A lipid-polymer hybrid nanoparticle carrier, its preparation method and application
By modifying PLGA with RGD peptides, the stability and targeting issues of lipid nanoparticle carriers were resolved, achieving efficient delivery and enhanced immunization effects of mRNA vaccines, and improving the safety and stability of mRNA vaccines.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JINLIN MEDICAL COLLEGE
- Filing Date
- 2026-06-12
- Publication Date
- 2026-07-21
AI Technical Summary
Existing lipid nanoparticle carriers face challenges in terms of stability, safety, and targeting, especially due to their tendency to aggregate and restructure in vitro and in vivo. This limits the storage, transportation, and administration of mRNA vaccines and makes release less controllable.
A lipid-polymer hybrid nanoparticle carrier modified with RGD peptide was developed. By combining PLGA with DSPC and DMG-PEG 2000 and modifying with RGD peptide, a stable nanoparticle structure was formed, which improved the uniformity of particle size distribution and mechanical stability, and enhanced the delivery effect of mRNA.
It significantly improves the mechanical stability of nanoparticles and the expression efficiency of antigen proteins, thereby enhancing the effectiveness of the delivery system and the immune effect. It also achieves precise control of expression sites by regulating the phase separation of components.
Smart Images
Figure CN122424162A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical preparation technology, specifically to a lipid-polymer hybrid nanoparticle carrier, its preparation method, and its application. Background Technology
[0002] mRNA vaccines stand out among numerous vaccine types due to their high efficacy, safety, ease of rapid development and production. Furthermore, mRNA vaccines have shown broad potential in infectious diseases, cancer, and autoimmune diseases. While mRNA vaccine development is rapid, free mRNA is extremely unstable and easily destroyed by hydrolysis and oxidation in liquid environments, leading to its inactivation. Therefore, the application of mRNA vaccines depends on the delivery system. Currently, the LNP system is the fastest developing, with three lipid nanoparticle (LNP)-based mRNA vaccines (BNT162b2, mRNA-1273, SYS6006) being the first to be approved for marketing. All three induced strong neutralizing antibodies (NAb) and Th1 skewed CD in non-primates. 4+ T-cell responses protect animals from SARS-CoV-2 attack.
[0003] While widely used lipid nanoparticles have successfully saved countless lives and effectively protect and deliver mRNA into cells, they are not without their flaws. Like all technologies, they face challenges in stability and safety. Pure lipid systems are prone to aggregation and structural remodeling both in vivo and in vitro, exhibiting poor stability, poor controllability of drug release, and a tendency for burst release. Furthermore, their inherent hepatic distribution and high sensitivity to shear stress pose bottlenecks to vaccine storage, transportation, and administration. Despite the significant success of LNP delivery systems, they still face challenges such as demanding storage conditions, potential inflammatory responses, and limited targeting. Therefore, developing novel LNPs and optimizing their composition are crucial for improving the safety, efficacy, and stability of mRNA vaccines.
[0004] PLGA is a synthetically produced polymer whose ester bonds hydrolyze to form lactic acid (LA) and glycolic acid (GA). These two products participate in the tricarboxylic acid cycle, ultimately converting into carbon dioxide and water and being metabolized and excreted from the body. Current research has reported the dual function of PLGA nanoparticles, exhibiting both antigen delivery and auxiliary properties. They enhance immunity by promoting the phagocytosis of antigens by dendritic cells (DCs), activating and maturing DCs, and inducing effective immune responses. Therefore, this study aims to construct PLGA-involved lipid-polymer hybrid nanoparticles (LPNs). This novel carrier combines the stability of polymer materials with the membrane fusion properties of lipids, laying a foundation for the continued development of mRNA nucleic acid vaccines with independent intellectual property rights. Summary of the Invention
[0005] The purpose of this invention is to provide a lipid-polymer hybrid nanoparticle carrier for RGD peptide-modified PLGA.
[0006] Another objective of this invention is to provide a method for preparing the aforementioned lipid-polymer hybrid nanoparticle carrier. By using RGD in conjunction with PEG and PLGA for modification, the particle size distribution range of the nanoparticles is synergistically reduced, and the stability of the nanoparticles is improved. This, in turn, enhances the uptake and transfection of nucleic acids in antigen-presenting cells, increases the expression efficiency of antigen proteins, and thus improves the immune response.
[0007] The objective of this invention is achieved through the following technical solution:
[0008] A lipid-polymer hybrid nanoparticle carrier modified with RGD peptide is characterized by: preparing LPNs from distearylphosphatidylcholine (DSPC), dimyristicoglycerol-polyethylene glycol 2000 (DMC-PEG2000) and polylactic acid-glycolic acid copolymer (PLGA), and modifying the LPNs with RGD peptide to obtain RGD-LPNs nanoparticles after carboxyl activation.
[0009] Furthermore, the LPNs are prepared by dissolving DSPC, DMG-PEG 2000 and PLGA in chloroform, adding RNase-free deionized water under magnetic stirring at 750-850 rpm, reacting for 5-8 min, then sonicating under ice bath and rotary evaporating to obtain LPNs.
[0010] Furthermore, the ratio of DSPC, DMG-PEG 2000, PLGA, chloroform, and RNase-free deionized water is 6.2~6.5mg: 3.1~3.3mg: 0.8~1.2mg: 1mL: 5mL.
[0011] Furthermore, the ultrasound is performed using an ultrasonic disruptor at a power of 100-120 W in an ice bath for 5-6 minutes, and the rotary evaporation is performed by slow rotary evaporation at a water bath temperature of 36-37 ℃ for 25-35 minutes to completely remove residual organic solvents, thereby obtaining LPNs.
[0012] Furthermore, the carboxyl activation involves dispersing LPNs in MES buffer at pH 5.0–6.0 at a concentration of 1–1.2 mg / mL, adding EDC and NHS, stirring at 120–160 rpm for 15–25 min at room temperature, centrifuging at 9000–12000 rpm for 10–15 min, and ultrafiltration to obtain the activated LPNs.
[0013] Furthermore, the mass ratio of LPNs, EDC and NHS is 1:1.2 to 1.4:1.
[0014] Further, the modification involves resuspending the activated LPNs in PBS buffer at pH 7.2-7.4, adding RGD peptide solution, reacting at room temperature for 4-10 h, then centrifuging at 10000-12000 rpm for 8-12 min at 4 °C, ultrafiltration, and washing three times with deionized water to obtain RGD-LPNs.
[0015] Furthermore, the RGD peptide solution is prepared with PBS at a concentration of 0.1~0.2 mg / mL, and the mass ratio of RGD peptide to LPNs in the solution is 1~1.5:0.8~1.
[0016] A method for preparing a lipid-polymer hybrid nanoparticle carrier, characterized by comprising the following steps: (1) Take DSPC, DMG-PEG 2000 and PLGA and dissolve them in chloroform. Then add deionized water without RNase to react. After sonication in an ice bath, rotate and evaporate to obtain LPNs. (2) Disperse LPNs in buffer solution and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for carboxyl activation; (3) The activated LPNs were resuspended in PBS buffer and RGD peptide solution was added to prepare RGD-LPNs nanoparticles.
[0017] Further, in step (1), the reaction involves taking DSPC, DMG-PEG 2000 and PLGA, dissolving them fully in chloroform, adding deionized water without RNase under magnetic stirring at 750~850 rpm, and reacting for 5~8 min.
[0018] Furthermore, the ratio of DSPC, DMG-PEG 2000, PLGA, chloroform, and RNase-free deionized water is 6.2~6.5mg: 3.1~3.3mg: 0.8~1.2mg: 1mL: 5mL.
[0019] Further, the carboxyl activation in step (2) involves dispersing LPNs in MES buffer at pH 5.0-6.0 at a concentration of 1-1.2 mg / mL, adding EDC and NHS, stirring at 120-160 rpm for 15-25 min at room temperature, centrifuging at 9000-12000 rpm for 10-15 min, and ultrafiltration to obtain activated LPNs.
[0020] Furthermore, the mass ratio of LPNs, EDC and NHS is 1:1.2 to 1.4:1.
[0021] Further, the modification in step (3) involves resuspending the activated LPNs in PBS buffer at pH 7.2-7.4, adding RGD peptide solution, reacting at room temperature for 4-10 h, then centrifuging at 10000-12000 rpm for 8-12 min at 4 ℃, ultrafiltration, and washing three times with deionized water to obtain RGD-LPNs.
[0022] Furthermore, the RGD peptide solution is prepared with PBS at a concentration of 0.1~0.2 mg / mL, and the mass ratio of RGD peptide to LPNs in the solution is 1~1.5:0.8~1.
[0023] Application of RGD-LPNs nanoparticles prepared by the above method in the preparation of COVID-19 vaccines loaded with mRNA.
[0024] Furthermore, the application involves reconstituted RGD-LPNs and mRNA separately using Ultra-MEM transfection-specific serum-reduced medium, and then incubating them together for 20-30 min to obtain RGD-mRNA-LPNs.
[0025] Most specifically, a method for preparing lipid-polymer hybrid nanoparticles loaded with mRNA is characterized by comprising the following steps: (1) Preparation of LPNs: DSPC, DMG-PEG 2000 and PLGA were placed in a round-bottom flask and dissolved in chloroform. Under magnetic stirring at 750-850 rpm, deionized water without RNase was added. After reacting for 5-8 min, the suspension was transferred to a 15 mL centrifuge tube and sonicated in an ice bath at 100-120 W for 5-6 min. The residual organic solvent was then completely removed by slow rotary evaporation in a water bath at 36-37 ℃ for 25-35 min to obtain LPNs. The LPNs were then dried in a vacuum drying oven for later use. The ratio of DSPC, DMG-PEG 2000, PLGA, chloroform and deionized water without RNase was 6.2-6.5 mg: 3.1-3.3 mg: 0.8-1.2 mg: 1 mL: 5 mL. (2) Activation of LPNs: Disperse LPNs in MES buffer at pH 5.0-6.0 at a concentration of 1-1.2 mg / mL, add EDC and NHS, stir at 120-160 rpm for 15-25 min at room temperature, then centrifuge at 9000-12000 rpm for 10-15 min, and ultrafilter to obtain activated LPNs. The mass ratio of LPNs, EDC and NHS is 1:1.2-1.4:1. (3) Modification of RGD: The activated LPNs were resuspended in PBS buffer at pH 7.2-7.4, and RGD peptide solution was added. The reaction was carried out at room temperature for 4-10 h. Then, the mixture was centrifuged at 10000-12000 rpm for 8-12 min at 4 °C. After ultrafiltration, the mixture was washed three times with deionized water to obtain RGD-LPNs. The RGD peptide solution was prepared with PBS at a concentration of 0.1-0.2 mg / mL. The mass ratio of RGD peptide to LPNs in the solution was 1-1.5:0.8-1. (4) Preparation of RGD-mRNA-LPNs by incubation: RGD-LPNs and mRNA were reconstituted using Ultra-MEM transfection-specific serum-reduced medium and incubated together for 20-30 min to obtain RGD-mRNA-LPNs.
[0026] The introduction of PLGA was intended to improve the mechanical and structural stability of nanoparticles and address the issues of poor stability of pure lipid systems, their tendency to aggregate and restructure in vivo and in vitro, their unsatisfactory particle size distribution, and their poor loading and encapsulation effects on mRNA, leading to burst release of mRNA during delivery due to poor nanoparticle structural stability. However, we found that this problem persisted even after the introduction of PLGA, resulting in low expression efficiency of antigen proteins and unsatisfactory immunogenicity of mRNA.
[0027] While RGD modification can further enhance the immune response, improper RGD modification can easily lead to off-target effects and ineffectiveness.
[0028] In this invention, lipid-polymer hybrid nanoparticles prepared by post-insertion coupling are activated and then modified with RGD peptides. Through the reaction of RGD with the activated carboxyl groups on the surface of PLGA, the modified RGD peptides are further inserted into the nanoparticles and anchored together with PLGA, forming a stable structure that is less prone to off-target effects. At the same time, through the interaction between RGD peptides and PLGA, a double protective layer is formed with hydrophilic PEG, forming a dense protective barrier that effectively prevents nanoparticle aggregation and sedimentation. Finally, after loading mRNA, the particle size uniformity of the nanoparticles is effectively improved and the distribution range is reduced.
[0029] The present invention has the following technical effects: In this invention, lipid-polymer hybrid nanoparticles (LPNs) are prepared using PLGA, then modified with RGD peptides, and finally loaded with mRNA to prepare RGD-mRNA-LPNs. Through the synergistic effect of RGD peptides and PLGA, the mechanical stability of the nanoparticles is significantly enhanced, improving the effectiveness of the delivery system. Furthermore, precise control of expression sites is achieved by regulating the phase separation of components. RGD peptides activate integrin receptors, increasing nucleic acid uptake and transfection in antigen-presenting cells, improving the expression efficiency of antigen proteins, and thus enhancing the immune effect. Attached Figure Description
[0030] Figure 1 TEM images of lipid-polymer hybrid nanoparticles prepared by different methods: (a): Example 1; (b): Comparative Example 1; (c): Example 2; (d): Comparative Example 2.
[0031] Figure 2 Particle size distribution of lipid-polymer hybrid nanoparticles prepared by different schemes; (a): Example 1; (b): Comparative Example 1; (c): Example 2; (d): Comparative Example 2.
[0032] Figure 3 Zeta potential distribution of lipid-polymer hybrid nanoparticles prepared by different methods.
[0033] Figure 4 Cytotoxicity results of mRNA-LPNs and RGD-mRNA-LPNs prepared in Examples 1 and 2.
[0034] Figure 5 The fluorescence expression of mRNA-LPNs and RGD-mRNA-LPNs prepared in Examples 1 and 2 in different cells.
[0035] Figure 6 Distribution of mRNA-LPNs and RGD-mRNA-LPNs prepared in Examples 1 and 2 in different tissues.
[0036] Figure 7 : Expression of S protein in various tissues after intramuscular injection of mRNA-LPNs and RGD-mRNA-LPNs prepared in Examples 1 and 2 for 28 days. Detailed Implementation
[0037] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.
[0038] The mRNA used in this invention is specifically SARS-CoV2.Spike-mRNA, and the PLGA used in this invention is specifically polylactic acid-glycolic acid copolymer 2000 (PLGA2000). Example 1 Preparation of lipid-polymer hybrid nanoparticles mRNA-LPNs: Accurately weigh 6.4 mg DSPC, 3.2 mg DMG-PEG2000, and 1 mg PLGA into a round-bottom flask, add 1 mL of chloroform and dissolve thoroughly. Under magnetic stirring at 800 rpm, add 5 mL of RNase-free deionized water and react for 5 min. Transfer the suspension to a 15 mL centrifuge tube and sonicate in an ice bath at 110 W for 5 min. Finally, use a rotary evaporator to slowly evaporate at 37 ℃ in a water bath for 30 min to completely remove residual organic solvent, obtaining high-concentration lipid-polymer hybrid nanoparticles (LPNs). Dry them in a vacuum drying oven for later use. LPNs and mRNA were reconstituted separately using Ultra-MEM transfection-specific serum-reduced medium, and then incubated together for 20 min to obtain mRNA-LPNs.
[0039] Example 2 A method for preparing lipid-polymer hybrid nanoparticles includes the following steps: (1) Preparation of LPNs: Accurately weigh 6.4 mg DSPC, 3.2 mg DMG-PEG 2000 and 1 mg PLGA into a round bottom flask, add 1 mL of chloroform to dissolve completely, add 5 mL of RNase-free deionized water under magnetic stirring at 800 rpm, react for 5 min and then transfer the suspension to a 15 mL centrifuge tube, use an ultrasonic homogenizer to sonicate in an ice bath at 110 W power for 5 min, use a rotary evaporator to slowly evaporate in a water bath at 37 ℃ for 30 min to completely remove residual organic solvents, obtain LPNs, put them in a vacuum drying oven for drying and use. (2) Activation of LPNs: Disperse LPNs in MES buffer at pH 5.5, add EDC and NHS, stir at 150 rpm for 20 min at room temperature, centrifuge at 10000 rpm for 12 min, and ultrafilter to obtain activated LPNs. The mass ratio of LPNs, EDC and NHS is 1:135:1. (3) Modification of RGD: The activated LPNs were resuspended in PBS buffer at pH 7.2, and RGD peptide solution was added. The reaction was carried out at room temperature for 6 h, and then centrifuged at 11000 rpm for 10 min at 4 °C. After ultrafiltration, the LPNs were washed three times with deionized water to obtain RGD-LPNs. The RGD peptide solution was prepared with PBS at a concentration of 0.15 mg / mL, and the mass ratio of RGD peptide to LPNs in the solution was 1.2:0.9. (4) Preparation of RGD-mRNA-LPNs by incubation: RGD-LPNs and mRNA were reconstituted separately using Ultra-MEM transfection-specific serum-reduced medium and incubated together for 25 min to obtain RGD-mRNA-LPNs.
[0040] The mRNA-LPNs and RGD-mRNA-LPNs prepared in Examples 1 and 2 can form uniformly dispersed nanoaggregates in aqueous solution.
[0041] Comparative Example 1 The preparation process of Example 1 was followed, except that DMG-PEG 2000 was replaced with DMG-PEG 2000-RGD, and the remaining steps were the same as in Example 1.
[0042] Comparative Example 2 Following the preparation process of Example 2, the RGD peptide was replaced with the CRGD peptide, and the other steps were the same as in Example 1.
[0043] The mRNA-LPNs prepared in Example 1, Example 2, and Comparative Examples 1 and 2 were observed using transmission electron microscopy. The results are as follows: Figure 1(Scale bar = 100 nm) As shown, (a) is the mRNA-LPNs prepared in Example 1, (b) is the RGD-mRNA-LPNs prepared in Comparative Example 1, and (c) and (d) are the RGD-mRNA-LPNs and CRGD-mRNA-LPNs prepared in Example 2 and Comparative Example 2, respectively. It can be seen that the mRNA-LPNs prepared in Example 1, as well as the RGD-mRNA-LPNs prepared in Comparative Example 1 and Example 2, are spherical with relatively rounded surfaces and clear membrane structures, while the CRGD-mRNA-LPNs prepared in Comparative Example 2 have a larger elliptical structure.
[0044] The particle size distributions of mRNA-LPNs and RGD-mRNA-LPNs were measured using a Malvern particle size analyzer, and the results are as follows: Figure 2 As shown. (a) is the mRNA-LPNs prepared in Example 1, with an average particle size of (113.7 ± 2.1) nm. (b) and (c) are the RGD-mRNA-LPNs prepared in Comparative Example 1 and Example 1, respectively, with average particle sizes of (121.3 ± 0.9) nm and (118.4 ± 0.7) nm, respectively. (d) is the CRGD-mRNA-LPNs prepared in Comparative Example 2, with an average particle size of (182.9 ± 0.4) nm.
[0045] The zeta potential changes of unbound mRNA LPNs, mRNA-LPNs prepared in Example 1, RGD-LPNs and RGD-mRNA-LPNs prepared in Comparative Example 1 (corresponding to RGD-LPNs (1) and RGD-mRNA-LPNs (1) in the figure), RGD-LPNs and RGD-mRNA-LPNs prepared in Example 2, and CRGD-LPNs and CRGD-mRNA-LPNs prepared in Comparative Example 2 were measured using a zeta potential analyzer. Figure 3As shown, the Zeta potential of the LPNs prepared in Example 1 is (8.4 ± 1.6) mV, and the Zeta potential of the RGD-LPNs (1) prepared in Comparative Example 1 is (11.8 ± 0.5) mV. After binding with mRNA, due to the negative charge of mRNA, the Zeta potential of mRNA-LNPs and RGD-mRNA-LPNs (1) decreases to -(1.0 ± 0.2) mV and -(5.0 ± 0.6) mV, respectively. In Example 1, due to the reaction between RGD and PLGA, the RGD penetrated deeper into the nanoparticles, resulting in a slight decrease in surface charge compared to Comparative Example 1. However, because the nanoparticles altered the spatial positioning of RGD, they achieved a higher mRNA loading capacity. The Zeta potentials of RGD-LPNs and RGD-mRNA-LPNs were (10.8±0.3) mV and -(7.1±0.6) mV, respectively. In Comparative Example 2, CRGD, being nearly electroneutrally neutral, had little effect on the nanoparticle potential after modification. However, the CRGD modification led to decreased nanoparticle uniformity and increased particle size, negatively impacting subsequent mRNA loading. The potentials of CRGD-LPNs and CRGD-mRNA-LPNs prepared in Comparative Example 2 were (8.7±0.7) mV and (1.9±0.4) mV, respectively. Encapsulation efficiency was determined using the following formula:
[0046] The results showed that the encapsulation rate of mRNA in the RGD-mRNA-LPNs prepared in Example 2 was about 94.1%, while the encapsulation rates of Comparative Examples 1 and 2 were only 89.8% and 84.7%, respectively. The experimental results indicate that the RGD-mRNA-LPNs prepared in Example 1 had a lower leakage rate, higher encapsulation stability, and the mRNA vaccine had superior physical and chemical properties.
[0047] Stability test: The mRNA-LPNs prepared in Example 1, the RGD-mRNA-LPNs prepared in Comparative Example 1 and Example 2 (comparative Example 1 is referred to as RGD-mRNA-LPNs (1)), and the CRGD-mRNA-LPNs prepared in Comparative Example 2 were stored at 4℃ and 37℃ for 28 days, respectively, and the final particle size and PDI changes were tested. The results are shown in Table 1.
[0048] Table 1:
[0049] As shown in the table above, although the average particle size and PDI of mRNA-LPNs without RGD modification were relatively small, both the particle size and PDI increased significantly during long-term storage. After further modification with RGD, although the initial average particle size increased, the structural stability was significantly improved. During long-term storage, the particle size and PDI of the nanoparticles changed less. The RGD modification in Example 1 showed better stability, while the CRDG modification in Comparative Example 2 not only significantly increased the initial average particle size, but also showed more obvious changes in particle size and PDI during long-term storage. The structural stability of the nanoparticles was not improved. The excessively large particle size of the nanoparticles made them difficult to be absorbed by cells, which significantly reduced the delivery efficiency.
[0050] To verify the difference in the effectiveness of nanoparticles before and after RGD modification, we used the mRNA-LPNs and RGD-mRNA-LPNs prepared in Examples 1 and 2 for further effectiveness verification.
[0051] Verification of the in vitro delivery effect of nanoparticles: 293T and HeLa cell lines were digested with trypsin and prepared into single-cell suspensions in antibiotic-free DMEM medium. These suspensions were then seeded into 96-well plates at a cell density of 2.0 × 10⁶ cells / well. 5 Cells / mL, let stand on the workbench for 10 min, then incubate overnight at 37 ℃ until the cell confluence is about 70%-80%, then discard the old culture medium.
[0052] The experiment was divided into two groups: a positive control group (transfection with Lipo2000+mRNA complex (mRNA-Lipo)) and a reagent treatment group. The reagent treatment group was treated with mRNA-LPNs prepared in Example 1 and RGD-mRNA-LPNs prepared in Example 2, respectively. The mRNA-LPNs and RGD-mRNA-LPNs groups used mRNAs carrying different concentrations (0.1, 0.2, 0.4, 0.8, and 1.6 μg / mL). After 24 h of treatment, 10 μL of 5 mg / mL MTT reagent was added to each well, incubated at 37 ℃ for 4 h, and then the solution was discarded. 150 μL of DMSO was added, shaken for 10 min to dissolve and crystallize the cells, and the absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Cell viability was calculated using the formula. Cell viability = [(OD value of experimental wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells)] × 100% The results are as follows Figure 4As shown, the cell viability of each group did not decrease significantly over time, and there was no significant difference compared with the control group. This indicates that both the prepared mRNA-LPNs and RGD-mRNA-LPNs exhibited weak cytotoxicity, and the cell viability was not significantly different from the blank control group. This demonstrates that the lipid-polymer hybrid nanoparticles prepared in this invention have biosafety.
[0053] Western blot analysis of S protein expression in 293T and HeLa cells after transfection with mRNA-LPNs and GRD-mRNA-LPNs: The total cytoplasmic protein content was obtained by quantifying the cell suspension using BCA protein. Subsequently, 30 μg of protein from each group was subjected to SDS-PAGE electrophoresis and membrane transfer. After blocking with 5% skim milk for 2 h, the cells were incubated with primary antibody (1:1000) at 4°C overnight. The next day, the cells were incubated with secondary antibody (1:5000) at room temperature for 2 h. The gray value was analyzed by ECL imaging.
[0054] The results are as follows Figure 5 As shown, clear black bands were observed on the membrane after transfection of mRNA-LPNs and RGD-mRNA-LPNs into HEK 293T cells and HeLa cells. In contrast, no clear bands were observed on the PVDF membrane in the untreated group. These results indicate that the mRNA vaccines mediated by mRNA-LPNs and RGD-mRNA-LPNs were successfully transfected into HEK 293T cells and HeLa cells. RGD-mRNA-LPNs showed higher S protein expression than mRNA-LPNs, and Western blot analysis further confirmed the effectiveness of the RGD-mRNA-LPNs delivery system.
[0055] Distribution of mRNA-LPNs and GRD-mRNA-LPNs in different tissues: Two administration methods were used: intramuscular injection and subcutaneous injection. Each administration method was further divided into mRNA-LPNs and GRD-mRNA-LPNs administration groups, with 10 mice in each group. The mice were immunized for 28 days, and each mouse was injected with 10 μg of mRNA-LPNs or GRD-mRNA-LPNs.
[0056] Mice were euthanized upon expiration. 500 μL of blood was collected from the eyeballs of each mouse and centrifuged at 4℃ and 4000 rpm for 15 minutes. The supernatant was collected and frozen at -80℃. Simultaneously, heart, liver, spleen, lung, kidney, and muscle tissues were dissected and collected. The distribution of mRNA-LPNs and GRD-mRNA-LPNs in different tissues was tested after 28 days. The results are as follows: Figure 6 As shown ( P < 0.05 P < 0.01).
[0057] The experiment was validated at the animal level. LPNs carrying SARS-CoV2.Spike-mRNA were used to detect the expression of the S protein in various sites in mice administered via intramuscular injection on day 28. Results are as follows: Figure 7 As shown, the S protein exhibits high expression levels in serum, muscle, and spleen. Experiments demonstrate that mRNA-LPNs, as a vaccine for COVID-19 translation, have a strong immunogenic effect.
[0058] Example 3 A method for preparing lipid-polymer hybrid nanoparticles includes the following steps: (1) Preparation of LPNs: Accurately weigh DSPC, DMG-PEG 2000 and PLGA and place them in a round bottom flask. Dissolve them completely in chloroform. Under magnetic stirring at 850 rpm, add RNase-free deionized water. After reacting for 6 min, transfer the suspension to a 15 mL centrifuge tube. Use an ultrasonic homogenizer to sonicate in an ice bath at 120 W for 5 min. Use a rotary evaporator to slowly evaporate in a water bath at 36 ℃ for 35 min to completely remove residual organic solvents and obtain LPNs. Place them in a vacuum drying oven for drying and use. The ratio of the amount of DSPC, DMG-PEG 2000, PLGA, chloroform and RNase-free deionized water is 6.2 mg: 3.1 mg: 0.8 mg: 1 mL: 5 mL. (2) Activation of LPNs: Disperse LPNs in MES buffer with pH 5.0~6.0 at a concentration of 1.1 mg / mL, add EDC and NHS, stir at 120 rpm for 25 min at room temperature, then centrifuge at 9000 rpm for 15 min, and ultrafilter to obtain activated LPNs. The mass ratio of LPNs, EDC and NHS is 1:1.2:1. (3) Modification of RGD: The activated LPNs were resuspended in PBS buffer at pH 7.3, and RGD peptide solution was added. The reaction was carried out at room temperature for 10 h, and then centrifuged at 10000 rpm for 12 min at 4 °C. After ultrafiltration, the LPNs were washed three times with deionized water to obtain RGD-LPNs. The RGD peptide solution was prepared with PBS at a concentration of 0.1 mg / mL, and the mass ratio of RGD peptide to LPNs in the solution was 1:0.8. (4) Preparation of RGD-mRNA-LPNs by incubation: RGD-LPNs and mRNA were reconstituted separately using Ultra-MEM transfection-specific serum-reduced medium and incubated together for 30 min to obtain RGD-mRNA-LPNs.
[0059] The average particle size of the RGD-mRNA-LPNs prepared in this embodiment is (120.4±0.7) nm, the Zeta potential is -(7.4±0.4) mV, and the encapsulation efficiency is 93.6%.
[0060] Example 4 A method for preparing lipid-polymer hybrid nanoparticles, characterized by comprising the following steps: (1) Preparation of LPNs: Accurately weigh DSPC, DMG-PEG 2000 and PLGA and place them in a round-bottom flask. Add chloroform and dissolve them completely. Under magnetic stirring at 750 rpm, add RNase-free deionized water. After reacting for 8 min, transfer the suspension to a 15 mL centrifuge tube. Use an ultrasonic homogenizer to sonicate in an ice bath at 100 W for 6 min. Use a rotary evaporator to slowly evaporate in a water bath at 37 ℃ for 25 min to completely remove residual organic solvents and obtain LPNs. Place them in a vacuum drying oven for drying and use. The ratio of the amount of DSPC, DMG-PEG 2000, PLGA, chloroform and RNase-free deionized water is 6.5 mg: 3.3 mg: 1.2 mg: 1 mL: 5 mL. (2) Activation of LPNs: LPNs were dispersed in MES buffer at pH 6.0 at a concentration of 1.2 mg / mL. EDC and NHS were added, and the mixture was stirred at 160 rpm for 15 min at room temperature. Then, it was centrifuged at 12000 rpm for 10 min and ultrafiltered to obtain activated LPNs. The mass ratio of LPNs, EDC and NHS was 1:1.4:1. (3) Modification of RGD: The activated LPNs were resuspended in PBS buffer at pH 7.4, and RGD peptide solution was added. The reaction was carried out at room temperature for 4 h, and then centrifuged at 12000 rpm for 8 min at 4 °C. After ultrafiltration, the LPNs were washed three times with deionized water to obtain RGD-LPNs. The RGD peptide solution was prepared with PBS at a concentration of 0.2 mg / mL, and the mass ratio of RGD peptide to LPNs in the solution was 1.5:1. (4) Preparation of RGD-mRNA-LPNs by incubation: RGD-LPNs and mRNA were reconstituted separately using Ultra-MEM transfection-specific serum-reduced medium and incubated together for 20 min to obtain RGD-mRNA-LPNs.
[0061] The RGD-mRNA-LPNs prepared in this embodiment had an average particle size of (119.8±0.6) nm, a Zeta potential of -(7.2±0.5) mV, and an encapsulation efficiency of 93.2%.
Claims
1. A lipid-polymer hybrid nanoparticle carrier for RGD peptide-modified PLGA, characterized in that: LPNs were prepared using DSPC, DMC-PEG2000 and PLGA as raw materials. After carboxyl activation of LPNs, RGD peptides were modified to obtain RGD-LPNs nanoparticles.
2. The lipid-polymer hybrid nanoparticle carrier of RGD peptide-modified PLGA as described in claim 1, characterized in that: The LPNs were prepared by dissolving DSPC, DMG-PEG 2000 and PLGA in chloroform, adding deionized water without RNase under magnetic stirring at 750-850 rpm, reacting for 5-8 min, and then sonicating under ice bath and rotary evaporating to obtain LPNs.
3. A lipid-polymer hybrid nanoparticle carrier for RGD peptide-modified PLGA as described in claim 1 or 2, characterized in that: The carboxyl activation involves dispersing LPNs in MES buffer at pH 5.0–6.0 at a concentration of 1–1.2 mg / mL, adding EDC and NHS, stirring at 120–160 rpm for 15–25 min at room temperature, centrifuging at 9000–12000 rpm for 10–15 min, and ultrafiltration to obtain the activated LPNs.
4. A lipid-polymer hybrid nanoparticle carrier for RGD peptide-modified PLGA as described in any one of claims 1-3, characterized in that: The modification involves resuspending the activated LPNs in PBS buffer at pH 7.2–7.4, adding RGD peptide solution, reacting at room temperature for 4–10 h, then centrifuging at 10,000–12,000 rpm for 8–12 min at 4 °C, ultrafiltration, and washing three times with deionized water to obtain RGD-LPNs.
5. A method for preparing a lipid-polymer hybrid nanoparticle carrier, characterized in that, Includes the following steps: (1) Take DSPC, DMG-PEG 2000 and PLGA and dissolve them in chloroform. Then add deionized water without RNase to react. After sonication in an ice bath, rotate and evaporate to obtain LPNs. (2) Disperse LPNs in buffer solution and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for carboxyl activation; (3) The activated LPNs were resuspended in PBS buffer and RGD peptide solution was added to prepare RGD-LPNs nanoparticles.
6. The method for preparing a lipid-polymer hybrid nanoparticle carrier as described in claim 5, characterized in that: In step (1), the reaction involves taking DSPC, DMG-PEG 2000 and PLGA, dissolving them fully in chloroform, adding deionized water without RNase under magnetic stirring at 750-850 rpm, and reacting for 5-8 minutes.
7. A method for preparing a lipid-polymer hybrid nanoparticle carrier as described in claim 5 or 6, characterized in that: The carboxyl activation in step (2) involves dispersing LPNs in MES buffer at pH 5.0-6.0 at a concentration of 1-1.2 mg / mL, adding EDC and NHS, stirring at 120-160 rpm for 15-25 min at room temperature, centrifuging at 9000-12000 rpm for 10-15 min, and ultrafiltration to obtain activated LPNs.
8. A method for preparing a lipid-polymer hybrid nanoparticle carrier as described in any one of claims 5-7, characterized in that: The modification in step (3) involves resuspending the activated LPNs in PBS buffer at pH 7.2-7.4, adding RGD peptide solution, reacting at room temperature for 4-10 h, then centrifuging at 10000-12000 rpm for 8-12 min at 4 ℃, ultrafiltration, and washing three times with deionized water to obtain RGD-LPNs.
9. The application of RGD-LPNs nanoparticles prepared by the method according to any one of claims 5-8 in the preparation of COVID-19 vaccines loaded with mRNA.
10. The application as described in claim 9, characterized in that: RGD-LPNs and mRNA were reconstituted using Ultra-MEM transfection-specific serum-reduced medium, and then incubated together for 20-30 min to obtain RGD-mRNA-LPNs.