CN115177739B - Tissue-cell-organelle three-level targeting bionic preparation, and preparation method and application thereof - Google Patents

Tissue-cell-organelle three-level targeting bionic preparation, and preparation method and application thereof Download PDF

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CN115177739B
CN115177739B CN202210161929.7A CN202210161929A CN115177739B CN 115177739 B CN115177739 B CN 115177739B CN 202210161929 A CN202210161929 A CN 202210161929A CN 115177739 B CN115177739 B CN 115177739B
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preparation
platelet
golgi
nanoparticles
targeting
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CN115177739A (en
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雷光华
邓彩凤
曾超
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Xiangya Hospital of Central South University
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Abstract

The invention discloses a three-level targeting bionic preparation of tissue-cell organelles, which comprises nanoparticles carrying bioactive substances and activated platelet membranes covered on the surfaces of the nanoparticles and modified by golgi targeting peptides, wherein the nanoparticles are polymer nanoparticles or liposomes, and the golgi targeting peptides comprise amino acid sequences SXYQRL, wherein X represents any amino acid. The preparation can realize three-level targeting drug delivery to arthritis-synovial fibroblasts-Golgi apparatus, and can block cytokines and enzymes which cause synovitis and bone erosion from being secreted by the synovial fibroblasts by disturbing the function of the Golgi apparatus, thereby finally realizing effective treatment of RA. The preparation method of the bionic preparation is simple to operate, low in cost and efficient in preparation.

Description

Tissue-cell-organelle three-level targeting bionic preparation, and preparation method and application thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a three-level targeting bionic preparation of tissue-cell-organelle, a preparation method and application thereof.
Background
Rheumatoid Arthritis (RA) is an autoimmune disease characterized by chronic erosive arthritis, which, as the disease progresses, causes degeneration and even disability of the patient's joints. Synovial macrophages are generally considered to be important immune cells in the synovium of RA joints, and play an important role in the development and progression of RA, often as therapeutic targets for RA, and drug delivery systems have been studied primarily around macrophages. Inhibition of synovial macrophage activation or blocking of its mediated cytokine signaling generally reduces RA synovitis to some extent, but these methods have limited protective effects on the sustained progression of synovitis and bone destruction. In addition, these treatments are ineffective for 30% of patients and there is still a need to find more effective treatments.
In recent years, researchers have come to appreciate that RA joint synovial fibroblasts can cause synovitis and bone erosion by secreting various cytokines and enzymes. Thus, pathological cytokines and enzymes that effectively deliver drugs to synovial fibroblasts and effectively block their secretion are of great interest for RA treatment.
Disclosure of Invention
The invention aims to solve the technical problems, overcome the defects and shortcomings in the background technology, and provide a bionic preparation capable of realizing three-level targeting of tissue-cell organelles, and finally realize effective treatment of RA.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
A three-level targeting bionic preparation of tissue-cell-organelle comprises nanoparticles carrying bioactive substances and activated platelet membranes coated on the surfaces of the nanoparticles and modified by golgi targeting peptides, wherein the nanoparticles are polymer nanoparticles or liposomes, and the golgi targeting peptides comprise an amino acid sequence SXYQRL, wherein X represents any amino acid, as shown in a sequence table SEQ ID NO: 1.
The bionic preparation can realize the targeting of inflammatory joints of RA by activating the tendency of platelet membranes to inflammation, targets synovial fibroblasts by endocytosis mediated by integrin alpha 2 beta 1, and finally is concentrated on the golgi apparatus of the synovial fibroblasts under the mediation of golgi targeting peptide (the polypeptide containing amino acid sequence SXYQRL can realize the reverse transport from endosome to golgi apparatus, thereby being positioned on the golgi apparatus). The bionic preparation is used for delivering a Golgi interfering agent (such as all-trans retinoic acid), and can specifically and efficiently destroy the Golgi structure of RA synovial fibroblasts so as to effectively block multiple pathogenic factors (such as inflammatory cytokines, chemokines, matrix degrading enzymes and the like) based on the synovial fibroblasts, thereby realizing the effective treatment of RA synovitis and bone erosion.
Preferably, the activated platelet membrane is a platelet membrane or a platelet microparticle membrane obtained after activation by a platelet activator; the polymer nanoparticles are PLGA nanoparticles; the bioactive substance is a golgi agent.
