Bionic drug-loaded nanoparticles as well as preparation method and application thereof
By preparing nanoparticles with a negatively charged PLGA shell and a positively charged polyethyleneimine layer, and combining them with gene editing plasmids and cell membrane layers, the problems of uneven particle size and insufficient targeting of PLGA nanoparticles were solved, enabling lysosomal escape and highly efficient disease treatment.
Patent Information
- Application Number
- CN202610034004.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-12
- Publication Date
- 2026-02-13
AI Technical Summary
Existing PLGA nanoparticles in drug delivery systems suffer from uneven particle size distribution, insufficient targeting ability, and lysosomal escape problems, leading to uneven drug distribution and degradation of drug activity in vivo.
Hydrophobic drugs were loaded onto a negatively charged PLGA shell, and a positively charged polyethyleneimine layer was modified on the outside to adsorb negatively charged gene-editing plasmids. The plasmids were then coated onto a cell membrane layer. Biomimetic drug-loaded nanoparticles were prepared by electrostatic interaction and liposome extrusion process.
This has resulted in nanoparticles with uniform particle size and precise targeting, which can effectively escape lysosomes and synergistically exert the dual effects of drug therapy and gene editing, thereby improving the treatment outcome of diseases.
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Figure CN121512972A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomaterials technology, specifically relating to a biomimetic drug-loaded nanoparticle, its preparation method, and its application. Background Technology
[0002] With the development of modern medicine, drug therapy remains one of the mainstream strategies for treating various diseases, especially malignant tumors, hereditary diseases, and refractory chronic diseases. However, traditional monotherapy faces significant limitations in clinical practice: on the one hand, the efficacy of a single drug is often limited, making it difficult to achieve a complete cure; on the other hand, long-term or high-dose use of a single drug easily induces drug resistance, leading to treatment failure or disease relapse. Therefore, developing new treatment strategies that can overcome drug resistance and achieve synergistic effects has become an important research direction in the current biomedical field. Among these, constructing multi-drug synergistic delivery systems, especially co-delivery systems capable of simultaneously delivering chemical drugs and gene drugs, shows enormous research and application potential.
[0003] In recent years, nanotechnology has provided new opportunities for the development of drug delivery systems. Nanoparticles prepared based on biodegradable materials, such as polylactic-co-glycolic acid copolymer (PLGA), have been widely studied as drug carriers due to their good biocompatibility and controllable release characteristics. However, conventional PLGA nanoparticles as drug carriers still face a series of problems that need to be solved: First, the preparation process often leads to uneven particle size distribution, affecting their in vivo distribution and pharmacokinetic behavior; second, these nanoparticles generally lack active targeting ability, mainly relying on enhanced penetration and retention (EPR) effects to accumulate at the lesion site, resulting in low targeting efficiency, and are easily captured and cleared by the mononuclear phagocyte system; more importantly, after entering cells via endocytosis, most of the nanoparticles are trapped in lysosomes, where they are degraded in the acidic environment and by various hydrolytic enzymes, causing the loaded drugs (especially bioactive molecules such as proteins and nucleic acids) to be inactivated before reaching the target site, i.e., the problem of lysosomal escape. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides a biomimetic drug-loaded nanoparticle, its preparation method, and its application. The prepared biomimetic drug-loaded nanoparticle has uniform particle size, high targeting precision, and lysosomal escape function.
[0005] This invention is specifically achieved through the following technical solutions: The first objective of this invention is to provide a biomimetic drug-loaded nanoparticle comprising a negatively charged PLGA shell, wherein a hydrophobic drug is loaded within the PLGA shell, and a positively charged polyethyleneimine layer is modified on the outside of the PLGA shell. The polyethyleneimine layer adsorbs a negatively charged gene-editing plasmid layer through electrostatic interaction, and the gene-editing plasmid layer is coated with a cell membrane layer.
