CN113521303A - Nano vesicle jointly loaded with PD-L1 antibody and STING agonist, and preparation method and application thereof - Google Patents

Nano vesicle jointly loaded with PD-L1 antibody and STING agonist, and preparation method and application thereof Download PDF

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CN113521303A
CN113521303A CN202110769402.8A CN202110769402A CN113521303A CN 113521303 A CN113521303 A CN 113521303A CN 202110769402 A CN202110769402 A CN 202110769402A CN 113521303 A CN113521303 A CN 113521303A
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antibody
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徐�明
黄金生
郭焕玲
张春阳
帅心涛
谭洋
谢晓燕
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First Affiliated Hospital of Sun Yat Sen University
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Abstract

The invention provides a nano vesicle loaded with a PD-L1 antibody and a STING stimulant, which structurally comprises a shell and an inner core, wherein the shell is prepared from a lipid bilayer membrane, the shell is coupled with MMP-2 sensitive polypeptide chains, the polypeptide chains are coupled with a PD-L1 antibody, and the shell is also coupled with PEG; the inner core is STING agonist. The nano vesicle takes long-chain PEG as a protective barrier of an antibody, and prevents the PD-L1 antigen on the normal tissue of the PD-L1 antibody from being subjected to nonspecific combination; the liposome also has double sensitivity of pH and MMP-2, and because the coat liposome is also coupled with an MMP-2 sensitive peptide chain, when the vesicle reaches a residual tumor microenvironment with acidity and high MMP-2 concentration, a PEG layer and a PD-L1 antibody can be released in a response manner, the surface charge of the vesicle is changed from negative to positive after the release, and the endocytosis of antigen presenting cells to the carrier carrying the STING activator can be effectively promoted.

Description

Nano vesicle jointly loaded with PD-L1 antibody and STING agonist, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of tumor diagnosis and treatment, and particularly relates to a nano vesicle commonly loaded with a PD-L1 antibody and a STING agonist, and a preparation method and application thereof.
Background
The STING activator can effectively inhibit tumor growth, however, the STING activator can induce PD-L1 to be up-regulated after STING activation, so that immune escape occurs, the curative effect is limited, and the curative effect of synergistic treatment of the STING activator and the PD-L1 antibody is obviously superior to that of single-drug treatment. The STING activating drugs on the market at present are hydrophilic small-molecule drugs, have the problems of easy degradation and difficult encystment, are limited to intratumoral injection treatment, and are not suitable for cancer treatment which cannot be directly injected. Currently, there have been several studies designed to load STING activators with different carriers for anti-tumor therapy.
The existing research reports that the loading of the STING activator system cannot realize the loading together with the antibody. STING activators have completely different physicochemical properties and pharmacokinetic profiles than PD-L1 antibody. Furthermore, the sites of action of STING agonists and α PD-L1 are located intracellularly and extracellularly, respectively, which makes it difficult to control the ratio and distribution at the tumor site after administration of each, and thus may affect the effect of synergistic therapy. And the free PD-L1 antibody can be non-specifically bound to normal tissues expressing PD-L1, thereby causing immune-related toxic and side effects.
Disclosure of Invention
In order to solve the technical problems, the invention provides a nano vesicle capable of simultaneously loading a PD-L1 antibody and a STING agonist. So as to achieve co-intravenous delivery of the STING activator and the PD-L1 antibody, facilitating synergistic treatment of both drugs.
The invention realizes the purpose of the invention by the following technical scheme:
a nanovesicle loaded with both PD-L1 antibody and STING agonist, the structure of the nanovesicle comprising an outer shell and an inner core, the outer shell being made of lipid bilayer membrane, the outer shell being coupled to MMP-2 sensitive polypeptide chain, which is further coupled to PD-L1 antibody, the outer shell being further coupled to PEG; the inner core is a hydrophilic small molecule STING agonist. As can be seen in fig. 1, the PD-L1 antibody was linked to the outer shell by conjugation to MMP-2 sensitive polypeptide chains.
Preferably, the hydrophilic small molecule STING agonist is a salt of 5, 6-dimethylxanthine-4-acetic acid (DMXAA) which is converted to a hydrophilic sodium salt of 5, 6-dimethylxanthine-4-acetic acid. In the present invention: DMXAAst.
