CN113797355B

CN113797355B - Preparation method and application of nano material with synovial inflammation inhibition function

Info

Publication number: CN113797355B
Application number: CN202111197427.1A
Authority: CN
Inventors: 杨成莉; 周凯; 胡丹蓉; 钱志勇
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2021-10-14
Filing date: 2021-10-14
Publication date: 2023-07-21
Anticipated expiration: 2041-10-14
Also published as: CN113797355A

Abstract

The invention discloses a preparation method and application of a nano material with a synovitis inhibiting function, belonging to the technical field of biology, and the technical scheme is as follows: the method comprises the following steps: s1, construction of shRNA-leptinR: inserting the synthesized shRNA-LeptinR fragment into a plasmid vector to obtain a recombinant plasmid vector, then converting the recombinant plasmid vector into escherichia coli, and extracting to obtain shRNA-LeptinR plasmid; s2, preparing a macrophage membrane; s3, preparing the gene-carrying nanoparticles M2H@RPK, and preparing the nanoparticles M2H@RPK by adopting an electrostatic adsorption method; and (3) wrapping the nanoparticle H@RPK by using the M2 macrophage membrane extracted in the step S2, and repeatedly passing through the polycarbonate membrane by adopting a micro extruder to obtain the nanoparticle M2H@RPK. The nanometer system is mainly used for reducing inflammatory cascade reaction in the OA process, targets cartilage matrix, and can effectively reduce joint injury and inhibit the severity of overall arthritis.

Description

Preparation method and application of nano material with synovial inflammation inhibition function

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method and application of a nano material with a synovitis inhibiting function.

Background

There is growing evidence that Osteoarthritis (OA) is an inflammatory-related immunological disorder in which the synovium of the joints plays an important role. The joint synovium can be divided into two layers, namely a synovial inner layer and a synovial lower layer which are thinner and close to the joint cavity, wherein the surface layer is provided with 2-3 layers of cells, and two types of cells can be confirmed under an electron microscope, but no obvious boundary exists between the two types of cells, namely macrophage-like synovial cells and fibroblast-like synovial cells.

During synovitis, the number of macrophages increases dramatically. This massive growth results in part from the original macrophages in the synovium escaping the apoptotic pathway, but more from the infiltrating macrophages during blood circulation. Macrophages are a highly variable cell, and mainly have two polarization forms, namely M1 type and M2 type, in response to the difference of microenvironments, M1 type macrophages are a main macrophage subgroup formed by osteoarthritic factors, cartilage degradation and osteophytes, and M2 type macrophages have a protective effect on osteoarthritis by secreting anti-inflammatory factors.

Synovium can secrete Leptin (Leptin) in large quantity, the expression level of Leptin is obviously related to the progress and severity of OA, leptin plays a regulating role through a receptor Leptin R, and participates in autoimmune inflammatory reaction through inducing proliferation and activation of mononuclear or macrophage, so that targeting and regulating Leptin signals provide a potential immune pathway for OA treatment.

OA is currently the most common joint disease in the elderly worldwide, is an important cause of affecting the quality of life of the elderly, and the major clinical manifestation of this disease is pain and gradual loss of function, and current treatments have certain limitations.

In order to solve the problems, the invention provides a preparation method and application of a nano material with a synovitis inhibiting function based on the prior art.

Disclosure of Invention

The invention aims to provide a preparation method of a nano material with a synovitis inhibiting function, and the nano material can effectively reduce joint injury and inhibit the severity of overall arthritis by constructing a shRNA-leptinR (Ob-Rb) nano system which is wrapped by a macrophage membrane and targets a cartilage matrix.

The technical aim of the invention is realized by the following technical scheme:

the preparation method of the nano material with the synovitis inhibiting function comprises the following steps:

s1, construction of shRNA-leptinR plasmid: and inserting the synthesized shRNA-LeptinR fragment into a plasmid vector to obtain a recombinant plasmid vector, then converting the recombinant plasmid vector into escherichia coli, and extracting to obtain the shRNA-LeptinR plasmid.

S2, preparing a macrophage membrane:

A. taking M0 type mouse mononuclear macrophage leukemia cells in logarithmic growth phase, adding IL-4 and IL-13 when the density reaches 70%, and collecting M2 type macrophages after 24h of action.

B. The cells were resuspended with pre-chilled Tris-magnesium salt buffer, then repeatedly extruded 20 times through a mini-extruder to destroy the cells, and then the cell membranes were extracted by sucrose gradient centrifugation.

