CN116172956B - Composition for targeted delivery of anti-HIV drug, pharmaceutical composition and preparation method - Google Patents

Composition for targeted delivery of anti-HIV drug, pharmaceutical composition and preparation method Download PDF

Info

Publication number
CN116172956B
CN116172956B CN202211440925.9A CN202211440925A CN116172956B CN 116172956 B CN116172956 B CN 116172956B CN 202211440925 A CN202211440925 A CN 202211440925A CN 116172956 B CN116172956 B CN 116172956B
Authority
CN
China
Prior art keywords
hiv
composition
sirna
cell
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202211440925.9A
Other languages
Chinese (zh)
Other versions
CN116172956A (en
Inventor
李志平
高春生
李林
杨阳
龚伟
王玉丽
杨美燕
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Academy of Military Medical Sciences AMMS of PLA
Original Assignee
Academy of Military Medical Sciences AMMS of PLA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Academy of Military Medical Sciences AMMS of PLA filed Critical Academy of Military Medical Sciences AMMS of PLA
Priority to CN202211440925.9A priority Critical patent/CN116172956B/en
Publication of CN116172956A publication Critical patent/CN116172956A/en
Application granted granted Critical
Publication of CN116172956B publication Critical patent/CN116172956B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Abstract

The invention belongs to the technical field of targeted drugs, and particularly relates to a composition for targeted delivery of an anti-HIV drug, a pharmaceutical composition and a preparation method thereof. The composition and the pharmaceutical composition can be strongly combined with gp120, and on one hand, can directly enrich, trap and neutralize free HIV and remarkably inhibit HIV; on one hand, the drug can be selectively targeted to HIV-infected cells and accurately delivered into HIV-infected cells, so that the curative effect of the drug is remarkably improved, the administration dosage is reduced, and the occurrence of toxic and side effects is reduced; meanwhile, the composition can be combined with gp120 protein to inhibit HIV-infected cells from killing bystander cells, so that the composition has obvious HIV inhibition effect and great potential in the aspect of AIDS treatment.