The nano vesicle can simultaneously load the PD-L1 antibody and the STING agonist because the PD-L1 antibody is coupled with the surface of the nano vesicle liposome through the MMP-2 sensitive short peptide chain, and finally the acid sensitive long-chain PEG is coupled on the surface of the outermost layer of the nano vesicle to be used as a shielding layer of the PD-L1 antibody and prevent the non-specific combination of the PD-L1 antibody and the PD-L1 molecule on a normal tissue.
The mechanism of the synergistic treatment of the tumor by combining the nano vesicle with the PD-L1 antibody and the STING agonist is as follows:
in one aspect, the acidic tumor microenvironment triggers acid sensitivity when the nanovesicles are enriched in tumor tissueWhen the PEG of the sensing shielding layer falls off, the MMP-2 protease which is overexpressed and exists in a tumor microenvironment can enzyme-cut the MMP-2 sensitive short peptide chain to release the PD-L1 antibody, so that the immunosuppression is relieved, and the anti-tumor immunity of T cells is reactivated. On the other hand, the release of PEG and antibody generates-NH2Causing the surface potential of the vesicle to switch from negative to positive, facilitating the endocytosis of the nanovesicle by macrophages and dendritic cells. In the acidic environment of intracellular lysosomes (pH 5.0), hydrophilic DMXAAst is converted into hydrophobic DMXAA, which is inserted into the liposomal membrane structure, inducing liposome reassembly, thereby rapidly releasing the drug (fig. 1), activating the STING pathway of macrophages and dendritic cells, and enhancing and maintaining tumor-specific T-immune responses after RFA surgery (radiofrequency ablation).
Preferably, the lipid bilayer membrane is made of distearoyl phosphatidylcholine (DSPC), Cholesterol (CHO), phospholipid-tetrapolyethylene glycol-diphenylcyclooctyne (DBCO-PEG)4-DSPE) and Distearoylphosphatidylethanolamine (DSPE), wherein the molar ratio of the four is, in order: 20:4:1:2.
Preferably, the amino acid sequence of the polypeptide chain coupled to the PD-L1 antibody comprises the sequence shown in SEQ ID NO. 1. The amino acid sequence shown in SEQ ID NO.1 is specifically: PLGVRG. The sequence 'PLGVRG' of the invention is the MMP-2 protease enzyme cutting site. Thus, it is only necessary that the polypeptide chain has the amino acid sequence shown in SEQ ID NO 1 that is recognized by the MMP-2 protease.
The invention also provides a preparation method of the nano-vesicle, which comprises the following steps:
step 1: under the action of a catalyst, carrying out esterification reaction on 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropionic acid (CDM) activated by oxalyl chloride and methoxy polyethylene glycol (mPEG-OH), after the reaction is finished, precipitating, carrying out suction filtration and washing on a reaction solution, and carrying out suction filtration again to obtain a polymer mPEG-CDM;
step 2: under the action of a catalyst, a peptide chain with an amino acid sequence shown as SEQ ID NO. 2 and 3-maleimidopropionic acid hydroxysuccinimide ester (3-Mal-NHS) are subjected to coupling reaction, and after the reaction is finished, reaction liquid is precipitated, dissolved and dialyzed, and is freeze-dried to obtain an MMP-2 sensitive short peptide chain;
and step 3: incubating the peptide chain in the step 2 with a PD-L1 antibody at room temperature, and coupling the peptide chain with a PD-L1 antibody to obtain an MMP-2 sensitive short peptide chain coupled with a PD-L1 antibody;
and 4, step 4: mixing distearoyl phosphatidylcholine (DSPC), Cholesterol (CHO), phospholipid-tetraethylene glycol-diphenyl cyclooctyne (DBCO-PEG)4-DSPE) and distearoyl phosphatidyl ethanolamine (DSPE) in a mixed solvent to prepare an outer shell of the nanovesicle;
adding 5, 6-dimethyl xanthine-4-sodium acetate (DMXAAst), performing ultrasonic treatment, and filtering to obtain a mixed solution A;
adding the short peptide chain in the step 3 into the mixed solution A, and performing vortex oscillation to obtain a mixed solution B;
adding a polymer mPEG-CDM into the mixed solution B, and performing vortex oscillation to obtain a mixed solution C;
after dialysis, ultrafiltration and concentration of the mixed solution C, nanovesicles according to claim 1 are obtained.
