Exosome-like nanovesicles for gene delivery and preparation method thereof

Abstract

The present invention relates to an exosome-like nanovesicle for gene delivery and a method for preparing the same. The exosome-like nanovesicle comprises a lipid phase and an inner aqueous phase encapsulated within the lipid phase. The raw materials for preparing the lipid phase include cholesterol, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and sphingomyelin; the inner aqueous phase contains a nucleic acid drug. The present invention arranges the raw material components of the lipid phase, avoiding the use of cationic lipids and ionizable lipids, thereby preparing anionic lipid vesicles that mimic the lipid components of exosomes. The lipid phase encapsulates the inner aqueous phase containing the nucleic acid drug, and the exosome-like nanovesicles have the characteristics of good stability, low toxicity, and high transfection efficiency. Not only do they have the advantages of natural exosomes with limited immunogenicity, high delivery efficiency, and enhanced stability during in vivo circulation, but the preparation process and final quality are controllable, making commercial production and clinical translation more easily achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to an exosome-like nanovesicle for gene delivery and a preparation method thereof. Background Art

[0002] In the field of gene delivery research, to avoid risks such as immune responses to viral vectors and inserted gene mutations, a growing number of studies are using non-viral vectors for gene delivery. Currently, lipid nanoparticles (LNPs) are a prominent example of these vectors, and mRNA vaccine products have been approved for marketing. Artificial exosomes, currently used to replace natural exosomes, offer advantages over endogenous exosomes, such as limited immunogenicity, high delivery efficiency, and enhanced stability during in vivo circulation. Furthermore, compared to natural exosomes, artificial exosomes offer controllable preparation and final quality, resulting in a "clean" product that is more readily commercially viable and clinically applicable. However, previous studies have often incorporated CLs or ILs into the prepared artificial exosomes, or used electroporation to load nucleic acids, which can result in relatively high cell lethality and consume significant amounts of DNA and cells.

[0003] For example, Chinese patent publication number CN115120572B provides a genetically engineered cell membrane-coated liposome nanovesicle, Adar1-LNPs@mPD1, composed of a biological cell membrane and a lipid mixture, with a particle size of 50 to 200 nm. The biological cell membrane surface is transfected with programmed death receptor 1 (PD1), and the lipid mixture is loaded with Adar1-si RNA (siAdar1). Adar1-si RNA is two reverse-complementary DNA strands, including a sense strand as shown in SEQ ID No. 4 and an antisense strand as shown in SEQ ID No. 5. The above-mentioned vesicles use LNPs as the lipid raw material, while the main components of commercial LNPs are still cationic lipids (CLs) and ionizable lipids (ILs). These positively charged lipids have problems with cytotoxicity, adverse reactions in the body, and poor in vitro stability.

[0004] Therefore, the existing technology has a large room for improvement. Summary of the Invention

[0005] The purpose of the present invention is to make up for the deficiencies of the prior art and to propose an exosome-like nanovesicle for gene delivery and a preparation method thereof, wherein the exosome-like nanovesicle has good stability, low toxicity and high transfection efficiency.

[0006] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0007] An exosome-like nanovesicle for gene delivery, comprising a lipid phase and an inner aqueous phase encapsulated within the lipid phase;

[0008] The raw materials for preparing the lipid phase include cholesterol, phosphatidylethanolamine (PE), phosphatidylserine, (PS) phosphatidylcholine (PC) and sphingomyelin (SM); the inner water phase includes nucleic acid drugs.

[0009] The present invention arranges the raw material components of the lipid phase, avoiding the use of cationic lipids and ionizable lipids, thereby preparing anionic lipid vesicles that simulate the lipid components of exosomes. The lipid phase encapsulates the inner aqueous phase containing nucleic acid drugs, and has the characteristics of good stability, low toxicity and high transfection efficiency. It not only has the advantages of limited immunogenicity of natural exosomes, high delivery efficiency, and enhanced stability during in vivo circulation, but also the preparation process and final quality are controllable, making it easier to achieve commercial production and clinical transformation.

[0010] According to the above scheme, the weight ratio of the cholesterol to the weight of the lipid phase is 5-40%, preferably 5-10%.

[0011] According to the above scheme, the weight ratio of the phosphatidylethanolamine to the weight of the lipid phase is 30-50%, preferably 40-45%.

[0012] According to the above scheme, the weight ratio of the phosphatidylserine to the weight of the lipid phase is 10-20%, preferably 14-16%.

[0013] According to the above scheme, the weight ratio of the phosphatidylcholine to the weight of the lipid phase is 10-20%, preferably 14-17%.

[0014] According to the above scheme, the weight ratio of the sphingomyelin to the weight of the lipid phase is 5-16%, preferably 8-12%.

[0015] According to the above scheme, the raw materials for preparing the nucleic acid drug include plasmid DNA (pDNA) and histone; the mass ratio of the plasmid DNA to histone is 1:2-1:7.

[0016] According to the above scheme, the particle size of the exosome-like nanovesicles is 20-500 nm.

[0017] The present invention provides a method for preparing exosome-like nanovesicles for gene delivery, comprising the following steps:

[0018] (1) Cholesterol, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and sphingomyelin are dissolved in an organic solvent to obtain an organic phase;

[0019] (2) Plasmid DNA and histones are added to pure water and incubated to obtain the inner aqueous phase;

[0020] (3) mixing the organic phase and the inner aqueous phase and homogenizing them at high speed to obtain colostrum;

[0021] (4) adding the colostrum obtained in step (3) to the external aqueous phase and homogenizing at high speed to obtain a double emulsion;

[0022] (5) Nitrogen gas is introduced into the emulsion to remove the organic solvent and obtain exosome-like nanovesicles for gene delivery.

[0023] The double emulsion method is used to allow the organic phase to encapsulate the inner aqueous phase, and then the organic solvent is removed to obtain exosome-like nanovesicles. The preparation method is simple and efficient.

[0024] In step (2), incubate at 37°C for 1 h.

[0025] According to the above scheme, the organic solvent is dichloromethane or chloroform.

[0026] According to the above protocol, the total concentration of lipid components in the organic phase is 20-80% W / V.

[0027] According to the above scheme, in step (3), the organic phase and the inner aqueous phase are mixed at a volume ratio of 2.5:1.

[0028] According to the above scheme, in step (4), the colostrum is injected into the external aqueous phase at a volume ratio of 1:4.

[0029] Those skilled in the art can select 3-4 wt % glucose solution as the external aqueous phase according to actual needs.

[0030] The beneficial effects of the present invention are:

[0031] The present invention arranges the raw material components of the lipid phase, avoiding the use of cationic lipids and ionizable lipids, thereby preparing anionic lipid vesicles that simulate the lipid components of exosomes. The lipid phase encapsulates the inner aqueous phase containing nucleic acid drugs, and has the characteristics of good stability, low toxicity and high transfection efficiency. It not only has the advantages of limited immunogenicity of natural exosomes, high delivery efficiency, and enhanced stability during in vivo circulation, but also the preparation process and final quality are controllable, making it easier to achieve commercial production and clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a transmission electron microscopy photograph of His-pDNA@EMNs obtained in Example 1;

[0033] Figure 2 This is a diagram showing the particle size of His-pDNA@EMNs obtained in Example 1;

[0034] Figure 3 Zeta potential diagram of His-pDNA@EMNs obtained in Example 1;

[0035] Figure 4 The in vitro cumulative release results of His-pDNA@EMNs obtained in Example 1;

[0036] Figure 5 The stability results of His-pDNA@EMNs obtained in Example 1;

[0037] Figure 6 The serum-induced stability results of His-pDNA@EMNs obtained in Example 1;

[0038] Figure 7 The cytotoxicity results of His-pDNA@EMNs obtained in Example 1;

[0039] Figure 8 The effect of His-pDNA@EMNs obtained in Example 1 on cell viability was qualitatively observed;

[0040] Figure 9 The effect of His-pDNA@EMNs obtained in Example 1 on the cell viability was quantitatively determined;

[0041] Figure 10 The His-pDNA@EMNs cell uptake results obtained in Example 1;

[0042] Figure 11 The His-pDNA@EMNs cell transfection results obtained in Example 1;

[0043] Figure 12 The expression results of PEDF protein in His-pDNA@EMNs cells obtained in Example 1;

[0044] Figure 13 The mouse tissue distribution results of His-pDNA@EMNs obtained in Example 1 were labeled with Cy5.5;

[0045] Figure 14 The pulmonary vascular distribution results of His-pDNA@EMNs mice obtained in Example 1 were labeled with Cy5.5;

[0046] Figure 15 The results of PEDF protein expression in His-pDNA@EMNs mouse tissue obtained in Example 1;

[0047] Figure 16 This is the preliminary safety evaluation result of His-pDNA@EMNs mouse tissue obtained in Example 1.

[0048] In the figure, EMNs are exosome-like nanovesicles in internal water that do not contain PEDF plasmids and histones. DETAILED DESCRIPTION

[0049] In order to better understand the present invention, the content of the present invention is further explained below with reference to the accompanying drawings and embodiments, but the present invention is not limited to the following embodiments.

[0050] Example 1

[0051] An exosome-like nanovesicle for gene delivery comprises a lipid phase and an inner aqueous phase encapsulated within the lipid phase; the lipid phase is prepared from raw materials such as cholesterol, phosphatidylethanolamine (PE), phosphatidylserine, (PS) phosphatidylcholine (PC), and sphingomyelin (SM); the inner aqueous phase comprises a nucleic acid drug; the nucleic acid drug comprises plasmid DNA (pDNA) containing the gene PEDF and histones.

[0052] The method for preparing exosome-like nanovesicles for gene delivery comprises the following steps:

[0053] (1) Dissolve 0.5 mg of cholesterol, 3.0 mg of phosphatidylethanolamine (PE), 0.5 mg of phosphatidylserine (PS), 1.1 mg of phosphatidylcholine (PC), and 0.9 mg of sphingomyelin (SM) in 1 mL of dichloromethane to obtain an organic phase;

[0054] (2) pDNA (15 μg) containing the PEDF gene and histones (45 μg) were added to 0.4 mL of pure water and incubated at 37°C for 1 h to obtain the inner aqueous phase;

[0055] (3) mixing the organic phase and the inner aqueous phase, and shearing them using a handheld high-speed homogenizer at 6000 rpm for 5 minutes to obtain colostrum; wherein the organic phase and the inner aqueous phase are mixed at a volume ratio of 2.5:1;

[0056] (4) adding the colostrum obtained in step (3) to 5 mL of a 4 wt% glucose solution, and shearing the mixture at 5000 rpm for 3 min using a handheld high-speed homogenizer to obtain a double emulsion; wherein the colostrum and the glucose solution are mixed at a volume ratio of 1:4;

[0057] (5) Nitrogen gas was introduced into the emulsion at 200 mL / s to remove dichloromethane, and exosome-like nanovesicles (abbreviated as His-pDNA@EMNs) for gene delivery were obtained.

[0058] The morphology, particle size distribution and potential of the prepared exosome-like nanovesicles were measured, and the results are shown in Figure 1-3 The test results showed that the particle size of the exosome-like nanovesicles was 102±2.4nm, the Zeta potential was -20±1.6mV, and they had a typical exosome cup-shaped structure.

[0059] Example 2

[0060] An exosome-like nanovesicle for gene delivery comprises a lipid phase and an inner aqueous phase encapsulated within the lipid phase; the raw materials for preparing the lipid phase include cholesterol, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and sphingomyelin; the inner aqueous phase comprises a nucleic acid drug; the nucleic acid drug comprises plasmid DNA (pDNA) containing the gene PEDF and histones.

[0061] The method for preparing exosome-like nanovesicles for gene delivery comprises the following steps:

[0062] (1) Cholesterol, phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), and sphingomyelin (SM) were dissolved in 1 mL of dichloromethane to obtain an organic phase;

[0063] (2) pDNA (15 μg) containing the PEDF gene and histones (45 μg) were added to 0.4 mL of pure water and incubated at 37°C for 1 h to obtain the inner aqueous phase;

[0064] (3) mixing the organic phase and the inner aqueous phase, and shearing them using a handheld high-speed homogenizer at 6000 rpm for 5 minutes to obtain colostrum; wherein the organic phase and the inner aqueous phase are mixed at a volume ratio of 2.5:1;

[0065] (4) adding the colostrum obtained in step (3) to a 4 wt % glucose solution, and shearing the mixture using a handheld high-speed homogenizer at a speed of 5000 rpm for 3 min to obtain a double emulsion; wherein the colostrum and the glucose solution are mixed in a volume ratio of 1:4;

[0066] (5) Nitrogen gas was introduced into the emulsion at 200 mL / s to remove dichloromethane and obtain exosome-like nanovesicles for gene delivery.

[0067] Various types of exosome nanovesicles were prepared by weighing the lipid phase preparation raw materials according to Table 1 below.

[0068] Table 1

[0069]

[0070]

[0071] Comparative Example 1:

[0072] A lipid vesicle comprises a lipid phase and an inner aqueous phase encapsulated within the lipid phase; the raw materials for preparing the lipid phase include cholesterol and lecithin; the inner aqueous phase includes a nucleic acid drug; the nucleic acid drug includes plasmid DNA (pDNA) containing the gene PEDF and histones, and the histones include histones H1, H2A, H2B, H3, and H4.

[0073] The method for preparing the lipid vesicles comprises the following steps:

[0074] (1) Dissolve 0.5 mg of cholesterol and 6.0 mg of lecithin in 1 mL of dichloromethane to obtain an organic phase;

[0075] (2) pDNA (15 μg) and histone (45 μg) containing PEDF were added to 0.4 mL of pure water and incubated at 37°C for 1 h to obtain the inner aqueous phase;

[0076] (3) mixing the organic phase and the inner aqueous phase, and shearing them using a handheld high-speed homogenizer at 6000 rpm for 5 minutes to obtain colostrum; wherein the organic phase and the inner aqueous phase are mixed at a volume ratio of 2.5:1;

[0077] (4) adding the colostrum obtained in step (3) to a 4 wt % glucose solution, and shearing the mixture using a handheld high-speed homogenizer at a speed of 5000 rpm for 3 min to obtain a double emulsion; wherein the colostrum and the glucose solution are mixed in a volume ratio of 1:4;

[0078] (5) Nitrogen gas was introduced into the emulsion at a rate of 200 mL / s to remove dichloromethane and obtain lipid vesicles (abbreviated as OL).

[0079] Comparative Example 2

[0080] An exosome-like nanovesicle comprises a lipid phase and an inner aqueous phase encapsulated within the lipid phase; the raw materials for preparing the lipid phase include cholesterol, dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylcholine (DOPC) and sphingomyelin; the inner aqueous phase comprises a nucleic acid drug; the nucleic acid drug comprises plasmid DNA (pDNA) containing the gene PEDF and histones.

[0081] The method for preparing exosome-like nanovesicles for gene delivery comprises the following steps:

[0082] (1) 0.5 mg of cholesterol, 3.0 mg of dioleoylphosphatidylethanolamine (DOPE), 0.5 mg of dioleoylphosphatidylcholine (DOPS), 1.1 mg of dioleoylphosphatidylcholine (DOPC), and 0.9 mg of sphingomyelin (SM) were dissolved in 1 mL of dichloromethane to obtain an organic phase;

[0083] (2) pDNA (15 μg) containing the PEDF gene and histones (45 μg) were added to 0.4 mL of pure water and incubated at 37°C for 1 h to obtain the inner aqueous phase;

[0084] (3) mixing the organic phase and the inner aqueous phase, and shearing them using a handheld high-speed homogenizer at 6000 rpm for 5 minutes to obtain colostrum; wherein the organic phase and the inner aqueous phase are mixed at a volume ratio of 2.5:1;

[0085] (4) adding the colostrum obtained in step (3) to a 4 wt % glucose solution, and shearing the mixture using a handheld high-speed homogenizer at a speed of 5000 rpm for 3 min to obtain a double emulsion; wherein the colostrum and the glucose solution are mixed in a volume ratio of 1:4;

[0086] (5) Nitrogen gas was introduced into the emulsion at 200 mL / s to remove dichloromethane and obtain exosome-like nanovesicles.

[0087] The vesicles obtained above were tested as follows:

[0088] 1. Determination of the encapsulation efficiency and in vitro cumulative release of exosome-like nanovesicles

[0089] To determine the vesicle encapsulation efficiency (EE), the His-pDNA@EMNs obtained in Example 1 were immersed in a suspension sample (1 mL) and centrifuged at 1000×g for 30 min to separate the free histones in the vesicles. The amount of histones in the supernatant was determined by BCA protein content determination. This value is the free histone amount (W 游离 After the vesicles were dissolved in acetone: water (3:1, V / V), the total amount of histones in the system (W 总 ).

[0090] The formula for EE is as follows: EE (%) = (W 总 -W 游离 ) / W 总 ×100%

[0091] The release of pDNA and histones from the vesicles was determined by dialysis. One mL of vesicles containing pDNA and histones was transferred to a dialysis bag with a molecular weight cutoff of 1000 kD. The bag was completely immersed in 10 mL of PBS (pH 7.4) and then placed in a shaker at 37°C, 100 rpm. At designated time intervals (0.5, 1, 2, 4, 8, 12, 24, 36, 48, 60, and 72 h), 0.5 mL of sample solution was removed and replaced with an equal volume of PBS. The free histone concentration was determined using the BCA protein assay, and the release profile of His-pDNA@EMNs was calculated based on the total histone concentration.

[0092] The calculated encapsulation efficiency of His-pDNA@EMNs was 54.34±4.77%, and the drug loading was 0.99±3.16%. Figure 4 As shown in the figure, His-pDNA@EMNs released rapidly in the first 24 h, with a cumulative release of 70.32%. The release was relatively slow from 24 to 96 h, and finally about 95% of His-pDNA was released from the vesicles within 48 h.

[0093] It can be seen that the exosome-like nanovesicles obtained in Example 1 have good stability and in vitro cumulative release efficiency.

[0094] 2. Stability investigation of exosome-like nanovesicles

[0095] The physical stability of the His-pDNA@EMNs obtained in Example 1 was studied for one month. The His-pDNA@EMNs were stored in a sealed glass bottle at 4-8°C. The particle size and zeta potential of the His-pDNA@EMNs were measured after 0, 5, 10, 15, 20, 25, and 30 days of storage. The test results are shown in Figure 2. Figure 5 As shown, the average particle size and Zeta potential of the vesicles did not change significantly within 1 month, indicating that the vesicles obtained in Example 1 have a good stability. Figure 5 EMNs are exosome-like nanovesicles in internal water that do not contain PEDF plasmids and histones.

[0096] To investigate the stability of the vesicles in serum, 100 μL of His-pDNA@EMNs obtained in Example 1 was incubated with 100 μL of fetal bovine serum (FBS) in a 96-well plate at 37°C. The absorbance at 630 nm was measured using a microplate reader at 0, 0.5, 1, 2, 4, 6, 8, 10, and 12 hours. Serum-induced aggregation was analyzed by turbidity, with the turbidity value of the vesicles designated as Z1. A negative control solution containing 100 μL of PBS (pH 7.4, 0.01 M) and 100 μL of FBS was used, with its turbidity designated as Z2. The relative turbidity of the vesicles is calculated as (Z1 - Z2) / Z2 × 100%.

[0097] The stability of vesicles in serum is shown in Figure 6 ,from Figure 6 It can be seen that the relative turbidity calculated based on the absorbance value does not change significantly over time, indicating that the vesicles do not aggregate in the serum; that is, the exosome-like nanovesicles obtained in Example 1 have good stability.

[0098] 3. Effects of exosome-like nanovesicles on HUVEC cell activity

[0099] 3.1. Detection of the toxicity of His-pDNA@EMNs obtained in Example 1 on HUVEC cells using CCK-8

[0100] According to 2×10 5 HUVEC were seeded at a density of 100 μg / well in 96-well plates. After 24 hours of culture, physiological saline, the transfection reagent Lipo6000 (0.2 μg / mL), exosome-like nanovesicles (EMNs) lacking the PEDF gene and histones, and various concentrations of His-pDNA@EMNs (1, 2.5, and 5 μg / mL) were added, with triplicate wells cultured for each concentration. After 1 hour of incubation, the absorbance was measured using a microplate reader (450 nm) to calculate the cytotoxicity of His-pDNA@EMNs and transfection reagent at different concentrations on HUVEC. The absorbance of blank cell culture medium was used as a 100% activity control.

[0101] Effects of His-pDNA@EMNs on HUVEC cell viability Figure 7 As shown, within 96 hours, cell viability of both blank EMNs and histones remained around 100%. However, at 24, 48, and 96 hours, cell viability in the Lipo6000 group decreased to 60.30%, 47.58%, and 37.40%, respectively, demonstrating the high cytotoxicity of cationic liposomes. Compared to the commercially available cationic liposome Lipo6000 for nucleic acid transfection, the cell viability of the EMNs@His-pDNA group at concentrations of 1-5 μg / mL approached 100%, demonstrating the low cytotoxicity of the His-pDNA@EMNs obtained in Example 1 towards HUVEC, demonstrating their enhanced safety.

[0102] 3.2. Effect of His-pDNA@EMNs obtained in Example 1 on the survival rate of HUVEC cells

[0103] HUVEC (2×10 5 / well), add 0.5mL DMEM medium (containing 10% FBS) overnight. Physiological saline, Lipo6000, histones, EMNs, and His-pDNA@EMNs were added to the wells and cultured for 24 and 48 hours. The culture medium was aspirated and the cells were washed three times with PBS. 250μL of Calcein AM / PI working solution was added and incubated at 37°C in the dark for 30 minutes. The staining effect was observed under a fluorescence microscope (Calcein AM is green fluorescence, Ex / Em = 494 / 517nm; PI is red fluorescence, Ex / Em = 535 / 617nm). By comparing the ratio of dead cells to live cells, the cell survival status of the control and treatment groups can be determined.

[0104] The cytotoxicity of His-pDNA@EMNs was qualitatively observed by staining living cells with calcein AM (green fluorescence) and dead cells with propidium iodide PI (red fluorescence). Figure 8 As shown, at 24 hours, the saline group, the blank EMNs group, and the His-pDNA@EMNs and histone groups showed a high number of live cells, with only sporadic dead cells. At 48 and 72 hours, the number of dead cells in these groups increased slightly. In the Lipo6000 group, the number of live cells decreased significantly between 24 and 48 hours, while the number of dead cells gradually increased.