S3, preparing the gene-carrying nanoparticles M2H@RPK, wherein the nanoparticles M2H@RPK are prepared by adopting an electrostatic adsorption method, and the specific steps are as follows:

a. mixing the shRNA-LeptinR plasmid in the step S1 with iRGD, and standing for 5min to form a blend of the iRGD and the shRNA-LeptinR plasmid; adding PEI and anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) to obtain compound RPK (KAFAK-iRGD-PEI-shRNA-leptinR), standing and mixing for 5min.

b. Adding HA into the compound RPK, uniformly mixing, and obtaining nano-particles H@RPK (HA@KAFAK-iRGD-PEI-shRNA-leptinR) through self-assembly.

c. And (3) wrapping the nanoparticle H@RPK by using the M2 macrophage membrane extracted in the step S2, repeatedly passing through the polycarbonate membrane by adopting a micro extruder to obtain the nanoparticle M2H@RPK, and then preserving at 4 ℃ and using as soon as possible.

By adopting the technical scheme, the synovium can secrete a large amount of Leptin (Leptin), the expression level of Leptin is obviously related to the progress and severity of OA, the Leptin plays a role in regulating through a receptor (Leptin R) of Leptin, and the shRNA is an RNA sequence with a tight clasp, can be used for RNA interference to silence the expression of a target gene, the shRNA is introduced into a plasmid vector, shRNA-Leptin R plasmid carrying the target Leptin R gene is obtained, and a promoter in the plasmid vector ensures that the shRNA is always expressed, and simultaneously silences the Leptin R gene, so that the effect of reducing the level of Leptin R protein is achieved, inflammatory cascade reaction in the OA process can be reduced, joint injury is reduced, and the severity of overall arthritis is inhibited.

The extracted M2 macrophage membrane is used for wrapping the nanoparticle M2H@RPK, so that the biocompatibility of the nanoparticle M2H@RPK is improved while the complex biological function of a cell membrane is maintained, the monitoring and the removal of an immune system of an organism can be effectively avoided, the targeting specificity is improved, the toxic and side effects are reduced, the targeting cartilage matrix of a nano system can be effectively realized, and the aims of reducing joint injury and inhibiting the severity of overall arthritis are fulfilled.

The adoption of the scheme can reduce the inflammatory cascade reaction in the OA process, and the nano system targets the cartilage matrix, so that the joint injury can be effectively reduced, and the severity of the whole arthritis can be inhibited.

Further, the forward sequence of the shRNA construction primer in step S1 is 5'-CACAAAGAAGTTACATCTT-3'; the reverse sequence of the constructed primer is 3'-AAGATGTAACTTCTTTGTG-5'.

Further, in step S3, shRNA-LeptinR plasmid: iRGD: PEI: KAFAK: HA was added in an amount of 1:5:5:20:30.

further, in the step S3, the mass ratio of the M2 cell membrane to the shRNA-leptinR plasmid addition amount is 48:10.

further, the pore diameter of the polycarbonate film in the S3 step was 400nm.

The invention also provides application of the preparation method of the nano material with the synovitis inhibiting function, and the preparation method of the nano material with the synovitis inhibiting function is applied to inhibiting synovitis.

In summary, the invention has the following beneficial effects:

1. according to the invention, an shRNA-leptinR (Ob-Rb) nano system wrapped by a macrophage membrane is constructed, so that inflammatory cascade reaction in the OA process can be reduced.

2. And the nano system targets the cartilage matrix, so that the joint injury can be effectively reduced, and the severity of the whole arthritis can be effectively inhibited.

Drawings

FIG. 1 is a graph of Western Blot results of in vitro down-regulation of the LeptinR protein by shRNA-LeptinR constructed in example 1;

FIG. 2 is a fluorescent staining verification chart of the M2 cell membrane coated nanosystems in example 1;

FIG. 3 is a graph of cell biosafety statistics of the nanosystems prepared in example 1;

FIG. 4 is a graph showing the detection of IL-10 and IL-1βTNF- α secretion from synovial cell supernatants by nanosystems in example 1;

FIG. 5 shows PCR detection of mRNA expression levels of synovial cell inflammatory factors IL1 beta, IL10 and TNF-alpha by the nanosystems of example 1.

