CN114699538A

CN114699538A - Core-shell type efficient gene drug delivery system and preparation method thereof

Info

Publication number: CN114699538A
Application number: CN202210350675.3A
Authority: CN
Inventors: 霍美蓉; 张盼; 刘楠; 周新源
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2022-07-05

Abstract

The invention provides a high-efficiency gene drug delivery system and a preparation method thereof. The delivery system consists of an inner core formed by protamine adsorption genes and an enzyme sensitive phospholipid shell. Protamine adsorbs genes to form a stable electropositive inner core, enzyme sensitive polypeptide is inserted into the electronegative or neutral phospholipid layer to form an electronegative or neutral outer shell, and the constructed core-shell nano system is beneficial to efficient delivery and transfection of gene drugs. Meanwhile, the phospholipid shell of the gene delivery system can be loaded with hydrophobic drugs according to needs, so that the efficient combined treatment effect of the gene drugs and the hydrophobic drugs is realized. The core-shell type efficient gene delivery system has the advantages of high biological safety, easy obtainment, simple preparation process, easy industrialization, low economic cost, environmental protection, effective promotion of efficient loading, stable transportation, targeted delivery and efficient transfection of gene drugs, good safety and effectiveness, and the possibility of joint co-delivery and cooperative treatment of the gene drugs and chemical drugs.

Description

Core-shell type efficient gene drug delivery system and preparation method thereof

Technical Field

The invention belongs to the technical field of drug gene carrier materials, and particularly relates to a high-efficiency gene drug delivery system and a preparation method thereof.

Background

With the improvement of living standard of people, the requirements of people on health are higher and higher. However, the incidence of various diseases such as cancer is high, and in the current clinical treatment, the chemical drug treatment still occupies the core position, but many diseases depend on only the chemical treatment with limited effect.

In recent years, gene therapy has been receiving increasing attention in clinical treatment of intractable diseases. The treatment mode is a fundamental treatment means for improving diseases by introducing external normal genes or therapeutic genes into target cells of a human body. The gene medicine can be used alone or combined with other chemical medicines with different treatment mechanisms to play a role in clinical treatment. However, most of the genetic drugs are composed of rigid polyanionic molecules with large molecular weight, are not easy to permeate through biological membranes, are very easy to degrade by nuclease, and can effectively exert the curative effect only by adopting a carrier rich in positive charge to effectively compress and efficiently transfect the gene. CN113683780A discloses a side chain guanidinylated polyamino acid cationic gene delivery vector, which can effectively compress genes, but has the problems of complex vector synthesis process and large toxicity in a positive charge carrier body. WeiweiWan et al (Journal of Controlled Release: 242 (2016: 71-79) report on grafting 25kDa polyethyleneimine (PEI25) and polyethylene glycol (PEG) onto poly [ (e-caprolactone) -co-glycoside ] (CG) using amphiphilic PEI-CG-PEI and PEG-CG block copolymers for micelle formation by self-assembly of PEI-CG-PEI or co-assembly of both copolymers for DNA and siRNA delivery. However, there are several serious problems in constructing a carrier by selecting a high molecular weight 25kDa cationic material PEI, such as cytotoxicity caused by strong positive charge and safety problem caused by the fact that high molecular weight PEI is not easily degraded in vivo.

On the basis of high-efficiency gene delivery, if the carrier can simultaneously load chemical drugs, feasibility is provided for stable co-delivery and synergistic treatment of the gene drugs and the chemical drugs. However, the gene drug is a water-soluble biological macromolecule drug, while a considerable part of the chemical drugs are hydrophobic small molecules, so that the problems of poor solubility and low absorption rate exist, and the effective co-delivery of the gene drug and the chemical drugs is often more challenging. Guan et al (Colloids and Surfaces B: Biointerfaces,162(2018):326-334) report a cationic micelle based on chitosan grafted poly (N-3-carbobenzyloxy-lysine) (CPCL) and modified with Cell Penetrating Peptide (CPP) for the co-delivery of chemical drug Doxorubicin (DOX) and gene drug p53 plasmid, the delivery system can effectively promote the uptake of target cells, but has the problems of complex vector synthesis process, uncontrollable drug release speed after the drug and the gene drug are inserted into cells, certain toxicity of the cationic vector and the like. Chinese patent CN104758952A also discloses a triphenylphosphine derivative-lonidamine prodrug carrier (TCPL) -siRNA-polyethylene glycol/polyacrylic acid (TCPL-siRNA-PPX) nano delivery system containing targeting ligand for co-delivery of drugs and genes. Although the delivery system can deliver the gene drug and the chemotherapeutic drug to a target site in a targeted manner, the delivery system has the defects of limited chemical coupling amount of the prodrug, very complicated synthesis process, low uptake efficiency of negatively charged carrier cells and the like.