Description

Composition for targeted delivery of anti-HIV drug, pharmaceutical composition and preparation method
Technical Field
The invention belongs to the technical field of targeted drugs, and particularly relates to a composition for targeted delivery of an anti-HIV drug, a pharmaceutical composition and a preparation method thereof.
Background
HIV is a major global public health problem.
High-potency retroviral therapy (HAART) achieves synergistic antiviral effects by combining three or more antiviral agents acting on different segments of viral replication, and is currently considered the most effective method of inhibiting HIV-1. However, the existing treatment methods mainly adopt systemic administration modes such as oral administration, and the like, and the proportion of the drugs actually reaching HIV and infected cells thereof is extremely low, so that the problems of large administration dosage, serious toxic and side effects, poor durability of patients and the like are caused. There is a great need to develop therapeutic approaches to HIV and its infected cells.
HIV invades human body in early stage mainly by means of its surface gp120 protein specificity to recognize and attack CD4 receptor protein expressing cell, and with the aid of auxiliary receptor such as CCR5 and CXCR4, it fuses with cell membrane of CD4+ T cell, virus nucleocapsid enters cell, HIV virus replicates in cell to form great amount of new virus particles, new virus is released from host cell by budding mode to infect other CD4+ T lymphocyte, so HIV gp120 protein is expressed on HIV infected cell surface.
Since CD4+ cells are the root of infection and proliferation of HIV, scientists extract CD4+ cell membrane modified vectors, and the trapping and neutralization of HIV are realized by constructing pseudo CD4+ cells, but the density of CD4 molecules on the surface of the vector obtained by the method is similar to that of CD4+ cells in vivo, and the vector formed by CD4+ cell membranes alone or the CD4+ cell membrane modified vector has no obvious competitive advantage compared with the CD4+ cells with huge quantity in vivo.
Disclosure of Invention
The invention aims to provide a composition for targeted delivery of an anti-HIV drug, a pharmaceutical composition and a preparation method thereof, which can greatly improve the density of gp120 targets on the surface of the composition/carrier, remarkably increase the competitive binding capacity of the composition/carrier, simultaneously realize targeted delivery of the drug, remarkably improve the curative effect, greatly reduce the dosage of the drug and reduce the toxic and side effects.
The invention provides a composition for targeted delivery of an anti-HIV drug, which comprises the following components: cd4+ cell membranes, distearoyl phosphatidylethanolamine polyethylene glycol 2000 modified with a ligand that selectively binds gp120 protein, and phospholipid material;
the CD4+ cell membrane is derived from a cell surface expressing a CD4 molecule.
Preferably, the cells comprise human T lymphocytes.
Preferably, the class of ligands that selectively bind gp120 protein comprises polypeptides, chemical small molecules, proteins or aptamers.
Preferably, the ligand which selectively binds gp120 protein comprises a polypeptide comprising 12p1 having the amino acid sequence shown in SEQ ID NO.1 or UM15 having the amino acid sequence shown in SEQ ID NO. 2.
Preferably, the phospholipid material comprises a phospholipid bilayer membrane forming material selected from one or more of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylphosphatides, phosphatidylserine and lysophosphatides.
Preferably, the composition further comprises cholesterol, and the weight ratio of the phospholipid material to the cholesterol to the distearoyl phosphatidylethanolamine polyethylene glycol 2000 modified by the ligand which selectively binds gp120 protein is 50-90: 0 to 18:0.5 to 18.
The invention also provides a pharmaceutical composition for targeting HIV and/or HIV-infected cells, comprising an anti-HIV drug and the composition.
Preferably, the anti-HIV drug includes one or more of a small molecule chemical drug, a nucleic acid drug, a polypeptide drug, and a protein drug.
The invention also provides a preparation method of the pharmaceutical composition, which comprises the following steps: the phospholipid material is prepared into liposome, and the liposome is used for wrapping the anti-HIV drug and fusing with CD4+ cell membranes.
Preferably, the method comprises the following steps: preparing a liposome from a phospholipid material and distearoyl phosphatidylethanolamine polyethylene glycol 2000 modified by a ligand which selectively binds gp120 protein, wrapping an anti-HIV drug by using the liposome, and fusing the anti-HIV drug with a CD4+ cell membrane to obtain the pharmaceutical composition;
or preparing phospholipid material into liposome, wrapping anti-HIV medicine with the liposome, and fusing with CD4+ cell membrane and distearoyl phosphatidylethanolamine polyethylene glycol 2000 modified by ligand which selectively binds gp120 protein to obtain the medicine composition.
The beneficial effects are that: the invention provides a composition for targeted delivery of anti-HIV drugs, and a pharmaceutical composition for targeted HIV and HIV-infected cells is prepared based on the composition, and has obvious advantages in aspects of in vivo long circulation, free HIV neutralization, virus inhibition in HIV-infected cells and the like, and specifically comprises the following steps:
(1) The composition can be strongly combined with gp120, on one hand, can directly enrich, trap and neutralize free HIV, and remarkably inhibit HIV; on one hand, the drug composition can selectively target HIV-infected cells, accurately deliver the drugs in the drug composition into the HIV-infected cells, remarkably improve the curative effect of the drugs, reduce the administration dosage and reduce the occurrence of toxic and side effects; meanwhile, the composition can be combined with gp120 protein to inhibit HIV-infected cells from killing bystander cells, so that the composition has obvious HIV inhibition effect and great potential in the aspect of AIDS treatment.
(2) The composition and the pharmaceutical composition mainly comprise a cell membrane rich in CD4 protein, a phospholipid modified ligand capable of being combined with gp120 and a phospholipid material, and the composition has stronger gp120 competitive binding capacity compared with a composition/carrier consisting of MT cell membrane or normal CD4+ cells in vivo; compared with a composition/carrier simply modified by targeting of polypeptide ligand and the like, the composition has an autologous recognition function due to the introduction of cell membrane, and can avoid the ingestion of mononuclear macrophages and the rapid clearance effect (ABC effect) caused by the introduction of PEGylation (for long circulation).
(3) The ligand contained in the composition and the pharmaceutical composition has special functions, such as UM15 and peptide triazole ligand have gp120 protein shedding function, so that the composition and the pharmaceutical composition have gp120 protein shedding function at the same time, and HIV inhibition function can be further enhanced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a laser confocal image of the binding of the vector to HIV-infected cells in example 1;
FIG. 2 is a laser confocal plot of the binding of the vector to HIV-infected cells in example 2;
FIG. 3 is a vector pair HIV-1 of example 1 NL4-3 Is a inhibition curve of (2);
FIG. 4 is a diagram of the vector pair HIV-1 of example 1 AD8 Is a inhibition curve of (2);
FIG. 5 is a vector pair HIV-1 of example 2 NL4-3 Is a inhibition curve of (2);
FIG. 6 is a vector pair HIV-1 of example 2 AD8 Is a inhibition curve of (2);
FIG. 7 is a confocal microscope examination of lysosomal escape effects of siRNA in the vector of example 1.
Detailed Description
The invention provides a composition for targeted delivery of an anti-HIV drug, which comprises the following components: cd4+ cell membranes, distearoyl phosphatidylethanolamine polyethylene glycol 2000 modified with a ligand that selectively binds gp120 protein, and phospholipid material;
