CN113521303B - Nanometer vesicle loaded with PD-L1 antibody and STING agonist together and preparation method and application thereof - Google Patents

Nanometer vesicle loaded with PD-L1 antibody and STING agonist together and preparation method and application thereof Download PDF

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CN113521303B
CN113521303B CN202110769402.8A CN202110769402A CN113521303B CN 113521303 B CN113521303 B CN 113521303B CN 202110769402 A CN202110769402 A CN 202110769402A CN 113521303 B CN113521303 B CN 113521303B
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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 agonist, which structurally comprises a shell and an inner core, wherein the shell is prepared from a lipid bilayer membrane, the shell is coupled with a polypeptide chain sensitive to MMP-2, the polypeptide chain is coupled with the 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 the antibody to prevent the non-specific binding of PD-L1 antigen on the normal tissue of the PD-L1 antibody; the liposome has double sensitivity of pH and MMP-2, and because the outer shell liposome is also coupled with a peptide chain sensitive to MMP-2, when the vesicle reaches the acidic and high MMP-2 concentration residual tumor microenvironment, the PEG layer and the PD-L1 antibody can be released in a responsive way, and after the PEG layer and the PD-L1 antibody are released, the surface charge of the vesicle changes from negative to positive, so that the endocytosis of an antigen presenting cell on a carrier carrying a STING activator can be effectively promoted.

Description

Nanometer vesicle loaded with PD-L1 antibody and STING agonist together 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 loaded with a PD-L1 antibody and a STING agonist together, and a preparation method and application thereof.
Background
The STING activator can effectively inhibit tumor growth, however, the STING can induce PD-L1 to be up-regulated after activation, and immune escape occurs, so that the curative effect is limited, and the curative effect of synergistic treatment of the STING activator and the PD-L1 antibody is obviously better than that of single-drug treatment. The STING activating medicines on the market at present are all hydrophilic micromolecular medicines, have the problems of easy degradation and difficult cell entry, are limited to intratumoral injection treatment, and are not suitable for the treatment of cancers which can not be directly injected. At present, various studies have been conducted to design different carriers for carrying STING activators for anti-tumor treatment.
Current studies report that STING activator systems cannot achieve co-loading with antibodies. STING activators have completely different physicochemical properties and pharmacokinetic characteristics than PD-L1 antibodies. Furthermore, the sites of action of STING agonists and αpd-L1 are located on the intracellular and extracellular membranes, respectively, which makes it difficult to control the proportion and distribution thereof at the tumor site after the respective administration, thereby possibly affecting the effect of the co-therapy. And the free PD-L1 antibody treatment can be non-specifically bound to normal tissues expressing PD-L1, thereby causing immune-related toxic side effects.
Disclosure of Invention
In order to solve the technical problems, the invention provides a nano vesicle capable of loading a PD-L1 antibody and a STING agonist simultaneously. So as to realize the common intravenous delivery of STING activator and PD-L1 antibody, and is beneficial to the cooperative treatment of the two medicaments.
The invention achieves the aim of the invention through the following technical scheme:
a nanovesicle loaded with both a PD-L1 antibody and a STING agonist, the structure of the nanovesicle comprising a shell and an inner core, the shell being prepared from a lipid bilayer membrane, the shell being coupled to an MMP-2 sensitive polypeptide chain, the polypeptide chain being further coupled to the PD-L1 antibody, the shell being further coupled to PEG; the inner core is a hydrophilic small molecule STING agonist. As can be seen from FIG. 1, the PD-L1 antibody is linked to the outer shell by conjugation to an MMP-2 sensitive polypeptide chain.
Preferably, the hydrophilic small molecule STING agonist is 5, 6-dimethylxanthine-4-acetate sodium salt salified from 5, 6-dimethylxanthine-4-acetate (DMXAA) to hydrophilic. In the present invention, it is noted that: dmxaash.
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 surface of the outermost layer of the nano vesicle is coupled with the acid sensitive long-chain PEG which is used as a shielding layer of the PD-L1 antibody to prevent the PD-L1 antibody from being non-specifically combined with the PD-L1 molecule on normal tissues.