Protamine (protamine) is used as a natural gene drug delivery material, and has strong gene adsorption capacity and high transfection efficiency. In addition, as a clinical use medicine, protamine has low toxic and side effects and cannot cause exogenous anaphylactic reaction. However, nanoparticles formed after protamine adsorbs genes have the characteristic of strong positive charges, are easy to adsorb nonspecific proteins, are extremely unstable in blood circulation, and are easy to be recognized and eliminated by a Mononuclear Phagocyte System (MPS), so that the nanoparticles cannot reach the action site. Ke Men et al (RSC Advances,8(2018):12104-12115) reported a cationic liposome prepared with cationic lipid material DOTAP to form a complex with protamine for the delivery of VSVMP mRNA (CLPP/mRNA). The system has very high positive charge density, and can effectively compress genes, but a large amount of positive charges can bring large toxic and side effects in vivo, and the genes are released too slowly and difficultly exert curative effects due to strong charge interaction with carrier materials. Vader et al (Journal of Controlled Release,160(2012):211-216) reported that a similar cationic lipid material, DOTAP, and protamine form a complex for the delivery of DNA and siRNA mixture, and further applied RGD-PEG-DSPE and PEG-DSPE for surface modification in order to shield the positive charge, although PEG can partially shield the positive charge, the problem of slow Release of gene drug is still not solved.

Based on the above, this patent developed a novel core-shell efficient gene delivery system consisting of an inner core formed by protamine adsorbing genes and an enzyme-responsive phospholipid shell. The system has the advantages that: 1) the useful raw and auxiliary materials are easy to obtain, and have the characteristics of good biocompatibility, biodegradability and no immunogenicity; 2) the system adopts a core-shell structure, and the positive charge core can efficiently compress genes; the neutral or electronegative shell can effectively shield the positive charge of the core and protect the gene to achieve the effect of stable transportation in vivo; 3) the system reaches a target site, and the shell can respond to an enzymolysis body existing in a target spot to expose the electropositive core, so that the electropositive core is easy to be absorbed by target cells, the transfection capability of genes is effectively improved, and the curative effect is exerted; 4) the phospholipid shell of the system can be further loaded with hydrophobic drugs according to needs, and the hydrophobic drugs can be rapidly released along with the enzyme triggering disintegration of the phospholipid shell, so that the purposes of stable co-loading and efficient combined treatment of gene drugs and hydrophobic drugs are achieved. The delivery system not only creates a platform for the efficient delivery and the effective transfection of genes, but also creates a platform for the efficient combined use of gene drugs and chemical drugs, can greatly improve the clinical treatment effect, has simple preparation process, controllable quality and good safety, and has higher clinical transformation potential.

Disclosure of Invention

The invention aims to provide a core-shell type efficient gene drug delivery system, which consists of an inner core formed by protamine adsorption genes and an enzyme sensitive phospholipid shell. Wherein the delivery system uses protamine compressed gene as natural gene delivery material, and has better degradability and lower immunogenicity compared with chemosynthetic cationic carrier. Meanwhile, the neutral or electronegative enzyme sensitive drug-loaded phospholipid is used for coating the core with positive charges, so that the positive charge effect of the gene carrier is shielded, the nonspecific adsorption of the preparation in a blood system is reduced, the stable transportation is realized, the phospholipid shell can respond to an enzymolysis body of a target spot and expose the core with positive charges, the uptake of the phospholipid by target cells is facilitated, and the better ground-based silencing effect is exerted. In addition, the phospholipid shell of the gene delivery system can be loaded with hydrophobic drugs according to needs, and under the stimulation of enzyme existing at an action target, the phospholipid shell can be decomposed in a responsive manner and quickly release the hydrophobic drugs, so that the precise co-delivery of the hydrophobic drugs and the gene drugs is realized, and the efficient synergistic treatment is achieved. The core-shell type efficient gene drug delivery system has the advantages of simple preparation process, controllable quality, realization of scale-up production by using an online liposome extruder conforming to cGMP production, and good commercial transformation prospect.

The second purpose of the invention is to provide a preparation method of the core-shell type high-efficiency gene drug delivery system.

The technical scheme is as follows: the invention provides a high-efficiency gene drug co-delivery system, which consists of an inner core formed by protamine adsorption genes and an enzyme sensitive phospholipid shell.

Specifically, the inner core formed by the protamine adsorption gene consists of protamine and the gene.

The enzyme sensitive phospholipid shell consists of enzyme sensitive polypeptide, cholesterol and neutral or electronegative phospholipid.

The enzyme sensitive phospholipid shell may be further loaded with hydrophobic drugs as required.

The above genes are any one or more of DNA, siRNA, shRNA, mRNA, miRNA, and CRISPR.

The enzyme sensitive polypeptide is any one or more of matrix metalloproteinase 2, matrix metalloproteinase 9, fibroblast activation protease, cathepsin B, secretory phospholipase A2, alpha-amylase, lysyl oxidase, and beta-glucuronidase sensitive polypeptide.

The neutral or electronegative phospholipid is any one or more of natural phospholipid, glycerol-3-Phosphorylcholine (PC), glycerol-3-Phosphorylethanolamine (PE), glycerol-3-phosphate-L-serine sodium salt (PS), phosphatidyl-DL-glycerol (PG), glycerol-3-phosphate sodium salt (PA) phospholipid and derivatives thereof.

The mass ratio of the enzyme sensitive phospholipid shell to the inner core formed by the protamine adsorption gene is 10:1-2000: 1.

The mass ratio of the protamine to the gene is 1:1-50: 1.

The mass ratio of the neutral or electronegative phospholipid to the cholesterol is 1:1-100: 1.

The mass ratio of the neutral or electronegative phospholipid to the enzyme response polypeptide is 2:1-100: 1.

The particle size of the core-shell type efficient gene delivery system is 10-1000 nm.