Preferably, the golgi disturbing agent is all-trans retinoic acid and its derivatives.
Preferably, the activated platelet membrane modified by the golgi targeting peptide is prepared by chemical synthesis or genetic engineering.
Based on a general inventive concept, the invention also provides a preparation method of the tissue-cell-organelle three-level targeting bionic preparation, which comprises the following steps:
(1) Incubating the Golgi apparatus targeting peptide activated by the N-hydroxysuccinimide with the activated platelet membrane to obtain an activated platelet membrane modified by the Golgi apparatus targeting peptide;
(2) Preparing a polymer nanoparticle suspension or a lipid body fluid;
(3) Incubating the activated platelet membrane modified by the Golgi apparatus targeting peptide and the polymer nanoparticle suspension or liposome liquid together, and preparing a bionic preparation by ultrasonic or extrusion through a microporous filter membrane.
In the above preparation method, preferably in the step (1), the co-incubation time is 2-6 hours, and unreacted golgi targeting peptide is removed by ultrafiltration; the activated platelet membrane is prepared by the following method: preparing platelets through gradient centrifugation, then adding platelet activating agent, incubating for 4-8h, centrifuging, taking supernatant rich in platelet particles, and removing the platelet particles content through repeated freeze thawing and gradient centrifugation; in addition, the activated platelet membrane can also be prepared by the following method: preparing platelets through gradient centrifugation, then adding platelet activating agent, incubating for 4-8 hours, and removing platelet contents through repeated freeze thawing and gradient centrifugation; the platelet activator is one or more of lipopolysaccharide, thrombin, collagen and calcium ion-containing reagent.
Preferably, in step (2), the polymer nanoparticle suspension is prepared by the following method: dissolving a polymer material and a bioactive substance in dichloromethane to obtain an organic phase; and in addition, taking PVA water solution with the mass-volume concentration of 1% as a water phase, adding the organic phase into the water phase, performing probe ultrasound of 200-400W for 5-15min, wherein the volume ratio of the organic phase to the water phase is 1:5-20, and obtaining the polymer nanoparticle suspension.
Preferably, in step (2), the lipid body fluid is prepared by the following method: dissolving phospholipid, cholesterol and bioactive substances in ethanol, removing organic reagent by rotary evaporation (rotary evaporator operation), removing membrane by adding water, and performing 200-400W probe ultrasound for 5-15min to obtain lipid body fluid.
Preferably, in step (3), the specific operation of co-incubation comprises the steps of: mixing the activated platelet membrane modified by the Golgi apparatus targeting peptide and the polymer nanoparticle suspension or liposome liquid to obtain a mixed liquid, and carrying out 200-400W probe ultrasonic treatment on the mixed liquid in an ice-water bath for 5-15min; or sequentially extruding the mixed solution through microporous filter membranes with diameters of 400nm, 200nm and 100 nm.
Based on a general inventive concept, the invention also provides application of the bionic preparation in preparing medicines for preventing and treating rheumatoid arthritis.
Compared with the prior art, the invention has the beneficial effects that:
1. The bionic preparation has the effect of targeting the golgi apparatus of the synovitis and the synovial fibroblast, can realize the three-stage targeting drug delivery of the arthritis-synovial fibroblast-golgi apparatus, and can block the secretion of cytokines and enzymes causing synovitis and bone erosion by the synovial fibroblast by disturbing the function of the golgi apparatus, thereby finally realizing the effective treatment of RA.
2. The preparation method provided by the invention is simple to operate, low in cost and high in preparation efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a representation of the biomimetic formulation prepared in example 1; wherein: a) Particle sizes of NPs, PMNPs and Gol-PMNPs; b) Zeta potentials of NPs, PMNPs and Gol-PMNPs; c) Polydisperse coefficients of NPs, PMNPs, and Gol-PMNPs particle sizes; d) Transmission electron microscopy images of NPs, PMNPs and Gol-PMNPs.