Preferably, the molar ratio of 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropionic acid (CDM) to methoxypolyethylene glycol (mPEG-OH) in step 1 is: 40: 1;
the molar ratio of the peptide chain to the 3-maleimidopropionic acid hydroxysuccinimide ester (3-Mal-NHS) in the step 2 is as follows: 1: 2.5;
the molar ratio of the peptide chain to the PD-L1 antibody in the step 3 is as follows: 1: 24;
the distearoyl phosphatidylcholine (DSPC), Cholesterol (CHO), phospholipid-tetrapolyethylene glycol-diphenyl cyclooctyne (DBCO-PEG) in the step 44-DSPE) and Distearoylphosphatidylethanolamine (DSPE) in the following order: 20:4:1: 2;
adding 5, 6-dimethyl xanthine-4-sodium acetate (DMXAAst) with a mass concentration of 4.1%;
adding the short peptide chain in the step 3 into the mixed solution A, wherein the mass concentration of the short peptide chain is 2%;
the mass concentration of the polymer mPEG-CDM added to the mixed solution B was 9.6%.
Preferably, the phospholipid-tetraethylene glycol-diphenylcyclooctyne (DBCO-PEG) in the step 44-DSPE) is prepared by the following process: under the action of a catalyst, dissolving distearoyl phosphatidyl ethanolamine (DSPE) and diphenylcyclooctyne-tetraethylene glycol-succinimide Ester (DBCO-PEG4-NHS Ester) in a mixed solvent, carrying out rotary evaporation, precipitating the reaction solution, centrifuging, collecting the precipitate, and carrying out vacuum drying to obtain the compound.
The invention also provides application of the nano-vesicle in preparation of a medicine for treating tumors.
The invention also provides a nano vesicle with double sensitivity of pH and MMP-2, which is characterized in that the structure of the nano vesicle comprises an outer shell and an inner core, wherein the outer shell is prepared from a lipid bilayer membrane, MMP-2 sensitive polypeptide chains are coupled on the outer shell, the polypeptide chains are coupled with a PD-L1 antibody, and the outer shell is also coupled with PEG; the inner core is an anti-tumor active drug.
Preferably, the polypeptide chain coupled to the PD-L1 antibody has an amino acid sequence comprising the sequence shown in SEQ ID NO. 1.
The invention has the beneficial effects that: the nano vesicle simultaneously loads an alpha PD-L1 antibody and DMXAAst, wherein the loading capacity of the PD-L1 antibody is 6.3%, the drug loading efficiency is 90.0%, the drug loading capacity of the DMXAAst is 10.3%, and the drug loading efficiency is 72.2%. The nano vesicle is subjected to long-chain PEG modification on the shell liposome, and the long-chain PEG is used as a protective barrier of an antibody to prevent the non-specific combination of a PD-L1 antibody and a PD-L1 molecule on a normal tissue, so that immune-related toxic and side effects in free PD-L1 antibody treatment are reduced; the nano vesicle also has double sensitivities of pH and MMP-2, and because the outer shell liposome is also coupled with the peptide chain sensitive to MMP-2, when the vesicle reaches the residual tumor microenvironment with acidity and high MMP-2 concentration, a PEG layer and a PD-L1 antibody can be released in a responsive way, the surface charge of the vesicle is changed from negative to positive after the release, the endocytosis of an antigen presenting cell to a STING activator can be effectively promoted, and the problem that free injection drugs are difficult to enter the vesicle is solved.
Drawings
FIG. 1 is a schematic diagram of the process of pH and MMP-2 double-sensitive nanovesicle PEG-CDM-alpha PD-L1/DMXAAst (noted as P-alpha PD-L1/D) releasing inner core drug in the tumor microenvironment.
Fig. 2 is a schematic diagram of pH and MMP-2 double sensitive nanovesicles PEG-CDM- α PD-L1/DMXAAst (P- α PD-L1/D) for tumor targeted co-delivery of α PD-L1 and DMXAAst and co-immunotherapy Radio Frequency Ablation (RFA) post-operative tumor recurrence and progression.