the CD4+ cell membrane is derived from a cell surface expressing a CD4 molecule.
The cells of the invention preferably comprise T lymphocytes of human originMore preferably, human lymphoma cells (MT 2 cells) and/or peripheral blood mononuclear cells (PBMC cells) are included. The CD4+ cell membrane is preferably obtained by extracting and purifying the humanized T lymphocyte. The method for extracting and purifying the cell membrane is not particularly limited, and preferably comprises one or more of a hypotonic method, an ultrasonic disruption method, a repeated freeze thawing method and an ultra-high speed centrifugation method. The cell membrane extracted by methods conventional in the art is preferably used at a concentration of 1X 10 6 Cell membrane of individual cells/ml-1×10 10 Cell membranes/ml of individual cells, more preferably 5X 10 7 Cell membrane of individual cells/ml-9X 10 8 Cell membranes per ml of individual cells.
The ligand-modified distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG 2000) that selectively binds gp120 protein of the present invention, wherein the class of ligand that selectively binds gp120 protein preferably comprises a polypeptide, a chemical small molecule, a protein or an aptamer, more preferably comprises a polypeptide comprising 12p1 (SEQ ID No.1: RINNIPWSEAMM) or UM15 (SEQ ID No.2: cys-Ile-Asn-Ile-X-Trp-NH 2), wherein X represents a ferrocene triazoloprolol amino acid residue (ferrocenyl-triazolePro amino acidresidue).
The phospholipid material of the present invention may form a phospholipid bilayer membrane, preferably one or more selected from Phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), acyl Phosphatidylcholines (PAs), phosphatidylserine (PS) and lysophospholipids, more preferably one or more selected from hydrogenated soybean lecithin (HSPC), egg yolk lecithin (EPC), dioleoyl lecithin (DOPC), dipalmitoyl lecithin (DPPC), distearoyl phosphatidylcholine (DSPC), 2-palmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-MPEG 2000) and derivatives thereof, further preferably one or more selected from egg yolk lecithin (EPC), dioleoyl lecithin (DOPC), distearoyl phosphatidylcholine (DPPC), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-MPEG 2000) and derivatives thereof.
The composition of the invention preferably further comprises cholesterol, and the total use concentration range of the phospholipid material, the cholesterol and the distearoyl phosphatidylethanolamine polyethylene glycol 2000 modified by the ligand is preferably 3mg/ml to 50mg/ml, more preferably 5mg/ml to 15mg/ml; the weight ratio of the phospholipid material, the cholesterol and the distearoyl phosphatidylethanolamine polyethylene glycol 2000 modified by the ligand which selectively binds gp120 protein is preferably 50-90: 0 to 18:0.5 to 18, more preferably 70 to 80:0 to 5:0.5 to 10, more preferably 75 to 85:0 to 1.5:0.5 to 5.
The composition provided by the invention has the advantages that on one hand, the characteristic that the surface of an in-vivo cell simultaneously expresses an autologous recognition molecule CD4 is utilized to realize the function that the formed composition/carrier evades the uptake and clearance of in-vivo mononuclear macrophages, the long circulation effect is realized, on the other hand, the characteristic of the HIV natural target of a CD4+ cell membrane is utilized, and the gp120 specific binding ligand is simultaneously introduced to jointly construct the composition/carrier, so that the gp120 target density on the surface of the composition/carrier is greatly improved, and the competitive binding capacity of the composition/carrier with free HIV of in-vivo normal CD4+ cells and infected cells of the composition/carrier is remarkably improved.
The invention also provides a pharmaceutical composition for targeting HIV and/or HIV-infected cells, comprising an anti-HIV drug and the composition.
The anti-HIV drugs of the present invention preferably include one or more of small molecule chemicals, nucleic acid drugs, polypeptide drugs, and protein drugs.
The invention also provides a preparation method of the pharmaceutical composition, which comprises the following steps: the phospholipid material is prepared into liposome, and the liposome is used for wrapping the anti-HIV drug and fusing with CD4+ cell membranes.
In the preparation of the pharmaceutical composition, the distearoyl phosphatidylethanolamine polyethylene glycol 2000 modified by the ligand which selectively binds gp120 protein can be applied to the process of preparing liposome and the process of fusing with CD4+ cell membrane, and when cholesterol is contained in the raw material, the raw material contains cholesterol for illustration, namely the preparation method comprises the following steps: preparing a liposome from a phospholipid material, cholesterol and distearoyl phosphatidylethanolamine polyethylene glycol 2000 modified by a ligand which selectively binds gp120 protein, wrapping an anti-HIV drug by using the liposome, and fusing the anti-HIV drug with a CD4+ cell membrane to obtain the pharmaceutical composition;
or preparing phospholipid material and cholesterol into liposome, wrapping anti-HIV medicine by using the liposome, and fusing with CD4+ cell membrane and distearoyl phosphatidylethanolamine polyethylene glycol 2000 modified by ligand which selectively binds gp120 protein to obtain the medicine composition.
The method for preparing the liposome and the method for fusing the liposome with a cell membrane are not particularly limited, and the liposome can be prepared by a conventional method in the art.
For further explanation of the present invention, a composition, a pharmaceutical composition and a preparation method for targeted delivery of an anti-HIV drug provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
All HIV pseudoviruses and live viruses in the examples below were provided by the institute of microbiology epidemics institute of military medical science AIDS detection center, and all experiments involving live viruses were performed in the institute of microbiology epidemics institute of military medical science AIDS detection center biosafety third-level (P3) laboratory.
Example 1
A pharmaceutical composition for targeting HIV and infected cells is formulated as shown in Table 1.
Table 1 pharmaceutical compositions
In Table 1, the sequence of Tat/rev siRNA:
sense strand (SEQ ID No. 3): 5 '-GCGGAGACAGCGAGGACGAAGAGCdTDT-3';
antisense strand (SEQ ID No. 4): 3 '-dTdTCGGCCUCUCUGGUCUCUCUCUCG-5';
12p1 is RINNIPWSEAMM.
The preparation process comprises the following steps:
1. extraction of MT2 cell membranes: separating and purifying lymphocyte membrane by hypotonic ultrasonic disruption-differential centrifugation. The specific experimental steps are as follows: a. the collected cells were washed three times with pre-chilled PBS and then further washed with 1X 10 solution -3 The M ethylenediamine tetraacetic acid (EDTA) and 1% Tris (Tris (hydroxymethyl) aminomethane) buffer of phenylmethylsulfonyl fluoride (PMSF) were resuspended in an ice-water bath, lymphocyte structures were disrupted by probe sonication (power 150W, working for 2s stopped 8s, for a total of 2 min) in an ice-water bath to give a cell homogenate, and hypotonic at 4℃for about 30min. b. Centrifuging at 4deg.C and 500×g for 10min with ultra-high speed centrifuge, discarding precipitate, collecting supernatant to remove incompletely broken and larger precipitate, centrifuging at 4deg.C and 20000×g for 20min, collecting supernatant, repeating the process twice, centrifuging at 100000×g for 50min, and collecting precipitate at bottom of centrifuge tube to obtain lymphocyte membrane. c. The collected lymphocyte membranes are washed and resuspended once by pre-cooled PBS buffer containing EDTA, and then are collected by centrifugation again, and then resuspended in PBS for later use.