FIG. 2 is a cell uptake experiment of the biomimetic preparation prepared in example 1; wherein: a) Observing the uptake of NPs, PMNPs and Gol-PMNPs on human synovial fibroblasts by a laser confocal microscope; b) Flow histograms of NPs, PMNPs and Gol-PMNPs uptake by human synovial fibroblasts; c) Flow quantitative analysis histogram of NPs, PMNPs and Gol-PMNPs uptake by human synovial fibroblasts (n=3), <0.01; d) Observing the co-localization of NPs, PMNPs and Gol-PMNPs with human synovial fibroblast Golgi body by a laser confocal microscope; e) NPs, PMNPs, and Gol-PMNPs have pearson correlation coefficients (n=3) co-localized with the golgi of human synovial fibroblasts, <0.01; f) Gol-PMNPs uptake pathway on human synovial fibroblasts (n=3), P <0.01, P <0.001.
FIG. 3 is an in vitro bioactivity assay of the biomimetic preparation prepared in example 1; wherein: a) Transmission electron microscope image of human synovial fibroblast golgi apparatus; b) A laser confocal map of human synovial fibroblast golgi apparatus; c) Flow quantitative histogram of green fluorescence in human synovial fibroblasts; d-j) protein concentration of cell culture supernatant 24h after drug treatment.
FIG. 4 is an in vivo distribution experiment of the biomimetic preparation prepared in example 1; wherein: a) In vivo fluorescence imaging of animals with NPs, PMNPs and Gol-PMNPs distributed in RA model rats (a, heart; B. liver; C. spleen; D. a lung; E. a kidney; F. blood; G. inflammatory joints); b) Semi-quantitative histogram of inflammatory joint distribution of NPs, PMNPs and Gol-PMNPs in RA model rats (n=6, 6 inflammatory joints of 3 animals), × P <0.001; c) Semi-quantitative histogram of primary organ distribution of NPs, PMNPs and Gol-PMNPs in RA model rats (n=3), P <0.05, P <0.01; d) NPs, PMNPs and Gol-PMNPs were co-localized with inflammatory joint synovial fibroblasts from RA model rats.
FIG. 5 is a pharmacodynamic experiment of the biomimetic formulation prepared in example 1; wherein: a) Schematic of dosing regimen; b) RA model rat hindlimb ankle diameter change plot (n=7), P <0.05; c) RA model rat hindlimb sole diameter change plot (n=7), P <0.01; d) 48H after the end of treatment, H & E (scale 100 μm), safranin-O (scale 200 μm) and toluidineblue (scale 200 μm) staining analyses of the hind limbs and sections of each group of rats; e) Micro-CT analysis charts of hind limb ankle joints of each group of rats after 48 hours of treatment; f-g) a bar graph of Micro-CT quantitative analysis of hindlimb ankle joints of rats of each group (n=3), P <0.05, P <0.01, P <0.001.
FIG. 6 is a cell uptake experiment of the biomimetic preparation prepared in example 2; wherein: a) Flow quantitative analysis histogram of Lip, PM-Lip and Gol-PM-Lip uptake by human synovial fibroblasts (n=3), P <0.001; b) The pearson correlation coefficients (n=3) for Lip, PM-Lip and Gol-PM-Lip co-localized with the golgi of human synovial fibroblasts are <0.01 and <0.001.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the invention, but the scope of the invention is not limited to the specific embodiments shown.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Example 1:
A three-stage targeting bionic preparation for tissue-cell-organelle comprises polymer nanoparticles (PLGA nanoparticles) and activated platelet membrane coated on the surface of the polymer nanoparticles and modified by golgi targeting peptide with amino acid sequence shown in SEQ ID NO. 2, and sequence abbreviated as ASDYQRLN.
The preparation method of the bionic preparation comprises the following steps:
1) Extraction of activated Platelet Membrane (PM)
Centrifuging the collected whole blood sample at 200g for 10min at 4 ℃, and taking the upper layer platelet-rich plasma; under the same conditions, repeating centrifugation for one time to purify, thus obtaining purified platelet-rich plasma; adding prostaglandin E1 into the purified platelet-rich plasma, and centrifuging 2000g at 4deg.C for 10min to obtain platelet precipitate; re-dispersing the platelet precipitate in physiological saline containing 1mM NaCl 2 and 5 μg/ml collagen, incubating for 4h, centrifuging at 4deg.C for 20min at 2000g, collecting supernatant rich in platelet particles, repeatedly freeze thawing, and removing platelet particle content by gradient centrifugation to obtain PM.
