WO2024065649A1 - Method for efficiently loading dna into exosome - Google Patents

Abstract

Provided is a method for efficiently loading DNA into an exosome, wherein the exosome is derived from mammalian cells or milk. A highly compressed DNA-polycationic compression agent complex is formed by combining a polycationic compression agent with a DNA, and the DNA-polycationic compression agent complex is loaded into the membrane of the exosome by means of a physical means, wherein the cationic compression agent may be selected from one or more of protamine, polylysine, polyarginine, and polyethyleneimine; therefore, efficient in vivo gene delivery and expression of large-fragment DNA are smoothly realized by utilizing the transportation function of the exosome. The exosome obtained by the method is expected to become an efficient tool for loading, transporting, and expressing large-fragment DNA in the field of gene therapy.

Description

A method for efficiently loading DNA into exosomes Technical Field

The present invention relates to the technical field of exosome loading, and specifically to a method for efficiently loading DNA into exosomes, wherein the exosomes are derived from mammalian cells or milk, a polycationic compressor is combined with DNA to form a highly compressed DNA-polycationic compressor complex, and the DNA-polycationic compressor complex is loaded into the exosome membrane by physical means, thereby successfully completing efficient loading of large-fragment DNA molecules and its gene delivery and expression in vivo.

Background technique

Gene therapy is an effective means to treat genetic diseases, malignant tumors and some degenerative diseases. DNA-mediated gene therapy still faces many problems that are difficult to effectively solve: (1) DNA not only needs to enter the cell, but also needs to enter the cell nucleus to be transcribed and expressed; 2) A fully functional DNA sequence needs to include multiple functional elements such as promoter, target fragment, polyA fragment, etc., and its size is usually much larger than the mRNA fragment used for treatment. Therefore, the current mainstream DNA-based gene therapy method is still based on adeno-associated virus AAV. However, AAV vectors themselves also have many serious problems, the most prominent of which is their safety risks in the human body. Clinical trials of gene therapy using AAV vectors have repeatedly caused patient deaths, and due to their immunogenicity, AAV drugs can only be used for single treatment and cannot guarantee long-term efficacy. Therefore, the field of gene therapy urgently needs a non-viral delivery vector that can achieve efficient in vivo DNA delivery.

Exosomes are a type of extracellular vesicles. Nucleic acid delivery is one of the natural functions of exosomes. In addition, exosomes can achieve tissue and cell-specific delivery through engineering modification. As drug transport carriers, they have outstanding advantages such as low immunogenicity, high transport efficiency, good stability, strong targeting, and the ability to cross the blood-brain barrier. If they can be used in the field of DNA delivery, exosomes will become better gene therapy vectors than AAV.

At present, the methods and technologies for loading RNA (mRNA, siRNA, etc.) into exosomes are relatively mature, while the common methods of loading RNA (mRNA, siRNA, etc.) into cell exosomes by incubation or electroporation can only be used to load small nucleic acid fragments such as miRNA, siRNA or ASO, and are powerless for mRNA or even large DNA fragments or DNA plasmids. Regarding the demand for loading large DNA fragments into exosomes, there are many technical difficulties that are difficult to overcome, including the large size of DNA fragments, which are difficult to load, and the low efficiency of DNA entering the nucleus. In the existing technology, there is a method of loading large nucleic acid fragments by placing a single cell in an electric field for electrical stimulation (https://doi.org/10.1038/s41551-019-0485-1), which is difficult to achieve large-scale industrial application and can only use adherent cells. The method developed by Carmine for loading plasmid DNA into red blood cell extracellular vesicles (RBC exosomes) (WO2021/145821 A1) is limited to exosomes derived from red blood cells, and the DNA loading reagents relied on by this method are difficult to circulate and obtain in many countries around the world, greatly limiting the application and advancement of the technology.

Therefore, in view of the technical difficulties in the above-mentioned fields, the present invention provides a method for efficiently loading DNA into mammalian cells or milk-derived exosomes and realizing in vivo gene delivery and expression. This technology not only uses reagents and excipients that have been included in the pharmacopoeias of various countries and are easy to obtain and circulate, but also simplifies the loading and preparation process, reduces costs, facilitates large-scale production, is compatible with existing biopharmaceutical production processes, and significantly improves the loading efficiency of large-fragment DNA molecules, providing a new and efficient tool for gene therapy.

Summary of the invention

The present invention provides a method for efficiently loading DNA into exosomes, wherein the exosomes are derived from mammalian cells or milk, a polycationic compressor is combined with DNA to form a highly compressed DNA-polycationic compressor complex, and the DNA-polycationic compressor complex is loaded into the exosome membrane by physical means, thereby successfully completing efficient DNA in vivo gene delivery and expression.

