CN110354096B

CN110354096B - Brain-targeted drug delivery system capable of improving drug-resistant brain glioma microenvironment and reversing drug resistance

Info

Publication number: CN110354096B
Application number: CN201910594699.1A
Authority: CN
Inventors: 周四元; 刘道洲; 成颖; 刘苗; 乔赛; 张邦乐; 纪奇峰
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2019-07-03
Filing date: 2019-07-03
Publication date: 2020-07-21
Anticipated expiration: 2039-07-03
Also published as: CN110354096A

Abstract

The invention discloses a brain targeting drug delivery system capable of improving a drug-resistant glioma microenvironment and reversing drug resistance, which is characterized in that Polyethyleneimine (PEI) with a small molecular weight is grafted on a Polyaspartic Acid (PASP) side chain through a disulfide bond to synthesize a novel cationic polymer PASP-G-PEI, the novel cationic polymer PASP-G-PEI is used for compressing sipD-L1, a complex formed by the sipD-L1 and the PASP-G-PEI is taken as a core, a lipid membrane formed by 2-deoxyglucose (Glu) modified distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), cholesterol, Dioleoylphosphatidylethanolamine (DOPE) and the like is taken as an outer shell, a membrane dispersion method is adopted to prepare a core-shell-shaped lipid polymer nanoparticle TMZ/sipD-L @ G L PN (PASP-G-PEI) carrying the sipD-L and TMZ together, the TMZ, the core-shell-shaped lipid polymer nanoparticle TMZ/sipD-L @ G L PN (the PASP-G-PEI) passes through a glucose transporter and enters drug-resistant glioma cells, and the drug-resistant glioma cells are rapidly killed under the GSH concentration condition, the brain colloidal glioma cytoplasm slurry releases the brain slurry, the brain body, the brain-resistant glioma cell is quickly, the brain tumor cell activation and the brain-resistant glioma-resistant system inhibits the brain tumor cell expression of TMPD-1, the brain tumor-resistant glioma, the brain tumor-resistant brain, the brain tumor cell, the brain tumor-resistant brain tumor pathway of TMPD.

Description

Brain-targeted drug delivery system capable of improving drug-resistant brain glioma microenvironment and reversing drug resistance

Technical Field

The invention relates to a novel brain-targeted drug delivery system, which can silence the expression of PD-L1 in drug-resistant brain glioma cells, improve the immune microenvironment of the drug-resistant brain glioma, reduce the content of MGMT in the drug-resistant brain glioma cells and reverse the drug resistance of the drug-resistant brain glioma to temozolomide.

Background

Malignant brain gliomas are the most common central nervous system tumors. Clinical studies have shown that after 3 courses of Temozolomide (TMZ) are administered in a standardized treatment, brain gliomas develop significant resistance to TMZ, which is a major cause of chemotherapy failure. The development of new methods that can effectively treat TMZ-resistant brain gliomas is an urgent clinical need.

The research shows that over-expression, Base Excision Repair (BER) and DNA mismatch repair (MMR) of O6-methylguanine-DNA-methyltransferase (MGMT) are important reasons for TMZ resistance of glioma, however, the recent research shows that TMZ resistance to glioma is not only related to the expression of brain glioma self-gene, but also promotes the generation of resistance by tumor microenvironment, so we find that ligand (PD-L1) of programmed death receptor-1 (PD-1) is highly expressed in TMZ-resistant glioma cells, and report that the high expression of PD-L leads to the continuous activation of PD-1/PD-L signaling pathway, so that tumor cells escape from the monitoring and monitoring of the immune system of the organism, promote the growth of tumor, and aggravate the drug resistance of tumor, when siRNA (sipD-L) with specific sequence inhibits the TMZ-resistant glioma cell PD-5821 from the expression, the expression of TMZ-resistant glioma cells, the inhibition effect of TMZ-MT-DNA-methyltransferase in the brain glioma cell pathway is obviously improved, and the inhibition effect of TMZ-resistant glioma cell expression of TMZ-DNA accumulation in the brain glioma cell is obviously improved by the inhibition of TMZ-resistant glioma-631, the blood brain glioma cell, the inhibition of the brain glioma-2, the inhibition of the brain glioma cell is obviously improved, and the inhibition of the brain glioma cell resistance of the brain glioma-resistant tumor cell resistance of the brain tumor cell-resistant glioma cell growth of the brain glioma-2, and the tumor cell resistance of the brain glioma accumulation of the tumor cell resistance of TMZ-resistant glioma-631, the tumor cell is obviously improved by the tumor cell resistance, the tumor cell resistance of the tumor cell-resistant glioma-resistant tumor cell-631, the tumor cell resistance of the tumor cell is obviously improved by the tumor cell, the tumor cell resistance of the tumor cell, the tumor cell resistance of the tumor cell-resistant tumor cell resistance of the tumor cell, the tumor cell resistance of TMZ-resistant tumor cell, the tumor cell.

Researches show that 2-deoxyglucose can be specifically combined with a glucose transporter on the surface of a brain capillary endothelial cell to mediate nanoparticles to pass through a blood brain barrier and accumulate in brain tissues. In addition, on the surface of the brain glioma cells, glucose transporters are highly expressed, which suggests that the 2-deoxyglucose-modified nanoparticles can efficiently enter the brain glioma cells through the mediation of the glucose transporters.

The cationic polymer carrier material for siRNA delivery mainly comprises Polyethyleneimine (PEI), polyamidoamine dendrimer (PAMAM) and the like. PEI has a large number of protonated amino groups, siRNA can be effectively compressed through electrostatic action, but PEI has obvious toxicity due to higher molecular weight and larger using dose, and the reduction of the molecular weight of PEI can reduce the toxicity of PEI, but also reduces the capability of PEI for compressing siRNA. Therefore, how to improve the gene delivery efficiency and reduce the toxicity of the gene delivery carrier material is a hot spot of the current siRNA delivery carrier material research.