[0006] Preferably, the particle size is 100-300 nm and the dispersion index is 0.01-0.3; the gene editing plasmid is Cas9 / sgFAK, Cas9 / sgHER2 or Cas9 / sgTBR1, and the cell membrane is SCC7 cell membrane, 4T1 cell membrane or MLF cell membrane.
[0007] A second objective of this invention is to provide a method for preparing the above-mentioned biomimetic drug-loaded nanoparticles, comprising the following steps: A hydrophobic drug and a PLGA solution were mixed to obtain an intermediate product; the intermediate product was then treated in a polyvinyl alcohol emulsion solution to obtain drug-loaded PLGA. Drug-loaded PLGA was placed in a polyethyleneimine solution, and the negatively charged PLGA combined with the positively charged polyethyleneimine to prepare drug-loaded PLGA-PEI nanoparticles. Drug-loaded PLGA-PEI nanoparticles and gene-editing plasmids were combined in HEPES, and the negatively charged gene-editing plasmids were adsorbed by electrostatic interaction to obtain drug-loaded PLGA-PEI-plasmid nanoparticles. Drug-loaded PLGA-PEI plasmid nanoparticles and cell membranes were ultrasonically combined in PBS, and then biomimetic drug-loaded nanoparticles were obtained by liposome extrusion.
[0008] Preferably, the concentration of the PLGA solution is 20 mg / mL, the concentration of the hydrophobic drug in the PLGA solution is 20 nM-5 mM, the mass ratio of drug-loaded PLGA to polyethyleneimine is 10:2.5-10, and the mixture is incubated at room temperature for 12-24 h.
[0009] Preferably, when using a compound gene editing plasmid, the mass ratio of the gene editing plasmid to the drug-loaded PLGA-PEI nanoparticles is 1:900.
[0010] Preferably, when composite cell membranes are used, the concentration of the cell membrane is 2-10 mg / mL, and the ratio of drug-loaded PLGA-PEI-plasmid nanoparticles to cell membranes is 17:1.
[0011] Preferably, during ultrasound, an ice-water bath is used with the following parameters: 100 W, 3 s on, 2 s off, 15 min.
[0012] Preferably, in the liposome extrusion process, a 400 μm membrane is extruded 20-40 times using a liposome extruder, and a 200 μm membrane is extruded 20-40 times.
[0013] A third objective of this invention is to provide the application of the above-mentioned biomimetic drug-loaded nanoparticles in the preparation of drugs for treating diseases.
[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes the excellent biocompatibility of PLGA with a negatively charged terminal carboxyl group to prepare nanoparticles as drug delivery carriers, internally loaded with hydrophobic drugs. Further, these nanoparticles are co-incubated with positively charged polyethyleneimine (PEI), leveraging PEI's lysosomal escape capability to enhance intracellular stability. Subsequently, negatively charged gene-editing plasmids are electrostatically adsorbed to knock out pathogenic genes at the gene level, fundamentally inhibiting their expression. Finally, utilizing the excellent biocompatibility, homology targeting, and immune escape characteristics of cell membranes, cell membrane-coated nanoparticles are prepared as drug delivery carriers. These nanoparticles fully utilize the homology targeting function of cell membranes, exhibiting excellent drug delivery performance and uniform particle size distribution. These biomimetic drug-loaded nanoparticles can exert dual therapeutic effects of drug therapy and pathogenic gene knockout, showing promising clinical application prospects. Specifically: In terms of material sourcing, this invention provides an effective plasmid-targeted delivery method. By exploring the conditions for plasmid loading onto particles, the effectiveness of plasmid loading onto nanoparticles is clarified. According to specific research objectives, selecting appropriate nucleic acid substances can effectively intervene in the expression of pathogenic genes and fundamentally inhibit disease progression. According to the purpose of disease treatment, selecting corresponding cell membranes can effectively improve the disease targeting of drug-loaded particles.
[0015] In terms of preparation process, this invention, through condition exploration, determines the conditions for PLGA incubation of PEI and the effective method for coating cell membranes, thereby efficiently achieving lysosomal escape of nanoparticles and good targeting of particles, while ensuring uniform particle size distribution. This preparation method is simple and has a wide range of applications.