2) Preparation of Gol-PM modified platelet membrane (Gol-PM) with Gol-targeting peptide
Adding NHS (N-hydroxysuccinimide) -activated golgi targeting peptide (amino acid sequence ASDYQRLN) into PBS containing platelet membrane, stirring at room temperature for 4 hr, and removing unreacted golgi targeting peptide by ultrafiltration.
3) Preparation of all-trans retinoic acid (ATRA) -loaded PLGA Nanoparticles (NPs)
Dissolving 5mg of all-trans retinoic acid and 500 mgPLGA/50 (average molecular weight 4 ten thousand) in dichloromethane to prepare an organic phase; PVA was dissolved in water to prepare a 1% mass-volume PVA aqueous solution as an aqueous phase. Mixing the two phases according to the volume ratio of the organic phase to the water phase of 1:10, performing ultrasonic treatment (300W, 8 min) on the mixture in an ice-water bath by using a probe, and removing the organic reagent by using a rotary evaporator to obtain the organic-phase/water-phase composite material.
4) Preparation of bionic preparation
Mixing the PM solution with NPs (the mass ratio of PM to PLGA is 1:2), and performing ultrasonic treatment (300W, 8 min) in an ice-water bath to obtain platelet membrane coated nanoparticles (PMNPs); the PM solution is changed into Gol-PM solution (the mass ratio of Gol-PM to PLGA is 1:2), and the bionic preparation (Gol-PMNPs) with the Gol matrix targeting is obtained by the same method.
5) Characterization of the formulation
Diluting the bionic preparation PMNPs and Gol-PMNPs with pure water until light blue opalescence exists, and measuring the particle size by a laser particle sizer; the zeta potential of the obtained preparation is directly measured without dilution; the morphology was observed by transmission electron microscopy.
As can be seen from FIG. 1, NPs are spherical-like particles with a particle size of about 150nm and a zeta potential of about-30 eV, and after the preparation of the bionic preparation PMNPs and Gol-PMNPs, the particle size is slightly increased, and the potential is increased to above-20 eV. As can be seen from the transmission electron microscope image (FIG. 1 d), the biomimetic preparation PMNPs and Gol-PMNPs have a distinct membrane structure on their surfaces. The results above all confirm the successful preparation of the biomimetic formulation.
6) In vitro uptake experiments
Taking joint synovium obtained after joint replacement operation of RA patient, cutting, digesting with type II collagen, passing digested tissue through 70 μm cell filter screen, and collecting filtered cell suspension; placing the cell suspension into a cell culture dish for culturing, removing non-adherent cells, inoculating the adherent fibroblasts onto a 12-hole cell culture plate, culturing for 24 hours, replacing the culture medium with a serum-free culture medium containing DiD marked NPs, PMNPs and Gol-PMNPs (DiD concentration is 1 mu/ml) after the cells are adherent, detecting the fluorescence intensity of the cells by using a flow cytometer after 4 hours of incubation, and observing and photographing the fluorescence distribution of the cells by using a laser confocal microscope. To verify the endocytic pathway of the nanoparticles, fibroblasts were pretreated with different inhibitors (25. Mu.M TCI-15, 30. Mu.M chlorpromazine, 50. Mu.M amiloride or 5mM M-. Beta. -CD) for 1h, followed by addition of DiD-labeled Gol-PMNPs, further incubation for 4h, and the inhibitor-free fibroblasts were used as control groups and the fluorescence intensity of the cells was measured by flow cytometry.
From FIG. 2, gol-PMNPs uptake on human synovial fibroblasts was significantly higher than NPs (FIGS. 2 a-c); further, gol-PMNPs has significantly higher co-localization ability with the fibroblast Golgi apparatus than NPs and PMNPs (FIGS. 2 d-e). In addition, gol-PMNPs uptake on synovial fibroblasts was significantly reduced after TCI-15 (integrin. Alpha. 2β1 inhibitor) pretreatment (FIG. 2 f). The results demonstrate that Gol-PMNPs can be efficiently taken up by synovial fibroblasts by integrin α2β1-mediated endocytosis and can be targeted for distribution on the golgi apparatus.