The mechanism of the synergistic treatment of tumor by combining the nano vesicle with the PD-L1 antibody and the STING agonist is as follows:
on the one hand, after the nano vesicles are enriched in tumor tissues, the acidic tumor microenvironment triggers the shedding of the acid sensitive shielding layer PEG, and the over-expressed MMP-2 protease existing in the tumor microenvironment can cleave the short peptide chain sensitive to the MMP-2 to release the PD-L1 antibody, so that the immunosuppression is relieved, and the anti-tumor immunity of the T cells is re-activated. On the other hand, release of PEG and antibodies produces-NH 2 The surface potential of the vesicle is reversed from electronegativity to electropositivity, and the endocytosis of the nano vesicle by macrophages and dendritic cells is promoted. In a fine mannerIn the acidic environment of the endolysosome (pH 5.0), hydrophilic dmxaash is converted to hydrophobic DMXAA, which is inserted into the liposome membrane structure, inducing liposome reassembly, thereby rapidly releasing the drug (fig. 1), activating STING pathways of macrophages and dendritic cells, and enhancing and maintaining tumor-specific T immune responses after RFA (radiofrequency ablation).
Preferably, the lipid bilayer membrane is composed of distearoyl phosphatidylcholine (DSPC), cholesterol (CHO), phospholipid-tetra-polyethylene glycol-diphenyl cyclooctyne (DBCO-PEG) 4 -DSPE) and distearoyl phosphatidylethanolamine (DSPE), wherein the molar ratio of the four is in turn: 20:4:1:2.
Preferably, the polypeptide chain coupled to the PD-L1 antibody has an amino acid sequence comprising a sequence as shown in SEQ ID NO. 1. The amino acid sequence shown in SEQ ID NO.1 is specifically: PLGVRG. The sequence of the invention "PLGVGG" is the MMP-2 protease cleavage site. Thus, the amino acid sequence shown in SEQ ID NO.1 can be recognized by MMP-2 protease as long as the polypeptide chain contains the amino acid sequence.
The invention also provides a preparation method of the nano vesicle, which comprises the following steps:
step 1: under the action of a catalyst, 2, 5-dihydroxyl-4-methyl-2, 5-dioxo-3-furanpropionic acid (CDM) activated by oxalyl chloride and methoxypolyethylene glycol (mPEG-OH) are subjected to esterification reaction, and after the reaction is finished, the reaction solution is precipitated, filtered and washed, and then filtered by suction 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) undergo a coupling reaction, and after the reaction is finished, the reaction solution is precipitated, dissolved and dialyzed, and then an MMP-2 sensitive short peptide chain is obtained after freeze drying;
step 3: incubating the peptide chain in the step 2 with a PD-L1 antibody at room temperature, and coupling the peptide chain with the PD-L1 antibody to obtain a short peptide chain sensitive to MMP-2 coupled with the PD-L1 antibody;
step 4: distearoyl phosphatidylcholine (DSPC), cholesterol (CHO), phospholipid-tetrapolyethylene glycol-diphenylcyclooctyne (DBCO-PEG) 4 -DSPE) and distearoyl phosphatidylethanolamine (DSPE) in a mixed solvent to produce the 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 carrying out vortex oscillation to obtain a mixed solution B;
adding a polymer mPEG-CDM into the mixed solution B, and carrying out 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-diphenylcyclooctyne (DBCO-PEG) described in step 4 4 -DSPE) and distearoyl phosphatidylethanolamine (DSPE) in the following order: 20:4:1:2;
adding 4.1% of 5, 6-dimethyl xanthine-4-sodium acetate (DMXAAst) by mass concentration;
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, in the step 4, the phospholipid-tetra-polyethylene glycol-diphenyl cyclooctyne (DBCO-PEG) 4 -DSPE) is prepared by the following method: under the action of a catalyst, distearoyl phosphatidylethanolamine (DSPE) and diphenyl cyclooctyne-tetra polyethylene glycol-succinimidyl Ester (DBCO-PEG 4-NHS Ester) are dissolved in a mixed solvent, and after rotary evaporation, the reaction solution is precipitated, and the precipitate is centrifugally collected and dried in vacuum, thus obtaining the catalyst.
The invention also provides application of the nano vesicle in preparing a medicament for treating tumors.
The invention also provides a pH and MMP-2 dual-sensitivity nano vesicle, which is characterized in that the structure of the nano vesicle comprises a shell and an inner core, wherein the shell is prepared from a lipid bilayer membrane, a polypeptide chain sensitive to MMP-2 is coupled on the shell, the polypeptide chain is coupled with a PD-L1 antibody, and the shell is also coupled with PEG; the inner core is an antitumor active drug.
Preferably, the polypeptide chain coupled to the PD-L1 antibody has an amino acid sequence comprising a sequence as shown in SEQ ID NO. 1.
The beneficial effects of the invention are as follows: the nano vesicle is loaded with the alpha PD-L1 antibody and the DMXAAst at the same time, 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 modified by long-chain PEG in the shell liposome, and long-chain PEG is used as a protective barrier of the antibody to prevent the PD-L1 antibody from non-specifically combining with the PD-L1 molecule on normal tissues, so that the immune-related toxic and side effects of free PD-L1 antibody treatment are reduced; the nano vesicle of the invention also has double sensitivity of pH and MMP-2, as the outer shell liposome is also coupled with a peptide chain sensitive to MMP-2, when the vesicle reaches the acidic and high MMP-2 concentration residual tumor microenvironment, the PEG layer and the PD-L1 antibody can be released in a responsive way, and the surface charge of the vesicle changes from negative to positive after the release, thereby effectively promoting the endocytosis of an antigen presenting cell to an STING activator and solving the problem that a free injection medicine is difficult to enter the cell.
Drawings
FIG. 1 is a schematic diagram of the release of core drug in tumor microenvironment by pH and MMP-2 double-sensitive nanovesicles PEG-CDM-alpha PD-L1/DMXAAst (denoted as P-alpha PD-L1/D).
FIG. 2 is a schematic representation of recurrence and progression of pH and MMP-2 double-sensitive nanovesicles PEG-CDM-alpha PD-L1/DMXAAst (P-alpha PD-L1/D) for tumor targeted co-delivery of alpha PD-L1 and DMXAAst and for co-immunotherapy radiofrequency ablation (radiofrequency ablation, RFA) post-operative tumors.
FIG. 3 is a fluorescence spectrum of P-. Alpha.PD-L1-Cy 3-FITC/DMXAAst nanovesicle solution. The excitation wavelength was 480nm of the maximum absorption wavelength of FITC. Incubation conditions were pH6.5+10nM MMP-2. The αpd-L1 and nanovesicles were labeled with Cy3 and FITC fluorophores, respectively. Pre refers to the nano-drug in a solution at pH 7.4 prior to addition of MMP-2 at pH6.5+10 nM.
FIG. 4 is a schematic diagram of cell uptake under different conditions of a laser confocal microscope (CLSM) study. Hydrophilic rhodamine 6G (Rho 6G) replaces DMXAAst to prepare P-alpha PD-L1/Rho6G nanovesicles. Bone Marrow Derived Macrophages (BMDMs) were incubated with PBS, free Rho6G or P-. Alpha.PD-L1/Rho 6G 1h. Rho6G excitation and emission were 550nm and 625nm, respectively. NPs refer to P-alpha PD-L1/Rho6G nanomedicines. Rho6G is shown in red light mode.
FIG. 5 is a tumor enrichment and biological profile of P-alpha PD-L1/ICG. (a) Tumor enrichment process of the nano vesicles P-alpha PD-L1/ICG and alpha PD-L1/ICG is tracked by in vivo fluorescence imaging. (b) Change in fluorescence intensity per unit area of tumor site at different time points. (c-d) fluorescent imaging of major organs and tumors of mice and (d) statistical plot of fluorescence intensity per unit area after 24h of tail vein injection of P-alpha PD-L1/ICG (indocyanine green (indocyanine green), ICG) and alpha PD-L1/ICG nanovesicles. Hydrophilic ICG fluorescent dye replaces DMXAAst to prepare P-alpha PD-L1/ICG and alpha PD-L1/ICG nano vesicles. ICG was injected at a dose of 0.5mg/kg of mouse body weight. CT26 subcutaneous tumors were indicated with red circles and yellow arrows indicate RFA ablation areas. The fluorescence intensities of the two groups P- αpd-L1/ICG and αpd-L1/ICG in panel b at the same time points were statistically analyzed, P <0.05, P <0.01 and P <0.0001.
Detailed Description
In order to more clearly demonstrate the technical scheme, objects and advantages of the present invention, the technical scheme of the present invention is described in detail below with reference to the specific embodiments. Unless otherwise specified, all reagents involved in the examples of the present invention are commercially available products and are commercially available.
Examples
The embodiment provides a preparation method of a nano vesicle 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 nanovesicle (designated mPEG-CDM)
(1) Activating 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropanoic acid:
0.37g of 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropionic acid (CDM) and 50. Mu. L N, N-dimethylformamide were dissolved in 5mL of dry dichloromethane and cooled in an ice-water bath. N (N) 2 Under an atmosphere, 1.27mL of oxalyl chloride was added dropwise to the reaction solution and stirred for 3 hours. The 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, degree of polymerization n of ethylene glycol of 114) was dissolved in 4mL of anhydrous dichloromethane, and then added dropwise to the pale yellow liquid of step (1), followed by cooling in an ice water bath. N (N) 2 Under an atmosphere, 12mL of toluene, 12mg of 4-dimethylaminopyridine and 0.35mL of triethylamine were added, respectively, to obtain a mixed solution. After stirring for 12h, the solids in the mixed solution are settled out by diethyl ether, and a filter cake is obtained by suction filtration. After dissolving the filter cake in 100mL of dichloromethane, 15mL of 0.5M aqueous hydrochloric acid and 15mL of saturated chlorination were used, respectivelySodium saltWashing with aqueous solution. Separating dichloromethane with MgSO 4 Drying, filtering, concentrating, adding excessive diethyl ether, and suction filtering to obtain pale yellow powder, namely mPEG-CDM (yield 82%), wherein the reaction formula is as follows:
2. synthesis of MMP-2 sensitive short peptide chain (Mal-GGPLGVRGG-K (N) 3 )-NH 2 )
92.1mg GGPLGVGRGGK (N) 3 )-NH 2 (SEQ ID NO: 2), 66.6mg of hydroxysuccinimide 3-maleimidopropionate (3-Mal-NHS) and 50. Mu. L N, N-diisopropylethylamine was dissolved in 1.5mL of N, N-Dimethylformamide (DMF) and stirred for 8h. The reaction solution was precipitated in an excessive amount of diethyl ether to obtain pale yellow powder. Dissolve pale yellow powder in 2mL H 2 O and 50 mu L of trifluoroacetic acid are added, and then the mixture is put into a dialysis bag and dialyzed by waterLyophilization yielded a white powder (yield: 75%) that was MMP-2 sensitive short peptide chains over 1 day. The reaction formula is as follows:
the PD-L1 antibody or the Iso antibody is labeled with Cy3, and the experimental steps are as follows: first, 60. Mu.L of saturated NaHCO 3 Aqueous solution and 367. Mu.g of sulfo-Cy3 NHS were added to PBS solution (pH 6.5) containing 3.0mg of alpha PD-L1 or Iso antibody and stirred at room temperature for 1h. Protein purification system equipped with 25 mL G25 HiTrap desalting columns in series connectionGE Healthcare) to synthesize Cy 3-labeled antibodies (. Alpha.PD-L1-Cy 3 or Iso-Cy 3). Next, 2.5mg of αPD-L1-Cy3 or αPD-L1 was added to the PBS solution containing EDTA (20mM PBS,30mM EDTA,pH 8.0), 45. Mu.g of tris (2-carboxyethyl) phosphine hydrochloride was further added thereto, and the mixture was stirred at room temperature for 1 hour. Finally, 430. Mu.g Mal-GGPLGVRGG-K (N) 3 )-NH 2 Stirring at room temperature for 6h, and adopting a protein purification system with 25 mL G25 HiTrap desalting columns connected in seriesGE Healthcare) to synthesize MMP-2 sensitive short peptide chain-labeled antibodies, the reaction formula (wherein the molar ratio of peptide chain to PD-L1 antibody is: 1:24):
4. synthesis of phospholipid-tetrapolyethylene glycol-diphenylcyclooctyne (DBCO-PEG) 4 -DSPE)