[0016] In terms of material function, the drug loaded inside the biomimetic nanoparticle PLGA of this invention can work synergistically with gene editing plasmids to achieve the purpose of disease treatment. Based on the specificity of the disease treatment purpose, a unique gene editing plasmid is selected to intervene in the expression of pathogenic genes, thereby greatly improving the treatment effect. Attached Figure Description
[0017] Figure 1 The image shows the morphology of the biomimetic drug-loaded nanoparticles prepared in Example 1.
[0018] Figure 2The image shows the potential diagram of the biomimetic nanoparticles prepared in Example 1, where P: PLGA; PP: PLGA-PEI; P-PC: PLGA-PEI-plasmid; P-PC@CM: PLGA-PEI-plasmid nanoparticles with cell membrane coating on the surface.
[0019] Figure 3 The particle size distribution of the biomimetic nanoparticles prepared in Example 1 is shown.
[0020] Figure 4 Lysosome escape experiment of biomimetic nanoparticles prepared in Example 1, wherein DiI labeled nanoparticles, lsyo-tracker Green labeled lysosomes, DAPI labeled cell nuclei, and Merge group of images.
[0021] Figure 5 The image shows the results of agarose gel electrophoresis analysis of the nucleic acid loading of nanoparticles. The mass ratio of plasmid to nanoparticles was 1:100-1:900.
[0022] Figure 6 The graph shows the inhibition of the target protein expression by the biomimetic nanoparticles prepared in Example 1.
[0023] Figure 7 This is a diagram showing the targeting test of the biomimetic nanoparticles (DiI-labeled) of Example 1 coated with SCC7 cell membrane.
[0024] Figure 8 This image shows the nucleic acid loading of nanoparticles analyzed by agarose gel electrophoresis. The mass ratio of plasmid to nanoparticles was 1:100-1:400.
[0025] Figure 9 This image shows the effect of agarose gel electrophoresis analysis of nanoparticles coated with cell membranes of different masses.
[0026] Figure 10 The diagram shows the testing methods used to explore the ways in which cell membranes encapsulate nanoparticles through ultrasound, ultrasound + extrusion, and extrusion. Detailed Implementation
[0027] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention. Unless otherwise specified, the experimental methods and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials described are commercially available.
[0028] The raw materials used in the following examples are sourced from (manufacturer, model, or product parameters): Polylactic acid-glycolic acid copolymer (PLGA): Jinan Daigang, molecular weight: 20000; Polyvinyl alcohol (PVA): Beyotime, molecular weight: 30,000-70,000; Polyethyleneimine (PEI): Maokang Biotechnology, molecular weight: 40,000; Gene editing plasmid Cas9 / sgFAK: constructed by Suzhou Hongxun Biotechnology Co., Ltd. HEPES: Beyotime (Item No.: SP / ST090-10g); SCC7 cell membrane: SCC7 cell membrane was extracted using a cell membrane protein and cytoplasmic protein extraction kit (catalog number: BL671B) (manufacturer: White Shark); Cas9 / sgHER2: Constructed by Suzhou Hongxun Biotechnology Co., Ltd. 4T1 cell membrane: The cell membrane of 4T1 cells was extracted using a cell membrane protein and cytoplasmic protein extraction kit (catalog number: BL671B) (manufacturer: White Shark); Gene editing plasmid Cas9 / sgTBR1: constructed by Suzhou Hongxun Biotechnology Co., Ltd.; MLF cell membrane: The cell membrane of MLF was extracted using a cell membrane protein and cytoplasmic protein extraction kit (catalog number: BL671B) (manufacturer: White Shark).