2. Preparation of siRNA/protamine complex: vortex-room temperature incubation method for preparing siRNA/protamine complex: salmon protamine was diluted to a concentration of 132 μg/mL in HEPES solution and added drop-wise to HEPES solution in which anti-HIV siRNA was dissolved, with a final mass ratio of 1:1. And (5) slightly swirling at room temperature for 20min to obtain the siRNA/protamine complex.
3. Preparation of siRNA/LP: precisely weighing three groups of lipid materials of HSPC, cholesterol and DSPE-PEG2000 (80:10:10, w/w/w), 20mg in a 50mL eggplant-shaped bottle, fully dissolving 1mL of chloroform, performing water bath vacuum rotary evaporation for more than 50min at 45 ℃ and 75rpm to form a lipid film, and then transferring the lipid film into a vacuum drying oven for drying for more than 12h to remove residual organic solvent. The lipid film after vacuum drying is hydrated for 40min at 55 ℃ by using 1ml of HEPES solution containing siRNA/protamine complex, DEPC water is added to supplement the weight loss, and then hydration is continued for 10min to uniformly mix, then the prepared lipid nano particles are subjected to ultrasonic probe (power 45% and total 2 min) in a water bath at 55 ℃ to obtain the lipid nano particles encapsulated with the siRNA/protamine complex, and then the lipid nano particles are repeatedly extruded for 21 times in a liposome extruder with a polycarbonate film with the aperture of 100nm, and the lipid nano particles containing the siRNA after homogenization treatment are labeled as the siRNA/LP.
4. Preparation of siRNA/MLP: MT2 cell membranes will be obtained, probe sonicated (100W, working for 2s at 8s, for a total of 2 min) in an ice-water bath to reduce the cell membrane vesicle size, and then the corresponding cell numbers will be approximately 1X 10 8 And mixing the lymphocyte membrane extracted from each cell with siRNA/LP in equal volume, and repeatedly extruding the mixed solution for 21 times in a liposome extruder with a polycarbonate membrane with the aperture of 400nm and 200nm in sequence to prepare the lipid nanoparticle siRNA/MLP wrapped by the lymphocyte membrane.
5. Preparation of siRNA/MLP-12p 1: weighing the prescribed amount DSPE-PEG2000-12p1, dissolving in HEPES buffer solution (pH=7.0) to prepare target mother liquor, and preserving at 4 ℃ for later use. And (3) transferring 200 mu L of target head mother liquor, mixing with 1.0mL of lipid nanoparticle siRNA/MLP wrapped with cell membrane, fully and uniformly mixing, and then incubating for 30min at 37 ℃ in a dark place, and inserting the target head into the surface of the lymphocyte membrane. Then transferring the mixture into a dialysis bag (MWCO: 8000-12000 Da), and dialyzing the mixture in DEPC-HEPES buffer solution (pH=7.0) in dark for overnight to remove the free target head, thus obtaining the siRNA/MLP-12p1.
Example 2
A composition for targeting HIV and infected cells, the prescription of which is shown in table 2.
Table 2 composition recipe
In Table 2, UM15 has the sequence Cys-Ile-Asn-Asn-Ile-X-Trp-NH2.
The preparation process comprises the following steps:
1. extraction of MT2 cell membranes: as in example 1.
2. Preparation of lipid nanoparticles (LP): precisely weighing three groups of lipid materials of HSPC, cholesterol and DSPE-PEG2000 (80:10:10, w/w/w), 20mg in a 50mL eggplant-shaped bottle, fully dissolving 1mL of chloroform, performing water bath vacuum rotary evaporation for more than 50min at 45 ℃ and 75rpm to form a lipid film, and then transferring the lipid film into a vacuum drying oven for drying for more than 12h to remove residual organic solvent. The lipid film after vacuum drying is hydrated for 40min at 55 ℃ with 1ml HEPES solution and 100rpm, DEPC water is added to supplement the weight loss, and then hydration is continued for 10min to uniformly mix, then the prepared lipid nano particles are subjected to probe ultrasound (power 45% and total 2 min) in a water bath at 55 ℃ to obtain the lipid nano particles, and then the lipid nano particles are repeatedly extruded for 21 times in a liposome extruder with a polycarbonate film with the aperture of 100nm, and the lipid nano particles after homogenization treatment are marked as LP.
3. Preparation of MT2 cell membrane-encapsulated lipid nanoparticles (MLPs): MT2 cell membranes will be obtained, probe sonicated (100W, working for 2s at 8s, for a total of 2 min) in an ice-water bath to reduce the cell membrane vesicle size, and then the corresponding cell numbers will be approximately 1X 10 8 The extracted lymphocyte membrane in individual cells is mixed with LP in equal volume, and the mixed solution is repeatedly extruded for 21 times in a liposome extruder with a polycarbonate membrane with the aperture of 400nm and 200nm, so as to prepare the lipid nanoparticle MLP wrapped by MT2 cell membrane.
4. Preparation of MT2 cell membranes and UM15 modified lipid nanoparticles (MLP-UM 15): weighing the prescription amount DSPE-PEG2000-UM15, dissolving in HEPES buffer solution (pH=7.0) to prepare target mother solution, and preserving at 4 ℃ for later use. And (3) transferring 200 mu L of target head mother liquor, mixing with 1.0mL of lipid nanoparticle MLP wrapped with cell membrane, fully and uniformly mixing, and then incubating for 30min at 37 ℃ in a dark place, and inserting the target head into the surface of the lymphocyte membrane. Transferring the sample into a dialysis bag (MWCO: 8000-12000 Da), and dialyzing overnight in HEPES buffer solution (pH=7.0) in the dark to remove the free target head, thereby obtaining the MLP-UM15.
Example 3
The pharmaceutical composition of the control formulation of example 1, liposome, is formulated as shown in table 3.
Table 3 pharmaceutical compositions
Sequence of Tat/rev siRNA:
sense strand (SEQ ID No. 3): 5 '-GCGGAGACAGCGAGGACGAAGAGCdTDT-3';
antisense strand (SEQ ID No. 4): 3 '-dTdTCGGCCUCUCUGGUCUCUCUCUCG-5';
12p1 is RINNIPWSEAMM.
The preparation process comprises the following steps:
1. preparation of siRNA/protamine complex: vortex-room temperature incubation method for preparing siRNA/protamine complex: salmon protamine was diluted to a concentration of 132 μg/mL in HEPES solution and added drop-wise to HEPES solution in which anti-HIV siRNA was dissolved, with a final mass ratio of 1:1. And (5) slightly swirling at room temperature for 20min to obtain the siRNA/protamine complex.
2. Preparation of siRNA/LP: precisely weighing three groups of lipid materials of HSPC, cholesterol and DSPE-PEG2000 (80:10:10, w/w/w), 20mg in a 50mL eggplant-shaped bottle, fully dissolving 1mL of chloroform, performing water bath vacuum rotary evaporation for more than 50min at 45 ℃ and 75rpm to form a lipid film, and then transferring the lipid film into a vacuum drying oven for drying for more than 12h to remove residual organic solvent. The lipid film after vacuum drying is hydrated for 40min at 55 ℃ by using 1ml of HEPES solution containing siRNA/protamine complex, DEPC water is added to supplement the weight loss, and then hydration is continued for 10min to uniformly mix, then the prepared lipid nano particles are subjected to ultrasonic probe (power 45% and total 2 min) in a water bath at 55 ℃ to obtain the lipid nano particles encapsulated with the siRNA/protamine complex, and then the lipid nano particles are repeatedly extruded for 21 times in a liposome extruder with a polycarbonate film with the aperture of 100nm, and the lipid nano particles containing the siRNA after homogenization treatment are labeled as the siRNA/LP.
Example 4
Example 1 control formulation-pharmaceutical composition of envelope liposome, the formulation of which is shown in table 4.
Table 4 pharmaceutical compositions
Sequence of Tat/rev siRNA:
sense strand (SEQ ID No. 3): 5 '-GCGGAGACAGCGAGGACGAAGAGCdTDT-3';
antisense strand (SEQ ID No. 4): 3 '-dTdTCGGCCUCUCUGGUCUCUCUCUCG-5';
12p1 is RINNIPWSEAMM.
The preparation process comprises the following steps:
1. extraction of MT2 cell membranes: separating and purifying lymphocyte membrane by hypotonic ultrasonic disruption-differential centrifugation. The specific experimental steps are as follows: a. the collected cells were washed three times with pre-chilled PBS and then further washed with 1X 10 solution -3 The M ethylenediamine tetraacetic acid (EDTA) and 1% Tris (Tris (hydroxymethyl) aminomethane) buffer of phenylmethylsulfonyl fluoride (PMSF) were resuspended in an ice-water bath, lymphocyte structures were disrupted by probe sonication (power 150W, working for 2s stopped 8s, for a total of 2 min) in an ice-water bath to give a cell homogenate, and hypotonic at 4℃for about 30min. b. Centrifuging at 4deg.C and 500×g for 10min with ultra-high speed centrifuge, discarding precipitate, collecting supernatant to remove incompletely broken and larger precipitate, centrifuging at 4deg.C and 20000×g for 20min, collecting supernatant, repeating the process twice, centrifuging at 100000×g for 50min, and collecting precipitate at bottom of centrifuge tube to obtain lymphocyte membrane. c. The collected lymphocyte membranes are washed and resuspended once by pre-cooled PBS buffer containing EDTA, and then are collected by centrifugation again, and then resuspended in PBS for later use.
2. Preparation of siRNA/protamine complex: vortex-room temperature incubation method for preparing siRNA/protamine complex: salmon protamine was diluted to a concentration of 132 μg/mL in HEPES solution and added drop-wise to HEPES solution in which anti-HIV siRNA was dissolved, with a final mass ratio of 1:1. And (5) slightly swirling at room temperature for 20min to obtain the siRNA/protamine complex.
3. Preparation of siRNA/LP: precisely weighing three groups of lipid materials of HSPC, cholesterol and DSPE-PEG2000 (80:10:10, w/w/w), 20mg in a 50mL eggplant-shaped bottle, fully dissolving 1mL of chloroform, performing water bath vacuum rotary evaporation for more than 50min at 45 ℃ and 75rpm to form a lipid film, and then transferring the lipid film into a vacuum drying oven for drying for more than 12h to remove residual organic solvent. The lipid film after vacuum drying is hydrated for 40min at 55 ℃ by using 1ml of HEPES solution containing siRNA/protamine complex, DEPC water is added to supplement the weight loss, and then hydration is continued for 10min to uniformly mix, then the prepared lipid nano particles are subjected to ultrasonic probe (power 45% and total 2 min) in a water bath at 55 ℃ to obtain the lipid nano particles encapsulated with the siRNA/protamine complex, and then the lipid nano particles are repeatedly extruded for 21 times in a liposome extruder with a polycarbonate film with the aperture of 100nm, and the lipid nano particles containing the siRNA after homogenization treatment are labeled as the siRNA/LP.
4. Preparation of siRNA/MLP: MT2 cell membranes will be obtained, probe sonicated (100W, working for 2s at 8s, for a total of 2 min) in an ice-water bath to reduce the cell membrane vesicle size, and then the corresponding cell numbers will be approximately 1X 10 8 And mixing the lymphocyte membrane extracted from each cell with siRNA/LP in equal volume, and repeatedly extruding the mixed solution for 21 times in a liposome extruder with a polycarbonate membrane with the aperture of 400nm and 200nm in sequence to prepare the lipid nanoparticle siRNA/MLP wrapped by the lymphocyte membrane.
Example 5
Example 1A blank formulation-coated and ligand double modified control composition, the formulation of which is shown in Table 5.
Table 5 pharmaceutical compositions
In table 5, the sequence of the Scrambled siRNA:
sense strand (SEQ ID No. 5): 5 '-GGGAGCACAGGGCGCAGACAGAdTdT-3';
antisense strand (SEQ ID No. 6): 3 '-dTdTCCCUCUGUCCGCGUCUGUCU-5';
the preparation process comprises the following steps:
1. extraction of MT2 cell membranes: separating and purifying lymphocyte membrane by hypotonic ultrasonic disruption-differential centrifugation. The specific experimental steps are as follows: a. the collected cells were washed three times with pre-chilled PBS and then further washed with 1X 10 solution -3 The M ethylenediamine tetraacetic acid (EDTA) and 1% Tris (Tris (hydroxymethyl) aminomethane) buffer of phenylmethylsulfonyl fluoride (PMSF) were resuspended in an ice-water bath, lymphocyte structures were disrupted by probe sonication (power 150W, working for 2s stopped 8s, for a total of 2 min) in an ice-water bath to give a cell homogenate, and hypotonic at 4℃for about 30min. b. Centrifuging at 4deg.C and 500×g for 10min with ultra-high speed centrifuge, discarding precipitate, collecting supernatant to remove incompletely broken and larger precipitate, centrifuging at 4deg.C and 20000×g for 20min, collecting supernatant, repeating the process twice, centrifuging at 100000×g for 50min, and collecting precipitate at bottom of centrifuge tube to obtain lymphocyte membrane. c. The collected lymphocyte membranes are washed and resuspended once by pre-cooled PBS buffer containing EDTA, and then are collected by centrifugation again, and then resuspended in PBS for later use.
2. Preparation of siRNA/protamine complex: vortex-room temperature incubation method for preparing siRNA/protamine complex: salmon protamine was diluted to a concentration of 132 μg/mL in HEPES solution and added drop-wise to HEPES solution in which anti-HIV siRNA was dissolved, with a final mass ratio of 1:1. And (5) slightly swirling at room temperature for 20min to obtain the siRNA/protamine complex.
3. Preparation of siRNA/LP: precisely weighing three groups of lipid materials of HSPC, cholesterol and DSPE-PEG2000 (80:10:10, w/w/w), 20mg in a 50mL eggplant-shaped bottle, fully dissolving 1mL of chloroform, performing water bath vacuum rotary evaporation for more than 50min at 45 ℃ and 75rpm to form a lipid film, and then transferring the lipid film into a vacuum drying oven for drying for more than 12h to remove residual organic solvent. The lipid film after vacuum drying is hydrated for 40min at 55 ℃ by using 1ml of HEPES solution containing siRNA/protamine complex, DEPC water is added to supplement the weight loss, and then hydration is continued for 10min to uniformly mix, then the prepared lipid nano particles are subjected to ultrasonic probe (power 45% and total 2 min) in a water bath at 55 ℃ to obtain the lipid nano particles encapsulated with the siRNA/protamine complex, and then the lipid nano particles are repeatedly extruded for 21 times in a liposome extruder with a polycarbonate film with the aperture of 100nm, and the lipid nano particles containing the siRNA after homogenization treatment are labeled as the siRNA/LP.
4. Preparation of siRNA/MLP: MT2 cell membranes will be obtained, probe sonicated (100W, working for 2s at 8s, for a total of 2 min) in an ice-water bath to reduce the cell membrane vesicle size, and then the corresponding cell numbers will be approximately 1X 10 8 And mixing the lymphocyte membrane extracted from each cell with siRNA/LP in equal volume, and repeatedly extruding the mixed solution for 21 times in a liposome extruder with a polycarbonate membrane with the aperture of 400nm and 200nm in sequence to prepare the lipid nanoparticle siRNA/MLP wrapped by the lymphocyte membrane.
5. Preparation of siRNA/MLP-12p 1: weighing the prescribed amount DSPE-PEG2000-12p1, dissolving in HEPES buffer solution (pH=7.0) to prepare target mother liquor, and preserving at 4 ℃ for later use. And (3) transferring 200 mu L of target head mother liquor, mixing with 1.0mL of lipid nanoparticle siRNA/MLP wrapped with cell membrane, fully and uniformly mixing, and then incubating for 30min at 37 ℃ in a dark place, and inserting the target head into the surface of the lymphocyte membrane. Then transferring the mixture into a dialysis bag (MWCO: 8000-12000 Da), and dialyzing the mixture in DEPC-HEPES buffer solution (pH=7.0) in dark for overnight to remove the free target head, thus obtaining the siRNA/MLP-12p1.
Example 6
The formulations of the control formulation-liposome compositions of example 2 are shown in Table 6.
Table 6 composition recipe
The preparation process comprises the following steps:
precisely weighing three groups of lipid materials of HSPC, cholesterol and DSPE-PEG2000 (80:10:10, w/w/w), 20mg in a 50mL eggplant-shaped bottle, fully dissolving 1mL of chloroform, performing water bath vacuum rotary evaporation for more than 50min at 45 ℃ and 75rpm to form a lipid film, and then transferring the lipid film into a vacuum drying oven for drying for more than 12h to remove residual organic solvent. Hydrating 1ml of a vacuum dried lipid film for 40min at 55 ℃ under the condition of 100rpm by using 1ml of HEPES solution, adding DEPC water to supplement the weight loss, continuing hydrating for 10min to uniformly mix the obtained lipid film, performing probe ultrasound (power 45% and total 2 min) on the obtained lipid nano-particles in a water bath at 55 ℃ to obtain the lipid nano-particles, repeatedly extruding the lipid nano-particles in a liposome extruder with a polycarbonate film with the aperture of 100nm for 21 times, and homogenizing the obtained lipid nano-particles to obtain the lipid nano-particles.
Example 7
Example 2 control formulation-coated liposome composition, the formulation of which is shown in table 7.
Table 7 composition recipe
The preparation process comprises the following steps:
1. extraction of MT2 cell membranes: as in example 1.
2. Preparation of liposomes: precisely weighing three groups of lipid materials of HSPC, cholesterol and DSPE-PEG2000 (80:10:10, w/w/w), 20mg in a 50mL eggplant-shaped bottle, fully dissolving 1mL of chloroform, performing water bath vacuum rotary evaporation for more than 50min at 45 ℃ and 75rpm to form a lipid film, and then transferring the lipid film into a vacuum drying oven for drying for more than 12h to remove residual organic solvent. Hydrating 1ml of a vacuum dried lipid film for 40min at 55 ℃ under the condition of 100rpm by using 1ml of HEPES solution, adding DEPC water to supplement the weight loss, continuing hydrating for 10min to uniformly mix the obtained lipid film, performing probe ultrasound (power 45% and total 2 min) on the obtained lipid nano-particles in a water bath at 55 ℃ to obtain the lipid nano-particles, repeatedly extruding the lipid nano-particles in a liposome extruder with a polycarbonate film with the aperture of 100nm for 21 times, and homogenizing the obtained lipid nano-particles to obtain the lipid nano-particles.
3. Preparation of MT2 cell membrane-encapsulated liposomes: MT2 cell membranes will be obtained, probe sonicated (100W, working for 2s at 8s, for a total of 2 min) in an ice-water bath to reduce the cell membrane vesicle size, and then the corresponding cell numbers will be approximately 1X 10 8 Mixing lymphocyte membrane extracted from individual cells with liposome in equal volume, and mixing the mixed solutionRepeatedly extruding for 21 times in a liposome extruder with a polycarbonate membrane with a pore size of 400nm and 200nm, and preparing the liposome wrapped by MT2 cell membrane.
Example 8
Gp120 protein binding assay
HIV-1 respectively MN (X4 phagocytosis) and HIV-1 BaL (R5 phagy) gp120 protein solution was treated with Na at a concentration of 0.05mol/L, pH =9.6 2 CO 3 ·NaHCO 3 Diluting the coating buffer solution into a solution with the concentration of 2 mug/mL, adding the solution into a 96-well ELISA plate after uniformly mixing, and coating protein in 100 mug/well at 4 ℃ in a refrigerator overnight; after coating, discarding the protein coating solution in the ELISA plate, and washing the PBST for 2 times; 200 mu L/well of 4% goat serum blocking solution is added, and the mixture is subjected to sealing incubation for 2h at 37 ℃; discarding the sealing liquid in the ELISA plate, and washing with PBST for 2 times; adding different carriers with fluorescent marks, diluting the mixture step by step, and incubating the mixture for 2 hours at 37 ℃ in a dark place after the mixture is mixed evenly by light shaking; after incubation is completed, discarding particle diluent in the ELISA plate, and washing with PBST for 3 times; the optical density value (excitation: 649 nm/emission: 680 nm) of the adsorbed nanoparticles was measured by a microplate reader, and the binding of each group of preparations to the target protein was analyzed. The results are shown below.
TABLE 8 results of gp120 protein binding experiments (Unit: fluorescence intensity) for example 1 and its control formulation
Liposome group Coated liposome group Example 1
HIV-1 MN 486±156 7519±438 13417±1401
HIV-1 BaL 498±185 6107±260 8844±652
TABLE 9 results of gp120 protein binding experiments (Unit: fluorescence intensity) for example 2 and its control formulation
Liposome group Coated liposome group Example 1
HIV-1 MN 481±88 5649±577 7725±724
HIV-1 BaL 334±126 7213±692 10585±773
Example 9
Vector binding experiments with HIV-1 infected cells
HIV-1 at an MOI of 0.01, respectively AD8 Strains and HIV-1 NL4-3 After the strain is infected with TZM-bl cells for 6 hours, washing off free viruses and culturing for 24 hours to obtain a cell model infected with the viruses; after washing free viruses, diluting different carriers of fluorescein cy5 fluorescent markers with serum-free DMEM high-sugar culture medium to have cy5 concentration of about 125nM, adding the carriers into a confocal dish inoculated with HIV-infected cells, and incubating for 2h in a dark place; removing the culture medium in the small dish, adding 2mLPBS, gently shaking to wash away unbound fluorescent-labeled nanoparticles, and repeating for 3 times every 5 min; adding 2mL of 4% paraformaldehyde tissue fixing solution, and fixing for 20min at room temperature; removing the fixed liquid, adding 2mLPBS, slightly shaking and washing, and repeating for 3 times every 5 min; after 10. Mu.g/mL Hoechst33258 staining the nuclei for about 10min, adding 2mLPBS, washing with gentle shaking to wash away unbound nuclei dye solution, repeating for 3 times every 5 min; adding 50% glycerol prepared from 1mL sterile PBS into each Confocal dish for sealing treatment and storing in dark; observations were made using a confocal laser microscope, and appropriate fields of view were selected for scanning and photographing and analysis of the results.
As a result, the vectors of example 1 and example 2 can specifically bind to HIV-infected cells as shown in FIGS. 1 and 2.
Example 10
Pseudovirus neutralization assay
The ability of the constructed biomimetic nano-delivery system to neutralize virus blocking to infect normal cells was examined with 8 pseudoviruses of AE34, B14, B16, B121, BC28, BC29, BC43, C11 and C15 (provided by the institute of microbiology epidemics institute aids detection center).
The specific experimental steps are as follows: firstly, transfecting HEK 293T cells with each pseudovirus plasmid, culturing for 48 hours, and then collecting and storing pseudovirus strains for later use at the temperature of minus 80 ℃; the compositions of examples 1 and 2 were diluted 3-fold, respectively, except for the cell control group and the virus control group; the diluted compositions were incubated with 200TCID pseudoviruses in medium containing 15. Mu.g/mL diethylin ethyl dextran, respectively2h, preparing an incubation liquid of PBS and 200TCID pseudoviruses by the same method; TZM-bl cells were plated in 10000 cells/well, and the bionic nano-delivery system/virus mixture in the above procedure was added to the cells so that the liquid per well was 100. Mu.L, an equivalent amount of PBS was added to a cell control group, a mixture of equivalent amount of PBS and 200TCID virus was added to a virus control group, 37℃C, 5% CO 2 Incubating and culturing for 6 hours under the condition; removing culture medium after 6 hr, adding PBS to wash cells, centrifuging at 37deg.C for 3 times for 10min, washing off free virus, adding 100 μl/hole of culture medium, culturing in cell culture box at 37deg.C under 5% CO 2 The method comprises the steps of carrying out a first treatment on the surface of the After 48h, the fluorescence intensity was measured and the IC80 value of siRNA/MLP-12p1 for each strain was calculated.
Specific results of the IC80 (μg/ml) inhibition of each pseudovirus subtype for each example are shown below.
TABLE 10 neutralization effects of HIV pseudoviruses by different vectors (IC 80)
AE34 B14 B16 B121 BC28 BC43 C11 C15
Example 1 19.1 47.0 17.1 23.1 50.7 25.5 30.1 30.8
Example 2 15.9 27.5 9.0 38.6 37.1 13.8 23.4 20.6