After the nanoparticles enter tumor cells, the nanoparticles can release free siRNA rapidly in cytoplasm to exert the due gene silencing effect. Research shows that the concentration of Glutathione (GSH) in brain glioma cell pulp is 100-1000 times of the extracellular concentration, which provides a new idea for responding to the release of drugs in the tumour cell pulp by the environment. Because the disulfide bond is very stable in a low-concentration GSH environment and can break in a high-concentration GSH environment, PEI with small molecular weight is grafted on the side chain of polyaspartic acid through the disulfide bond, so that the compression capability of the gene transfer carrier material on siRNA can be ensured, the in vitro and in vivo toxicity of the gene transfer carrier material can be expected to be reduced, more importantly, the gene transfer carrier material also has the redox property, can be depolymerized in a high-concentration GSH environment in brain glioma cell paste, loses the compound capability on the siRNA, quickly dissociates the siRNA, and plays a gene silencing effect.

Disclosure of Invention

Based on the research background, the invention aims to screen out a siPD-L1 sequence which can block a PD-1/PD-L1 signal channel in a drug-resistant brain glioma microenvironment and reverse the drug-resistant property of the drug-resistant brain glioma, and prepare a lipid polymer nanoparticle which is loaded with siPD-L1 and TMZ together, wherein the nanoparticle can penetrate through a blood brain barrier and efficiently enter drug-resistant brain glioma cells through a glucose transporter on the surface of a brain capillary endothelial cell, so that siPD-L1 and TMZ are released, the expression of PD-L1 in the drug-resistant brain glioma cells is silenced, the killing effect of an organism immune system on the brain glioma is enhanced, the expression of MGMT is reduced, the sensitivity of the drug-resistant brain glioma on TMZ is improved, and the growth of the drug-resistant brain glioma is inhibited in a dual way.

Based on the research background, the technical scheme of the invention is as follows:

①, a sequence of siPD-L1 is designed and synthesized, the sequence number is cd274-mus-362 (sense chain: 5'-GAAGGGAAAUGCUGCCCUUTT-3', antisense chain: 5'-AAGGGCAUUUCCCUUCTT-3'), the sequence can not only silence the expression of PD-L1 in drug-resistant brain glioma cells, block PD-1/PD-L1 signal channels in a drug-resistant brain glioma microenvironment, enhance the killing effect of an organism immune system on brain glioma, but also reduce the expression of MGMT and improve the sensitivity of the drug-resistant brain glioma on TMZ.

② a small molecular weight Polyethyleneimine (PEI) is grafted to a side chain of Polyaspartic Acid (PASP) through a disulfide bond to synthesize a novel cationic polymer PASP-G-PEI, the novel cationic polymer PASP-G-PEI is used for compressing the SIPD-L1, a complex formed by the SIPD-L and the PASP-G-PEI is taken as a core, a lipid film formed by 2-deoxyglucose (Glu) modified distearoyl phosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG), cholesterol, dioleoyl phosphatidyl ethanolamine (DOPE) and the like is taken as an outer shell, a film dispersion method is adopted to prepare the core-shell lipid polymer nanoparticle TMZ/SIPD-L @ 563281G 7 PN (PASP-G-PEI) co-loaded with the SIPD-L1 and the TMZ, the core-shell lipid polymer nanoparticle TMZ/SIPD-L @ L PN (the PASP-G-PEI) passes through a blood brain barrier and enters drug-resistant glioma cells through a glucose transporter, the glucose transporter mediates the blood brain barrier and enters the drug-resistant glioma cells, the brain glioma cells are rapidly killed under the condition that GSH concentration of the colloidal glioma cell slurry releases the TMPD-351, the dual drug-resistant glioma cell, the brain tumor cell expression pathway of the TMPD-resistant glioma cells is reduced, and the dual-resistant glioma-resistant brain tumor cell is simultaneously, the inhibitory system, the dual-resistant brain tumor cell expression of the TMPD-resistant brain tumor cell is increased, the TMPD-resistant glioma, and the TMPD-resistant brain tumor system.

① designs and synthesizes a siPD-L1 sequence, the siPD-L1 not only can silence the expression of PD-L1 in drug-resistant brain glioma cells, block PD-1/PD-L1 signal pathways in drug-resistant brain glioma microenvironment, but also can reduce the expression of MGMT in the drug-resistant brain glioma cells and reverse the drug resistance of the drug-resistant brain glioma to TMZ ② constructs a brain-targeted drug delivery system which can efficiently pass through a blood brain barrier and directly deliver the siPD-L1 into the drug-resistant brain glioma cell pulp.

Drawings

FIG. 1 is C₆/TR cells and C₆Expression of PD-L1 in cells n-3,

**P<0.01, and C₆Comparison with TR cells.

FIG. 2 is a diagram of different sequences siPD-L1 vs C₆CD274-mus-44 for A, CD274-mus-88 for B, CD274-mus-674 for C, CD274-mus-444 for D, CD274-mus-788 for E, CD274-mus-362 for F, CD274-mus-890 for G, CD274-mus-890 for H, CD274-mus-258 for n-3,

*P<0.05，**P<0.01, compared to the control group.

FIG. 3 is the complexing ability of polymeric nanoparticles to siPD-L1.

FIG. 4 is a graph of the particle size distribution and zeta potential of the polymeric nanoparticles.

FIG. 5 is a graph of the particle size distribution and zeta potential of the lipopolymer nanoparticles.

FIG. 6 is a scanning electron micrograph of a lipopolymer nanoparticle

FIG. 7 shows the lipid polymer nanoparticles in C₆Distribution in/TR cells (60 ×). A TMZ/siPD-L1 @ L PN (PASP-g-PEI)₁₈₀₀)；B：TMZ/siPD-L1@GLPN(PASP-g-PEI₁₈₀₀). Blue indicates the nucleus, red indicates the Cy7.5 labeled nanoparticle, green indicates the lysosome, and yellow indicates that the nanoparticle is locked in the lysosome.