[0029] This invention provides nanoparticles for disease treatment and their preparation method. Utilizing the principle of electrostatic adsorption, negatively charged PLGA nanoparticles are loaded with therapeutic hydrophobic drugs. By exploring different incubation times between PEI solution and PLGA nanoparticles, the effectiveness of adsorbing positively charged PEI on the nanoparticle surface is clarified, enabling lysosomal escape. Furthermore, different mass ratios of nanoparticles co-incubated with gene-editing plasmids are investigated to explore the maximum plasmid loading capacity and efficiently inhibit pathogenic gene expression. Different methods for coating nanoparticles with cell membranes are explored, including ultrasonication, liposome extrusion, and ultrasonic-assisted extrusion, to achieve good cell targeting.
[0030] The experimental steps and parameter selection range are as follows: Step 1: 10 mg of PLGA was dissolved in 500 μL of dichloromethane, and paclitaxel, tamoxifen, cisplatin, pirfenidone, and other drugs were added until completely dissolved, with a drug concentration of 20 nM-5 mM in the mixture, to obtain an intermediate product. To stabilize the nanoparticles, the intermediate product was sonicated in an ice bath in 2 mL of 5% polyvinyl alcohol (PVA) emulsifier solution, followed by the addition of 8 mL of PVA. The mixture was then magnetically stirred at room temperature for 6 h, and the precipitate was washed with PBS or ddH2O to obtain drug-loaded PLGA.
[0031] Add 2.5-10 mL of prepared PEI (1 mg / mL, 1 mg PEI dissolved in 1 mL HEPES aqueous solution and pH adjusted to 7) to 10 mg drug-loaded PLGA and incubate on a horizontal shaker at room temperature for 12-24 h; then centrifuge at 12000 rpm for 10-30 min and resuspend in HEPES solution to obtain drug-loaded PLGA-PEI nanoparticles.
[0032] Step 2: Take 1-10 mg of the drug-loaded PLGA-PEI nanoparticles prepared in Step 1, add 2-10 μg of gene editing plasmid, with a plasmid:PLGA-PEI ratio of 1:100-1:10000, and add HEPES solution to make up to 200 μL. Incubate at room temperature for 4-12 h. Then centrifuge (12000 rpm, 10-30 min) to collect the precipitate and obtain the drug-loaded PLGA-PEI-plasmid nanoparticles.
[0033] Step 3: Dissolve 1-10 mg of the drug-loaded PLGA-PEI plasmid nanoparticles prepared in Step 2 in PBS, add 0.01-1 mg of cell membrane (such as breast cancer cell membrane, 4T1 cell membrane, head and neck squamous cell carcinoma cell membrane, SCC7 cell membrane, lung fibroblast cell membrane, MLF cell membrane, etc.), and finally make up the cell membrane concentration to 2-10 mg / mL. Process the system by sonication in an ice-water bath (100 w, 3 son, 2 s off, 15 min), or by extruding a 400 μm membrane 20-40 times and a 200 μm membrane 20-40 times using a liposome extruder, or by using a combination of sonication and a liposome extruder. Finally, centrifuge at 12000 rpm and resuspend in PBS for later use.
[0034] Based on the above, the present invention will be specifically described below through the following embodiments and comparative examples.
[0035] Example 1 To treat oral squamous cell carcinoma, biomimetic nanoparticles loaded with paclitaxel were prepared, along with a plasmid that knocks out the pathogenic gene, focal adhesion kinase (FAK). The nanoparticles were then coated with the cell membrane of squamous cell carcinoma cells (SCC7) to achieve biomimetic camouflage. The specific preparation method includes the following steps: Step 1: 10 mg of PLGA was dissolved in 500 μL of dichloromethane, and paclitaxel was added until completely dissolved. The concentration of paclitaxel in the mixture was 1 mM, yielding an intermediate product. The intermediate product was sonicated in 2 mL of 5% polyvinyl alcohol (PVA) solution in an ice bath, followed by the addition of 8 mL of PVA. The mixture was magnetically stirred at room temperature for 6 h, and the precipitate was washed with ddH2O to obtain drug-loaded PLGA. The prepared drug-loaded PLGA was added to 2.5 mL of prepared PEI (1 mg / mL, 1 mg PEI dissolved in 1 mL of HEPES aqueous solution and adjusted to pH=7), and incubated on a horizontal shaker at room temperature for 12 h. Afterward, the mixture was centrifuged at 12000 rpm for 15 min and resuspended in HEPES to obtain drug-loaded PLGA-PEI nanoparticles.
