to obtain (GGGGS) N-Pluronic micelles;

(2) adding 1-ethyl-3 (3-dimethylaminopropyl) -carbodiimide (EDC) and N- [ maleimidocaproic acid ] hydrazide trifluoroacetic acid (EMCH) into (GGGGS) N-Pluronic micelles, rotating and stirring at 200rpm for 15min to activate carboxyl, and adding TCEP reduced RSPO1 into the micelle solution under the protection of N2; oscillating at normal temperature for 2h to obtain the final product RSPO1- (GGGGS) n-Pluronic.

Preparation and in vitro targeting study of RSPO1 modified liposome:

RSPO1 modified doxorubicin liposomes were prepared and subjected to in vitro tumor cell targeting studies, according to the method of example 5. The result shows that the RSPO1 modified adriamycin liposome has stronger growth inhibition effect on colorectal cancer LoVo cells. Specifically, the inhibitory effect of the RSPO1 modified adriamycin liposome on colorectal cancer LoVo cells is 5 times higher than that of unmodified liposome.

Example 11 preparation of RSPO1-CpG ODN-lauric acid and tumor cell targeting study

Preparation of RSPO1-CpG ODN-lauric acid:

(1) and dissolving the alkynyl-modified CpG ODN, the copper sulfate and the sodium ascorbate powder in ultrapure water respectively, and shaking and mixing uniformly.

(2) And respectively dissolving azido lauric acid and TBTA powder in DMF, and shaking and mixing uniformly.

(3) And sequentially adding a copper sulfate solution, a TBTA solution, a triethylamine acetic acid buffer solution, a CpG ODN solution, an azido lauric acid solution and a sodium ascorbate solution into a centrifugal tube, shaking and uniformly mixing after adding each substance, sealing after completely adding, and shaking and reacting at room temperature overnight to obtain the CpG ODN-lauric acid.

(4) Mixing CpG ODN-lauric acid and PMPI (N- [ p-maleimide benzene ] isocyanate) solution, and adding RSPO1 after TCEP reduction; oscillating at normal temperature under the condition of the rotating speed of 500rpm, and reacting for 2 hours to obtain the final product RSPO1-CpG ODN-lauric acid.

Preparation and in vitro targeting study of RSPO1 modified liposome:

The inventor of the present invention has found, through the extensive and intensive studies, that a drug carrier is modified by coupling RSPO1 with a carrier molecule, and particularly, the RSPO1 and the carrier molecule are linked by a thioether bond, and the molar ratio of the RSPO1 to the carrier molecule is 1:1, it is particularly critical for RSPO1 to become an efficient "targeting head" for coupling to a carrier molecule. A large number of researches show that the RSPO1 on the surface of the drug carrier and the target drug delivery system containing the RSPO1 can directionally transport the drug carrier to Lgr5 high-expression tumor tissues with characteristics and high efficiency, the drug carrier distributed in the target tissues can be combined with target proteins on the surface of tumor cells, and the processes of endocytosis or drug release of the drug carrier and the like are induced, so that the drug effect is exerted. The RSPO1 modified targeting drug delivery system has stronger growth inhibition effect on Lgr5 high-expression tumor cells. Specifically, the RSPO1 modified targeting drug delivery system has about 5 times of the inhibition effect on Lgr5 high-expression tumor cells without the RSPO1 modified drug delivery system. The invention is not repeated for other carrier molecules in RSPO1 coupled carrier molecules, other drug carriers in targeted drug delivery systems and other drugs.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Claims

1. An RSPO1 conjugated carrier molecule having the formula: RSPO 1-carrier molecule, wherein the RSPO1 is connected with the carrier molecule through covalent bond, and the RSPO1 coupling carrier molecule has the structural formula: RSPO1-linker-R, wherein the RSPO1 is connected with the linker through a thioether bond, the linker is connected with the sulfhydryl group of the cysteine at the 6 th position at the N end of the RSPO1, the linker and the R are connected through covalent bonds, and the molar ratio of the RSPO1 to the linker to the R is 1: 1:1, the linker is selected from any one of PEG, short peptide and oligonucleotide, and the R is selected from any one of phospholipid, fatty acid and block copolymer.

2. The RSPO1 coupled carrier molecule according to claim 1, wherein the phospholipid is selected from any one of distearoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, heptadecylphosphatidylethanolamine, erucylphosphatidylethanolamine, hydrogenated soy phosphatidylethanolamine, hydrogenated egg phosphatidylethanolamine, soy phosphatidylethanolamine, or egg phosphatidylethanolamine.

3. The RSPO1 conjugated carrier molecule according to claim 1, wherein the fatty acid is selected from any one of caprylic acid C8, capric acid C10, lauric acid C12, myristic acid C14, palmitic acid C16, stearic acid C18.