Figure 8 is the efficiency of lipopolymer nanoparticles to penetrate the blood brain barrier in vitro. A: schematic representation of an in vitro blood brain barrier model; b: penetration efficiency of nanoparticles. n is 3, and n is 3,

**P<0.01, and scrambled siRNA @ L PN (PASP-g-PEI)₁₈₀₀) And (4) comparing.

FIG. 9 shows lipopolymer nanoparticles in bEnd3 cells (A) and C₆Accumulation in/TR cells (B).

Fig. 10 is the distribution of lipopolymer nanoparticles in normal mice. A: in vivo imaging observation results of lipid polymer nanoparticles distributed in mice, B: statistics of mean fluorescence intensity in brain tissue. n is 5, and n is 5,

**P<0.01, TMZ/siPD-L1 @ L PN (PASP-g-PEI) at the same time point₁₈₀₀) And (4) comparing.

FIG. 11 shows lipopolymer nanoparticles on C-loading₆Distribution of/TR cells in situ glioma mice.

A: the in vivo imaging observation result of the lipid polymer nanoparticles distributed in tumor-bearing mice, B: statistics of mean fluorescence intensity in brain tissue. n is 5, and n is 5,

FIG. 12 shows a greaseGeopolymer nanoparticle pair C₆Inhibition of growth of in situ brain glioma by luc/TR cells A in vivo imaging with saline, B in vivo imaging with 50mg/kg TMZ administration, C in vivo imaging with sipD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) (siPD-L1: 0.5mg/kg) group administration for in vivo imaging, D: TMZ/siPD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) (siPD-L1: 0.5mg/kg, TMZ:44mg/kg) administration group living body imaging, E: TMZ/siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) (siPD-L1: 0.5mg/kg, TMZ:44mg/kg) administration group was imaged in vivo, F: statistical result of tumor fluorescence intensity. n. 5,

**P<0.01, compared with the normal saline group,^##P<0.01, compared to the TMZ group,^&&P<0.01, with TMZ/siPD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) And (4) comparing.

FIG. 13 shows lipopolymer nanoparticle pair C₆-the effect of expression of PD-L1 in tumor tissue of luc/TR cells in situ glioma mice.n-3,

**P<0.01, compared with the normal saline group,^#P<0.05，^##P<0.01, with TMZ/siPD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) Group phase comparison

FIG. 14 lipopolymer nanoparticle vs. Charge C₆-effect of MGMT expression in luc/TR cells in situ glioma tissue. n is 3, and n is 3,

**P<0.01, compared with the normal saline group,^#P<0.05, with TMZ/siPD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) Group comparison.

Detailed Description

1 research method:

1.1 SiPD-L1 base pair sequence Activity Screen

Synthesis of siPD-L1 of different sequences was designed, and siPD-L1 of different sequences was transfected into C using lipofectamine 2000 transfection reagent₆in/TR cells, a western blot examined the sequence pairs C of each siPD-L1 sequence₆Silencing of PD-L1 in/TR cells.

1.1.1 drug treatment

Taking C in logarithmic growth phase₆washing/TR cells with PBS buffer, adding 0.25% trypsin solution 2ml, digesting for 1min, removing trypsin solution, adding 4ml DMEM complete culture solution, blowing off cells, calculating cell concentration with cell counting plate, adding DMEM complete culture solution to dilute cell concentration to 1x10⁶Adding the cells/ml into a six-well plate, adding 2ml into each well, culturing in a carbon dioxide constant temperature incubator for 24h, then sucking out the culture solution, adding a siPD-L1 @ lipofectamine 2000 compound (prepared by serum-free DMEM culture solution) with siPD-L1 concentration of 50, 100 and 160nM, incubating in the carbon dioxide constant temperature incubator for 8h, replacing the culture solution with fresh DMEM complete culture solution, and continuing culturing for 40 h.

1.1.2 extraction of Total protein

Collecting the above cells with a cell scraper, adding into a 2ml centrifuge tube, centrifuging for 15min (12000g, 4 deg.C), collecting the lower layer cells, adding 100 μ l RAPI cell lysate, blowing off the cells, performing ice bath lysis for 15min, and performing continuous lysis for 15min after shaking. Centrifuging for 15min (12000g, 4 deg.C), collecting supernatant 85 μ l, adding supernatant 5 μ l into Coomassie brilliant blue solution 195 μ l, shaking for 10min, detecting absorbance value of each group with enzyme linked immunosorbent assay (detection wavelength is 570nm), and calculating relative concentration of total protein in each group of lysate. Adding 20 μ l SDS-protein loading buffer into the rest supernatant, mixing, boiling for 10min, and placing in a refrigerator at 4 deg.C for use.

1.1.3 Western blot examination of different siPD-L1 sequence pairs C₆Gene silencing of PD-L1 in TR cells

(1) Solution preparation

Preparing an electrophoresis buffer solution: 14.42g of glycine, 1g of Sodium Dodecyl Sulfate (SDS) and 3.03g of Tris (hydroxymethyl) aminomethane (Tris) were weighed and added to 1000ml of distilled water, and sufficiently stirred until completely dissolved for use.

Preparing a membrane transfer buffer solution: weighing 14.42g of glycine and 3.03g of Tris (hydroxymethyl) aminomethane (Tris) into 800ml of distilled water, fully stirring until the glycine and the Tris (hydroxymethyl) aminomethane are completely dissolved, adding 200ml of methanol, uniformly mixing, and precooling in a refrigerator at 4 ℃ for later use.

Preparation of PBST lotion: adding 1ml of Tween-20 into each liter of PBS buffer solution with the pH value of 7.4, and shaking up to obtain the final product.

Preparing a sealing liquid: a5% skim milk powder solution was prepared using PBS buffer pH 7.4.