FIG. 3 is a fluorescence spectrum of P-alpha PD-L1-Cy3-FITC/DMXAAst nano-vesicle solution. The excitation wavelength is the maximum absorption wavelength of FITC of 480 nm. Incubation conditions were pH6.5+10nM MMP-2. The α PD-L1 and nanovesicles were labeled with Cy3 and FITC fluorophores, respectively. Pre means that the nano-drug is in solution at pH 7.4 before the addition of MMP-2 at pH6.5+10 nM.
FIG. 4 is a schematic diagram of laser confocal microscopy (CLSM) study of cellular uptake under various conditions. Hydrophilic rhodamine 6G (Rho6G) replaces DMXAAst to prepare P-alpha PD-L1/Rho6G nano vesicles. Bone Marrow Derived Macrophages (BMDMs) were mixed with PBS, free Rho6G or P-alpha PD-L1/Rho6G 1 h. The excitation and emission light of Rho6G were 550nm and 625nm, respectively. NPs refer to P-alpha PD-L1/Rho6G nano-drugs. Rho6G is displayed in red mode.
FIG. 5 is a tumor enrichment and biodistribution map of P- α PD-L1/ICG. (a) In-vivo fluorescence imaging traces the tumor enrichment process of the nano vesicles P-alpha PD-L1/ICG and alpha PD-L1/ICG. (b) Change in fluorescence intensity per unit area at tumor sites at different time points. (c-d) after injecting P-alpha PD-L1/ICG (indocyanine green, ICG) and alpha PD-L1/ICG nano vesicle for 24h in tail vein, (c) fluorescence imaging graph of mouse major organs and tumor and (d) statistical graph of fluorescence intensity per unit area. And (3) preparing two nano vesicles, namely P-alpha PD-L1/ICG and alpha PD-L1/ICG, by using a hydrophilic ICG fluorescent dye instead of DMXAAst. The injection dosage of ICG was 0.5mg/kg mouse body weight. CT26 subcutaneous transplanted tumor is indicated with red circles and yellow arrows refer to RFA ablated regions. Statistical analysis in b, fluorescence intensity at the same time point for both groups P- α PD-L1/ICG and α PD-L1/ICG, # P <0.05, # P <0.01 and # P < 0.0001.
Detailed Description
In order to show technical solutions, purposes and advantages of the present invention more concisely and clearly, the technical solutions of the present invention are described in detail below with reference to specific embodiments. Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Examples
The embodiment provides a preparation method of a nanovesicle loaded with a PD-L1 antibody (alpha PD-L1) and a STING agonist together, which specifically comprises the following steps:
1. preparation of PEG coupled to the outer shell of the Nanobubule (noted: mPEG-CDM)
(1) Activating 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropionic acid:
0.37g of 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropionic acid (CDM) and 50. mu. L N, N-dimethylformamide are dissolved in 5mL of dry dichloromethane and cooled in an ice-water bath. N is a radical of2Under an atmosphere, 1.27mL of oxalyl chloride was added dropwise to the reaction solution and stirred for 3 h. Dichloromethane and excess oxalyl chloride were removed by rotary evaporation to give a pale yellow liquid.
(2) 0.25g of methoxypolyethylene glycol (mPEG-OH, 5KDa, the polymerization degree n of ethylene glycol is 114) is dissolved in 4mL of anhydrous dichloromethane, and then the solution is dripped into the light yellow liquid in the step (1), and the mixture is cooled in an ice-water bath. N is a radical of2Under the atmosphere, 12mL of toluene, 12mg of 4-dimethylaminopyridine and 0.35mL of triethylamine were added to obtain mixed solutions, respectively. And after stirring for 12 hours, settling out the solid in the mixed solution by using ether, and performing suction filtration to obtain a filter cake. The filter cake was dissolved in 100mL of dichloromethane and then chlorinated with 15mL of 0.5M aqueous hydrochloric acid and 15mL of saturated hydrochloric acid, respectivelySodium saltAnd (4) washing with an aqueous solution. The dichloromethane after the liquid separation is respectively MgSO4Drying, filtering, concentrating, adding excessive ether, and performing suction filtration to obtain light yellow powder, namely mPEG-CDM (yield is 82%), wherein the reaction formula is as follows:
Figure BDA0003152200810000061
2. synthesis of MMP-2 sensitive short peptide chain (Mal-GGPLGVRGG-K (N)3)-NH2)
Taking 92.1mg GGPLGGRGK (N)3)-NH2(SEQ ID NO:2), 66.6mg of 3-maleimidopropanoic acid hydroxysuccinimide ester (3-Mal-NHS) and 50. mu. L N, N-diisopropylethylamine were dissolved in 1.5mL of N, N-Dimethylformamide (DMF) and stirred for 8 h. The reaction solution was precipitated in excess ether to give a pale yellow powder. The pale yellow powder was dissolved in 2mL of H2Adding 50 mu L of trifluoroacetic acid into the mixture, then putting the mixture into a dialysis bag, dialyzing the mixture for 1 day by using water, and freeze-drying the mixture to obtain white powder (yield: 75%), wherein the white powder is the MMP-2 sensitive short peptide chain. The reaction formula is as follows:
Figure BDA0003152200810000071
the PD-L1 antibody or Iso antibody was labeled with Cy3, and the experimental procedure was as follows: first, 60. mu.L of saturated NaHCO3The aqueous solution and 367. mu.g of sulfo-Cy3 NHS were added to a PBS solution (pH 6.5) containing 3.0mg of. alpha.PD-L1 or Iso antibody and stirred at room temperature for 1 h. A protein purification System comprising 2G 25 HiTrap desalting columns of 5mL in tandem (
Figure BDA0003152200810000073
GE Healthcare) to synthesize Cy 3-labeled antibodies (α PD-L1-Cy3 or Iso-Cy 3). Next, 2.5mg of. alpha.PD-L1-Cy 3 or. alpha.PD-L1 was added to the EDTA-containing PBS solution (20mM PBS, 30mM EDTA, pH 8.0), and 45. mu.g of tris (2-carboxyethyl) hydrogenophosphate was added thereto, followed by stirring at room temperature for 1 hour. Finally, 430. mu.g of Mal-GGPLGVRGG-K (N) was added3)-NH2Stirring at room temperature for 6h, and using a protein purification system equipped with 2G 25 HiTrap desalting columns of 5mL in series (
Figure BDA0003152200810000074
GE Healthcare) to synthesize MMP-2 sensitive short peptide chain labeled antibody according to the following reaction formula (wherein the molar ratio of peptide chain to PD-L1 antibody is: 1:24):
Figure BDA0003152200810000072
4. synthesis of phospholipid-Tetrapolyethylene glycol-Diphenylcyclooctyne (DBCO-PEG)4-DSPE)
195mg Distearoylphosphatidylethanolamine (DSPE), 187mg diphenylcyclooctyne-tetraethylene glycol-succinimidyl ester (DBCO-PEG)4-NHS Ester), 2mL dichloromethane, 2mL methanol and 83. mu. L N, N-diisopropylethylamine were transferred to the reaction flask and stirred for 12 h. After removing dichloromethane by rotary evaporation, excessive n-hexane and ether (v/v,1/1) are added dropwise; the precipitate was collected by centrifugation and dried under vacuum (yield 78%) to obtain phospholipid-tetraethylene glycol-diphenylcyclooctyne (DBCO-PEG)4-DSPE). The reaction formula is as follows:
Figure BDA0003152200810000081
5. preparation of nano-vesicles loaded with PD-L1 antibody and STING agonist
15.1mg Distearoylphosphatidylcholine (DSPC), 1.5mg Cholesterol (CHO), 1.23mg phospholipid-tetraethylene glycol-diphenylcyclooctyne (DBCO-PEG)4-DSPE) and 1.44mg Distearoylphosphatidylethanolamine (DSPE) in 20mL of dichloromethane/methanol/H2O (15/4/1, v/v/v). After removal of the solvent by rotary evaporation, 6mL of PBS (pH 7.4) containing 4.1mg of 5, 6-dimethylxanthine-4-acetic acid sodium salt (DMXAAst) was added and sonicated for 10 min. The sample solution loaded with DMXAAst was filtered through a filter membrane with a pore size of 400nm to remove large particle aggregates. Secondly, 2.0mg of alpha PD-L1-peptide-N is added3Or Iso-peptide-N3Vortex at room temperature for 8 h. Again, 9.6mg mPEG-CDM was dissolved in 3mL H2After O, add to the above solution and continue vortexing at room temperature for 4 h. Finally, the solution was dialyzed against PBS to remove free drug and mPEG, concentrated by ultrafiltration, and stored at 4 ℃ until use. Obtaining the nano-vesicle of the invention loaded with alpha PD-L1 and DMXAAst together.