The invention provides a preparation method of the high-efficiency core-shell type gene drug delivery system, which comprises the following steps:

1) weighing protamine powder, dissolving with HEPES solution completely, and mixing with gene medicine solution to obtain protamine adsorption gene inner core;

2) weighing neutral or electronegative phospholipid, cholesterol and hydrophobic drug (or not), dissolving with organic solvent completely, placing into a reaction bottle, adding enzyme sensitive polypeptide, dissolving completely, and evaporating organic solvent under reduced pressure to obtain a uniform thin film layer; adding phosphate buffer solution for hydration for a period of time, extruding the obtained suspension to pass through a membrane, adding the protamine adsorption gene kernel into the obtained liquid, uniformly mixing, extruding again to pass through the membrane, and finally preparing a core-shell type efficient gene delivery system or a hydrophobic drug and gene drug co-delivery system;

the organic solvent used in the step 2) is any one or more of dichloromethane, dimethyl sulfoxide, methanol, ethanol, ethyl acetate, N-dimethylformamide or N-hexane.

More specifically, the preparation method of the core-shell type efficient gene drug delivery system comprises the following steps:

1) accurately weighing protamine powder, fully dissolving with HEPES solution (10mM, pH 7.2), selecting appropriate solution according to different gene drugs to prepare gene drug solution, and mixing the two solutions according to a certain proportion to obtain protamine adsorption gene inner core;

2) the neutral or electronegative phospholipid, cholesterol and hydrophobic drugs (or none) are precisely weighed, are completely dissolved by using a proper amount of organic solvent, are placed in a reaction bottle, are added with a certain amount of enzyme sensitive polypeptide to be completely dissolved, and are subjected to reduced pressure evaporation of the organic solvent under the condition of reaching the reduced pressure boiling point temperature of the organic solvent to form a uniform film layer on the bottle wall. The phospholipid membrane was hydrated for 30min by adding an appropriate volume of PBS (pH 7.4). The resulting suspension was extruded back and forth 10-30 times each using a liposome extruder with 0.4 μm and 0.2 μm pore size etched pore membranes. Then adding the inner core solution of protamine adsorption genes according to a certain mass ratio, uniformly mixing, and extruding back and forth for 10-30 times by using liposome extruders with pore membranes etched by the apertures of 0.4 mu m and 0.2 mu m respectively, finally preparing the core-shell type efficient gene delivery system or the hydrophobic drug and gene drug co-delivery system.

The invention provides another preparation method of the core-shell type efficient gene drug delivery system, which comprises the following steps:

1) weighing protamine powder, dissolving with HEPES solution completely, and mixing with gene solution to obtain protamine adsorbed gene core;

2) weighing neutral or electronegative phospholipid, cholesterol and hydrophobic drug (or not), dissolving with organic solvent completely, placing into a reaction bottle, adding enzyme sensitive polypeptide, dissolving completely, and evaporating organic solvent under reduced pressure to obtain a uniform thin film layer; adding the inner core solution of protamine adsorbing gene, hydrating for some time, extruding to pass through membrane, and finally obtaining the core-shell gene medicine delivery system or hydrophobic medicine and gene medicine co-delivery system.

More specifically, the preparation method of the efficient core-shell type gene drug delivery system comprises the following steps:

1) accurately weighing protamine powder, fully dissolving with HEPES solution (10mM, pH 7.2), selecting appropriate solution according to different gene drugs to prepare gene solution, and mixing the two solutions according to a certain proportion to obtain an inner core of protamine adsorption gene;

2) the neutral or electronegative phospholipid, cholesterol and hydrophobic drugs (or none) are precisely weighed, are completely dissolved by using a proper amount of organic solvent, are placed in a reaction bottle, are added with a certain amount of enzyme sensitive polypeptide to be completely dissolved, and are subjected to reduced pressure evaporation of the organic solvent under the condition of reaching the reduced pressure boiling point temperature of the organic solvent to form a uniform film layer on the bottle wall. Adding a core solution with a proper volume of protamine adsorption genes into a phospholipid membrane, hydrating for 30min, then extruding back and forth for 10-30 times by using a liposome extruder with a pore membrane etched by 0.4 mu m and 0.2 mu m, and finally preparing the core-shell type gene drug delivery system or the hydrophobic drug and gene drug co-delivery system.

The invention has the following function principle:

the action principle of the core-shell efficient gene drug delivery system is as follows: after intravenous injection administration, the delivery system is stably transported in vivo due to the nano-scale structure and the surface neutral or electronegativity characteristic, reaches a target tissue, and under the stimulation of specific enzyme of the target tissue, the outer-layer phospholipid is disintegrated to expose a gene inner core rich in positive charges, which is beneficial to being taken by target cells, effectively improves the gene transfection capability and exerts curative effect. Meanwhile, the outer phospholipid layer of the gene delivery system can be loaded with hydrophobic drugs according to needs, and the phospholipid layer can rapidly release the hydrophobic drugs to exert the pharmacological activity of the hydrophobic drugs and the gene drugs to exert the maximum synergistic treatment effect under the stimulation of target enzyme. The delivery system can effectively realize stable loading and high-efficiency transfection of the gene drug, and provides possibility for combined delivery and synergy of the gene drug and the hydrophobic drug.