Preparation of 10% separation gel:

preparation of 5% concentrated gum:

(2) western blot

Electrophoresis: preparing 10% separation gel, mixing, quickly adding into glass plates, adding about 4.5ml, adding distilled water into the upper layer, standing at room temperature for about 30min, pouring out distilled water between the glass plates, quickly adding 5% concentrated gel, inserting into a comb, and standing at room temperature for about 30min until the concentrated gel naturally coagulates. Soaking the glass plate in electrophoresis buffer solution, pulling out the comb, adding 15 μ l of total protein extracted from each well, performing 80V constant voltage electrophoresis, adjusting the total protein to 120V constant voltage electrophoresis after the total protein enters the separation gel, and turning off the power supply after the indicator bromophenol blue runs out of the separation gel.

Film transfer: one layer of sponge and three layers of filter paper were laid on the bottom plate of the film rotator, the above gel and the wetted NC film were gently placed on the filter paper in this order (taking care to prevent the generation of air bubbles), and three layers of filter paper and one layer of sponge were continued to be laid on the NC film. The gel faces to the negative electrode, the NC membrane faces to the positive electrode, the voltage is adjusted to be 100mV under the ice bath condition, the membrane is rotated for 50min, and the power supply is turned off.

Negative dyeing: after the completion of the membrane transfer, the NC membrane was taken out, added to the ponceau staining solution and stained for 10min, the bands on the NC membrane were observed, the observation results were recorded, the NC membrane was transferred to the PBST buffer solution and rinsed 3 times (7 min/time), and ponceau on the NC membrane was removed.

And (3) sealing: the NC membrane was transferred to 150ml of blocking solution, placed on a shaker and shaken slowly for 80min, and then transferred to PBST buffer and rinsed 3 times (7 min/time).

And (3) incubating the antibody, namely cutting the NC membrane, adding the anti-PD-L1 antibody, standing overnight in a refrigerator at 4 ℃, rinsing PBST for 3 times, transferring the NC membrane into a secondary antibody (1:5000) diluted by PBST, and shaking for 2h at normal temperature.

Detection NC membranes were transferred to PBST buffer, rinsed 3 times (7 min/time), and the expression of PD-L1 was detected with a bioluminescent instrument.

1.2 preparation and characterization of Co-loaded siPD-L1 and temozolomide lipopolymer nanoparticles

1.2.1 preparation and characterization of Polymer nanoparticles Supported with siPD-L1

(1) Dissolving 10OD siPD-L1 in 2880 μ l HEPES (10mM, pH 7.4) buffer solution to obtain siPD-L1 solution with concentration of 8.7 μ M, and weighing 2mg PASP-g-PEI₁₈₀₀The conjugate was dissolved in 1ml of HEPES (10mM, pH 7.4) buffer solution and prepared to 100. mu.M PASP-g-PEI₁₈₀₀The solution was diluted with HEPES (10mM, pH 7.4) buffer solution to 10, 7.5, 6.5, 5.5, 4.5, 3.5, 2.5, 1.5, 0.5. mu.M PASP-g-PEI₁₈₀₀Taking the prepared siPD-L1 and the PASP-g-PEI with different concentrations₁₈₀₀Respectively 125 mu l of the solution, mixing uniformly, whirling for 30s, standing at room temperature for 30min, and preparing the siPD-L1 polymer nanoparticles (siPD-L1 @ PASP-g-PEI) with N/P of 20, 15, 13, 11, 9, 7, 5, 3 and 1₁₈₀₀). With PEI_25KThe same method is adopted to prepare siPD-L1 @ PEI as the raw material_25K。

(2) Weighing 600mg agarose, dispersing in 40ml TBE buffer solution, heating in microwave oven for 1min to completely melt, adding 4 μ l gel signal^TMAnd mixing the green uniformly, adding the mixture into an electrophoresis tank in which a comb is inserted in advance, standing for 30min to obtain 1.5% agarose gel, taking 8.5 mu l of the prepared siPD-L1 polymer nanoparticle solution, adding 1.5 mu l of 5 × RNA sample loading buffer solution, mixing uniformly, loading the sample, placing the sample into TBE buffer solution, performing 90V constant-pressure electrophoresis for about 30min, and observing the capability of the cationic polymer for compressing the siPD-L1 by using a gel imager.

(3) 100 mu l of the prepared optimal N/P siPD-L1 polymer nanoparticle solution is added with 1.5ml of distilled water for dilution, and a nano-particle size and zeta potential analyzer is adopted to measure the particle size, zeta potential and polydispersity index (PDI) of the siPD-L1 polymer nanoparticles.

1.2.2 preparation and characterization of Co-loaded SiPD-L1 and temozolomide lipopolymer nanoparticles

(1) Fixing the molar ratio of phospholipid and cholesterol at 9:2, Glu-PEG-DSPE and DOPE at 7.65:1.35, preparing lipid thin film, weighing DOPE316mg, cholesterol at 54mg and Glu-PEG-DSPE at 210mg, adding 2ml of chloroform/methanol (V/V ═ 3:1) mixed solution, dissolving, removing chloroform/methanol solution by rotary evaporation at 35 deg.C under reduced pressure to form uniform thin film, and preparing SiPD-L1 @ PASP-g-PEI of N/P ═ 9₁₈₀₀(2ml, containing siPD-L150 mug) and a saturated TMZ aqueous solution (2ml) are added into a lipid film, stirred at room temperature overnight, subjected to ultrasonic treatment for 1min, and then transferred into a dialysis bag with the molecular weight cutoff of 1000Da, dialyzed in distilled water for 2h, the distilled water is replaced every 0.5h, placed in a refrigerator at-80 ℃ for 24h, and subjected to freeze drying for 24h to obtain the lipopolymer nanoparticles TMZ/siPD-L1 @ G L PN (PASP-G-PEI) carrying the siPD-L1 and the TMZ together₁₈₀₀) Solid powder of siPD-L1 @ PASP-g-PEI₁₂₀₀And siPD-L1 @ PEI_25KThe same method is adopted to prepare TMZ/siPD-L1 @ G L PN (PASP-G-PEI) as raw material₁₂₀₀) And TMZ/siPD-L1 @ G L PN (PEI)_25K) The PEG-DSPE is adopted to prepare the lipid polymer nanoparticles TMZ/siPD-L1 @ L PN (PASP-g-PEI) without 2-deoxyglucose modification₁₈₀₀)。