7) In vitro biological Activity investigation
Taking joint synovium obtained after joint replacement operation of RA patient, cutting, digesting with type II collagen, passing digested tissue through 70 μm cell filter screen, and collecting filtered cell suspension; culturing the cell suspension in a cell culture dish, removing non-adherent cells, inoculating the adherent fibroblasts on a 12-hole cell culture plate, culturing for 24 hours, respectively adding ATRA-carrying NPs, PMNPs and Gol-PMNPs (ATRA concentration is 200ng/mL, non-medicated culture medium treatment is used as a control) into a culture medium after the adherent fibroblasts are adhered, incubating for 24 hours, taking supernatant, and measuring the concentrations of collagense, MMP-1, CX3CL, IL-8, IL-6, MCP-1 and MMP-13 by using a ELLISA method; observing the morphology of the golgi apparatus of each group of cells by transmission electron microscopy; the cell golgi apparatus was labeled with GM130 antibody with green fluorescence, and the fluorescence intensity of the cells was observed by a confocal laser microscope and measured using a flow cytometer.
As can be seen from fig. 3, the ATRA-Gol-PMNPs treatment was more effective in disrupting the morphology of the golgi apparatus and significantly reducing the secretory capacity of the fibroblast proteins relative to other treatments. These results further demonstrate that Gol-PMNPs can effectively target the synovial fibroblast golgi apparatus and achieve effective drug delivery.
8) RA rat model
And injecting the complete Freund adjuvant into the root of the rat tail by subcutaneous injection, and obtaining the RA rat model when the joint 'ankylosis' phenomenon occurs in the hind limb of the rat.
9) In vivo distribution experiments
DiD solution and DiD-labeled NPs, PMNPs and Gol-PMNPs were injected into rats via tail vein, respectively, and after 24 hours, fluorescence intensity at inflammatory joints of rats was observed by a small animal biopsy imager and photographed. After the observation of the living body imager of the small animals is finished, the rats are sacrificed, the synovium of the inflammatory joint of each group of rats is separated, frozen and sectioned, then the cell nuclei and the synovial fibroblasts of the cells in the sections are respectively stained with DAPI and green fluorescent-labeled vimentin antibodies, and the cells are observed and photographed by a laser confocal microscope.
From FIG. 4, it can be seen that Gol-PMNPs can effectively target inflammatory joints distributed in RA rats and at locations enriched in synovial fibroblasts relative to NPs. These results indicate that Gol-PMNPs can achieve targeting of arthritis and synovial fibroblasts.
10 Pharmacodynamic study
RA rats were treated by tail vein injection with saline, ATRA solution, ATRA-NPs, ATRA-PMNPs, and ATRA-Gol-PMNPs, respectively. ATRA was administered at a dose of 1mg/kg, once every two days, 5 times in total. From the first day of administration, the diameters of ankle and sole thickness of each group of rats were measured once every other day.
From fig. 5, it is clear that ATRA-Gol-PMNPs treatment is more effective in alleviating joint inflammation and achieving better bone protection in RA rats than others. The results fully demonstrate that the effectiveness of the arthritis-fibroblast-Golgi body three-level targeting drug delivery on RA treatment is realized, and the bionic preparation has good application effect in preparing the drug for preventing and treating rheumatoid arthritis.
Example 2: bionic liposome
A three-stage targeting bionic preparation for tissue-cell-organelle comprises bionic liposome and activated platelet membrane coated on the surface of the bionic liposome and modified by golgi targeting peptide, wherein the amino acid sequence of the golgi targeting peptide is shown as SEQ ID NO. 2, and the sequence is ASDYQRLN.
The preparation method of the bionic preparation comprises the following steps:
1) Extraction of activated Platelet Membrane (PM)
Centrifuging the collected whole blood sample at 200g for 10min at 4 ℃, and taking the upper layer platelet-rich plasma; under the same conditions, repeating centrifugation for one time to purify, thus obtaining purified platelet-rich plasma; adding prostaglandin E1 into the purified platelet-rich plasma, and centrifuging 2000g at 4deg.C for 10min to obtain platelet precipitate; re-dispersing the platelet precipitate in 100ng/ml collagen-containing physiological saline, incubating for 8h, repeatedly freezing and thawing, and removing platelet content by gradient centrifugation to obtain PM.
2) Preparation of Gol-PM modified platelet membrane (Gol-PM) with Gol-targeting peptide
Adding NHS (N-hydroxysuccinimide) -activated golgi targeting peptide (amino acid sequence ASDYQRLN) into PBS containing platelet membrane, stirring at room temperature for 24 hr, and removing unreacted golgi targeting peptide by ultrafiltration.
3) Preparation of DiD-labeled liposomes (Lip)
Dissolving DiD, phospholipid, cholesterol and all-trans retinoic acid in ethanol, removing organic reagent by rotary evaporator, removing film by adding water, and ultrasonic treating with probe in ice water bath (350W, 6 min). The particle size and potential of the particles are 118.5+ -6.5 nm and-6.1+ -1.4 mV respectively as measured by a laser particle sizer.
4) Preparation of bionic preparation
Mixing the PM solution with Lip (the mass ratio of PM to lipid is 1:5), sequentially extruding the mixed solution through microporous filter membranes with diameters of 400nm, 200nm and 100nm to obtain platelet membrane-coated liposome (PM-Lip), and measuring the particle size and the potential of the liposome by a laser particle sizer to be 126.3+/-4.2 nm and-14.4+/-3.1 mV respectively; the PM solution is changed into Gol-PM solution (the mass ratio of Gol-PM to lipid is 1:5), and the bionic preparation (Gol-PM-Lip) with Gol matrix targeting is obtained by the same method, and the particle size and the potential of the bionic preparation are measured to be 127.1+/-3.5 nm and-15.2+/-2.8 mV by a laser particle sizer.
5) In vitro uptake experiments
Taking joint synovium obtained after joint replacement operation of RA patient, cutting up, digesting with type II collagen, passing digested tissue through 70 μm cell filter screen, and collecting filtered cell suspension; placing the cell suspension into a cell culture dish for culturing, removing non-adherent cells, inoculating the adherent fibroblasts onto a 12-hole cell culture plate, culturing for 24 hours, replacing a culture medium with a serum-free culture medium containing DiD marked Lip, PM-Lip and Gol-PM-Lip (DiD concentration is 1 mu/ml) after the adherent fibroblasts are attached, detecting the fluorescence intensity of the cells by using a flow cytometer after incubating for 4 hours, and observing the fluorescence distribution of the cells and calculating the Golgi co-localization coefficient of the liposome by using a laser confocal microscope.
From FIG. 6, gol-PM-Lip uptake on human synovial fibroblasts was significantly higher than Lip (FIG. 6 a); further, gol-PM-Lip co-localizes with the fibroblast golgi apparatus significantly higher than Lip and PM-Lip (fig. 6 b). The results show that Gol-PM-Lip not only can be more efficiently taken up by synovial fibroblasts, but also can be distributed on the Golgi apparatus in a targeted manner.
In conclusion, the bionic preparation provided by the invention has the advantages that the bionic preparation has the engineering platelet membrane bionic preparation targeted by the synovial fibroblast golgi body in arthritis, the three-level targeted drug delivery to the arthritis-synovial fibroblast golgi body can be realized, and the functions of the golgi body can be disturbed, so that cytokines and enzymes causing synovitis and bone erosion are blocked from being secreted by the synovial fibroblast, and finally, the effective treatment of RA is realized. The preparation method is simple to operate, low in cost and efficient in preparation.
Sequence listing
<110> Xiangya Hospital at university of south China
<120> A tissue-cell-organelle three-level targeting bionic preparation, and preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 1
Ser Xaa Tyr Gln Arg Leu
1 5
<210> 2
<211> 8
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 2
Ala Ser Asp Tyr Gln Arg Leu Asn
1 5

Claims (9)

1. The three-level targeting bionic preparation for tissue-cell-organelles is characterized by comprising nanoparticles carrying bioactive substances and activated platelet membranes which are covered on the surfaces of the nanoparticles and are modified by golgi targeting peptides, wherein the nanoparticles are polymer nanoparticles or liposomes, and the golgi targeting peptides are Ala Ser Asp Tyr Gln Arg Leu Asn;
The bioactive substance is all-trans retinoic acid and its derivatives.
2. The three-level targeted biomimetic formulation of claim 1, wherein the activated platelet membrane is a platelet membrane obtained after activation by a platelet activator; the polymer nanoparticles are PLGA nanoparticles.
3. A method of preparing a three-stage targeted biomimetic formulation of any one of claims 1-2, comprising the steps of:
(1) Incubating the Golgi apparatus targeting peptide activated by the N-hydroxysuccinimide with the activated platelet membrane to obtain an activated platelet membrane modified by the Golgi apparatus targeting peptide;
(2) Preparing a polymer nanoparticle suspension or a lipid body fluid carrying a bioactive substance;
(3) Incubating the activated platelet membrane modified by the Golgi apparatus targeting peptide and the polymer nanoparticle suspension or liposome liquid together, and preparing a bionic preparation by ultrasonic or extrusion through a microporous filter membrane.
4. A method according to claim 3, wherein in step (1), the activated platelet membrane is prepared by: preparing platelets through gradient centrifugation, then adding platelet activating agent, incubating for 4-8h, centrifuging, taking supernatant rich in platelet particles, and removing the platelet particles content through repeated freeze thawing and gradient centrifugation; the platelet activator is one or more of lipopolysaccharide, thrombin, collagen and a reagent containing calcium ions; the co-incubation time is 2-6h, and unreacted golgi targeting peptide is removed by ultrafiltration.
5. A method according to claim 3, wherein in step (1), the activated platelet membrane is prepared by: preparing platelets through gradient centrifugation, then adding platelet activating agent, incubating for 4-8 hours, and removing platelet contents through repeated freeze thawing and gradient centrifugation; the platelet activator is one or more of lipopolysaccharide, thrombin, collagen and a reagent containing calcium ions; the co-incubation time is 2-6h, and unreacted golgi targeting peptide is removed by ultrafiltration.
6. A method of preparation according to claim 3, wherein in step (2) the polymer nanoparticle suspension is prepared by: dissolving a polymer material and a bioactive substance in dichloromethane to obtain an organic phase; and in addition, taking PVA water solution with the mass-volume concentration of 1% as a water phase, adding the organic phase into the water phase, performing probe ultrasound of 200-400W for 5-15min, wherein the volume ratio of the organic phase to the water phase is 1:5-20, and obtaining the polymer nanoparticle suspension.
7. A method of preparation according to claim 3, wherein in step (2) the lipid body fluid is prepared by: dissolving phospholipid, cholesterol and bioactive substances in ethanol, removing organic reagent by rotary evaporation, removing membrane by adding water, and performing 200-400W probe ultrasound for 5-15min to obtain lipid body fluid.
8. A method according to claim 3, wherein in step (3), the specific procedure of co-incubation comprises the steps of: mixing the activated platelet membrane modified by the Golgi apparatus targeting peptide and the polymer nanoparticle suspension or liposome liquid to obtain a mixed liquid, and carrying out 200-400W probe ultrasonic treatment on the mixed liquid in an ice-water bath for 5-15min; or sequentially extruding the mixed solution through microporous filter membranes with diameters of 400nm, 200nm and 100 nm.
9. Use of a biomimetic formulation according to any one of claims 1-2 or a biomimetic formulation prepared by a method according to any one of claims 3-8 in the manufacture of a medicament for the prevention and treatment of rheumatoid arthritis.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111317715A (en) * 2018-12-13 2020-06-23 四川大学 Golgi-targeted mucopolysaccharide nano-micelle and preparation method and application thereof
CN113173955A (en) * 2021-03-11 2021-07-27 济南大学 Two-photon diagnosis and treatment prodrug with targeted Golgi pH response and preparation thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8647673B2 (en) * 2007-07-24 2014-02-11 Wayne State University Nanoparticles for imaging and treating chlamydial infection
JP6790057B2 (en) * 2015-07-09 2020-11-25 ブイアイビー ブイゼットダブリュＶｉｂ Ｖｚｗ Cells producing glycoproteins with modified N- and O-glycosylation patterns and methods and uses thereof
US20190046497A1 (en) * 2016-02-14 2019-02-14 Yeda Research And Development Co., Ltd. Methods of modulating protein exocytosis and uses of same in therapy

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111317715A (en) * 2018-12-13 2020-06-23 四川大学 Golgi-targeted mucopolysaccharide nano-micelle and preparation method and application thereof
CN113173955A (en) * 2021-03-11 2021-07-27 济南大学 Two-photon diagnosis and treatment prodrug with targeted Golgi pH response and preparation thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
The uptake and intracellular fate of PLGA nanoparticles in epithelial cells;Cartiera, MS等;《BIOMATERIALS》;20090531;第30卷(第14期);第2790-2798页 *

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