Advantageous effects

How to effectively and efficiently deliver gene drugs to a target site is always a technical problem in the field, and the nano delivery system adopted by the prior art solution still has the following problems: 1) the positive charge adjuvant is adopted to compress the gene, if the positive charge is too strong, the gene can be effectively compressed and can be efficiently transfected, but the gene has larger toxicity in vivo and is easy to be cleared by a phagocytosis system; if the positive charge is too little, the gene is loaded unstably and is easily degraded by nuclease, esterase and the like in vivo in blood circulation, and the half-life period is short; 2) if the strong positive charge of the carrier is covered by the auxiliary material with negative charge, although the safety can be improved, the problems of difficult cell entry and low gene transfection efficiency can be caused on the electronegative surface of the carrier; 3) the current gene delivery system can not effectively load hydrophobic drugs with different properties and is difficult to meet the requirements of combined treatment. The efficient gene drug delivery system with the core-shell structure is a brand new delivery system, can effectively solve the problems, and related in vitro, cell and animal experiments effectively prove that the delivery system has excellent stability in blood circulation, can effectively deliver genes to target sites stably, realizes efficient transfection, and can load hydrophobic drugs as required to perform effective combined treatment.

1. The delivery system consists of an inner core made of protamine and an outer shell made of electronegative or neutral phospholipid, wherein the cationic inner core can realize effective compression and loading of gene drugs, and the neutral or electronegative phospholipid outer shell can shield positive charges of the inner core and can protect stable loading and stable in vivo transportation of the gene drugs of the inner core.

2. Under the stimulation of enzyme in the action target, the phospholipid shell can be decomposed in a responsive manner, and the gene inner core rich in positive charges is exposed, so that the phospholipid shell is taken up by target cells, and the gene transfection is effectively improved.

3. The delivery system can load hydrophobic drugs on the phospholipid shell according to the needs of drug combination, can realize effective release and efficient transfection of the hydrophobic drugs and genes at target points, exerts the synergistic interaction of the hydrophobic drugs and the genes to the maximum extent, greatly improves the clinical treatment effect, and provides a new design idea for exploring more drug combination modes.

4. The core-shell type efficient gene delivery system has the advantages of high biological safety, easy obtainment, simple preparation process, easy industrialization, low economic cost and environmental protection.

Drawings

FIG. 1 shows MSL/Pro_-siPD-L1A transmission electron microscope image of the delivery system, wherein A is an original image, and B is a partially enlarged image;

FIG. 2 is MSL/Pro_-siPD-L1A map of the responsive morphometric changes of the delivery system, wherein a is an electron micrograph without incubation with MMP2 enzyme and B is an electron micrograph with incubation with MMP2 enzyme;

FIG. 3 shows MSL/Pro_-siPD-L1A delivery system responsive charge reversal map;

FIG. 4 shows MSL/Pro_-siPD-L1A delivery system serum stability profile;

FIG. 5 is MSL/Pro-_siNCThe cytotoxic consequences of the delivery system;

FIG. 6 shows MSL/Pro-_sicy5A graph of cellular uptake results for the delivery system;

FIG. 7 shows MSL/Pro-_siLucResults plot of gene transfection efficiency for delivery system;

FIG. 8 is a MSL^-LY/Pro_-siPD-L1A delivery system response in vitro drug release profile;

FIG. 9 is MSL^-LY/Pro_-siPD-L1Tumor profile following treatment of breast cancer tumor-bearing mice with the delivery system;

FIG. 10 is a MSL^-LY/Pro_-siPD-L1Tumor volume-time plot after treatment of breast cancer tumor-bearing mice with the delivery system;

FIG. 11 is a MSL^-LY/Pro_-siPD-L1Tumor mass maps following treatment of breast cancer tumor-bearing mice with the delivery system;

FIG. 12 is a MSL^-LY/Pro_-siPD-L1The result graph of tumor PD-L1 protein expression after the delivery system treats the breast cancer tumor-bearing mice, wherein A is a tumor tissue PD-L1 protein immunohistochemical graph, and B is a tumor tissue PD-L1 protein semi-quantitative result graph.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the claims.

Example 1

Taking siRNA (siPD-L1) for silencing PD-L1 protein as an example, a core-shell type high-efficiency gene is constructedDue to the drug delivery system MSL/Pro_-siPD-L1It was prepared and characterized as follows:

(1) preparation of core-shell type high-efficiency gene delivery system

1) Weighing appropriate amount of protamine powder, dissolving with HEPES solution (10mM, pH 7.2), preparing siPD-L1 solution with concentration of 20mM with DEPC solution, mixing the two solutions at a mass ratio of 3:1 by vortex for 5min to obtain protamine adsorbed PD-L1 siRNA nano-core (Pro-siPD-L1)

2) The components are precisely weighed according to the mass ratio of 1, 2-dioleoyl-sn-glycerol-3-phosphorylcholine (DOPC) to cholesterol (w: w:) ═ 10:1, and after the components are completely dissolved by dichloromethane, 2mg of miniPEG-G (C14) PLGIAgQ (C14) -miniPEG (MMP2 enzyme-responsive polypeptide) is added. The organic solvent was removed under reduced pressure at 37 ℃ to form a uniform thin film layer on the wall of the bottle. Adding phosphate buffer solution with pH 7.4 into phospholipid membrane, and hydrating for 30 min. Squeezing the obtained suspension with liposome extruder with pore membrane etched with 0.4 μm and 0.2 μm pore diameter for 20 times, adding Pro-siPD-L1 nanometer core solution according to the mass ratio of MSL: Pro-siPD-L1(w: w) ═ 200:1, mixing, squeezing with liposome extruder with pore membrane etched with 0.4 μm and 0.2 μm pore diameter for 20 times, and making into MSL/Pro-shell type high-efficiency gene drug delivery system with core-shell structure_-siPD-L1。

(2) Characterization of delivery systems

The particle size and potential of the samples were measured using a Zetasizer 3000HS Instrument (Malvern Instrument, Malvern, UK) at 633nm, 25 ℃ and He-Ne laser, and the results are given in Table 1.

TABLE 1MSL/Pro_-siPD-L1Characterization of the NanoDeliver System

Taking 1 drop of MSL/Pro_-siPD-L1The solution was dropped onto a copper mesh and counterstained with a 2% tungsten phosphate solution, air dried at room temperature and the particle morphology was photographed using a Transmission Electron Microscope (TEM). As shown in FIG. 1, MSL/Pro_-siPD-L1The nanoparticles exhibit a typical "core-shell" morphologyThe middle is a sphere-like Pro-siPD-L1 nano core, the outer layer is a white phospholipid shell, the thickness of the outer phospholipid shell is about 10nm, and compared with the size of the Pro-siPD-L1 nano core observed in TEM, the MSL/Pro is_-siPD-L1The added thickness is just the thickness of the phospholipid layer. This is also consistent with the particle size measured for both in solution, indicating the successful construction of a highly efficient core-shell gene drug delivery system loaded with siPD-L1.

Example 2

MSL/Pro_-siPD-L1In vitro responsive morphological Change assessment of delivery systems

MSL/Pro prepared in example 1_-siPD-L1Delivery system, incubated for 6h with PBS (pH 7.4), MMP2 enzyme solution (pH 7.4), respectively. And then dropping the preparation on a copper net, counterdyeing by using 2% tungsten phosphate, standing at room temperature, airing, and observing the change of particle size morphology by using a transmission electron microscope.

As shown in FIG. 2, responsive MSL/Pro without incubation with MMP2 enzyme_-siPD-L1The shell of (a) is clearly visible (fig. 2A), but the phospholipid shell dissociates after incubation with MMP2 enzyme, exposing the positively charged nanocore (fig. 2B). The above results further demonstrate that MSL/Pro was achieved upon incubation with MMP2 enzyme_-siPD-L1The delivery system has the ability to enzymatically respond to the removal of the phospholipid shell.

Example 3

MSL/Pro_-siPD-L1In vitro responsive charge reversal evaluation of delivery systems

To investigate in depth the MSL/Pro prepared in example 1_-siPD-L1The enzyme response capability of the delivery system. Will react with MSL/Pro-_siPD-L1Incubation with MMP2 enzyme was performed and Zeta potential of the nano delivery system was measured at 0, 1,2, 4, 6h, respectively.

As shown in FIG. 3, the responsiveness MSL/Pro_-siPD-L1The surface charge of the nano delivery system is-3.8 mV, and the zeta potential is helpful for the stable transportation of the nano particles in vivo. The charge of the nanosystems gradually increased from-3.8 mV to +13.2mV within 6h of incubation with MMP2 enzyme, indicating that MSL/Pro was under the action of MMP2 enzyme_-siPD-L1The charge reversal was achieved, indicating MSL/Pro with incubation of MMP2 enzyme_-siPD-L1The nano delivery system has the capability of rapidly responding to enzyme and removing phospholipid shells, exposes the nano inner core with positive charges, is favorable for improving the capability of taking the nano inner core by cells and enhancing the delivery efficiency of gene drugs.

Example 4

MSL/Pro_-siPD-L1Delivery System serum stability Studies

Mixing MSL/Pro_-siPD-L1Serum stability of the delivery system was evaluated by incubating with 10% serum (FBS) for 2, 4, 6, 8, 12, 16 and 24h, respectively, and taking solutions at different time points to measure particle size and potential.

As shown in FIG. 4, MSL/Pro_-siPD-L1When the delivery system is incubated with 10% FBS, no obvious change occurs in the particle size and the potential within 24h, which indicates that the delivery system has better serum stability.

Example 5

MSL/Pro_-siNCDelivery System cytotoxicity Studies

MSL/Pro was prepared according to the preparation method in example 1, replacing siPD-L1 with siNC_-siNCDelivery system to investigate the cytotoxicity of the blank gene delivery system. Respectively planting murine breast cancer 4T1 cells and mouse fibroblast NIH 3T3 cells in a 96-well plate according to the density of 5000 cells/well, after the cells adhere to the wall overnight, removing the culture medium, adding a fresh complete culture medium, adding MMP2 enzyme, incubating for 4h, and adding MSL/Pro with different concentrations_-siNCAnd setting a control group, setting 6 replicates in each group, and culturing for 48 hours in a carbon dioxide incubator. When the culture is terminated, 20 mu L of MTT solution is added into each hole, the culture is continued in the incubator for 4h, then the solution in each hole is discarded, 150 mu L of DMSO solution is added, the hole plate is placed in a horizontal shaking table and is uniformly mixed at 800rpm for 4min to promote the dissolution of crystal violet, and the absorbance of each hole is measured at 570nm by using a microplate reader to calculate the survival rate of the cells.

As shown by the results in FIG. 5, MSL/Pro_-siNCThe delivery system has no obvious toxicity to two cells, and shows excellent safety.

Example 6

MSL/Pro_-siCy5Delivery System cellular uptake Capacity Studies

The fluorescent enzyme-responsive MSL/Pro was prepared according to the preparation method of example 1, substituting siPD-L1 for siCy5_-siCy5A delivery system. Replacing miniPEG-G (C14) PLGIAGQ (C14) -miniPEG (MMP2 enzyme response polypeptide) with miniPEG-G (C14) GGPALIQ (C14) -miniPEG (non-MMP 2 enzyme response polypeptide) to prepare the fluorescent non-enzyme response MNL/Pro_-siCy5A delivery system.

4T1 cells were plated at 1X 10⁶The density of each hole is planted in a confocal dish, after the cells adhere to the wall overnight, the culture medium is discarded, and the 1640 culture medium without serum is added. Then adding MSL/Pro respectively_-siCy5And MMP2 enzyme-pretreated MSL/Pro_-sicy5And MNL/Pro_-sicy5Incubate with cells for 4 h. After 4h, the cells were washed three times with pre-cooled PBS, fixed with 4% paraformaldehyde for 10min, washed again with PBS after completion, and the nuclei were stained with DAPI staining solution for 10min in the dark, followed by PBS washing and CLSM for Cy5 fluorescence intensity.

As shown in FIG. 6, after enzymatic pretreatment with MMP2, the cells were paired for MSL/Pro_-siCy5Has a greatly increased intake of MSL/Pro_-siCy5The uptake of the protein is higher than MNL/Pro_-siCy5Group, this is due to MSL/Pro after MMP2 enzyme incubation_-Cy5The outer shell can be removed in an enzyme response way, the Pro-siCy5 nano-core with positive charge is exposed, the cell uptake capacity of the cell to the Pro-siCy5 nano-core is obviously improved, and the MNL/Pro is_-siCy5Enzyme-responsive coat removal is not achieved and the uptake capacity of the cells is weak. The results prove that the MMP2 enzyme response core-shell delivery system constructed in the embodiment shows good MMP2 enzyme response uncoating capacity, and the rapid response of the MMP2 enzyme is benefited to crack the outer shell, so that the capacity of a cell for taking up the nanometer inner core is improved, and higher gene transfection efficiency is predicted.

Example 7

MSL/Pro_-siLucExamination of Gene transfection efficiency of delivery System

The fluorescent enzyme-responsive MSL/Pro was prepared by replacing siPD-L1 with siLuc according to the preparation method in example 1_-siLucA delivery system. Mixing miniPEG-G (C14) PLGIAGQ (C14) -miniPEG (M)MP2 enzyme response polypeptide) is replaced by miniPEG-G (C14) GGPALIQ (C14) -miniPEG (non-MMP 2 enzyme response polypeptide) to prepare fluorescent non-enzyme response MNL/Pro_-siLucA delivery system.

The luciferase reporter gene is a commonly used positive gene, and catalyzes substrate oxidation to generate oxyluciferin under the action of luciferase, so that the effectiveness of carrier transfection siRNA can be verified by silencing the luciferase reporter gene. The dual-luciferase detection kit can detect the luminescence intensity of firefly luciferase and sea cucumber luciferase respectively, and eliminate errors caused by different cell numbers by using the luminescence intensity of the sea cucumber luciferase as an internal reference. 4T1-Luc biotin fluorescent cells were arranged at 1X 10⁴Inoculating the cells/well in a 12-well plate, replacing a fresh culture medium and medicines after 12-18 h of cell adherence, and setting a blank group and MSL/Pro_-siNCGroup, free siLuc group (Naked siLuc), MNL/Pro_-siLucGroup, MSL/Pro_-siLucAnd (4) grouping. MMP2 response groups were pretreated with MMP2 enzyme for 4h before administration, the final concentration of siLuc in each group was 100nM, after 48h of culture in an incubator, a dual-luciferase reporter gene detection kit was used, after cell lysis, 20. mu.L of lysate was placed in a dark 96-well plate, firefly luciferase substrate was added, firefly luciferin reporter gene activity was detected at 560nM using a microplate reader, then 100. mu.L of freshly prepared renilla luciferase substrate was added to the reaction solution, and renilla luciferase reporter gene activity was detected at 465nM using a microplate reader. And calculating the relative expression strength of the firefly luciferase by taking the activity of the sea cucumber luciferase as an internal reference.

FIG. 7 Experimental results show that MMP 2-responsive MSL/Pro of transfection negative gene_-siNCAnd free siLuc were unable to silence the luciferase reporter. MNL/Pro_-siLucThe silencing effect on luciferase reporter gene is only about 31.2 percent, which is probably because under the action of MMP2 enzyme, responsive uncoating can not be realized due to the lack of enzyme response polypeptide, the nano nucleus for releasing delivery gene is less, the gene silencing effect is limited, and MMP2 responsive MSL/Pro for transfecting positive gene_-siLucThe silencing effect on the luciferase reporter gene reaches 72.1 percent. FromThe results show that the MMP2 enzyme-responsive core-shell delivery system constructed in this example can improve the ability of cells to take up gene-loaded cores under the action of MMP2 enzyme, has high gene transfection efficiency, and can exert excellent gene silencing effects.