The DMXAAst is prepared by converting DMXAA into sodium carboxylate thereof, and the method comprises the following steps:
282.29mg of DMXAA (5, 6-dimethylxanthine-4-acetic acid) was dissolved in 5mL of DMF (N, N-dimethylformamide), 40mg of NaOH (sodium hydroxide) and 20. mu.L of water were added thereto, the mixture was stirred for 5min, 50mL of diethyl ether was added thereto, and the precipitate was collected by centrifugation, washed with diethyl ether, and dried in vacuo to give DMXAAst (5, 6-dimethylxanthine-4-acetic acid sodium salt). DMXAA commodity information: merchant selelck, CAS No. 117570-53-3.
Experiment one: detecting drug loading rate of nano vesicle PD-L1 antibody and DMXAAst
The nano-vesicles are prepared by using Cy 3-labeled alpha PD-L1 (namely alpha PD-L1-Cy3), and the loading capacity of the alpha PD-L1 is 6.3% and the drug loading efficiency is 90.0% as measured by a fluorescence spectrophotometer (excitation and emission wavelengths are respectively set to be 530nm and 560 nm). The drug loading rate of DMXAAst is 10.3% and the drug loading efficiency is 72.2% by HPLC.
Experiment two: detecting whether the PD-L1 antibody on the nano vesicle is successfully linked to the nano vesicle through MMP-2 sensitive peptide Surface of
A solution of P- α PD-L1-Cy3-FITC/DMXAAst (pH6.5+10nM MMP-2) was prepared by the following procedure: 15.1mg distearoylphosphatidylcholine, 1.5mg cholesterol, 1.23mg phospholipid-tetraethylene glycol-diphenylcyclooctyne, and 1.44mg FITC-labeled distearoylphosphatidylethanolamine were dissolved in 20mL dichloromethane/methanol/H2O (15/4/1, v/v/v). After removal of the solvent by rotary evaporation, 6mL of PBS (pH 7.4) containing 4.1mg of DMXAAst was added and sonicated for 10 min. The sample solution loaded with DMXAAst was filtered through a filter membrane with a pore size of 400nm to remove large particle aggregates. Next, 2.0mg Cy 3-labeled α PD-L1-peptide-N was added3Vortex at room temperature for 8 h. Again, 9.6mg mPEG-CDM was dissolved in 3mL H2After O, add to the above solution and continue vortexing at room temperature for 4 h. Finally, the solution was dialyzed against PBS to remove free drug and mPEG, concentrated by ultrafiltration, and stored at 4 ℃ until use. The nano-vesicle jointly loaded with alpha PD-L1 and DMXAAst is obtained, the PD-L1 antibody in the vesicle is labeled by Cy3, and the outer shell of the nano-vesicle is labeled by FITC.
Fluorescence spectra of the nano vesicle solution FITC and Cy3 under irradiation of FITC with laser with the maximum excitation wavelength (480nm) were analyzed by a Fluorescence Resonance Energy Transfer (FRET) principle using a fluorescence spectrophotometer. The results are shown in FIG. 3As shown, at the start of incubation (0h), the fluorescence spectrum shows both FTIC (520nm) and Cy3(570nm) fluorescence peaks, indicating the occurrence of FRET, due to the fact that the distance between. alpha.PD-L1-Cy 3 and DSPE-FITC is smaller than that of
Figure BDA0003152200810000091
So that the result is; while the fluorescence intensity of FITC gradually increased and the fluorescence intensity of Cy3 gradually decreased with the increase of the incubation time until the detection became undetectable, indicating that FRET gradually failed due to the increase of the distance between α PD-L1-Cy3 and DSPE-FITC. This demonstrates that at the beginning of incubation, the antibody was successfully coupled to the nanovesicle via the short peptide of MMP-2, and after cleavage by MMP-2, the sensitive peptide was cleaved and α PD-L1-Cy3 was released, and thus FRET no longer occurred.