(2) Taking a proper amount of solid lipide polymer nano-particle powder, adding 1ml of distilled water for dispersion, and then measuring the particle size, the zeta potential and the polydispersity index (PDI) of the lipide polymer nano-particle by adopting a nano-particle size and zeta potential analyzer. Meanwhile, a small amount of lipid polymer nanoparticle solution is dropwise added on a glass sheet, the glass sheet is placed in a vacuum drying oven at 35 ℃ for drying for 2 hours, gold is plated, and the morphology of the lipid polymer nanoparticles is observed by adopting a field emission scanning electron microscope.

1.3 Effect of lipid Polymer nanoparticles on the expression of PD-L1 and MGMT in drug-resistant glioma cells

Logarithmic growth phase C₆the/TR cells were washed with PBS buffer and addedAdding 0.25% trypsin solution 2ml, digesting for 1min, removing the trypsin solution, adding DMEM complete culture solution 4ml, blowing off cells, calculating cell concentration with cell counting plate, adding DMEM complete culture solution, and diluting to 1x10 cell concentration⁶Adding into six-well plate, adding 2ml per well, culturing in carbon dioxide incubator for 24 hr, and changing the culture solution to siPD-L1 @ lipofectamine 2000, siPD-L1 @ PASP-g-PEI₁₈₀₀、siPD-L1@LPN(PASP-g-PEI₁₈₀₀) And siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) Diluting the solution (with serum-free DMEM culture solution, siPD-L1 concentration of 160nM), incubating in carbon dioxide constant temperature incubator for 8h, replacing the culture solution with fresh DMEM complete culture solution, culturing for 40h, extracting total protein, and detecting C with western blot₆Expression of PD-L1 and MGMT in TR cells.

1.4 lipid Polymer nanoparticles in C₆Dynamic distribution in/TR cells

PASP-g-PEI marked by Cy7.5₁₈₀₀Observing the lipid polymer nanoparticles in the sample C by using a laser confocal microscope₆Dynamic distribution within/TR cells. Taking C in logarithmic growth phase₆washing/TR cells with PBS buffer, adding 0.25% trypsin solution 2ml, digesting for 1min, removing trypsin solution, adding DMEM complete culture solution 4ml, blowing off cells, calculating cell concentration with cell counting plate, adding DMEM complete culture solution, diluting to 1x10 cell concentration⁵Adding into 24-well plate (bottom is covered with glass cover), adding 1ml per well, culturing in carbon dioxide incubator for 24 hr, and changing the culture solution to TMZ/siPD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) And TMZ/siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) Adding 500 μ l of solution (diluted by serum-free DMEM culture solution) into each well, keeping the concentration of siPD-L1 at 25nM, incubating in a carbon dioxide constant temperature incubator for 0.5, 2 and 4 hours, replacing the culture solution with PBS buffer solution with pH7.4 at 37 ℃, placing in the carbon dioxide constant temperature incubator for 10min, removing residual nanoparticle solution, repeating the washing operation for 3 times, adding 500 μ l of lysotracker green solution (with concentration of 600nM), staining in the carbon dioxide constant temperature incubator for 20min, replacing the lysotracker green solution with 37 deg.CThe pH7.4 PBS buffer solution is placed in a carbon dioxide constant temperature incubator for 10min, the residual lysotracker red solution is removed, the washing operation is repeated for 3 times, 500 mu l of DAPI solution with the concentration of 500nM is added, the dyeing is carried out in the carbon dioxide constant temperature incubator for 10min, the DAPI solution is replaced by the pH7.4 PBS buffer solution with the temperature of 37 ℃, the placing is carried out in the carbon dioxide constant temperature incubator for 10min, the residual DAPI solution is removed, the washing operation is repeated for 3 times, 1ml of 4 percent paraformaldehyde is added, the fixation is carried out in the carbon dioxide constant temperature incubator for 10min, a cover glass is reversely buckled on the glass slide (1 drop of glycerol is previously dropped on the glass slide), and the observation of the lipid polymer nanoparticles on the C is carried out by adopting a laser₆Dynamic distribution within/TR cells.

1.5 efficiency of lipopolymer nanoparticles to penetrate the in vitro blood brain barrier

In order to not destroy the integrity of the in vitro blood brain barrier model, in this experiment, scramble siRNA was used to prepare the lipid polymer nanoparticles without TMZ (scrambled siRNA is non-functional mismatched siPD-L1, the same below).

1.5.1 in vitro blood brain Barrier model establishment

Cleaning bEnd3 cells in logarithmic growth phase with PBS buffer solution, adding 0.25% trypsin solution 2ml, digesting for 1min, removing trypsin solution, adding DMEM complete culture solution 4ml, blowing off cells, calculating cell concentration with cell counting plate, adding DMEM complete culture solution, diluting to 1x10 cell concentration⁵One cell/ml was inoculated into a transwell supply cell, DMEM complete medium was added to the receiving cell, and the medium was changed every other day. After culturing for 7 days, detecting the resistance value between a transwell supply pool and a transwell receiving pool by using a resistance meter, and when the resistance value is more than 200 omega/cm²In time, the in vitro blood brain barrier model is successfully constructed.