Detailed Description

The invention is described in further detail below with reference to the attached drawings and embodiments:

example 1: the preparation method of the nano material with the synovitis inhibiting function comprises the following steps:

s1, construction of shRNA-leptinR plasmid: inserting the synthesized shRNA-LeptinR fragment into a Plko.1-EGFP-PURO plasmid vector to obtain a recombinant plasmid vector, then converting the recombinant plasmid vector into escherichia coli, extracting to obtain shRNA-LeptinR plasmid which contains green fluorescent protein labeled fragments and has ampicillin resistance, and transferring the shRNA-LeptinR plasmid into the escherichia coli, wherein the forward sequence of a adopted construction primer is 5'-CACAAAGAAGTTACATCTT-3'; the reverse sequence of the constructed primer is 3'-AAGATGTAACTTCTTTGTG-5'.

S2, preparing a macrophage membrane:

A. taking M0 type mouse mononuclear macrophage leukemia cells in logarithmic phase, adding 20ng.mL when density reaches 70% ^-1 IL-4 and IL-13, after 24h of action, M2 type macrophages were collected.

B. The cells were resuspended with pre-chilled Tris-magnesium salt buffer (TM buffer, pH 7.4,0.01M Tris and 0.001M MgCl) to a cell concentration of 2X 107 cells/mL, and then repeatedly extruded 20 times through a mini-extruder to destroy the cells, followed by sucrose gradient centrifugation to extract the cell membranes. The sucrose gradient centrifugation method comprises the following specific steps:

diluting the TM buffer solution to the concentration of 0.25M sucrose by adopting 1M sucrose solution, and centrifuging for 10min at 4 ℃ with 2000 g; then collecting supernatant, centrifuging at 4000g and 4 ℃ for 30min, re-suspending and washing the lower precipitate with TM buffer of 0.25M sucrose, centrifuging at 4000g and 4 ℃ for 30min again, collecting cell membranes, and then adopting 100W power ultrasound for 2min to store the obtained cell membranes for later use. Protein quantification of the extracted M2 macrophage membrane using BCA protein quantification showed a protein content of about 0.24mg per 108 cells.

a. mixing 10 mu g shRNA-LeptinR plasmid in the step S1 with 48 mu g giRGD, and standing for 5min to form a blend of iRGD and shRNA-LeptinR plasmid; then 50. Mu.g PEI and 200. Mu.g anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) were added to obtain a complex RPK (KAFAK-iRGD-PEI-shRNA-leptinR), and the mixture was allowed to stand and mixed for 5 minutes.

b. Adding 300 mu g of HA into the composite RPK, uniformly mixing, and obtaining the nanoparticle H@RPK (HA@KAFAK-iRGD-PEI-shRNA-leptinR) through self-assembly.

c. And (3) wrapping the nanoparticle H@RPK by using the M2 macrophage membrane extracted in the step (S2), adding 0.048mg M2 cell membrane into every 10 mug shRNA-leptin R plasmid, repeatedly passing through 400 polycarbonate membranes by adopting a micro extruder to obtain the nanoparticle M2H@RPK, wherein the extrusion times are 20 times, and then preserving at 4 ℃ and using as soon as possible.

The invention adopts western blotting to evaluate the down-regulation condition of the leptin R protein, and comprises the following steps:

cells were collected at the end of transfection, washed with cold PBS and then resuspended in 100. Mu.L of RIPA lysis buffer containing cocktail.

After incubation on ice for 30min, lysed cells were collected and centrifuged at 12000g for 15min. The BCA kit was used to determine the total protein concentration of each group. Total proteins were separated on a 15% SDS-PAGE gel (120V voltage, 2 h), transferred to PVDF membrane (80V voltage, 2 h) and blocked in 5% skim milk for 2h.

Incubation with LeptinR rabbit monoclonal antibody overnight at 4 ℃. After washing the PVDF membrane twice with TBST buffer, the PVDF membrane (goat anti-rabbit) was incubated with horseradish peroxidase conjugated secondary antibody, and analyzed with a chemiluminescent detection system.

The invention characterizes the physicochemical properties of the nano particles by a plurality of characterization modes.

d. Particle size, potential

The particle size, polydispersity (PDI) and Zeta potential of H@RPK, M2H@RPK were determined using a Markov laser scattering particle sizer. The test was repeated 3 times at 25 ℃.

e. Appearance and morphology determination

The morphology of H@RPK, M2H@RPK was observed using TEM. Before observation, the prepared sample dispersion liquid is dripped on a copper mesh covered by nitrocellulose, and after being negatively dyed by 2% phosphotungstic acid, the sample dispersion liquid is dried at room temperature, and the shape of the sample dispersion liquid is observed.

f.DNA gel blocking experiment

In order to determine the protection effect of the nanoparticles on shRNA-leptinR, a gel blocking experiment was performed. The shRNA-LeptinR, RR, RP, RPK, H@RPK and M2H@RPK are diluted by deionized water, adjusted to the same amount of shRNA-leptinR (200 ng of shRNA-leptinR is added into each hole), placed in agarose gel with the concentration of 1.0%, electrophoresed for 40 minutes under the constant pressure of 110V by adopting TAE electrophoresis buffer solution, and imaged by adopting a nucleic acid gel imaging system.