Example 8

Taking siRNA (siPD-L1) silencing PD-L1 protein and TGF-beta small molecule inhibitor LY3200882 as examples, loading hydrophobic drug LY3200882 on phospholipid shell to prepare MSL (mesenchymal stem cell) of high-efficiency core-shell hydrophobic drug and gene drug co-delivery system^-LY/Pro_-siPD-L1The preparation and in vitro responsive drug release evaluation are as follows:

(1) preparation of delivery systems

1) Weighing appropriate amount of protamine powder, dissolving with HEPES solution (10mM, pH 7.2), preparing siPD-L1 solution with concentration of 20mM with DEPC solution, mixing the two solutions at a mass ratio of 5:1 by vortex for 5min to obtain protamine adsorbed PD-L1 siRNA nano-core (Pro-siPD-L1)

2) Precisely weighing each component according to the mass ratio of 1, 2-dioleoyl-sn-glycerol-3-phosphorylcholine (DOPC) to cholesterol: LY3200882(w: w: w) to 50:4:1, dissolving the components completely by using dichloromethane, and adding 3mg of miniPEG-G (C14) PLGIAgQ (C14) -miniPEG (MMP2 enzyme-responsive polypeptide). The organic solvent was removed under reduced pressure at 37 ℃ to form a uniform thin film layer on the wall of the bottle. Adding phosphate buffer solution with pH 7.4 into phospholipid membrane, and hydrating for 30 min. Extruding the obtained suspension back and forth 20 times by using a liposome extruder with pore diameter of 0.4 mu m and pore diameter of 0.2 mu m respectively, then adding Pro-siPD-L1 nano-core solution according to the mass ratio of MSL-LY to Pro-siPD-L1(w: w) being 500:1, mixing uniformly, extruding back and forth 20 times by using a liposome extruder with pore diameter of 0.4 mu m and pore diameter of 0.2 mu m respectively, and finally preparing the MSL with a core-shell type hydrophobic drug and gene drug co-delivery system^-LY/Pro_-siPD-L1。

(2) In vitro responsive drug release evaluation

According to the preparation method, the miniPEG-G (C14) PLGIAgQ (C14) -miniPEG (MMP2 enzyme response polypeptide) is replaced by miniPEG-G (C14) GGPALIQ (C14) -miniPEG (non-MMP 2 enzyme response polypeptide) to prepare the non-enzyme response MNL^-LY/Pro_-siPD-L1A delivery system. Mixing MSL^-LY/Pro_-siPD-L1And MNL^-LY/Pro_-siPD-L1The nano delivery systems were separately loaded into dialysis bags (MWCO 3500D) and MMP2 enzyme was added at a final concentration of 10 μ M, the air was excluded as much as possible, and the bags were placed in 50mL jars containing dialysis media (PBS solution containing 0.3% Tween 20, pH 7.4), the horizontal shaker was turned on, the temperature was set at 37 ℃, the rotation speed was 100 ± 5rpm, and 1mL of release media was taken at different time points (0, 1,2, 4, 6, 12, 24h) and an equal amount of freshly prepared release media was added to restore the original volume. The amount of LY3200882 in the release medium was determined and a cumulative release curve was calculated and generated.

As shown in FIG. 8, non-enzyme responsive MNLs^-LY/Pro_-siPD-L1The delivery system also only released 35% of LY within 24h, and did not achieve rapid release of LY, whereas the enzyme responded to MSL^-LY/Pro_-siPD-L1Delivery system released 63.1% of LY3200882 within 6h, achieving a fast response release of LY 3200882. The results show that MSL^-LY/Pro_-siPD-L1The delivery system has the capability of quick enzyme response to release medicine.

Example 9

MSL^-LY/Pro_-siPD-L1Combination therapy pharmacodynamic evaluation of nano drug delivery systems

According to the preparation method in example 8, the miniPEG-G (C14) PLGIAGQ (C14) -miniPEG (MMP2 enzyme response polypeptide) is replaced by miniPEG-G (C14) GGPALIQ (C14) -miniPEG (non-MMP 2 enzyme response polypeptide) to obtain the non-enzyme-sensitive control co-delivery vector MNL-^LY/Pro_-siPD-L1A delivery system.

Female mice were treated as 1X 10⁶Mouse-derived breast cancer 4T1 cells and 5X 10 cells⁵The dosage of each fibroblast NIH/3T3 cell is inoculated in the left mammary fat pad of the mouse, and then a mouse in-situ breast cancer tumor model is established. When the tumor volume increases to 200mm³At the same time, the patients were randomly divided into 4 groups of 5 individuals, and the groups were injected with normal saline, MSL/Pro separately into tail vein_-siPD-L1、MNL^-LY/Pro_-siPD-L1And MSL^-LY/Pro_-siPD-L1WhereinThe dose of PD-L1 siRNA was 1mg/kg, and the content of LY3200882 was 20mg/kg, and the administration was once every 3 days for 6 times in a row. Periodically measuring the major diameter a (mm) and the minor diameter b (mm) of the Tumor by using vernier calipers and adopting the formula Tumor volume (mm)³)＝a×b²In situ Tumor volume (Tumor volume, mm) was calculated³) From the start of dosing, mice orthotopic Tumor volume was monitored and recorded every 3 days and Tumor volume (Tumor volume, mm) was plotted³) -Time (Time, day) curve. After the last administration, mice were sacrificed, tumor tissue was dissected, weighed and photographed.

MSL/Pro as shown in FIGS. 9-11_-siPD-L1The targeted delivery of gene can only silence the expression of PD-L1, and cannot achieve a good tumor inhibition effect. After the high-efficiency core-shell type gene drug delivery system is loaded with hydrophobic drug LY3200882, non-enzyme-sensitive MNL^-LY/Pro_-siPD-L1And sensitive group MSL^-LY/Pro_-siPD-L1The group showed good tumor inhibition effect, and benefited from the combination treatment of gene therapy and hydrophobic drugs, the responsive MSL^-LY/Pro_-siPD-L1The delivery system exhibited the strongest antitumor effect, the slowest tumor volume growth rate, the smallest tumor volume and the smallest tumor weight. The reason for this is the responsive MSL^-LY/Pro_-siPD-L1The TGF-beta receptor inhibitor LY3200882 can be quickly released to weaken the tumor matrix barrier, the tumor immunosuppression microenvironment is remodeled, and the gene inner core with positive charge is exposed after the enzyme sensitive phospholipid shell is removed, so that the permeation of the Pro-sipD-L1 nano core is further enhanced, and the remarkable synergistic antitumor effects of the two are effectively exerted.

The dissected tumor tissue was further subjected to PD-L1 protein immunohistochemical analysis. As shown in FIG. 12, the benefit of shielding positive charge of the core by the neutral or electronegative phospholipid shell, the stable loading and in vivo stable transport of the core gene drug can be realized, so that MSL/Pro_-siPD-L1、MNL^-LY/Pro_-siPD-L1And MSL^-LY/Pro_-siPD-L1All can effectively reduce the expression of PD-L1, but MNL^-LY/Pro_-siPD-L1LY3200882 cannot be released rapidly to play a role in remodeling tumor minicircles due to the inability to efficiently achieve enzyme-responsive extracellular uncoatingThe medicine effect is good, the positive charge gene kernel can not be exposed to promote the uptake, the gene kernel uptake is still hindered, and the gene transfection efficiency is higher than that of the MSL^-LY/Pro_-siPD-L1Low, but MSL^-LY/Pro_-siPD-L1By virtue of the effective co-delivery of LY3200882 and PD-L1 siRNA by the core-shell nano system, the expression of PD-L1 protein in tumor tissues can be remarkably reduced.

Claims

1. A core-shell type high-efficiency gene drug delivery system is characterized in that the delivery system consists of an inner core formed by protamine adsorbing gene drugs and an enzyme sensitive phospholipid shell.

2. The core-shell type highly efficient gene drug delivery system according to claim 1, wherein: the inner core formed by protamine adsorbing gene medicine consists of protamine and gene medicine.

3. The core-shell type highly efficient gene drug delivery system according to claim 1, wherein: the enzyme sensitive phospholipid shell consists of enzyme sensitive polypeptide, cholesterol and neutral or negative phospholipid.

4. The core-shell type highly potent gene drug co-delivery system according to claim 1, wherein: the enzyme sensitive phospholipid shell can be loaded with hydrophobic drugs according to the needs of drug combination.

5. The core-shell type highly potent gene drug co-delivery system according to claim 1, wherein: the gene medicine is any one or more of DNA, siRNA, shRNA, mRNA, miRNA or CRISPR.

6. The core-shell type highly efficient gene drug delivery system according to claim 1, wherein: the enzyme sensitive polypeptide is any one or more of matrix metalloproteinase 2, matrix metalloproteinase 9, fibroblast activation protease, cathepsin B, secreted phospholipase A2, alpha-amylase, lysyl oxidase, and beta-glucuronidase sensitive polypeptide.

7. The core-shell efficient gene drug delivery system of claim 1, wherein: the neutral or electronegative phospholipid is any one or more of natural phospholipid, glycerol-3-phosphorylcholine, glycerol-3-phosphoethanolamine, glycerol-3-phosphate-L-serine sodium salt, phosphatidyl-DL-glycerol, glycerol-3-sodium phosphate phospholipid and derivatives thereof.

8. The core-shell type efficient gene drug delivery system of claim 1, wherein the mass ratio of the enzyme sensitive phospholipid shell to the inner core formed by protamine adsorption genes is 10:1-2000: 1; the mass ratio of the protamine to the gene is 1:1-50: 1; the mass ratio of the neutral or electronegative phospholipid to the cholesterol is 1:1-100: 1; the mass ratio of the neutral or electronegative phospholipid to the enzyme response polypeptide is 2:1-100: 1.

9. The core-shell type highly potent gene drug delivery system according to any of claims 1 to 7, characterized in that the particle size of the delivery system is 10 to 1000 nm.

10. The method for preparing a core-shell type highly efficient gene drug delivery system according to any one of claims 1 to 7, comprising the steps of:

2) weighing neutral or electronegative phospholipid, cholesterol and hydrophobic drug (or not), dissolving with organic solvent completely, placing into a reaction bottle, adding enzyme sensitive polypeptide, dissolving completely, and evaporating organic solvent under reduced pressure to obtain a uniform thin film layer; adding phosphate buffer solution for hydration for a period of time, extruding the obtained suspension to pass through a membrane, adding the obtained liquid into the kernel of the protamine adsorption gene, uniformly mixing, extruding the membrane again, and finally preparing a core-shell type gene drug delivery system or a co-delivery system of hydrophobic drugs and gene drugs;