1.5.2 Lipopolymer nanoparticles transport across the blood brain barrier in vitro

(1) Taking C in logarithmic growth phase₆washing/TR cells with PBS buffer, adding 0.25% trypsin solution 2ml, digesting for 1min, removing trypsin solution, adding DMEM complete culture solution4ml, blow off cells, calculate cell concentration using cell counting plate, add DMEM complete medium, dilute cell concentration to 1X10⁶Adding the cells/ml into a six-well plate, adding 2ml into each well, and placing the plate in a carbon dioxide constant temperature incubator for 24 hours. The transwell feed cell was placed in a six well plate and labeled with Cy7.5 PASP-g-PEI₁₈₀₀The supply pool was filled with scrambled siRNA @ L PN (PASP-g-PEI)₁₈₀₀) And scrambled siRNA @ G L PN (PASP-G-PEI)₁₈₀₀) The concentration of the nanoparticles is 5mg/ml, after incubation for 2, 4 and 8 hours, the concentration of Cy7.5 in the receiving pool is detected by a fluorescence spectrophotometer, and the transport efficiency across the blood brain barrier of the lipid polymer nanoparticles is calculated, wherein the transport efficiency (%) (the fluorescence intensity of the culture solution in the transwell receiving pool/the fluorescence intensity of the nanoparticle solution before administration) is × 100%.

(2) After 8 hours of incubation according to the procedure of (1), the bEnd3 cells in the transwell donor pool and C in the recipient pool were collected₆and/TR cells, and detecting the accumulation of the lipid polymer nanoparticles in the two cells by adopting flow cytometry.

1.6 Lipopolymer nanoparticles in vivo anti-glioma Activity

1.6.1 establishment of C6/TR-luc cell in situ glioma model

Logarithmic growth phase C₆washing/TR-luc cells with PBS buffer, adding 0.25% trypsin solution 2ml, digesting for 1min, removing trypsin solution, adding DMEM complete culture solution 4ml, blowing off cells, calculating cell concentration with cell counting plate, centrifuging, adding serum-free DMEM culture solution to obtain cell concentration of 1 × 10⁸The cell suspension of (3). ICR mice were injected with 10% chloral hydrate intraperitoneally, after anesthesia, the mice were fixed and drilled at the junction of the mouse's medial border line and the sagittal midline of the head, 1.8mm to the left of bregma and 1mm to the front. Aspirate C with a 5. mu.l microinjector₆Fixing 5 μ l/TR-luc cell suspension on mouse brain stereotaxic apparatus, vertically inserting needle depth 4mm along bore hole, withdrawing needle 1mm, and mixing with C₆Slowly injecting the/TR-luc cell suspension into the mouse brain, staying for 5min after injection, pulling out the microinjector after the cells are completely precipitated, and suturing the incision.

1.6.2 distribution of lipopolymer nanoparticles in situ glioma mice and Normal mice

(1) Taking normal mice and C₆Model mouse of/TR cell orthotopic brain glioma, tail vein injection Cy7.5 labeled TMZ/siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) And Cy7.5-labeled TMZ/siPD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) (the dose of the nanoparticles is 5mg/kg), and the distribution of the nanoparticles in the mouse body is observed by using a living body imager after 12 hours and 24 hours. After the mice are sacrificed, organ tissues such as brain, heart, liver, spleen, lung, kidney and the like are taken out, and the accumulation of the nanoparticles in each organ tissue of the mice is observed by a living body imager.

1.6.3 inhibition of drug-resistant brain glioma growth by lipopolymer nanoparticles

C₆-luc cells and C ₆7 days after the inoculation of/TR-luc cells, tumor-bearing mice were administered by tail vein injection 1 time every 3 days for 4 consecutive doses, and TMZ/sipD-L1 @ G L PN (PASP-G-PEI) was examined₁₈₀₀) And the treatment effect of different medicines on the brain glioma.

(1) On

days

7, 12, 17 and 22 after tumor inoculation, luciferase substrate is injected into the abdominal cavity of the tumor-bearing mouse, the dose is 150mg/kg, and after injection for 15min, the size of the glioma of the tumor-bearing mouse is observed by using a living body imaging instrument after isoflurane anesthesia.

(2) Taking 5 tumor-bearing mice from each group, killing the mice on the 2 nd day after the last administration to obtain the tumor area of the brain tissue of the mice, uniformly grinding, adding 150 mu l of RAPI cell lysate into the RAPI cell lysate, cracking the RAPI cell lysate in ice bath for 30min, centrifuging the RAPI cell lysate at 12000g and 4 ℃ for 20min, collecting supernatant, taking 5 mu l of supernatant, adding the supernatant into 195 mu l of Coomassie brilliant blue solution, shaking for 10min, detecting the absorbance value of the solution at 570nm by an enzyme linked immunosorbent assay detector, calculating the relative concentration of proteins in each group, adding the remaining supernatant into 20 percent SDS-protein loading buffer, uniformly mixing, boiling for 10min, and detecting the expression of PD-L1 and MGMT in the brain glioma tissue by adopting western blot.

2, experimental results:

2.1 different siPD-L1 sequence pairs C₆Silencing of PD-L1 in TR cells

C₆Cells and C₆Expression of PD-L1 in TR cellsHorizontal as shown in FIG. 1, see C₆The expression of PD-L1 in/TR cells is significantly higher than that of C₆A cell.

To screen for effective silencing of C₆The experimental design synthesizes 8 siPD-L sequences, and siPD-L sequences with different transfection are transfected to C by lipofectamine 2000 transfection reagent₆in/TR cells, a western blot examined the different siPD-L1 sequence pairs C₆Silencing of PD-L1 in TR cells the results are shown in FIG. 2, with the siPD-L1 sequence cd274-mus-362 vs C₆The expression of PD-L1 in/TR cells has better silencing effect, and C has better silencing effect when the concentration of cd274-mus-362 is 160nM₆The expression level of PD-L1 in/TR cells was 24% of that of the control group, and cd274-mus-362 was paired with C₆PD-L1 silencing effects were clearly concentration dependent in/TR cells.

2.2 characterization of the Supported siPD-L1 Polymer nanoparticles

PEI was investigated using a gel retardation experiment_25KAnd PASP-g-PEI₁₈₀₀The efficiency of compressing siPD-L1 is shown in fig. 3. the results show that PASP-g-PEI when N/P is 9₁₈₀₀Can be completely compounded with siPD-L1, and has compression effect and PEI_25KAre substantially equivalent.