From the above results, it can be seen that the carrier modified by MT2 cell membrane and targeting ligand has lower IC80, i.e. has stronger virus neutralization capability; however, the effect of adding siRNA into the vector on the neutralization ability of virus is not great, mainly because the experiment adopts an operation mode of combining the vector with pseudovirus and then infecting cells, and the investigation period is short, while the siRNA needs to target virus infected cells for its effect, enter the virus infected cells, escape from the endosome of the infected cells into the cytoplasm, form RNA-induced silencing complex (RISC) in the cytoplasm by the siRNA, regulate gene silencing, inhibit viral protein expression, and other complex processes, so that a long time is required for the therapeutic effect. The literature reports that the peak time of siRNA concentration and the peak time of therapeutic effect have very long time lags, and clinical data are usually from several days to several months. Therefore, the siRNA does not exert curative effect in the experimental process and is normal.
Example 11
Neutralization assay for different subtypes of HIV-1 virus
By HIV-1 AD8 And HIV-1 NL4-3 Viruses represent R5 and X4 ecotropic virus strains respectively, and the capability of the constructed bionic nano-system and the viruses for blocking infection of normal cells is examined.
The specific experimental steps are as follows: the carriers of example 1 and example 2 were diluted 3-fold, respectively, for a total of 9 gradients, except for the cell control group and the virus control group; incubating the diluted bionic nano-delivery system with two viruses of 200TCID in a medium containing 15 mug/mL diethyl aminoethyl dextran for 2 hours respectively, and preparing incubation solutions of the two viruses of PBS and 200TCID by the same method; TZM-bl cells were plated at 25000/well in 225. Mu.L/well, and 25. Mu.L of vector/virus mixture from the previous step was added to the cells so that 250. Mu.L of liquid was present per well, and an equal amount of PBS was added to the cell control group; adding a mixture of equal amount of PBS and 200TCID viruses into a virus control group, and incubating and culturing for 6 hours at 37 ℃ under the condition of 5% CO 2; removing culture medium after 6 hr, adding PBS to wash cells, centrifuging at 37deg.C for 3 times for 10min, washing off free virus, adding 250 μl/hole of culture medium, culturing in cell culture box at 37deg.C under 5% CO 2 The method comprises the steps of carrying out a first treatment on the surface of the After 48 hours, the amount of p24 in the culture supernatant was measured to determine the replication efficiency of the virus and the inhibitory effect of the vector on the virus, and IC80 of the vector on each strain was calculated.
According to the results of the above experiments for measuring the neutralization ability of the carrier viruses in example 1 and example 2, a carrier concentration having a virus inhibition ratio of about 80% was selected, and control carriers (simple cell membrane-modified carrier, simple ligand-modified carrier) were prepared in respective concentrations, and incubated with 200TCID HIV-1NL4-3 virus and AD8 virus in a medium containing low concentration of diethylin for 2 hours, respectively. Subsequent experiments were then performed following the procedure described above to evaluate the effect of the vectors in reducing the infectivity of the eukaryotic virus, resulting in the blocking of normal cells by both viruses by different pairs of vectors.
The specific experimental results are shown in FIG. 3-graph6 and tables 11-12, the HIV-1 of example 1 was obtained by calculation from the above results AD8 And HIV-1 NL4-3 Virus neutralization IC 80 16.4. Mu.g/mL and 64.2. Mu.g/mL, respectively, example 2HIV-1 AD8 And HIV-1 NL4-3 The virus neutralization IC80 is 20.6 mug/mL and 34.4 mug/mL respectively, and the vector (the vector in the example 1 and the example 2) is co-modified by MT2 cell membrane and ligand, and has stronger virus neutralization capability than the simple enveloped liposome.
TABLE 11 HIV virus neutralization inhibition test results (viral infection rate,%)
Table 12 Experimental results of neutralization inhibition of HIV virus (viral infection Rate,%)
Control group Liposome group Coated liposome group Example 1
HIV-1 NL4 -3 100±2 92±1 39±3 16±4
HIV-1 AD8 100±3 93±4 37±9 18±4
Example 12
Inhibition assay for HIV-1 envelope protein induced bystander T cell apoptosis
In order to verify that the prepared vector can inhibit the killing effect of virus gp120 protein on bystander T cells after being combined with virus envelope protein, different vectors are respectively mixed with HIV-1gp120 protein for incubation and then added into Naive CD4+ cells, and the relative activity of final cells of each group of cells is compared to determine the inhibiting effect.
The specific experimental process is as follows: after incubation of the HIV-1gp120 protein with different vectors at equal concentrations for 2h, 10. Mu.L each was added to a solution containing 5X 10 5 Culturing in 96-well plates of individual/well Naive cd4+ cells; after 24h, 10. Mu.L of CCK-8 reagent was added to each 100. Mu.L of medium per well and incubated in an incubator at 37℃for about 4h; after 4 hours, using a multifunctional enzyme-labeled instrument to set 450nm as a detection wavelength and 650nm as a reference wavelength, carrying out a dual-wavelength method to measure the OD absorbance of each group of samples and calculating. And (3) result judgment: cell viability (cellViability) is expressed as a percentage of control (untreated) cells as follows: cell viability= (OD Experiment -OD Background of the invention )/(OD Control -OD Background of the invention )×100％。
The specific experimental results are as follows:
TABLE 13 inhibition experiments (cell Activity,%)
Control group Liposome group Coated liposome group Blank carrier set Example 1
HIV-1 MN 50.2±1.7 51.6±2.1 83.6±3.5 92.9±5.7 94.2±2.2
HIV-1 BaL 40.7±2.2 43.8±1.7 75.9±3.2 91.1±4.8 89.7±7.1
TABLE 14 inhibition experiments (cell Activity,%)
From the above results, it was found that example 1, example 2 and the coated control liposome thereof all had an effect of improving cell activity, and the cell activity improvement was most remarkable in the carriers (examples 1 and 2) modified with both cell membranes and ligands.
Example 13
Lysosomal escape effect evaluation of delivery vehicle
At 2X 10 5 Density of individuals/wells RAW264.7 cells in log phase were seeded in a cofocal dish and placed in a cell incubator for overnight culture; diluting the vector coated with cy5 labeled siRNA in example 1 to 125nM in serum-free DMEM medium, adding 2mL into a confocal dish carrying cells, and incubating for 1h, 3h and 6h in a cell incubator under dark condition; taking out the small dish after reaching the established incubation time, discarding the culture solution, adding 2mL of PBS, slightly shaking and washing to remove the non-ingested carrier, 5min each time, adding 2mL of LysoTracker Red dye solution preheated at 37 ℃ for 3 times, and placing the cell culture box in a cell incubator to be cultured for 30min in a dark place; taking out the small dish after the culture is finished, discarding the culture solution, adding 2mL of PBS, slightly shaking and washing to remove redundant dye solution, adding 3 times each time for 5min, adding 2mL of 4% paraformaldehyde tissue fixing solution, fixing at room temperature for 20min, adding 2mL of PBS, slightly shaking and washing, and repeating 3 times each time for 5 min; adding 2mL of Hoechst33258 dye solution with the concentration of 10 mug/mL to dye the cell nuclei, discarding the dye solution after 10min, adding 2mL of PBS, gently shaking to wash away unbound cell nucleus dye, and repeating for 3 times every 5 min; finally, 50% glycerol prepared by 1mL of sterile PBS is added into each Confocal petri dish for sealing treatment and light-shielding storage; observations were made using a confocal laser microscope, and appropriate fields of view were selected for scanning and photographing and analysis of the results.
As shown in FIG. 7, the siRNA in the constructed vector can realize lysosome escape after endocytosis, and the siRNA is successfully released into the cytoplasm, so that the gene silencing effect of the siRNA is normally exerted.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (2)