[0105] The ratio of dead cells to live cells is shown in Figure 9 ,from Figure 9 It can be seen that the dead cell ratio of the His-pDNA@EMNs group was 2.13 times lower than that of the Lipo6000 group (P<0.0001). Except for the Lipo6000 group, the survival rates of the other groups were all above 95%, further demonstrating that the His-pDNA@EMNs obtained in Example 1 had low cytotoxicity to HUVEC and had higher safety.

[0106] 4. Testing the effect of exosome-like nanovesicles on HUVEC cell uptake

[0107] The His-pDNA@EMNs obtained in Example 1 and the OL obtained in Comparative Example 1 were labeled with equal amounts of coumarin 6. The endothelial cells in the logarithmic growth phase with good growth status were taken and the cell density of the cell suspension was adjusted to 1×10 5 Cells were plated at a density of 100 cells / mL. 1 mL of cell suspension was added to each well of a 24-well plate and cultured for 24 hours. After cell attachment, all medium was aspirated and the cells were washed three times with saline. Labeled His-pDNA@EMNs and labeled OLs were added at 0, 2, and 4 hours, respectively. The OL group served as the control group. Returned the cells to the incubator and cultured for another 2 hours. Fixation with 4% paraformaldehyde and staining with DAPI for 10 minutes were performed. The cells were washed three times with saline and observed under an inverted fluorescence microscope in the dark. Images were taken and recorded under the same conditions.

[0108] like Figure 10 The cellular uptake of His-pDNA@EMNs obtained in Example 1 and OLs obtained in Comparative Example 1 at different times was compared. Under serum-free conditions, His-pDNA@EMNs were significantly taken up by cells at 2 hours, with no significant change at 4 or 6 hours, indicating that cellular uptake was essentially complete by 2 hours. Compared to OLs, His-pDNA@EMNs exhibited 92% higher uptake at 2 hours, and the difference in uptake between the two at 4 and 6 hours was also significant, with His-pDNA@EMNs exhibiting higher cellular uptake, indicating that His-pDNA@EMNs containing exosome lipid components possessed better affinity.

[0109] In the presence of serum, the cellular uptake of His-pDNA@EMNs was similar to that in the absence of serum, indicating that the presence of serum does not affect uptake. While this has no direct impact on drug delivery via aerosol inhalation, it does open up the possibility of lipid vesicles as injectable medications. However, OL uptake decreased by 30.68% compared to that in the presence of serum, indicating that serum affects OL uptake, while His-pDNA@EMNs exhibit relatively good stability.

[0110] 5. Effect of exosome-like nanovesicles on HUVEC cell transfection

[0111] HUVEC cells in the logarithmic growth phase with good growth status were seeded into 24-well culture plates (1×10 4 Cells were plated in a 0.5 mL DMEM medium (containing 10% FBS) overnight. After 24 hours, the serum-free medium was replaced with the His-pDNA@EMNs obtained in Example 1 to a pDNA concentration of 2.5 μg / mL. The cells were then incubated at 37°C overnight. After 6 hours, the serum-free medium containing the His-pDNA@EMNs was removed and replaced with DMEM containing 10% FBS. Fluorescence intensity was measured at 48 hours. Comparative transfections were performed using the Lipo6000 transfection reagent according to the manufacturer's instructions. Transfection efficacy was assessed using an inverted fluorescence microscope.

[0112] In the cell transfection experiment, the transfection performance of His-pDNA@EMNs, Lipo6000, and the histone-pDNA complex was compared. The red fluorescence comes from the red fluorescent protein gene in the plasmid. Figure 11 (A) In the absence of serum, the fluorescence intensity of His-pDNA@EMNs transfection was higher than that of Lipo6000, and the fluorescence intensity of the complex of histone and pDNA was weaker than that of both His-pDNA@EMNs and Lipo6000. Figure 11 (B) Quantitative data showed that the fluorescence intensity of His-pDNA@EMNs transfection was 18.74% higher than that of Lipo6000 (P < 0.001), and the fluorescence intensity of Lipo6000 was 67.33% higher than that of the histone-pDNA complex (P < 0.0001), both of which were statistically significant. These results indicate that the histone-pDNA complex can be successfully transfected, but its difficulty in crossing the cell barrier results in a low transfection rate. His-pDNA@EMNs and Lipo6000 can carry the plasmid across the cell barrier, enabling successful transfection, and His-pDNA@EMNs is more effective than Lipo6000.

[0113] The transfection efficiency of His-pDNA@EMNs remained essentially unchanged in the serum-treated group, while that of Lipo6000 decreased. This is because proteins in serum can affect the transfection efficiency of cationic liposomes, indicating that His-pDNA@EMNs cell transfection is not affected by serum.

[0114] Figure 11 (C) Comparison of transfection efficiencies at 24, 48, and 72 h showed that the fluorescence intensity of His-pDNA@EMNs reached its highest at 48 h, and there was no significant change in fluorescence intensity when the time was extended to 72 h. Therefore, 48 h was preliminarily judged to be the final transfection time.

[0115] 6. Effect of exosome-like nanovesicles on PEDF expression in HUVEC cells

[0116] Take the cells transfected with His-pDNA@EMNs obtained in Example 1 for 48 hours, discard the cell culture medium, wash three times with PBS, add the appropriate amount of lysis buffer (Ripa: phosphatase inhibitor: protease inhibitor = 100:1:1), place on ice for lysis for 40 minutes, vortex once every 10 minutes, centrifuge at 5180×g, 4°C, and take the supernatant. Use the BCA kit to determine the protein content, add the remaining supernatant to the loading buffer according to the proportion, and inactivate the protein at 100°C. Use 10% PAGE gel electrophoresis at 80V for 40 minutes and 100V for 100 minutes, then transfer the protein on the gel to NC membrane with a constant current of 0.3mA. The NC membrane is blocked and washed after blocking. Incubate the primary antibody at 4°C overnight, then wash the membrane and incubate the secondary antibody. After washing the membrane, detect.

[0117] Extract normal HUVEC cell proteins and transfected cell proteins for WB analysis, such as Figure 12 The expression level of PEDF protein in the transfected cells was significantly increased, 1.5 times that of the blank control cell group, indicating that the gene delivered by His-pDNA@EMNs obtained in Example 1 was successfully expressed.

[0118] 7. Testing the distribution of exosome-like nanovesicles in mouse lung tissue

[0119] Two samples were prepared by labeling histones with Cy5.5 from the His-pDNA@EMNs obtained in Example 1 and the OLs obtained in Comparative Example 1. Mice were then exposed to the Cy5.5-labeled samples for 1 hour (10 mL, 50 μg / mL pDNA concentration, in a 12 cm × 12 cm × 16 cm inhalation chamber). This process delivered approximately 5 μg / mL of pDNA to the lungs of the mice. The biodistribution of His-pDNA@EMNs in mice was observed using a visible light small animal in vivo imaging system at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, and 120 h). At the corresponding time points after administration, one mouse was randomly selected from each group and sacrificed by cervical dislocation. The heart, liver, spleen, lung, and kidney were observed and analyzed for tissue photon intensity (excitation wavelength: 673 nm, emission wavelength: 695 nm).

[0120] like Figure 13 As shown in the figure, after administration of each group, qualitative observation of the distribution of His-pDNA@EMNs and OL in the lungs revealed significant fluorescence in the lungs of mice after inhalation of OL and His-pDNA@EMNs. The fluorescence gradually increased over time, reaching its peak at 8 hours in both groups. This may be due to the gradual inhalation of lipid vesicles from the nasal cavity and airways into the lungs over time. In the OL group, lung fluorescence gradually weakened after 8 hours and disappeared at 24 hours. However, fluorescence in the lungs of mice in the His-pDNA@EMNs group was still observed at 120 hours, indicating that His-pDNA@EMNs have a longer retention time in the lungs than OL.

[0121] After inhalation of OL, significant fluorescence accumulated in the mouse liver and kidneys within 0.5 hours, and residual fluorescence was still present in the liver and kidneys 24 hours later. In contrast, after inhalation of His-pDNA@EMNs, only a small amount of fluorescence appeared in the liver at 0.5 hours, and in the kidneys only after 24 hours. This further validates the long-term retention of His-pDNA@EMNs in the mouse lungs.

[0122] 8. Testing the distribution of exosome-like nanovesicles in mouse pulmonary vasculature

[0123] Lung tissues of mice 4 h after inhalation in the above-mentioned in vivo experiments were lavaged and cryosectioned. Lung cryosections were obtained using a microtome, and the pulmonary vascular endothelium was stained with FITC fluorescent-labeled anti-CD31 antibody (Servicebio, Wuhan, China). The drug distribution was observed under an inverted fluorescence microscope.

[0124] Since the site of action of His-pDNA@EMNs is vascular endothelial cells and the drug is administered by aerosol inhalation, in order to verify that the drug can successfully reach the pulmonary blood vessels, frozen sections were processed 4 hours after inhalation and immunofluorescence staining was performed for the vascular marker CD31, which was green fluorescence, and Cy5.5-labeled His-pDNA@EMNs were distributed in red. Figure 14 It can be seen that the red fluorescence-labeled His-pDNA@EMNs are widely distributed in the lungs, and the red fluorescence of a large number of His-pDNA@EMNs overlaps with the green fluorescence of the vascular endothelium, proving that it can successfully reach the vascular endothelial cells and exert its effect.

[0125] 9. Test the distribution of PEDF protein expression in mouse lung tissue by exosome-like nanovesicles

[0126] An appropriate amount of lung tissue was collected and added with ten times the volume of lysis buffer (RIPA: phosphatase inhibitor: protease inhibitor = 100:1:1). The tissue was thoroughly lysed using a tissue homogenizer and centrifuged at 6000×g for 5 min at 4°C. The supernatant was then subjected to Western blotting.

[0127] The lung tissues of mice were extracted after modeling, and the PEDF content in the lung tissues of the NC group (blank group), Model group (model group), and His-pDNA@EMNs group was compared. Figure 15 The results showed that the PEDF content in the lung tissue of mice in the Model group was significantly lower than that in the NC group, while the PEDF content in the lung tissue of mice that inhaled His-pDNA@EMNs was significantly increased. This result, together with all the above pharmacodynamic results, indicates that His-pDNA@EMNs increased PEDF expression in the lungs of mice under hypoxic conditions, thereby exerting a positive intervention effect on the core symptoms of HAPE in mice.

[0128] 10. Safety evaluation of aerosol inhalation of exosome-like nanovesicles in mice

[0129] Mice were inhaled for 1 hour in a nebulizer inhalation chamber (12 cm × 12 cm × 16 cm) containing 10 mL of His-pDNA@EMNs (prepared in Example 1) at a pDNA concentration of 50 μg / mL. After 3 and 5 consecutive days of inhalation, the mice were sacrificed. The lungs, liver, heart, spleen, and kidneys were removed. Tissues were fixed with 4% paraformaldehyde, sectioned, and stained with hematoxylin and eosin for histopathological examination.

[0130] After continuous inhalation of His-pDNA@EMNs for 3 and 5 days, pathological examinations of the heart, liver, spleen, lung, and kidney of mice were performed, e.g. Figure 16In the mouse lungs, alveolar epithelial cells were neatly arranged, alveoli were evenly distributed, and no inflammatory cell infiltration was observed. Cardiac myocytes were neatly arranged and morphologically normal. The central veins of the liver lobules were distinct, with neatly arranged hepatocyte cords surrounding them, abundant cytoplasm, and clear nucleoli. The spleen had a distinct cortical-medullary junction and dense lymphocytes. The renal tubular epithelial cells were neatly arranged and structurally normal. No significant histopathological changes were observed in the mouse lungs, livers, hearts, spleens, or kidneys, demonstrating the good biocompatibility of His-pDNA@EMNs.

[0131] 11. Test the encapsulation efficiency of exosome-like nanovesicles

[0132] The exosome-like nanovesicles obtained in Example 1 and Comparative Example 2 were compared to test their encapsulation efficiency. The exosome-like nanovesicles obtained in Example 1 achieved an encapsulation efficiency of over 50%. However, the exosome-like nanovesicles obtained in Comparative Example 2, which incorporated dioleoyl groups, had a lower encapsulation efficiency of only 3%, making it difficult to achieve gene delivery.

[0133] It is speculated that due to the strong lipophilicity of the dioleoyl group, the volatilization of nitrogen during lipid formation is slow, causing the newly formed and unstable lipid vesicles to rupture, resulting in serious leakage, thus greatly reducing the encapsulation efficiency and making it difficult to load drugs.

[0134] The present invention arranges the raw materials for preparing the lipid phase so that the obtained exosome-like nanovesicles have a higher encapsulation efficiency and can effectively deliver nucleic acids to the diseased area.

[0135] 13. Effect of cholesterol content on the particle size and stability of exosome-like nanovesicles

[0136] The particle sizes of the samples of experimental groups 1-1 to 1-5 in Example 2 were measured, and the results are shown in Table 2 below.

[0137] Table 2

[0138] Experimental group 1-1 1-2 1-3 1-4 1-5 Cholesterol mass percentage 3％ 5％ 7％ 10％ 15％ Particle size (nm) Unformed 86 97 120 432

[0139] As can be seen from the table above, when the cholesterol concentration is in the range of 5-10% (W / W), uniform and appropriate exosome-like nanovesicles are obtained. When the cholesterol concentration is too low or too high, the particle size will be too small or too large. Therefore, in order to make the exosome-like nanovesicles have a more appropriate particle size distribution, the cholesterol concentration is preferably 5-10% (W / W) of the total mass.

[0140] 14. Effects of PE and DOPE Content on Particle Size and Escape Rate of Exosome-like Nanovesicles

[0141] The PE in the raw materials of experimental groups 2-1 to 2-8 in Example 2 was replaced with DOPE to obtain samples of comparative groups 6-1 to 6-8.

[0142] The particle sizes of the experimental group 2-1 to 2-8 samples and the comparative group 6-1 to 6-8 samples in Example 2 were measured, and the lysosomal escape rates were determined according to the following method: HUVEC cells were seeded into a confocal culture dish (1×10 5 / dish) overnight. Coumarin 6 was used to label the lipid membrane of the exosome-like vesicles, and LysoTracker was used to label lysosomes. Cells were incubated with coumarin 6-labeled exosome-like vesicles (2.5 μg / mL) for 2 h. After reaching the incubation time point, the cells were aspirated and rinsed, and LysoTracker (75 mmol / L) was added to label the lysosomes for 30 min. The cells were rinsed twice with PBS and incubated with DAPI for 10 min to label the cell nuclei. A laser scanning confocal microscope (STELLARIS 5, Leica, Wetzlar, Germany) was used for observation and photography, and Image J software was used to calculate the percentage of non-overlapping green fluorescence of exosome-like vesicles and red fluorescence of lysosomes, which was recorded as the lysosomal escape rate. The results are shown in Table 3.

[0143] Table 3

[0144]

[0145]

[0146] As can be seen from the above table, the lysosomal escape rate of PE-incorporated is generally higher than that of DOPE-incorporated, and in the range of 30-50% (W / W), exosome-like nanovesicles with appropriate particle size and high lysosomal escape rate are obtained. When the ratio is too low or too high, the particle size is too large and the escape rate is reduced. Therefore, in order to make the exosome-like nanovesicles have a more appropriate particle size distribution, PE is preferably incorporated with phospholipids, and PE is preferably 40-45% (W / W) of the total mass.

[0147] 15. Effects of PS and DOPS Content on Particle Size and Escape Rate of Exosome-like Nanovesicles

[0148] The PS in the raw materials of experimental groups 3-1 to 3-8 in Example 2 was replaced with DOPS to obtain samples of comparative groups 7-1 to 7-8.

[0149] The particle size, lysosomal escape rate and nuclear entry number of the experimental group 3-1 to 3-8 samples and the control group 7-1 to 7-8 samples in Example 2 were measured. The nuclear entry number was determined as follows: HUVEC cells were seeded into a confocal culture dish (1×10 5The cells were incubated overnight in a 5% PBS container (100 μg / dish) with coumarin 6 to label the lipid membrane of the exosome-like vesicles. Cells were incubated with coumarin 6-labeled exosome-like vesicles (2.5 μg / mL) for 2 h. After the incubation time, the cells were aspirated and rinsed. The cells were then rinsed twice with PBS and incubated with DAPI for 10 min to label the cell nuclei. Observation and photography were performed using a laser scanning confocal microscope (STELLARIS 5, Leica, Wetzlar, Germany). Image J software was used to count the total amount of green fluorescent photons emitted by exosome-like vesicles within the blue fluorescent area of ​​the nucleus, which was recorded as the number of exosome-like vesicles entering the nucleus. The results are shown in Table 4.

[0150] Table 4

[0151]

[0152]

[0153] As can be seen from the table above, the lysosomal escape rate of PS-incorporated vesicles is generally higher than the lysosomal escape rate and nuclear entry rate of DOPS-incorporated vesicles. In the range of 10-20% (W / W), exosome-like nanovesicles with a higher lysosomal escape rate are obtained. When the PS incorporation amount is too low or too high, the escape rate will decrease. When the PS incorporation amount is 14% and 16%, the exosome-like nanovesicles have a higher nuclear entry efficiency. Therefore, in order to make the exosome-like nanovesicles have a more appropriate particle size distribution, lysosomal escape efficiency, and nuclear entry efficiency, it is preferred that PS be incorporated into phospholipids, and PS is preferably 14%-16% (W / W) of the total mass.

[0154] 16. Effects of PC and DOPC Content on the Uptake and Stability of Exosome-like Nanovesicles

[0155] The PC in the raw materials of experimental groups 4-1 to 4-8 in Example 2 was replaced with DOPC to obtain samples of comparative groups 8-1 to 8-8.

[0156] The quantitative uptake fluorescence intensity and stability of the experimental group 4-1 to 4-8 samples and the control group 8-1 to 8-8 samples in Example 2 were measured. The quantitative uptake measurement method is as follows: HUVEC cells were seeded into a confocal culture dish (1×10 5 The cells were incubated overnight in a 5% PBS container (100 μg / dish). Coumarin 6 was used to label the lipid membrane of the exosome-like vesicles. Cells were incubated with coumarin 6-labeled exosome-like vesicles (2.5 μg / mL) for 2 h. After the incubation time, the cells were aspirated and rinsed, then rinsed twice with PBS. Laser scanning confocal microscopy (STELLARIS 5, Leica, Wetzlar, Germany) was used for observation and photography. Image J software was used to calculate the total amount of green fluorescence photons from the exosome-like vesicles, which was recorded as the uptake fluorescence intensity. The results are shown in Table 5.

[0157] Table 5

[0158]

[0159]

[0160] As can be seen from the above table, the stability of exosome-like nanovesicles incorporated with PC is better than that of exosome-like nanovesicles incorporated with DOPC, and within the range of 10-20% (W / W), exosome-like nanovesicles with generally higher PC incorporation uptake and good stability are obtained. Too low or too high will lead to poor stability and reduced uptake. Therefore, in order to make the exosome-like nanovesicles have better uptake and stability, PC is preferably incorporated into phospholipids, and PC is preferably 14%-17% (W / W) of the total mass.

[0161] 17. Effect of SM content on the transfection efficiency and escape rate of exosome-like nanovesicles

[0162] The lysosomal escape efficiency of 5-1 to 5-8 in Example 2 was determined. The results are shown in Table 6.

[0163] Table 6

[0164]

[0165] As can be seen from the above table, the incorporation of SM in the range of 5-16% (W / W) can obtain exosome-like nanovesicles with higher lysosomal escape efficiency. When it is too low or too high, the trans-lysosomal escape rate will be reduced. Therefore, in order to make the exosome-like nanovesicles have a higher lysosomal escape efficiency, SM is preferably 8%-12% (W / W) of the total mass.

[0166] The above description is only a preferred embodiment of the present invention. Therefore, any equivalent changes or modifications made according to the structure, characteristics and principles described in the scope of the patent application of the present invention are included in the scope of the patent application of the present invention.

Claims

1. An exosome-like nanovesicle for gene delivery, comprising a lipid phase and an inner aqueous phase encapsulated within the lipid phase, characterized in that: The raw materials for preparing the lipid phase include cholesterol, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and sphingomyelin; The inner aqueous phase includes nucleic acid drugs; The weight of the cholesterol accounts for 5-10% of the weight of the lipid phase; The weight of the phosphatidylethanolamine accounts for 40-45% of the weight of the lipid phase; The weight of the phosphatidylserine accounts for 14-16% of the weight of the lipid phase; The weight of the phosphatidylcholine accounts for 14-17% of the weight of the lipid phase; The weight ratio of the sphingomyelin to the weight of the lipid phase is 8-12%.

2. The exosome-like nanovesicle for gene delivery according to claim 1, characterized in that The raw materials for preparing the nucleic acid drug include plasmid DNA and histone; the mass ratio of plasmid DNA to histone is 1:2-1:

7.

3. The exosome-like nanovesicle for gene delivery according to claim 1, characterized in that The particle size of the exosome-like nanovesicles is 20-500 nm.

4. A method for preparing exosome-like nanovesicles for gene delivery according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Cholesterol, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and sphingomyelin are dissolved in an organic solvent to obtain an organic phase; (2) Plasmid DNA and histones are added to pure water and incubated to obtain the inner aqueous phase; (3) mixing the organic phase and the inner aqueous phase and homogenizing them at high speed to obtain colostrum; (4) adding the colostrum obtained in step (3) to the external aqueous phase and homogenizing at high speed to obtain a double emulsion; (5) Nitrogen gas is introduced into the emulsion to remove the organic solvent and obtain exosome-like nanovesicles for gene delivery.

5. The method for preparing exosome-like nanovesicles for gene delivery according to claim 4, characterized in that: The total concentration of lipid components in the organic phase is 20-80% W / V; in step (3), the organic phase and the inner aqueous phase are mixed at a volume ratio of 2.5:1; in step (4), the colostrum is injected into the outer aqueous phase at a volume ratio of 1:4.

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