The particle size and zeta potential of the nanoparticles were measured by a nanometer particle size and zeta potential analyzer, and the results are shown in FIG. 4. under the optimum N/P conditions, siPD-L1 @ PASP-g-PEI₁₈₀₀The particle diameter of (2) was 69nm, and the zeta potential was +26mV, respectively.

2.3 characterization of Co-loaded siPD-L1 and temozolomide lipopolymer nanoparticles

The particle size and potential of the co-supported siPD-L1 and TMZ lipopolymer nanoparticles were measured by a nanometer particle size and zeta potential analyzer, and the results are shown in FIG. 5. under the optimal N/P conditions, TMZ/siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) The particle size of the nanoparticle is 92nm, the zeta potential is-33 mV., the morphology of the lipopolymer nanoparticle is observed by adopting a field emission scanning electron microscope, and the result is shown in figure 6. TMZ/siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) The particle diameter of (A) is 80 nm.

2.4 Lipopolymer nanoparticles in C₆Dynamic distribution in/TR cells

After entering tumor cells, siRNA is delivered by an siRNA delivery system, the siRNA needs to be prevented from being degraded by lysosomes as much as possible and is rapidly distributed in cytoplasm, so that the gene silencing effect of the siRNA can be better exerted. Therefore, the experiment adopts a laser confocal microscope to observe the lipid polymer nanoparticles in the sample C₆Dynamic distribution in/TR cells the results are shown in FIG. 7, red labeled TMZ/siPD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) Quilt C₆The majority of the uptake by the/TR cells was blocked in green-labeled lysosomes, which appear yellow in the figure, with increasing incubation time, TMZ/sipD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) Can partially escape from lysosomes, and TMZ/siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) After entering the C6/TR cells, only a small fraction is distributed in lysosomes and a large fraction is distributed in the cytoplasm. This result suggests that 2-deoxyglucose modification can deliver lipopolymer nanoparticles directly into the cytosol of drug-resistant glioma cells.

2.5 transport efficiency of lipopolymer nanoparticles across the in vitro blood brain barrier

To fully examine the scrambled siRNA @ G L PN (PASP-G-PEI)₁₈₀₀) The transfer efficiency of the cross-body blood brain barrier is detected by the experiment through a fluorescence spectrophotometer, and the scrambled siRNA @ G L PN (PASP-G-PEI)₁₈₀₀) The concentration of the blood brain barrier penetrated is detected by adopting a flow cytometer to detect the bEnd3 cells and C₆/TR intracellular scambled siRNA @ G L PN (PASP-G-PEI)₁₈₀₀) The fluorescence spectrophotometer assay result is shown in FIG. 8, and the scrambled siRNA @ G L PN (PASP-G-PEI)₁₈₀₀) Can pass through blood brain barrier in a time-dependent manner, when the incubation time is 8h, the fluorescence intensity in a transwell receiving pool is 10.6 percent of that of a nanoparticle solution before administration, and the transport efficiency is obviously higher than that of scarmbled siRNA @ L PN (PASP-g-PEI)₁₈₀₀) The flow cytometry results are shown in FIG. 9, and scrambled siRNA @ G L PN (PASP-G-PEI)₁₈₀₀) In bEnd3 cells and C₆The accumulation in the/TR cells is obviously more than that of the scrambled siRNA @ L PN (PASP-g-PEI)₁₈₀₀). The above results all show that 2-deoxyglucose modification can increase the efficiency of lipopolymer nanoparticles in crossing the blood brain barrier, due to capillary blood in brainA large number of glucose transporters exist on the surface of endothelial cells of the tube, and can mediate 2-deoxyglucose modified nanoparticles to pass through a blood brain barrier.

2.6 distribution of lipopolymer nanoparticles in situ glioma mice and Normal mice

In order to examine the ability of the lipopolymer nanoparticles to cross the blood brain barrier, the lipopolymer nanoparticles were injected into the tail vein of a normal mouse, and the accumulation of the lipopolymer nanoparticles in the brain tissue was observed by using a living body imager, the whole animal living body imaging is shown in FIG. 10, and the results show that TMZ/sipD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) Hardly crosses the blood-brain barrier, only a very small amount of nanoparticles accumulate in brain tissue at 12h and 24h, while TMZ/siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) The accumulation in brain tissue is obviously increased, and the fluorescence intensity reaches 6.83 × e at 12h⁷p/sec/cm2/sr, and after 24h, TMZ/siPD-L1 @ G L PN (PASP-G-PEI) can still be observed in the brain₁₈₀₀) Accumulation in brain tissue and fluorescence intensity of 2.35 × e⁷p/sec/cm2/sr。

To investigate the distribution of the lipopolymer nanoparticles in brain gliomas after crossing the blood brain barrier, the charge C₆The method comprises the steps of injecting lipid polymer nanoparticles into mice with/TR cells in situ by mouse tail vein, and observing the accumulation of the lipid polymer nanoparticles in brain glioma tissues by adopting a living body imager. The whole animal living body imaging result is shown in FIG. 11, and the lipid polymer nanoparticles are in the glioma tissue of tumor-bearing mice (C)₆the/TR brain glioma was inoculated in mice on the left side of bregma at the junction of the medial border line and the sagittal midline of the head) with significant accumulation, and at 12h and 24h after administration, TMZ/sipD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) The accumulation in brain glioma reached 8.25 × e⁷p/sec/cm2/sr and 4.95 × e⁷p/sec/cm2/sr, significantly more than TMZ/sipD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) And (4) administration groups. The above results suggest that the 2-deoxyglucose-modified lipopolymer nanoparticles can not only effectively cross the blood brain barrier, but also effectively accumulate in the brain glioma tissue.