1. A composition for targeted delivery of an anti-HIV drug, comprising: preparing liposome by using HSPC, cholesterol and DSPE-PEG2000, wrapping the liposome by using MT2 cell membrane, and adding 12p1 or UM15 modified DSPE-PEG2000 to obtain a composition with targeting effect;
the amino acid sequence of 12p1 is shown as SEQ ID NO.1, and the amino acid sequence of UM15 is shown as SEQ ID NO. 2.
2. A pharmaceutical composition for targeting HIV and/or HIV-infected cells, comprising an anti-HIV agent comprising siRNA and the composition of claim 1.
CN202211440925.9A 2022-11-17 2022-11-17 Composition for targeted delivery of anti-HIV drug, pharmaceutical composition and preparation method Active CN116172956B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211440925.9A CN116172956B (en) 2022-11-17 2022-11-17 Composition for targeted delivery of anti-HIV drug, pharmaceutical composition and preparation method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211440925.9A CN116172956B (en) 2022-11-17 2022-11-17 Composition for targeted delivery of anti-HIV drug, pharmaceutical composition and preparation method

Publications (2)

Publication Number Publication Date
CN116172956A CN116172956A (en) 2023-05-30
CN116172956B true CN116172956B (en) 2023-09-22

Family

ID=86439109

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211440925.9A Active CN116172956B (en) 2022-11-17 2022-11-17 Composition for targeted delivery of anti-HIV drug, pharmaceutical composition and preparation method

Country Status (1)

Country Link
CN (1) CN116172956B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0432693A1 (en) * 1989-12-11 1991-06-19 Sumitomo Chemical Company, Limited Liposomes cytotoxic to virus-infected cells
EP1666056A1 (en) * 2004-12-06 2006-06-07 Bestewil Holding B.V. Cancer vaccine vesicles
CN103550783A (en) * 2013-04-27 2014-02-05 中国人民解放军军事医学科学院放射与辐射医学研究所 Nucleic acid drug targeting delivery system and preparation method thereof
CN115006368A (en) * 2022-07-01 2022-09-06 重庆大学 Cell membrane coated nano-drug and application thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030232738A1 (en) * 1998-04-20 2003-12-18 National Jewish Medical And Research Center Method and composition for increasing CD4+ T lymphocyte immune responsiveness
US7556808B2 (en) * 2004-12-16 2009-07-07 Philadelphia Health And Education Corporation Dual inhibitors of HIV-1 gp-120 interactions
WO2010151675A1 (en) * 2009-06-24 2010-12-29 Philadelphia Health & Education Corporation D/B/A Drexel University College Of Medicine Active cores of peptide triazole hiv-1 entry inhibitors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0432693A1 (en) * 1989-12-11 1991-06-19 Sumitomo Chemical Company, Limited Liposomes cytotoxic to virus-infected cells
EP1666056A1 (en) * 2004-12-06 2006-06-07 Bestewil Holding B.V. Cancer vaccine vesicles
CN103550783A (en) * 2013-04-27 2014-02-05 中国人民解放军军事医学科学院放射与辐射医学研究所 Nucleic acid drug targeting delivery system and preparation method thereof
CN115006368A (en) * 2022-07-01 2022-09-06 重庆大学 Cell membrane coated nano-drug and application thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CD4 ⴙ T Cell-Mimicking Nanoparticles Broadly Neutralize HIV-1 and Suppress Viral Replication through Autophagy;Gang Zhang等;American society for microbiology;第11卷(第5期);e00903-20 *
半乳糖神经酰胺脂质体对HIV-1感染性的影响;王茜, 郑永唐, 戎隆富, 刘广杰;免疫学杂志(02);128-131 *
抗HIV巨噬细胞靶向给药系统的研究进展;杜丽娜;金义光;;国际药学研究杂志(01);45-48 *
杜丽娜 ; 金义光 ; .抗HIV巨噬细胞靶向给药系统的研究进展.国际药学研究杂志.2013,(01),45-48. *
王茜,郑永唐,戎隆富,刘广杰.半乳糖神经酰胺脂质体对HIV-1感染性的影响.免疫学杂志.2002,(02),128-131. *

Also Published As

Publication number Publication date
CN116172956A (en) 2023-05-30

Similar Documents

Publication Publication Date Title
Miller et al. Alterations in cell membrane permeability by the lentivirus lytic peptide (LLP-1) of HIV-1 transmembrane protein
EP0298280B1 (en) Animal derived cell with antigenic protein introduced therein
Zou et al. Extracellular vesicles expressing a single-chain variable fragment of an HIV-1 specific antibody selectively target Env+ tissues
Röcker et al. Structure, function and antagonism of semen amyloids
CN105209617B (en) Microcapsule bubble and its manufacturing method
Asadikaram et al. Niosomal virosome derived by vesicular stomatitis virus glycoprotein as a new gene carrier
Gomara et al. Peptide assembly on the membrane determines the HIV-1 inhibitory activity of dual-targeting fusion inhibitor peptides
CN116172956B (en) Composition for targeted delivery of anti-HIV drug, pharmaceutical composition and preparation method
CN114292848A (en) Engineered exosome with high expression of cancer-suppressing miRNA and targeting lung cancer
CN103626846A (en) Ligand polypeptide specifically combined with MDSCs (Myeloid-Derived Suppressor Cells) and drug delivery system
Moreno et al. Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan
CN105770912B (en) Load medicine ATP sensitive liposome with tumour near-infrared fluorescent image displaying function and preparation method thereof
CN105031623B (en) Biological agent and the application in the medicine for preparing prevention and control Ebola virus
Shen et al. Exosomal vaccine loading T cell epitope peptides of SARS-CoV-2 induces robust CD8+ T cell response in HLA-A transgenic mice
JPH06504429A (en) Inhibition of viral replication
CN104546726A (en) Self-assembled nucleic acid nanotube preparation as well as preparation method and application thereof
CN112472791A (en) Application of CsA liposome in preparation of anti-SARS-CoV-2 medicine
Zhang et al. 3-hydroxyphthalic anhydride-modified rabbit anti-PAP IgG as a potential bifunctional HIV-1 entry inhibitor
CN106110422B (en) AIDS immunization therapy absorber
CN108586580A (en) Cell-penetrating peptides DHYHPFS and as the intracellular purposes for transporting carrier
CN108727472A (en) Negatively charged cell-penetrating peptides and the purposes as intracellular transport carrier
CN108752426A (en) Cell-penetrating peptides with neutral charge and the purposes as intracellular transport carrier
Düzguneş et al. Liposome-mediated therapy of human immunodeficiency virus type-1 and Mycobacterium infections
Caobi UNDERSTANDING EXOSOMAL EXTRACELLULAR VESICLES AND MORPHINE IN THE NEUROPATHOLOGY OF HUMAN IMMUNODEFICIENCY VIRUS AND DIFFERENTIAL ZIKA VIRUS STRAIN-ASSOCIATED PATHOLOGY
RU2701959C1 (en) Method of controlling production of a vaccine against influenza

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant