An exosome / cationic branched poly(beta-amino ester) hybrid carrier, and a preparation method and application thereof

Abstract

The present application relates to the technical field of biomedical materials, and particularly relates to an exosome / cationic branched poly(beta-amino ester) hybrid carrier as well as a preparation method and application thereof. The hybrid carrier of the present application comprises an exosome and a branched poly(beta-amino ester); the concentration of the exosome is 2x10 5 / µL~2x10 6 / µL; and the concentration of the branched poly(beta-amino ester) is 10~15 kg / mol. The hybrid carrier of the present application can safely and efficiently mediate mRNA transfection, and provides an important platform technology for clinical anti-aging, anti-wrinkle and other transformations.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to an exosome / cationically branched poly(β-amino ester) hybrid carrier, its preparation method, and its application. Background Technology

[0002] Gene therapy refers to the delivery of genetic material such as DNA or RNA with specific functions into target tissues or cells using specific methods to regulate functional proteins and achieve the goal of treating diseases. mRNA therapy, in particular, shows great promise for treating diseases such as aging and wrinkles. However, naked RNA is difficult to cross the negatively charged and hydrophobic cell membrane and is easily degraded by nucleases and restriction endonucleases in the blood or cytoplasm. Therefore, gene therapy requires the use of vectors to compress and protect DNA and RNA. The lack of safe and efficient gene delivery vectors severely limits the clinical translation of gene therapy.

[0003] Exosomes (Exos) are nanoscale lipid bilayer vesicles actively secreted by cells. Compared to synthetic carriers such as lipid nanoparticles and liposomes, exosomes are structurally more complex and contain a richer variety of biological components, including lipids, proteins, and nucleic acids. The inherent low immunogenicity, high physicochemical stability, high tissue penetration, and innate transport capabilities of exosomes make them a promising next-generation gene / mRNA drug delivery carrier.

[0004] However, exosomes themselves are negatively charged, and their drug loading capacity, circulating half-life, and targeting efficiency are still inferior to some synthetic vectors. Currently, exosomes have a poor ability to load exogenous functionalized mRNA, and efficiently loading mRNA presents a significant challenge. Electroporation technology creates temporary hydrophilic pores on the exosome membrane by applying a pulsed electric field, thereby promoting mRNA permeation into the exosome. However, high-voltage pulses can damage the exosome membrane, and some exosomes cannot complete membrane repair, resulting in low loading rates. This significantly limits the efficient delivery of functional mRNA and the clinical translational applications of exosomes. Summary of the Invention

[0005] This invention utilizes the self-assembly of exosomes and branched poly(β-amino esters) to form a hybrid vector, enhancing the interaction between exosomes and mRNA, improving mRNA encapsulation efficiency, and thus efficiently mediating mRNA transfection efficiency. This provides an important platform technology for exosome-based clinical gene therapy for anti-aging and anti-wrinkle purposes. Specifically, it relates to an exosome / cationic branched poly(β-amino ester) hybrid vector, its preparation method, and its applications.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a hybrid carrier comprising exosomes and branched poly(β-amino esters). The concentration of the exosomes was 2 × 10⁻⁶. 5 pcs / μL ~2×10 6 pcs / μL; The concentration of the branched poly(β-amino ester) is 10~15 kg / mol.

[0007] The present invention also provides the application of the hybrid vector in the preparation of products loaded with highly efficient mRNA.

[0008] This invention also provides the application of the hybrid vector in the preparation of products with improved mRNA encapsulation efficiency and transfection efficiency.

[0009] The present invention also provides a method for preparing a hybrid support, comprising the following steps: Exosomes were self-assembled with branched poly(β-amino ester) in a buffer solution.

[0010] Preferably, the buffer solution is a sodium acetate solution.

[0011] The present invention also provides a hybrid composite nanoparticle comprising the following components: Exosomes, branched poly(β-amino esters), and mRNA; The concentration of the exosomes was 2 × 10⁻⁶. 5 pcs / μL ~2×10 6 pcs / μL; The mass ratio of branched poly(β-amino ester) to mRNA is 28~32:1.

[0012] Preferably, the particle size of the hybrid composite nanoparticles is 140~300nm.

[0013] This invention also provides a method for preparing hybrid composite nanoparticles, comprising the following steps: The exosomes, branched poly(β-amino esters), and mRNA were mixed to obtain the desired result.

[0014] The present invention also provides the application of the hybrid composite nanoparticles described above or the hybrid composite nanoparticles prepared by the described preparation method in the preparation of anti-aging products.

[0015] The present invention also provides the application of the hybrid composite nanoparticles described herein or the hybrid composite nanoparticles prepared by the described preparation method in the preparation of anti-wrinkle products.

[0016] Beneficial effects

[0017] This invention provides an exosome / cationically branched poly(β-amino ester) hybrid carrier, its preparation method, and its applications. The invention utilizes exosomes, which have high biocompatibility and deep tissue penetration capabilities, as a substrate, and assembles them with cationic branched poly(β-amino ester) to form a hybrid carrier. This enhances the interaction between exosomes and mRNA, improving mRNA encapsulation efficiency. In vitro transfection results from various tissues and cells show that the exosome-cationically branched poly(β-amino ester) hybrid carrier can safely and efficiently mediate mRNA transfection. Its safety and transfection efficiency significantly optimize commercial mRNA transfection reagents, further confirming the accuracy and broad applicability of this invention. The branched polymer monomers used in this invention are widely available, simple to synthesize, inexpensive, and exhibit excellent transfection performance. The hybrid carrier mRNA delivery scheme of this invention provides an important platform technology for clinical anti-aging and anti-wrinkle applications. Attached Figure Description

[0018] Figure 1 This is a gel permeation chromatography (GPC) curve of cationic branched poly(β-amino ester).

[0019] Figure 2 Cationic branched poly(β-amino ester) 1 H NMR spectrum.

[0020] Figure 3 Example 2 illustrates the affinity of the exosome-cationic poly(β-amino ester) hybrid vector for mRNA.

[0021] Figure 4 The particle size of the hybrid complex nanoparticles formed by compressing mRNA with exosome-cationic poly(β-amino ester) hybrid carrier in Example 2 is shown.

[0022] Figure 5 The zeta potential of the hybrid complex nanoparticles formed by compressing mRNA with exosome-cationic poly(β-amino ester) hybrid carrier in Example 2.

[0023] Figure 6 The affinity of the hybrid complex nanoparticles prepared in Example 3 for mRNA is shown.

[0024] Figure 7 The particle size of the hybrid composite nanoparticles prepared in Example 3 is shown.

[0025] Figure 8 The zeta potential is shown for the hybrid composite nanoparticles prepared in Example 3.

[0026] Figure 9 For the qualitative evaluation of the GFP mRNA transfection performance mediated by the hybrid vector of Example 2 in A549 cells (fluorescence results).

[0027] Figure 10 For qualitative evaluation of the GFP mRNA transfection performance mediated by the hybrid vector of Example 2 in A549 cells (flow cytometry histogram).

[0028] Figure 11 To quantitatively evaluate the transfection performance of the hybrid vector mediated by Example 2 for GFP mRNA in A549 cells, the percentage of positive cells after transfection was measured (flow cytometry results).

[0029] Figure 12 To quantitatively evaluate the transfection performance of the hybrid vector mediated by Example 2 in A549 cells, the fluorescence intensity of the transfected cells was measured (flow cytometry results).

[0030] Figure 13 To evaluate cell viability in A549 cells after transfection with the hybrid vector mediated by Example 2 for GFP mRNA.

[0031] Figure 14 For qualitative evaluation of the GFP mRNA transfection performance mediated by the hybrid vector of Example 2 in L929 cells (fluorescence results).

[0032] Figure 15 For evaluating the transfection performance of the hybrid vector mediated by Example 2 in L929 cells (flow cytometry histogram).

[0033] Figure 16 To quantitatively evaluate the transfection performance of the hybrid vector mediated by Example 2 in L929 cells, the percentage of positive cells after transfection was measured (flow cytometry results).

[0034] Figure 17 To quantitatively evaluate the transfection performance of the hybrid vector mediated by Example 2 in L929 cells, the fluorescence intensity of the transfected cells was measured (flow cytometry results).

[0035] Figure 18 To evaluate cell viability in L929 cells after transfection with the hybrid vector mediated by Example 2 for GFP mRNA.

[0036] Figure 19 Qualitative evaluation of the transfection performance (fluorescence results) of the hybrid complex nanoparticles of Example 3 in A549 cells.

[0037] Figure 20 Qualitative evaluation of the transfection performance of the hybrid complex nanoparticles of Example 3 in A549 cells (flow cytometry histogram).

[0038] Figure 21To quantitatively evaluate the transfection performance of the hybrid complex nanoparticles of Example 3 in A549 cells, the positive cell rate after transfection was measured (flow cytometry results).

[0039] Figure 22 To quantitatively evaluate the transfection performance of the hybrid complex nanoparticles of Example 3 in A549 cells, the fluorescence intensity of the transfected cells was measured (flow cytometry results).

[0040] Figure 23 Qualitative evaluation of the transfection performance (fluorescence results) of the hybrid complex nanoparticles of Example 3 in L929 cells.

[0041] Figure 24 Qualitative evaluation of the transfection performance of the hybrid complex nanoparticles of Example 3 in L929 cells (flow cytometry histogram).

[0042] Figure 25 To quantitatively evaluate the transfection performance of the hybrid complex nanoparticles of Example 3 in L929 cells, the positive cell rate after transfection was measured (flow cytometry results).

[0043] Figure 26 To quantitatively evaluate the transfection performance of the hybrid complex nanoparticles of Example 3 in L929 cells, the fluorescence intensity of the transfected cells was measured (flow cytometry results). Detailed Implementation

[0044] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] The exosomes derived from induced pluripotent stem cells (IPSCs) and exosomes derived from umbilical cords in the embodiments of this invention are both derived from Shenzhen Danlun Gene Technology Co., Ltd.

[0046] Example 1

[0047] Preparation of cationic branched poly(β-amino ester)

[0048] 1,4-Butanediol diacrylate (12 mmol, 2.37 g) and 5-amino-1-pentanol (10 mmol, 1.03 g) were dissolved in dimethyl sulfoxide (DMSO) (2 mL) and reacted at 90 °C for 10 h. After the system reached room temperature, DMSO was added to terminate the reaction, and a linear poly(β-amino ester) with a double bond at the end was synthesized.

[0049] Then, linear poly(β-amino ester) with terminal double bonds and propylenediamine were dissolved in the organic solvent DMSO, wherein the concentration of linear poly(β-amino ester) with terminal double bonds was 500 mg / mL. Polymerization was carried out at 60 °C under magnetic stirring, followed by the addition of excess N-(3-aminopropyl)morpholine for end-capping. Finally, precipitation was performed in excess diethyl ether, followed by freeze-drying to obtain cationic branched poly(β-amino ester). The molecular weight of the polymer was determined using GPC, and the measured values ​​were recorded. 1 H NMR spectra, results as follows Figures 1-2 As shown.

[0050] Figures 1-2 The final molecular weight of the cationic branched poly(β-amino ester) was 13.2 kg / mol. The cationic branched poly(β-amino ester) of this invention was successfully synthesized.

[0051] The branched poly(β-amino ester) used in the following experiments is the cationic branched poly(β-amino ester) prepared in this example.

[0052] Example 2

[0053] Hybrid carriers of exosomes / cationically branched poly(β-amino esters) and hybrid complex nanoparticles

[0054] Cationic branched poly(β-amino ester) (molecular weight 13.2 kg / mol) and exosomes derived from IPSC were electrostatically recombine in sodium acetate solution to form an exosome / cationic branched poly(β-amino ester) hybrid carrier, wherein the amount of exosomes used was 2 × 10⁻⁶. 5 cells / μL, 4×10 5 cells / μL, 1×10 6 cells / μL, 2×10 6 The hybrid vector was then mixed with EGFP mRNA at a mass ratio of 30:1 (when the amount of EGFP mRNA was 100 ng, the exosome / cationically branched poly(β-amino ester) hybrid vector was rapidly added to the GFP mRNA). The mixture was vortexed for 30 s and then allowed to stand for 10 min to form hybrid complex nanoparticles. The hybrid complex nanoparticles were then diluted to 100 μL with serum-free medium, and 100 μL of RiboGreen working solution was added. The excitation wavelength was 480 nm, and the emission wavelength was 520 nm. The particle size and surface zeta potential of the hybrid complex nanoparticles were measured using dynamic light scattering (DLS).

[0055] Figure 3The results of the affinity test of the exosome / cationically branched poly(β-amino ester) hybrid vector for EGFP mRNA show that at a mass ratio of 30:1, the exosome / cationically branched poly(β-amino ester) hybrid vector exhibits a high affinity efficiency for EGFP mRNA, exceeding 70%. This confirms that the prepared exosome / cationically branched poly(β-amino ester) hybrid vector has a high interaction with EGFP mRNA, thus providing better protection efficiency for EGFP mRNA.

[0056] Figure 4 Particle size was measured for the hybrid complex nanoparticles of exosomes / cationically branched poly(β-amino ester) hybrid carrier and EGFP mRNA. DLS results confirmed that the particle size was within acceptable limits when exosome dosage was 2 × 10⁻⁶. 5 cells / μL, 4×10 5 cells / μL, 1×10 6 cells / μL, 2×10 6 At a density of 1 / μL, the particle size range of the hybrid complex nanoparticles prepared by exosome / cationically branched poly(β-amino ester) hybrid carrier and mRNA ranges from 140 nm to 300 nm, which is significantly larger than the particle size of exosomes (100 nm). This demonstrates that the exosome / cationically branched poly(β-amino ester) hybrid carrier successfully encapsulates EGFP mRNA, and the resulting complex nanoparticles are easily taken up by cells.

[0057] Figure 5 Zeta potential was measured for the hybrid complex nanoparticles of exosomes / cationically branched poly(β-amino ester) hybrid carriers and EGFP mRNA. The results confirmed that the zeta potential was achieved with exosome amounts of 2 × 10⁻⁶. 5 cells / μL, 4×10 5 cells / μL, 1×10 6 cells / μL, 2×10 6 At a concentration of 1 / μL, the exosome / cationically branched poly(β-amino ester) hybrid carrier can compress the hybrid complex nanoparticles formed by mRNA and effectively shield the negative potential of mRNA and exosomes themselves. Furthermore, the potential on the surface of the hybrid complex nanoparticles is greater than +8mV, which further promotes the interaction between the hybrid complex nanoparticles and the cell membrane and cell uptake, providing the necessary conditions for subsequent mRNA transfection.

[0058] Example 3

[0059] Hybrid composite nanoparticles

[0060] Cationic branched poly(β-amino ester) (molecular weight 13.2 kg / mol) was vortexed with EGFP mRNA for 30 s, then allowed to stand for 5 min to form a complex nanoparticle solution. The mass ratio of cationic branched poly(β-amino ester) to EGFP mRNA was 30:1, and the amount of EGFP mRNA was 100 ng. The complex nanoparticle solution was then electrostatically recombinated with umbilical cord-derived exosomes and incubated for 5 min to obtain hybrid complex nanoparticles. The amount of exosomes used was 2 × 10⁻⁶. 5 cells / μL, 4×10 5 cells / μL, 1×10 6 cells / μL, 2×10 6 The number of nanoparticles per μL was determined. The hybrid composite nanoparticles were diluted to 100 μL with serum-free medium, and then 100 μL of RiboGreen working solution was added. The excitation fluorescence intensity was detected at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The particle size and surface zeta potential of the composite nanoparticles were measured using DLS.