[0036] Step 2: Take the drug-loaded PLGA-PEI nanoparticles prepared in Step 1, add 2 μg of gene editing plasmid Cas9 / sgFAK, with a mass ratio of plasmid:PLGA-PEI of 1:900, and add HEPES to make up to 200 μL. Incubate at room temperature for 4 h. Then centrifuge (12000 rpm, 15 min) to collect the precipitate and obtain drug-loaded PLGA-PEI-Cas9 / sgFAK nanoparticles.
[0037] Step 3: Dissolve 3 mg of drug-loaded PLGA-PEI-Cas9 / sgFAK nanoparticles prepared in Step 2 in PBS, add 0.176 mg of SCC7 cell membrane, and finally make up the cell membrane concentration to 2 mg / mL. Sonicate the system in an ice-water bath (100 w, 3 son, 2 s off, 15 min). Use a liposome extruder to extrude 400 μm membrane 20 times and 200 μm membrane 20 times. Finally, centrifuge at 12000 rpm to collect the precipitate, resuspend it in PBS for later use, and obtain biomimetic drug-loaded nanoparticles.
[0038] Comparative Example 1 The difference from Example 1 is that in step 1, PLGA and PEI were incubated for 9 hours. Because the incubation time was too short, the negatively charged PLGA failed to fully contact and bind with the positively charged PEI, resulting in some negative charge being exposed. The results showed that the final potential was -1.13 ± 0.058 mV, which did not meet the surface positive potential requirement; therefore, this condition could not be chosen.
[0039] Comparative Example 2 The difference from Example 1 is that in step 2, the plasmid:PLGA-PEI ratio was 1:100. Due to insufficient PLGA-PEI, the plasmid was not completely adsorbed, resulting in some plasmid being leaked out without being encapsulated. Agarose gel electrophoresis results showed ( Figure 8When free plasmid bands appear, the nanoparticles cannot completely adsorb the plasmids, so this condition cannot be selected.
[0040] Comparative Example 3 The difference from Example 1 is that in step 2, the plasmid:PLGA-PEI ratio was 1:200. Due to insufficient PLGA-PEI, the plasmid was not completely adsorbed, resulting in some plasmid being leaked out without being encapsulated. Agarose gel electrophoresis results showed ( Figure 8 When free plasmid bands appear, the nanoparticles cannot completely adsorb the plasmids, so this condition cannot be selected.
[0041] Comparative Example 4 The difference from Example 1 is that in step 2, the plasmid:PLGA-PEI ratio was 1:400. Due to insufficient PLGA-PEI, the plasmid was not completely adsorbed, resulting in some plasmid being leaked out without being encapsulated. Agarose gel electrophoresis results showed ( Figure 8 When free plasmid bands appear, the nanoparticles cannot completely adsorb the plasmids, so this condition cannot be selected.
[0042] Comparative Example 5 The difference from Example 1 is that in step 2, the plasmid:PLGA-PEI ratio is 1:300.
[0043] Comparative Example 6 The difference from Example 1 is that in step 2, the plasmid:PLGA-PEI ratio is 1:500.
[0044] Comparative Example 7 The difference from Example 1 is that in step 2, the plasmid:PLGA-PEI ratio is 1:700.
[0045] Comparative Example 8 The difference from Example 1 is that in step 3, the drug-loaded PLGA-PEI-Cas9 / sgFAK nanoparticles are dissolved in PBS and 0.1 mg of SCC7 cell membrane is added.
[0046] Comparative Example 9 The difference from Example 1 is that in step 3, the drug-loaded PLGA-PEI-Cas / sgFAK nanoparticles are dissolved in PBS and 0.05 mg of SCC7 cell membrane is added.