195mg distearoyl phosphatidylethanolamine (DSPE), 187mg diphenylcyclooctyne-tetrapolyethylene glycol-succinimidyl ester (DBCO-PEG) 4 NHS Ester), 2mL of dichloromethane, 2mL of methanol and 83. Mu. L N, N-diisopropylethylamine was transferred to the reaction flask and stirred for 12h. RotatingAfter removing the methylene chloride by evaporation, excess n-hexane and diethyl ether (v/v, 1/1) were added dropwise; the precipitate was collected by centrifugation and dried in vacuo (yield 78%) to give phospholipid-tetrapolyethylene glycol-diphenylcyclooctyne (DBCO-PEG) 4 -DSPE). The reaction formula is as follows:
5. preparation of nanovesicles loaded with PD-L1 antibody and STING agonist
15.1mg distearoyl phosphatidylcholine (DSPC), 1.5mg Cholesterol (CHO), 1.23mg phospholipid-tetrapolyethylene glycol-diphenylcyclooctyne (DBCO-PEG) 4 -DSPE) and 1.44mg distearoyl phosphatidylethanolamine (DSPE) in 20mL dichloromethane/methanol/H 2 O (15/4/1, v/v/v) in a mixed solvent. After removal of the solvent by rotary evaporation, 6mL PBS (pH 7.4) containing 4.1mg of 5, 6-dimethylxanthine-4-acetate sodium salt (DMXAAst) was added and sonicated for 10min. The dmxaash loaded sample solution was filtered through a 400nm pore size filter to remove large particle aggregates. Next, 2.0mg of alpha PD-L1-peptide-N is added 3 Or Iso-peptide-N 3 Vortex was continued for 8h at room temperature. Again, 9.6mg of mPEG-CDM was dissolved in 3mL H 2 After O, add to the solution and continue to vortex for 4h at room temperature. Finally, the solution is dialyzed in PBS to remove free drugs and mPEG, ultrafiltered and concentrated, and stored at 4 ℃ for standby. The nano vesicles of the invention loaded with alpha PD-L1 and DMXAAst together are obtained.
The DMXAAst is prepared by converting DMXAA into sodium carboxylate, and the method comprises the following steps:
after 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, stirred for 5 minutes, 50mL of diethyl ether was added, and the precipitate was collected by centrifugation, washed with diethyl ether and dried under vacuum to give DMXAAst (5, 6-dimethylxanthine-4-acetic acid sodium salt). DMXAA commodity information: merchant selectk, CAS No.117570-53-3.
Experiment one: detection of drug-loading amount of nanovesicle PD-L1 antibody and DMXAAst
The nano vesicle is prepared by adopting Cy 3-marked alpha PD-L1 (namely alpha PD-L1-Cy 3), the loading capacity of the alpha PD-L1 is 6.3 percent and the drug loading efficiency is 90.0 percent measured by a fluorescence spectrophotometer (the excitation wavelength and the emission wavelength are respectively set to 530nm and 560 nm). And the drug loading rate of DMXAAst is 10.3% and the drug loading efficiency is 72.2% as measured by HPLC.
Experiment II: detection of whether PD-L1 antibodies on nanovesicles were successfully linked to nanovesicles by MMP-2 sensitive peptides Surface of the body
P-. Alpha.PD-L1-Cy 3-FITC/DMXAAst solution (pH6.5+10 nM MMP-2) was prepared as follows: 15.1mg distearoyl phosphatidylcholine, 1.5mg cholesterol, 1.23mg phospholipid-tetrapolyethylene glycol-diphenylcyclooctyne and 1.44mg FITC-labeled distearoyl phosphatidylethanolamine were dissolved in 20mL dichloromethane/methanol/H 2 O (15/4/1, v/v/v) in a mixed solvent. After removal of the solvent by rotary evaporation, 6mL PBS (pH 7.4) containing 4.1mg DMXAAst was added and sonicated for 10min. The dmxaash loaded sample solution was filtered through a 400nm pore size filter to remove large particle aggregates. Next, 2.0mg of Cy3-labeled alpha PD-L1-peptide-N was added 3 Vortex was continued for 8h at room temperature. Again, 9.6mg of mPEG-CDM was dissolved in 3mL H 2 After O, add to the solution and continue to vortex for 4h at room temperature. Finally, the solution is dialyzed in PBS to remove free drugs and mPEG, ultrafiltered and concentrated, and stored at 4 ℃ for standby. The nano vesicles loaded with alpha PD-L1 and DMXAAst together are obtained, PD-L1 antibodies in the vesicles are marked by Cy3, and the outer shells of the nano vesicles are marked by FITC.
Fluorescence spectra of the nanovesicle solutions FITC and Cy3 were analyzed by fluorescence spectrophotometer under irradiation of laser at the maximum excitation wavelength of FITC (480 nm) using Fluorescence Resonance Energy Transfer (FRET) principle. As a result, as shown in FIG. 3, at the beginning of incubation (0 h), fluorescence spectra simultaneously show fluorescence peaks of FTIC (520 nm) and Cy3 (570 nm), indicating the occurrence of FRET, since the distance between αPD-L1-Cy3 and DSPE-FITC is smaller thanResulting in that; whereas, as the incubation time is prolonged, the fluorescence intensity of FITC is gradually increased,the fluorescence intensity of Cy3 gradually decreased until undetectable, indicating that FRET gradually failed due to the increasing distance between αPD-L1-Cy3 and DSPE-FITC. This demonstrates that at the beginning of incubation, the antibody was successfully coupled to the nanovesicles via MMP-2 short peptide, whereas after cleavage by MMP-2, the sensitive peptide was cleaved and alpha PD-L1-Cy3 was released, so FRET no longer occurs.
Experiment III: detecting the influence of nanovesicles on intracellular drug uptake
P-alpha PD-L1/Rho6G was prepared using the hydrophilic small molecule fluorescent dye rhodamine 6G (Rho 6G) instead of DMXAAst to investigate whether P-alpha PD-L1/Rho6G could effectively deliver hydrophilic drugs into Bone Marrow Derived Macrophages (BMDMs) under tumor mimetic conditions. The results are shown in FIG. 4. The CLSM observations showed that nanocarriers deliver drug that significantly increased intracellular drug aggregation compared to free Rho 6G.
Experiment IV: detection of tumor accumulation and biodistribution of nanovesicles in vivo
The hydrophilic fluorescent molecule indocyanine green ICG is coated in polyethylene glycol PEG modified and PEG-free nano vesicles instead of DMXAAst, wherein the injection dosages of P-alpha PD-L1/ICG and alpha PD-L1/ICG are respectively 0.5mg/kg body weight of the mouse. An incomplete ablation model of the subcutaneous tumor of the mouse is established, and the distribution of the nano vesicles in the mouse is tracked by using an In Vivo Fluorescence Imaging System (IVFIS). As a result, as shown in FIG. 5, tumor Fluorescence Intensities (FI) in mice of the P-. Alpha.PD-L1/ICG group and mice of the αPD-L1/ICG group were maximized 9h after the tail vein injection. The fluorescence intensity of the P-alpha PD-L1/ICG group is obviously higher than that of the alpha PD-L1/ICG group. The reason for this is that the PEG shell in P-alpha PD-L1/ICG can significantly reduce the non-targeting effect of alpha PD-L1 and phagocytic function of macrophages in circulation, thereby prolonging circulation time.
Mice were sacrificed 9h after intravenous injection, tumors and vital organs were dissected and imaged ex vivo. As shown in FIG. 5, the fluorescence intensity (mean FL) per unit area of the tumors in the P-alpha PD-L1/ICG group is obviously higher than that in the alpha PD-L1/ICG group, and the mean value of the livers and spleens FI is obviously lower than that in the alpha PD-L1/ICG group. These results indicate that PEG modification is effective in protecting PD-L1 antibody (αpd-L1), reducing its binding to normal organs expressing PD-L1, thereby reducing non-targeted delivery of PD-L1 antibody-loaded nanovesicles.
Based on the experimental results of the experiments two to four, the nano vesicles loaded with the PD-L1 antibody and the STING agonist simultaneously take long-chain PEG as a protective barrier of the antibody, prevent the PD-L1 antibody from being non-specifically combined with PD-L1 molecules on normal tissues, and reduce non-targeted delivery of the nano vesicles loaded with the PD-L1 antibody; because PEG and antibody coupled on the shell are coupled with MMP-2 sensitive peptide chains, when the nano vesicle reaches the acidic and high MMP-2 concentration residual tumor microenvironment, the nano vesicle can responsively release the PEG layer and the PD-L1 antibody, the surface charge of the released vesicle is changed from negative to positive, the endocytosis of antigen presenting cells on the nano vesicle is effectively promoted, and in the acidic environment (pH 5.0) of an intracellular lysosome, hydrophilic DMXAAst is converted into hydrophobic DMXAA and is inserted into a liposome membrane structure to induce liposome to reassemble, so that the medicine is quickly released, and the problem that free injection medicine is difficult to enter cells is solved.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> university of Zhongshan affiliated first hospital
<120> a nano vesicle loaded with PD-L1 antibody and STING agonist together, and preparation method and application thereof
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Claims (5)

1. The nano vesicle is characterized in that the nano vesicle is loaded with a PD-L1 antibody and a STING agonist at the same time, the structure of the nano vesicle comprises a shell and an inner core, the shell is prepared from a lipid bilayer membrane, the shell is coupled with an MMP-2 sensitive polypeptide chain, the polypeptide chain is also coupled with the PD-L1 antibody, and the shell is also coupled with PEG; the inner core is a hydrophilic small molecule STING agonist;
the lipid bilayer membrane consists of distearoyl phosphatidylcholine, cholesterol, phospholipid-tetra-polyethylene glycol-diphenyl cyclooctyne and distearoyl phosphatidylethanolamine, wherein the molar ratio of the distearoyl phosphatidylcholine to the phospholipid-tetra-polyethylene glycol-diphenyl cyclooctyne is as follows: 20:4:1:2;
the hydrophilic small molecule STING agonist is 5, 6-dimethyl xanthine-4-acetic acid sodium salt;
the polypeptide chain coupled with the PD-L1 antibody has an amino acid sequence shown in SEQ ID NO. 1.
2. A method of preparing the nanovesicle of claim 1, comprising the steps of:
step 1: under the action of a catalyst, 2, 5-dihydroxyl-4-methyl-2, 5-dioxo-3-furanpropionic acid activated by oxalyl chloride and methoxy polyethylene glycol undergo esterification reaction, and after the reaction is finished, the reaction solution is precipitated, filtered and washed, and then filtered by suction to obtain 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 undergo a coupling reaction, and after the reaction is finished, the reaction solution is precipitated, dissolved and dialyzed, and freeze-dried to obtain an MMP-2 sensitive short peptide chain;
step 3: incubating the MMP-2 sensitive short peptide chain in the step 2 with a PD-L1 antibody at room temperature, and coupling the MMP-2 sensitive short peptide chain with the PD-L1 antibody to obtain the MMP-2 sensitive short peptide chain coupled with the PD-L1 antibody;
step 4: dissolving distearoyl phosphatidylcholine, cholesterol, phospholipid-tetra-polyethylene glycol-diphenyl cyclooctyne and distearoyl phosphatidylethanolamine in a mixed solvent to prepare the shell of the nano vesicle;
adding 5, 6-dimethyl xanthine-4-acetic acid sodium salt, carrying out ultrasonic treatment, and filtering to obtain a mixed solution A;
adding the MMP-2 sensitive short peptide chain coupled with the PD-L1 antibody in the step 3 into the mixed solution A, and carrying out vortex oscillation to obtain a mixed solution B;
adding the mPEG-CDM in the step 1 into the mixed solution B, and carrying out vortex oscillation to obtain a mixed solution C;
and (3) dialyzing, ultrafiltering and concentrating the mixed solution C to obtain the nano vesicle.
3. The method according to claim 2, 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 with the amino acid sequence shown in SEQ ID NO. 2 to the 3-maleimidopropionic acid hydroxysuccinimide ester in the step 2 is as follows: 1:2.5;
the molar ratio of the MMP-2 sensitive short peptide chain to the PD-L1 antibody in the step 3 is as follows: 1:24;
adding 4.1% of 5, 6-dimethyl xanthine-4-sodium acetate;
adding the MMP-2 sensitive short peptide chain coupled with the PD-L1 antibody in the step 3 into the mixed solution A, wherein the mass concentration of the MMP-2 sensitive short peptide chain coupled with the PD-L1 antibody is 2%;
the mass concentration of mPEG-CDM described in step 1 was 9.6% to the mixed solution B.
4. The method of claim 2, wherein the phospholipid-tetra-polyethylene glycol-diphenyl cyclooctyne in step 4 is prepared by the following method: under the action of a catalyst, distearoyl phosphatidylethanolamine and diphenyl cyclooctyne-tetra polyethylene glycol-succinimidyl ester are dissolved in a mixed solvent, and after rotary evaporation, the reaction solution is precipitated, and the precipitate is centrifugally collected and dried in vacuum, thus obtaining the catalyst.
5. Use of a nanovesicle according to claim 1 for the preparation of a medicament for the treatment of a tumor.
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