Experiment three: detecting the influence of nanovesicles on intracellular drug uptake
P-alpha PD-L1/Rho6G was prepared by replacing DMXAAst with a hydrophilic small molecule fluorescent dye rhodamine 6G (Rho6G) to investigate whether P-alpha PD-L1/Rho6G could effectively deliver hydrophilic drugs to bone marrow-derived macrophages (BMDMs) under tumor-mimicking conditions. The results are shown in FIG. 4. CLSM observations showed that nanocarrier-delivered drug resulted in a significant increase in intracellular drug aggregation compared to free Rho 6G.
Experiment four: detection of tumor accumulation and biodistribution of nanovesicles in vivo
Hydrophilic fluorescent molecule indocyanine green ICG is wrapped in polyethylene glycol PEG modified and PEG-free nano vesicles instead of DMXAAst, wherein P-alpha PD-L1/ICG and alpha PD-L1/ICG are adopted, and the injection dosage of ICG is 0.5mg/kg of mouse body weight. Establishing a mouse subcutaneous tumor incomplete ablation model, and tracking the distribution of the nano vesicles in the mouse by using an In Vivo Fluorescence Imaging System (IVFIS). As shown in FIG. 5, the tumor Fluorescence Intensity (FI) reached a maximum at 9h after tail vein injection in P- α PD-L1/ICG group mice and α PD-L1/ICG group mice. The fluorescence intensity of the P-alpha PD-L1/ICG group is significantly higher than that of the alpha PD-L1/ICG group. The reason is that the PEG shell layer in the P-alpha PD-L1/ICG can obviously reduce the non-targeting effect of the alpha PD-L1 and the phagocytic function of macrophages in circulation, thereby prolonging the circulation time.
Mice were sacrificed 9h after intravenous injection, tumors and vital organs dissected and imaged ex vivo. As shown in FIG. 5, the fluorescence intensity per unit area (mean FL) of the tumor in the P-alpha PD-L1/ICG group is significantly higher than that in the alpha PD-L1/ICG group, while the FI mean of the liver and spleen is significantly lower than that in the alpha PD-L1/ICG group. These results indicate that PEG modification can effectively protect PD-L1 antibody (α PD-L1), reduce its binding to normal organs expressing PD-L1, and thereby reduce non-targeted delivery of PD-L1 antibody-loaded nanovesicles.
Based on the experimental results of the second to fourth experiments, the invention shows that the nano vesicle loaded with the PD-L1 antibody and the STING agonist simultaneously takes the long-chain PEG as the protective barrier of the antibody, prevents the PD-L1 antibody from being non-specifically combined with the PD-L1 molecule on the normal tissue, and reduces the non-targeted delivery of the nano vesicle loaded with the PD-L1 antibody; because the PEG coupled on the outer shell and the antibody are coupled with the MMP-2 sensitive peptide chain, when the nano vesicle reaches a residual tumor microenvironment with acidity and high MMP-2 concentration, the nano vesicle can responsively release a PEG layer and a PD-L1 antibody, the surface charge of the released vesicle is changed from negative to positive, the endocytosis of the nano vesicle by an antigen presenting cell is effectively promoted, hydrophilic DMXAAst is converted into hydrophobic DMXAA in the acidic environment (pH 5.0) of an intracellular lysosome and is inserted into a liposome membrane structure to induce the liposome to be reassembled, so that the drug is rapidly released, and the problem that the free injected drug is difficult to enter the cell is solved.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> secondary first hospital of Zhongshan university
<120> nano vesicle jointly loaded with PD-L1 antibody and STING agonist, and preparation method and application thereof
<130> 6.23
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<170> PatentIn version 3.3
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Pro Leu Gly Val Arg Gly
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Gly Gly Pro Leu Gly Val Arg Gly Gly Xaa
1 5 10

Claims (10)

1. A nanovesicle, wherein the nanovesicle is loaded with a PD-L1 antibody and a STING agonist at the same time, and the structure of the nanovesicle comprises an outer shell and an inner core, wherein the outer shell is prepared from a lipid bilayer membrane, the outer shell is coupled with a polypeptide chain sensitive to MMP-2, the polypeptide chain is further coupled with a PD-L1 antibody, and the outer shell is further coupled with PEG; the inner core is a hydrophilic small molecule STING agonist.
2. The nanovesicle of claim 1, wherein said hydrophilic small molecule STING agonist is 5, 6-dimethylxanthine-4-acetic acid sodium salt.
3. The nanovesicle of claim 1, wherein said lipid bilayer membrane consists of distearoylphosphatidylcholine, cholesterol, phospholipid-tetraethylene glycol-diphenylcyclooctyne, and distearoylphosphatidylethanolamine, wherein the molar ratio of the four is, in order: 20:4:1:2.
4. The nanovesicle of claim 1, wherein the polypeptide chain coupled to the PD-L1 antibody comprises an amino acid sequence comprising the sequence set forth in SEQ ID No. 1.
5. A method for preparing the nanovesicle of any one of claims 1-4, comprising the steps of:
step 1: under the action of a catalyst, carrying out esterification reaction on 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropionic acid activated by oxalyl chloride and methoxypolyethylene glycol, precipitating, filtering, washing and filtering a reaction solution to obtain mPEG-CDM after the reaction is finished;
step 2: under the action of a catalyst, a peptide chain with an amino acid sequence shown as SEQ ID NO. 2 and 3-maleimide hydroxyl propionic acid succinimide ester are subjected to coupling reaction, and after the reaction is finished, reaction liquid is precipitated, dissolved, dialyzed and freeze-dried to obtain an MMP-2 sensitive short peptide chain;
and step 3: incubating the peptide chain in the step 2 with a PD-L1 antibody at room temperature, and coupling the peptide chain with a PD-L1 antibody to obtain an MMP-2 sensitive short peptide chain coupled with a PD-L1 antibody;
and 4, step 4: dissolving distearoyl phosphatidylcholine, cholesterol, phospholipid-tetraethylene glycol-diphenyl cyclooctyne and distearoyl phosphatidylethanolamine in a mixed solvent to prepare the shell of the nano vesicle;
adding 5, 6-dimethyl xanthine-4-sodium acetate, performing ultrasonic treatment, and filtering to obtain a mixed solution A;
adding the short peptide chain in the step 3 into the mixed solution A, and performing vortex oscillation to obtain a mixed solution B;
adding mPEG-CDM into the mixed solution B, and performing vortex oscillation to obtain a mixed solution C;
and (3) dialyzing, ultrafiltering and concentrating the mixed solution C to obtain the nano vesicles.
6. The method of claim 5, wherein the molar ratio of 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropionic acid to methoxypolyethylene glycol in step 1 is: 40: 1;
the molar ratio of the peptide chain to the 3-maleimidopropionic acid hydroxysuccinimide ester in the step 2 is as follows: 1: 2.5;
the molar ratio of the peptide chain to the PD-L1 antibody in the step 3 is as follows: 1: 24;
the molar ratios of the distearoyl phosphatidylcholine, the cholesterol, the phospholipid-tetraethylene glycol-diphenyl cyclooctyne and the distearoyl phosphatidylethanolamine in the step 4 are as follows in sequence: 20:4:1: 2;
adding 5, 6-dimethyl xanthine-4-sodium acetate with mass concentration of 4.1%;
adding the short peptide chain in the step 3 into the mixed solution A, wherein the mass concentration of the short peptide chain is 2%;
the mass concentration of the polymer mPEG-CDM added to the mixed solution B was 9.6%.
7. The method according to claim 5, wherein the phospholipid-tetrapolyethylene glycol-diphenylcyclooctyne in the step 4 is prepared by: under the action of a catalyst, dissolving distearoyl phosphatidyl ethanolamine and diphenyl cyclooctyne-tetraethylene glycol-succinimide ester in a mixed solvent, carrying out rotary evaporation, precipitating the reaction solution, centrifuging, collecting the precipitate, and carrying out vacuum drying to obtain the distearoyl phosphatidyl ethanolamine.
8. Use of nanovesicles of claim 1 in the preparation of a medicament for the treatment of a tumor.
9. A nano-vesicle with double sensitivity to pH and MMP-2 is characterized in that the structure of the nano-vesicle comprises an outer shell and an inner core, wherein the outer shell is prepared from a lipid bilayer membrane, MMP-2 sensitive polypeptide chains are coupled to the outer shell, the polypeptide chains are coupled with a PD-L1 antibody, and the outer shell is also coupled with PEG; the inner core is hydrophilic anti-tumor active medicine.
10. The nanovesicle of claim 8, wherein the polypeptide chain coupled to the PD-L1 antibody comprises an amino acid sequence as set forth in SEQ ID No. 1.
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