[0047] Displayed by SDS-PAGE electrophoresis ( Figure 9 The effect of 0.1 mg or 0.05 mg of SCC7 cell membrane on the encapsulation of drug-loaded nanoparticles is limited, so this condition cannot be selected.
[0048] Comparative Example 10 The difference from Example 1 is that the method for loading the cell membrane was a single sonication method. The results were displayed by SDS-PAGE electrophoresis. Figure 10 Since the effects of ultrasound or liposome extrusion on the encapsulation of drug-loaded nanoparticles by cell membranes are limited, this method cannot be chosen.
[0049] Comparative Example 11 The difference from Example 1 is that the method for loading the cell membrane was a single liposome extrusion method. SDS-PAGE electrophoresis showed (…). Figure 10 Since the effects of ultrasound or liposome extrusion on the encapsulation of drug-loaded nanoparticles by cell membranes are limited, this method cannot be chosen.
[0050] Example 2 To treat breast cancer, biomimetic nanoparticles loaded with tamoxifen were prepared, which simultaneously inhibited the expression of the oncogene HER2. The nanoparticles were then coated with the cell membrane of breast cancer cells (4T1) to achieve biomimetic camouflage.
[0051] Step 1: 10 mg of PLGA was dissolved in 500 μL of dichloromethane, and tamoxifen was added until completely dissolved, with a tamoxifen concentration of 1 mM in the mixture, to obtain an intermediate product. The intermediate product was ultrasonically dissolved in 2 mL of 5% polyvinyl alcohol (PVA) solution in an ice bath, followed by the addition of 8 mL of PVA. The mixture was magnetically stirred at room temperature for 6 h, and the precipitate was washed with ddH2O to obtain drug-loaded PLGA. The prepared drug-loaded PLGA was added to 2.5 mL of prepared PEI (1 mg / mL, 1 mg PEI dissolved in 1 mL of HEPES aqueous solution and adjusted to pH=7), and incubated on a horizontal shaker at room temperature for 12 h. Afterward, the mixture was centrifuged at 12000 rpm for 15 min and resuspended in HEPES to obtain drug-loaded PLGA-PEI nanoparticles.
[0052] Step 2: Take the drug-loaded PLGA-PEI nanoparticles prepared in Step 1, add 2 μg of gene editing plasmid Cas9 / sgHER2, with a mass ratio of plasmid:PLGA-PEI of 1:900, and add HEPES to make up to 200 μL. Incubate at room temperature for 4 h. Then centrifuge (12000 rpm, 15 min) to collect the precipitate and obtain drug-loaded PLGA-PEI-Cas9 / sgHER2 nanoparticles.
[0053] Step 3: Dissolve the drug-loaded PLGA-PEI-Cas9 / sgHER2 nanoparticles prepared in Step 2 in PBS, add 0.176 mg of 4T1 cell membrane, and finally make up the cell membrane concentration to 2 mg / mL. Sonicate the system in an ice-water bath (100 w, 3 s on, 2 s off, 15 min). Use a liposome extruder to extrude 400 μm membrane 20 times and 200 μm membrane 20 times. Finally, centrifuge at 12000 rpm to collect the precipitate, resuspend it in PBS for later use, and obtain biomimetic drug-loaded nanoparticles.
[0054] Example 3 To treat pulmonary fibrosis, biomimetic nanoparticles loaded with pirfenidone were prepared, which simultaneously inhibited the expression of the fibrosis gene TGF-β1 receptor (TBR1). The nanoparticles were then coated with lung fibroblast (MLF) membranes to achieve biomimetic camouflage.
[0055] Step 1: 10 mg of PLGA was dissolved in 500 μL of dichloromethane, and pirfenidone was added until completely dissolved. The concentration of pirfenidone in the mixture was 1 mM, yielding an intermediate product. The intermediate product was ultrasonically dissolved in 2 mL of 5% polyvinyl alcohol (PVA) solution in an ice bath, followed by the addition of 8 mL of PVA. The mixture was magnetically stirred at room temperature for 6 h, and the precipitate was washed with ddH2O to obtain drug-loaded PLGA. The prepared drug-loaded PLGA was added to 2.5 mL of prepared PEI (1 mg / mL, 1 mg PEI dissolved in 1 mL of HEPES aqueous solution and adjusted to pH=7), and incubated on a horizontal shaker at room temperature for 12 h. Afterward, the mixture was centrifuged at 12000 rpm for 15 min and resuspended in HEPES to obtain drug-loaded PLGA-PEI nanoparticles.