Identification of M2 cell membrane coating fluorescent staining

To visually identify whether the M2 cell membrane is successfully coated on the surface of the H@RPK nanoparticle. The invention adopts a cell membrane green fluorescent dye PKH67 to mark an M2 cell membrane, connects a red fluorescent dye cy5 with PEI to mark H@RPK nanoparticles, and observes the coating condition of the M2 cell membrane by observing the co-localization of red and green fluorescence, and comprises the following specific steps: centrifuging to collect polarized M2 cell membrane, washing with serum-free medium, and sucking to dry the cell to ensure that the residue is not more than 25 μL; then, M2 cell membranes were green fluorescent labeled with 2X 107 cells stained with 4. Mu.L PKH67 ethanol stock solution according to PKH67 cell stain kit; meanwhile, the cy5-NHS and PEI (PEI-cy 5) are connected through an amide bond, and H@RPK nanoparticles marked by the red fluorescent dye cy5 are prepared; then repeatedly passing through a 400nm polycarbonate film (20 times) through a micro extruder to prepare a fluorescent marker M2H@RPK; meanwhile, nanoparticles of M2 cell membranes which are not marked by PKH67 are prepared as a research control; finally, taking the prepared nanoparticles by using synovial cells, and imaging by using a confocal microscope.

The inhibition effect of the M2H@RPK nano system on osteoarthritis synovitis is also evaluated through various experiments:

(1) Nanoparticle cytotoxicity investigation

To examine the safety of different components of the nanoparticle to synovial membrane and chondrocyte in joint cavity. Seeding synovial membrane and chondrocytes in 96-well plates at a density of 3500 per well; after 24h PEI-containing (final concentrations 2, 4, 7.8, 15.6, 31.2, 62.5, 125, 250, 500. Mu.g.mL) was added ^-1 ) KAFAK (final concentrations 2, 4, 7.8, 15.6, 31.2, 62.5, 125, 250, 500. Mu.g.mL) ^-1 ) M2 (final concentration 0.25, 0.5, 1, 2, 3.75, 7.5, 15, 30, 60. Mu.g.multidot.mL) ^-1 ) M2H@RPK (final concentration 7.5, 15, 30, 60, 120, 225, 450, 900, 1800. Mu.g.mL) ^-1 ) Continuously culturing for 24 hours; mu.L MTT (5 mg. Multidot.mL) was added to each well ^-1 pH 7.4), incubation at 37℃for 4h; the supernatant was pipetted off and 150. Mu.L of DMSO was added to each well to completely dissolve the bottom crystals and absorbance was read at 570 nm. Cell viability was calculated from the blank wells (cells incubated without drug medium) and zeroed wells (medium alone without cells) set up, with 6 duplicate wells per sample.

(2) Dynamic observation of synovial cell uptake

To evaluate the effect of cell uptake time on cell uptake rate, synovial cells were seeded at 5×104 cells/well into a 24-well cell culture plate at the bottom of glass, after 24h, the cells were lysosomally labeled with a green fluorescent lysosome probe dye (lyso-tracker), after 2h staining, the medium was discarded, PBS was slightly washed twice, serum-Free medium containing Free cy5, PEI-cy5, cy5 labeled h@rpk and m2h@rpk nanoparticles was added, and subjected to real-time photographing observation under a confocal microscope.

(3) Intracellular positioning observation

To further examine the effect of measuring synovial cells on the intracellular distribution of Free cy5, PEI-cy5, cy 5-labeled H@RPK and M2H@RPK nanoparticles, synovial cells were seeded at 5X 104 cells/well into 24-well plates; after 24 hours, the culture medium is discarded and replaced by a serum-free culture medium containing the components; 2h after dosing, PBS was washed 2 times, fixed with 4% paraformaldehyde for 15min, PBS was washed 2 times, membrane disruption with 0.2% Triton X-100 for 10min, PBS was washed 3 times, then blocked with 1% BSA for 30min, PBS was washed 2 times, after washing with phalloidin for 1h, PBS was washed 2 times, finally after washing with 1. Mu.g.mL-1 DAPI for 10min, PBS was washed 3 times, and cell images were observed and obtained under confocal microscope.