[0061] Figure 6 To test the affinity performance of the hybrid complex nanoparticles for mRNA, the results showed that when the mass ratio of cationic branched poly(β-amino ester) to EGFP mRNA was 30:1, the hybrid complex nanoparticles exhibited a high mRNA affinity efficiency of 62%, confirming that the prepared hybrid complex nanoparticles have a high interaction with mRNA and thus have a better protective efficiency for mRNA.

[0062] Figure 7 The particle size of the hybrid composite nanoparticles was measured. DLS results confirmed that the exosome dosage was 2×10⁻⁶. 5 cells / μL, 4×10 5 cells / μL, 1×10 6 cells / μL, 2×10 6 When the particle size of the hybrid complex nanoparticles is less than 200 nm, the particle size is significantly larger than that of exosomes (100 nm), which proves that the prepared hybrid complex nanoparticles are easily taken up by cells.

[0063] Figure 8 The surface potential of the hybrid composite nanoparticles was measured. DLS results confirmed that the surface potential of the exosomes was 2 × 10⁻⁶. 5 cells / μL, 4×10 5 cells / μL, 1×10 6 cells / μL, 2×10 6At a concentration of 1 / μL, the hybrid complex nanoparticles can effectively shield the negative potential of mRNA and exosomes themselves, and the surface potential of the hybrid complex nanoparticles exceeds +9 mV, which further promotes the interaction between the hybrid complex nanoparticles and the cell membrane and cellular uptake, providing the necessary conditions for subsequent gene transfection.

[0064] Example 4

[0065] A549 cells and L929 cells were respectively fed with 2.0 × 10⁻⁶ cells. 4 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured overnight at 37°C. The exosome / cationically branched poly(β-amino ester) hybrid vector prepared in Example 2 was compounded with EGFP mRNA to form hybrid nanoparticles, and transfection efficiency was measured during transfection. The mass ratio of cationicly branched poly(β-amino ester) to EGFP mRNA was 30:1. After standing for 15 min, the mixture was added to serum-free medium, thoroughly mixed, and then slowly added to the cells. The medium was replaced after 4 h, and the cells were cultured for another 44 h. Finally, the transfection efficiency and cell viability were evaluated.

[0066] Figure 9 To qualitatively evaluate (fluorescence results) the transfection performance of EGFP mRNA mediated by the exosome / cationically branched poly(β-amino ester) hybrid vector in Example 2 in A549 cells, and to confirm its excellent mRNA transfection efficiency.

[0067] Figure 10 For qualitative evaluation (flow cytometry histogram) of the EGFP mRNA transfection performance mediated by the exosome / cationically branched poly(β-amino ester) hybrid vector in Example 2 in A549 cells, a significant rightward shift in the histogram confirms its excellent mRNA transfection efficiency.

[0068] Figure 11 To quantitatively evaluate the transfection performance of EGFP mRNA mediated by the exosome / cationically branched poly(β-amino ester) hybrid vector in Example 2 in A549 cells. The positive cell rate after transfection (flow cytometry results) showed a transfection efficiency exceeding 80%, confirming its excellent mRNA transfection efficiency.

[0069] Figure 12 To quantitatively evaluate the transfection performance of EGFP mRNA mediated by the exosome / cationically branched poly(β-amino ester) hybrid vector in Example 2 in A549 cells. Flow cytometry results showed that the fluorescence intensity of the transfected cells was significantly higher than that of the commercial transfection reagent jetPEI, confirming its excellent mRNA transfection efficiency.

[0070] Figure 13To evaluate cell viability in A549 cells after transfection with EGFP mRNA mediated by the exosome / cationically branched poly(β-amino ester) hybrid vector in Example 2, CCK8 results showed that cells maintained high viability (greater than 60%) after transfection, confirming its excellent biosafety.

[0071] Figure 14 To qualitatively evaluate (fluorescence results) the transfection performance of EGFP mRNA mediated by the exosome / cationically branched poly(β-amino ester) hybrid vector in Example 2 in L929 cells, and to confirm its excellent mRNA transfection efficiency.

[0072] Figure 15 To evaluate the transfection performance of the exosome / cationically branched poly(β-amino ester) hybrid vector mediated by Example 2 in L929 cells, a significant rightward shift in the histogram of positive cells after transfection confirmed its excellent mRNA transfection efficiency.

[0073] Figure 16 To quantitatively evaluate the transfection performance of EGFP mRNA mediated by the exosome / cationically branched poly(β-amino ester) hybrid vector in L929 cells, the positive cell rate after transfection (flow cytometry results) confirmed its excellent mRNA transfection efficiency, which reached 79%.

[0074] Figure 17 To quantitatively evaluate the transfection performance of EGFP mRNA mediated by the exosome / cationically branched poly(β-amino ester) hybrid vector in Example 2 in L929 cells. Flow cytometry results showed that the fluorescence intensity of transfected cells was significantly better than that of the exosome group alone, confirming that the exosome / cationically branched poly(β-amino ester) hybrid vector efficiently mediates mRNA transfection.

[0075] Figure 18 To evaluate cell viability in L929 cells after transfection with EGFP mRNA mediated by the exosome / cationically branched poly(β-amino ester) hybrid vector in Example 2, CCK8 results showed that the cell viability after transfection with the exosome / cationically branched poly(β-amino ester) hybrid vector exceeded 81%, reaching a maximum of 100%. This was mainly due to the excellent biodegradability and biocompatibility of the exosome / cationically branched poly(β-amino ester) hybrid vector, further confirming the broad commercial application potential of the exosome / cationically branched poly(β-amino ester) hybrid vector prepared in this invention.

[0076] Example 5

[0077] A549 cells and L929 cells were respectively fed with 2.0 × 10⁻⁶ cells. 4Cells were seeded at a density of 100 cells / well in 96-well plates and cultured overnight at 37°C. The transfection performance of the hybrid complex nanoparticles prepared in Example 3 was evaluated. The mass ratio of cationic branched poly(β-amino ester) to EGFP mRNA was 30:1. The hybrid complex nanoparticles were allowed to stand for 15 min, then added to serum-free culture medium, thoroughly mixed, and slowly added to the cells. The culture medium was replaced after 4 h, and the cells were cultured for another 44 h. Finally, the mRNA transfection performance was evaluated.

[0078] Figure 19 To qualitatively evaluate the transfection performance of the exosome / cationically branched poly(β-amino ester) / mRNA hybrid nanoparticles in Example 3 in A549 cells (fluorescence results), their excellent mRNA transfection efficiency was confirmed.

[0079] Figure 20 To qualitatively evaluate the transfection performance of the exosome hybrid / cationically branched poly(β-amino ester) / mRNA hybrid nanoparticles in Example 3 in A549 cells, a significant rightward shift in the histogram of positive cells after transfection confirmed its excellent mRNA transfection efficiency.

[0080] Figure 21 To quantitatively evaluate the transfection performance of the exosome / cationically branched poly(β-amino ester) / mRNA hybrid nanoparticles in Example 3 in A549 cells, flow cytometry results showed a positive cell rate of 40% after transfection, confirming its excellent mRNA transfection efficiency.

[0081] Figure 22 To quantitatively evaluate the transfection performance of the exosome hybrid / cationically branched poly(β-amino ester) / mRNA hybrid nanoparticles in Example 3 in A549 cells. Flow cytometry results showed that the fluorescence intensity of transfected cells was significantly better than that of the exosome group alone, confirming that the exosome hybrid nanoparticles efficiently mediated superior mRNA transfection.

[0082] Figure 23 To qualitatively evaluate the transfection performance of the exosome / cationically branched poly(β-amino ester) / mRNA hybrid nanoparticles in Example 3 in L929 cells (fluorescence results), their excellent mRNA transfection efficiency was confirmed.

[0083] Figure 24 To qualitatively evaluate the transfection performance of the exosome hybrid / cationically branched poly(β-amino ester) / mRNA hybrid nanoparticles in Example 3 in L929 cells, a significant rightward shift in the histogram of positive cells after transfection confirmed its excellent mRNA transfection efficiency.

[0084] Figure 25To quantitatively evaluate the transfection performance of the exosome / cationically branched poly(β-amino ester) / mRNA hybrid nanoparticles in Example 3 in L929 cells, flow cytometry results showed a positive cell rate of 58% after transfection, confirming its excellent mRNA transfection efficiency.

[0085] Figure 26 To quantitatively evaluate the transfection performance of the exosome / cationically branched poly(β-amino ester) / mRNA hybrid nanoparticles in L929 cells in Example 3, flow cytometry results showed that the fluorescence intensity of transfected cells was significantly better than that of the exosome group alone, confirming that the exosome hybrid nanoparticles efficiently mediate superior mRNA transfection. These results further confirm the broad commercial application potential of the exosome / cationically branched poly(β-amino ester) / mRNA hybrid nanoparticles prepared in this invention.

[0086] In summary, this invention provides an exosome / cationically branched poly(β-amino ester) hybrid carrier, its preparation method, and its applications. This invention utilizes exosomes, which possess high biocompatibility and deep tissue penetration capabilities, as a substrate, and assembles them with cationic branched poly(β-amino ester) to form a hybrid carrier. This enhances the interaction between exosomes and mRNA, improving mRNA encapsulation efficiency. In vitro transfection results from various tissues and cells demonstrate that the exosome-cationically branched poly(β-amino ester) hybrid carrier can safely and efficiently mediate mRNA transfection. The branched polymer monomers used in this invention are widely available, simple to synthesize, inexpensive, and exhibit excellent transfection performance. This invention's hybrid carrier mRNA delivery scheme provides an important platform technology for clinical anti-aging and anti-wrinkle applications.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hybrid carrier, characterized in that, The hybrid carrier includes exosomes and branched poly(β-amino esters). The concentration of the exosomes was 2 × 10⁻⁶. 5 pcs / μL ~2×10 6 pcs / μL; The concentration of the branched poly(β-amino ester) is 10~15 kg / mol.

2. The use of the hybrid vector according to claim 1 in the preparation of products loaded with highly efficient mRNA.

3. The use of the hybrid vector according to claim 1 in the preparation of products with improved mRNA encapsulation efficiency and transfection efficiency.

4. A method for preparing a hybrid support, characterized in that, Includes the following steps: Exosomes were self-assembled with branched poly(β-amino ester) in a buffer solution.

5. The preparation method according to claim 4, characterized in that, The buffer solution is a sodium acetate solution.

6. A hybrid composite nanoparticle, characterized in that, It includes the following components: Exosomes, branched poly(β-amino esters), and mRNA; The concentration of the exosomes was 2 × 10⁻⁶. 5 pcs / μL ~2×10 6 pcs / μL; The mass ratio of branched poly(β-amino ester) to mRNA is 28~32:

1.

7. The hybrid composite nanoparticles according to claim 6, characterized in that, The hybrid composite nanoparticles have a particle size of 140~300 nm.

8. A method for preparing hybrid composite nanoparticles, characterized in that, Includes the following steps: The exosomes, branched poly(β-amino esters), and mRNA were mixed to obtain the desired result.

9. The application of the hybrid composite nanoparticles according to any one of claims 6 to 7 or the hybrid composite nanoparticles prepared by the preparation method according to claim 8 in the preparation of anti-aging products.

10. The use of the hybrid composite nanoparticles according to any one of claims 6 to 7 or the hybrid composite nanoparticles prepared by the preparation method according to claim 8 in the preparation of anti-wrinkle products.

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