[0056] Step 2: Take the drug-loaded PLGA-PEI nanoparticles prepared in Step 1, add 2 μg of gene editing plasmid Cas9 / sgTBR1, with a mass ratio of plasmid:PLGA-PEI of 1:900, and add HEPES to make up to 200 μL. Incubate at room temperature for 4 h. Then centrifuge (12000 rpm, 15 min) to collect the precipitate and obtain drug-loaded PLGA-PEI-Cas9 / sgTBR1 nanoparticles.
[0057] Step 3: Dissolve the drug-loaded PLGA-PEI-Cas9 / sgTBR1 nanoparticles prepared in Step 2 in PBS, add 0.176 mg of MLF cell membrane, and finally make up the cell membrane concentration to 2 mg / mL. Sonicate the system in an ice-water bath (100 w, 3 s on, 2 s off, 15 min). Use a liposome extruder to extrude 400 μm membrane 20 times and 200 μm membrane 20 times. Finally, centrifuge at 12000 rpm to collect the precipitate, resuspend it in PBS for later use, and obtain biomimetic drug-loaded nanoparticles.
[0058] Figure 1 The image shows the morphology of the biomimetic drug-loaded nanoparticles prepared in Example 1, indicating that the particle size is uniform and stable.
[0059] Figure 2 The image shows the potential diagram of the biomimetic nanoparticles prepared in Example 1, where P: PLGA; PP: PLGA-PEI; P-PC: PLGA-PEI-plasmid; P-PC@CM: PLGA-PEI-plasmid nanoparticles with cell membrane coating on the surface.
[0060] Figure 3 The particle size distribution of the biomimetic nanoparticles prepared in Example 1 is shown. The particles exhibit good uniformity, with a particle size of 251±8.42 nm and a dispersion index of 0.18±0.042.
[0061] Figure 4 For the lysosome escape experiment, the specific method was as follows: biomimetic nanoparticles were stained red with DiI and treated with SCC7 cells. Then, lysosomes were stained green with Lyso-tracker Green. After 2 hours, observation using laser confocal microscopy revealed that the red biomimetic nanoparticles and green lysosomes co-localized, indicating that the biomimetic nanoparticles were captured by the lysosomes. After 6 hours of cell treatment with biomimetic nanoparticles, the co-localization of red nanoparticles and green lysosomes disappeared, indicating that the nanoparticles could effectively escape from the lysosomes. Among them, DiI labeled nanoparticles, Lyso-tracker Green labeled lysosomes, DAPI labeled cell nuclei, and Merge is a series of images.
[0062] Figure 5 To analyze the nucleic acid loading of nanoparticles (plasmid to nanoparticle mass ratio of 1:100–1:900) using agarose gel electrophoresis, when the mass ratio was 1:900, the nanoparticles completely adsorbed the plasmid. However, when the ratio was 1:100–1:700, free plasmid bands appeared, indicating that the nanoparticles did not completely adsorb the plasmid. More specifically… Figure 8 To analyze the nucleic acid loading of nanoparticles by agarose gel electrophoresis, the mass ratio of plasmid to nanoparticles was 1:100-1:400, and the nanoparticles could not completely adsorb the plasmid.
[0063] Figure 6 The figure shows the inhibition of the target protein expression by the biomimetic nanoparticles prepared in Example 1. P-PC@CM can effectively inhibit the expression of the pathogenic gene FAK.
[0064] Figure 7 The targeting test of SCC7 cells by biomimetic nanoparticles (DiI-labeled) coated with SCC7 cell membrane in Example 1 showed that the biomimetic nanoparticles (DiI-labeled) coated with SCC7 cell membrane had high targeting ability for SCC7 cells, but poor targeting ability for 3T3 and 4T1 cells, indicating that the targeting ability of nanoparticles is improved after biomimetic camouflage with cell membrane.
[0065] Figure 9 To analyze the effect of different cell membrane masses on nanoparticle coating using agarose gel electrophoresis, it was found that the coating effect of 0.1 mg and 0.05 mg cell membranes was poor.
[0066] Figure 10 To explore the methods of cell membrane coating of nanoparticles using ultrasound, ultrasound + extrusion, and extrusion, the results showed that the effect of cell membrane coating of nanoparticles using ultrasound alone or extrusion alone was far less than the effect of using the two methods in combination.
[0067] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, it is intended to include any modifications and variations that fall within the scope of the claims and their equivalents.
Claims
1. A biomimetic drug-loaded nanoparticle, characterized in that, It includes a negatively charged PLGA shell, a hydrophobic drug loaded inside the PLGA shell, a positively charged polyethyleneimine layer modified on the outside of the PLGA shell, a negatively charged gene-editing plasmid layer adsorbed by the polyethyleneimine layer through electrostatic interaction, and a cell membrane layer covering the outside of the gene-editing plasmid layer.
2. The biomimetic drug-loaded nanoparticles according to claim 1, characterized in that, The particle size is 100~300 nm, and the dispersion index is 0.01~0.3; the gene editing plasmid is Cas9 / sgFAK, Cas9 / sgHER2 or Cas9 / sgTBR1, and the cell membrane is SCC7 cell membrane, 4T1 cell membrane or MLF cell membrane.
3. A method for preparing biomimetic drug-loaded nanoparticles according to claim 1, characterized in that, Includes the following steps: The hydrophobic drug and PLGA solution were mixed to obtain an intermediate product; The intermediate product was treated with a polyvinyl alcohol emulsifier solution to obtain drug-loaded PLGA; Drug-loaded PLGA was placed in a polyethyleneimine solution, and the negatively charged PLGA combined with the positively charged polyethyleneimine to prepare drug-loaded PLGA-PEI nanoparticles. Drug-loaded PLGA-PEI nanoparticles and gene-editing plasmids were combined in HEPES, and the negatively charged gene-editing plasmids were adsorbed by electrostatic interaction to obtain drug-loaded PLGA-PEI-plasmid nanoparticles. Drug-loaded PLGA-PEI plasmid nanoparticles and cell membranes were ultrasonically combined in PBS, and then biomimetic drug-loaded nanoparticles were obtained by liposome extrusion.
4. The preparation method according to claim 3, characterized in that, The concentration of the PLGA solution was 20 mg / mL, and the concentration of the hydrophobic drug in the PLGA solution was 20 nM to 5 mM; the mass ratio of drug-loaded PLGA to polyethyleneimine was 10:2.5 to 10, and the mixture was incubated at room temperature for 12 to 24 h.
5. The preparation method according to claim 3, characterized in that, When using compound gene editing plasmids, the mass ratio of gene editing plasmid to drug-loaded PLGA-PEI nanoparticles is 1:
900.
6. The preparation method according to claim 3, characterized in that, When using composite cell membranes, the concentration of the cell membrane is 2~10 mg / mL, and the ratio of drug-loaded PLGA-PEI-plasmid nanoparticles to cell membranes is 17:
1.
7. The preparation method according to claim 3, characterized in that, During ultrasound, an ice-water bath was used with the following parameters: power of 100 W, time interval of 3 s on and 2 s off, and ultrasound duration of 15 min.
8. The preparation method according to claim 3, characterized in that, In the liposome extrusion process, a 400 μm membrane is extruded 20-40 times using a liposome extruder, and a 200 μm membrane is extruded 20-40 times.
9. The application of the biomimetic drug-loaded nanoparticles according to claim 1 in the preparation of drugs for treating diseases.