4. The RSPO 1-coupled carrier molecule according to claim 1, wherein the block copolymer material is selected from any one of PLA, PLGA, PLG, Pluronic, PEO, PEI, PPO, PEG or a composite of two or more thereof.

5. The RSPO 1-coupled carrier molecule according to claim 1, wherein the molecular weight range of the polyethylene glycol is 400-8000 Da.

6. The RSPO 1-coupled carrier molecule according to claim 1, wherein the short peptide is selected from any one of Gn, (GGS) n, (GGGS) n, (GGGGS) n, RGD.

7. The RSPO1 coupled carrier molecule according to claim 1, wherein the molecular weight range of the oligonucleotide is 600-8000 Da.

8. A method for preparing the RSPO 1-coupled carrier molecule according to any one of claims 1 to 7, comprising the steps of: the preparation method comprises the following steps of carrying out covalent bond coupling reaction on RSPO1 and a carrier molecule linker-R.

9. Use of the RSPO1 conjugated carrier molecule according to any one of claims 1 to 7 in the preparation of a targeted drug delivery carrier or a targeted drug delivery system.

10. A targeted drug delivery carrier, which contains the RSPO1 coupled carrier molecule as described in any one of claims 1-7.

11. The targeted drug delivery carrier according to claim 10, which comprises: RSPO1 is coupled to carrier molecules as well as drug carrier particles.

12. The targeted drug delivery carrier of claim 11, wherein the drug carrier particles have RSPO1 on the surface.

13. The targeted drug delivery carrier of claim 11, wherein the surface of each drug carrier particle is 5-10000 RSPO 1.

14. The targeted drug delivery carrier according to claim 11, wherein the particle size of the drug carrier particle is in the range of 10 to 1000 nm.

15. The targeted drug delivery carrier according to claim 11, wherein the drug carrier is selected from any one of liposome, lipid nanoparticle and polymer nanoparticle.

16. The targeted delivery vehicle of claim 15, wherein the liposome is comprised of a member selected from the group consisting of egg phospholipids, hydrogenated soy phosphatidylcholine, hydrogenated egg phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1-stearoyl-2-linoleoyl phosphatidylcholine, dioleoyl phosphatidylcholine, hydrogenated dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, hydrogenated soy phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, Dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, brain phosphatidylserine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, egg phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, sphingomyelin, dipalmitoyl sphingomyelin or distearoyl sphingomyelin, cholesterol, dioleoxypropyltrimethylammonium chloride (DOTAP), dioleoylchlorotrimethylammonium chloride (DOTMA), dioctadecyldimethylammonium bromide (DDAB), dimethylaminoethylpropionyl-cholesterol (DC-Chol), octacosylamide 5-carboxyamidoacetate (DOGS), Dioleoylsuccinylcholinesteramide (DOSC), dioleoylcarboxylamidoghyldimethylammonium trifluoroacetate (DOSPA), or two or more thereof Combinations of (a) and (b).

17. The targeted drug delivery carrier of claim 15, wherein the polymeric nanoparticle comprises the following components: any one of PLA, PLGA, PLG, Pluronic, PEO, PEI, PPO, PEG, or a combination of two or more thereof.

18. The method for constructing the targeted drug delivery carrier according to any one of claims 10 to 17, which is selected from any one of the following:

the method comprises the following steps:

the second method, post-insertion method, includes the following steps:

(1) firstly, constructing a drug carrier;

(2) mixing RSPO1 coupled carrier molecule with the constructed drug carrier;

19. Use of a targeted drug delivery vector according to any one of claims 10 to 17 in the preparation of a targeted drug delivery system.

20. A targeted drug delivery system comprising the targeted drug delivery carrier according to any one of claims 10 to 17 and a drug molecule.

21. The targeted drug delivery system of claim 20, wherein the drug is selected from the group consisting of an anti-tumor drug; a photosensitizer; anti-inflammatory agents; an immunosuppressant; an antiviral drug.

22. The targeted drug delivery system of claim 20, wherein the drug is selected from the group consisting of a target gene, an antisense gene, a suicide gene, an apoptosis gene, a cytokine gene, siRNA, mRNA, and combinations thereof, or eukaryotic expression vector DNA containing the same.

23. A method of constructing a targeted delivery system as claimed in any one of claims 20 to 22, wherein any one of the following is selected:

the method comprises the following steps:

the second method, post-insertion method, includes the following steps:

(1) firstly, constructing a drug carrier carrying drugs;

24. Use of a targeted drug delivery system as claimed in any one of claims 20 to 22 in the preparation of ultrasound contrast agents, radiological contrast agents, nuclear medicine contrast agents.