2.7 therapeutic Effect of lipid Polymer nanoparticles on TMZ-resistant in situ brain glioma

Free TMZ to C₆Inhibition of in situ glioma growth of-luc/TR cells As shown in FIG. 12B, TMZ failed to inhibit C₆-growth of luc/TR cells in situ gliomas, with a tumor volume of 89.8% of that of the saline group on day 22 of tumor inoculation when a dose of 50mg/kg was administered. Lipopolymer nanoparticle pair C₆The inhibition of in situ glioma growth of-luc/TR cells is shown in FIGS. 12C-F, where the dose of siPD-L1 was 0.5mg/kg and TMZ was 44mg/kg, siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) The tumor volume of the administration group at the 22 th day of tumor inoculation was 30.5% of that of the normal saline group, and TMZ/sipD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) The tumor volume of the group administered at day 22 of tumor inoculation was 47.6% of that of the saline group, TMZ/siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) The tumor volume in the group administered was only 4.34% of that in the group administered with physiological saline on day 22 of tumor inoculation, the above results indicate that siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀)、TMZ/siPD-L1@LPN(PASP-g-PEI₁₈₀₀) And TMZ/siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) All can inhibit C₆Growth of luc/TR cells in situ glioma and TMZ/sipD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) Inhibition of C₆The growth effect of the-luc/TR cell in situ glioma is obviously stronger than that of sipD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) And TMZ/siPD-L1 @ L PN (PASP-g-PEI)₁₈₀₀)。

2.8 Effect of lipid Polymer nanoparticles on the expression of glioma tissue PD-L1 and MGMT

As shown in FIG. 13, at C₆Luc cells in situ glioma tissue and C₆In situ glioma tissue of/TR-luc cells, TMZ/sipD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) Can obviously inhibit the expression of PD-L1, and the inhibition effect is obviously better than that of TMZ/siPD-L1 @ L PN (PASP-g-PEI)₁₈₀₀)。

Earlier studies showed that the expression of MGMT in TMZ-resistant glioma cells was increased, while the lipopolymer nanoparticles were able to reduce the expression of MGMT in TMZ-resistant glioma cells, therefore, this part of experiments examined the effect of the lipopolymer nanoparticles on the expression of MGMT in TMZ-resistant glioma tissues, the results are shown in FIG. 14, TMZ/sipD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) Can obviously reduce the expression of MGMT in TMZ-resistant in-situ glioma, the expression level is only 58 percent of that of MGMT in brain glioma tissues of a physiological saline group, and the inhibition effect is obviously stronger than that of TMZ/sipD-L1 @ L PN (PASP-g-PEI)₁₈₀₀) And (4) administration groups.

And 3, conclusion:

TMZ/siPD-L1@GLPN(PASP-g-PEI₁₈₀₀) Can effectively compress siPD-L1 and improve the stability of siPD-L1 in serum, TMZ/siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) Can be covered with C₆Cells and C₆Uptake by/TR cells, release free siPD-L1. siPD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) Can cross blood brain barrier and effectively accumulate in brain glioma tissues TMZ/sipD-L1 @ G L PN (PASP-G-PEI)₁₈₀₀) The expression of PD-L1 in silent drug-resistant brain glioma not only can inhibit the activation of PD-1/PD-L1 pathway in the tumor microenvironment and improve the killing effect of the immune system of the organism on brain glioma, but also can improve the sensitivity of drug-resistant brain glioma cells to TMZ by reducing the expression of MGMT, enhance the toxicity of TMZ on drug-resistant brain glioma cells, inhibit the growth of drug-resistant brain glioma in double ways and improve the treatment effect on drug-resistant brain glioma.

Sequence listing

<110> the fourth military medical university of the Chinese people liberation army

<120> brain targeting drug delivery system for improving drug-resistant brain glioma microenvironment and reversing drug resistance

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>21

<212>RNA

<213> Artificial sequence (unknown)

<220>

<221>misc_RNA

<222>(10，13，18，19)

<223> y = t/u or c

<400>1

gaagggaaay gcygcccyyt t 21

<210>2

<211>18

<212>RNA

<213> Artificial sequence (unknown)

<220>

<221>misc_RNA

<222>(8，9，10，14，15)

<223> y = t/u or c

<400>2

aagggcayyy cccyyctt 18

Claims

1. A preparation method of core-shell lipopolymer nanoparticles co-loaded with siPD-L1 and temozolomide is characterized in that polyethyleneimine with the molecular weight of 1800 Da is grafted on a side chain of polyaspartic acid through a disulfide bond to synthesize a novel cationic polymer PASP-G-PEI for compressing siPD-L1, a compound formed by the siPD-L1 and the PASP-G-PEI is taken as a core, a lipid film formed by 2-deoxyglucose modified distearoylphosphatidylethanolamine-polyethylene glycol, cholesterol and dioleoylphosphatidylethanolamine is taken as an outer shell, a saturated TMZ aqueous solution is added into the lipid film, and a film dispersion method is adopted to prepare the core-shell lipopolymer nanoparticles TMZ/siPD-L1 @ G L PN co-loaded with the siPD-L1 and TMZ;

the lipid polymer nanoparticle TMZ/siPD-L1 @ G L PN passes through a blood brain barrier and enters drug-resistant glioma cells through a glucose transporter mediation, naked siPD-L1 and TMZ are rapidly released under the condition of high GSH concentration in drug-resistant glioma cell paste, the expression of PD-L1 in the TMZ-resistant glioma cells is silenced, a PD-1/PD-L1 pathway in the TMZ-resistant glioma cells is blocked, the killing effect of an organism immune system on the glioma is activated, the expression of MGMT is reduced, the sensitivity of the TMZ-resistant glioma cells to TMZ is improved, and the growth of the drug-resistant glioma is inhibited through dual pathways;

wherein the sequence of siPD-L1 is shown in SEQ ID: No.1, TMZ is temozolomide, MGMT is O6-methylguanine-DNA-methyltransferase, and GSH is glutathione.