(4) Synovial cell inflammatory factor detection

1) Synovial cell supernatant inflammatory factor detection

Synovial cells were seeded at 5X 104/well into 24-well cell culture plates and, after 24h, serum-free culture with 10 ng.mL-1 IL-1β was added for 24h to stimulate synovial cell inflammation formation. Inflammatory factor IL-10IL-1β was detected by the Meso QuickPlex SQ 120 kit (Meso Scale Discovery, rockville, md., USA) following the PBS, shRNA-LeptinR, KAFAK, M2, H@RPK, M2H@RPK packet interventions, collecting supernatant after 0.5d, 1d, 3d, 5d days.

2) Synovial cell inflammatory factor qPCR detection

The mRNA level of Ptpn2 in mouse tumor tissues is detected by adopting real-time fluorescence quantitative PCR (qPCR), and the specific steps are as follows: firstly extracting total RNA, performing grouping intervention, and adding Trizol lysate to lyse synovial cells after 0.5d, 1d, 3d and 5 d; adding 1/5 volume chloroform solution of Trizol, shaking, mixing, centrifuging at 12000rpm for 15min, and collecting supernatant; adding equal volume of 75% ethanol, centrifuging at 12000rpm for 15min, and collecting supernatant; then, the mixture was centrifuged at 12000rpm for 15min, the supernatant was discarded, and the precipitate was washed once with 0.5mL of 75% ethanol; the supernatant was discarded after centrifugation at 12000rpm for 15min again, and the total RNA concentration was determined by ultra clean bench air drying. Next, RNA was reverse transcribed into DNA according to the instructions of the RNA reverse transcription kit, and the DNA concentration was measured. Finally, qPCR was performed according to the primer sequences of the factors of Table 1.

TABLE 1 qPCR experimental primer sequences

In the embodiment of the invention, the shRNA-leptinR (Ob-Rb) nano system wrapped by the macrophage membrane is constructed, and the nano system targets the cartilage matrix, so that the joint injury can be effectively reduced, and the severity of the whole arthritis can be effectively inhibited.

The present embodiment is only for explanation of the present invention and is not to be construed as limiting the present invention, and modifications to the present embodiment, which may not creatively contribute to the present invention as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present invention.

Claims

1. The preparation method of the nano material with the synovitis inhibiting function is characterized by comprising the following steps: the method comprises the following steps:

s1, construction of shRNA-leptinR plasmid: inserting the synthesized shRNA-LeptinR fragment into a plasmid vector to obtain a recombinant plasmid vector, then converting the recombinant plasmid vector into escherichia coli, and extracting to obtain shRNA-LeptinR plasmid;

s2, preparing a macrophage membrane:

A. taking M0 type mouse mononuclear macrophage leukemia cells in logarithmic growth phase, adding IL-4 and IL-13 when the density reaches 70%, and collecting M2 type macrophages after 24 hours of action;

B. re-suspending cells by adopting precooled Tris-magnesium salt buffer salt, repeatedly extruding for 20 times by a micro extruder to destroy the cells, and extracting cell membranes by adopting a sucrose gradient centrifugation method;

a. mixing the shRNA-LeptinR plasmid in the step S1 with iRGD, and standing for 5min to form a blend of the iRGD and the shRNA-LeptinR plasmid; adding PEI and anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) to obtain composite RPK (KAFAK-iRGD-PEI-shRNA-leptinR), standing and mixing for 5min;

b. adding HA into the composite RPK, uniformly mixing, and obtaining nano particles H@RPK (HA@KAFAK-iRGD-PEI-shRNA-leptinR) through self-assembly;

2. The method for preparing a nanomaterial with synovitis inhibiting function according to claim 1, characterized in that: the forward sequence of the shRNA construction primer in the S1 step is 5'-CACAAAGAAGTTACATCTT-3'; the reverse sequence of the constructed primer is 3'-AAGATGTAACTTCTTTGTG-5'.

3. The method for preparing a nanomaterial with synovitis inhibiting function according to claim 1, characterized in that: in the S3 step, shRNA-LeptinR plasmid: iRGD: PEI: KAFAK: HA was added in an amount of 1:5:5:20:30.

4. the method for preparing a nanomaterial with synovitis inhibiting function according to claim 1, characterized in that: in the S3 step, the mass ratio of the M2 cell membrane to the shRNA-leptinR plasmid addition amount is 48:10.

5. the method for preparing a nanomaterial with synovitis inhibiting function according to claim 1, characterized in that: the pore diameter of the polycarbonate film in the S3 step was 400nm.