The present invention provides a method for efficiently loading DNA into exosomes, wherein the exosomes are derived from mammalian cells or milk, and the specific steps are:

(1) thoroughly mixing the polycationic compacting agent with DNA;

(2) adding the polycation-DNA complex to mammalian cell or milk-derived exosomes and mixing them evenly;

(3) Complete DNA loading.

Further, the method may include step (4) of removing unloaded DNA and fragments;

Furthermore, in step (4), heparin is added to dissociate the unloaded DNA-protamine complex, and the method for removing the unloaded free DNA can be selected from: one or more of high-salt nuclease, DNAseI treatment, and Benzonase treatment, and the removal of nucleic acid fragments can be selected from anion exchange chromatography column purification and/or Capto Core 700 chromatography column purification method;

Further, the method may include step (5), verifying the loading effect using a qPCR method;

Furthermore, in the step (1), the exosomes derived from mammalian cells or milk may be unmodified exosomes or engineered exosomes;

Furthermore, the engineered exosomes are exosomes engineered with syncytin-1;

Furthermore, the engineered exosomes are exosomes modified with single-chain integrin Integrin αLβ2;

Furthermore, the engineered exosomes are exosomes modified with single-chain integrin Integrin αDβ2;

Furthermore, in step (1), the ratio of DNA:exosomes is 3-20:1;

Further, the polycationic compressing agent may be selected from one or more of protamine, polylysine, polyarginine, and polyethyleneimine (PEI);

Furthermore, the polycationic compression agent may be protamine, and the ratio of protamine to DNA is 2-10:1;

Furthermore, the polycationic compression agent may be protamine, and the ratio of protamine to DNA is 7:1;

Furthermore, in the step (2), after the protamine is mixed with the exosomes and DNA, the resulting precipitate can be separated from the supernatant by centrifugation;

Furthermore, in step (3), the DNA loading method may be an ultrasound method and/or a heat shock/thermal cycle method;

Furthermore, the number of cycles of the heat shock/thermal cycle loading method is 1-20 times;

Furthermore, the number of cycles of the heat shock/thermal cycle loading method is 10-20 times;

Furthermore, the heat shock temperature of the heat shock/thermal cycle loading method may be 25-60°C, and the cooling temperature may be 0°C or 4°C;

Furthermore, the heat shock temperature for loading milk exosomes was 60°C, the cooling temperature was 0°C, the number of heat shocks was 20 times, and the heat shock temperature for loading cell exosomes was 60°C;

Furthermore, the exosomes are pretreated before DNA loading, such as adjusting the pH;

Furthermore, in the step (3), auxiliary molecules are added to promote the loading of DNA into the exosome membrane, and the auxiliary factors can be selected from one or two of SM102 and DLin-MC3-DMA;

Furthermore, the auxiliary molecule addition ratio is SM102/DLin-MC3-DMA: protamine: DNA = 3: 1: 1 (w/w) or 6: 2: 1 (w/w);

Furthermore, when the auxiliary molecule is DLin-MC3-DMA, the ratio of DNA to protamine is 1:2;

Furthermore, the size of the loaded DNA is 1.9 kbp-9 kbp;

The present invention provides an exosome with high efficiency of loading DNA prepared by the above method, wherein the exosome is derived from mammalian cells or milk;

The present invention proposes the use of exosomes efficiently loaded with DNA prepared by the above method in the preparation of drugs;

Furthermore, the drug is a drug used as a non-viral vector for in vivo DNA delivery in gene therapy.

The present invention provides a medicine, which comprises exosomes prepared by the preparation method of the present invention.

The present invention also provides a gene therapy method, which comprises administering the drug of the present invention.

Beneficial effects:

(1) The method for efficiently loading DNA into exosomes provided by the present invention realizes the efficient loading of large DNA molecules, and its efficient loading range can be from 1.9 kbp to 9 kbp, which effectively solves the problem of inefficient loading of large DNA molecules in the field, so that when exosomes are used as gene therapy tools, they can carry large pieces of target DNA, expand the range of target gene selection for gene therapy, provide exosomes with a broader drug potential, and provide a powerful tool for gene therapy.

(2) The present invention uses one or more of protamine, polylysine, polyarginine, and polyethyleneimine to help DNA molecules fold and thus achieve efficient loading. The above ingredients are all widely used drugs or reagents, thus achieving significant savings in experimental costs and even future treatment costs. It also achieves the highly difficult loading of large DNA molecules in the most conventional loading method, and has significant advantages in efficiency and economy in industrial applications.

(3) The present invention further creatively adds one or more auxiliary factors of SM102 and DLin-MC3-DMA to the DNA exosome loading, thereby further improving the loading effect and stability, providing strong support for the application of the exosomes obtained by the above loading method in gene therapy.

(4) The exosomes loaded by the present invention may be exosomes modified with syncytin-1 and/or single-chain integrin Integrin αLβ2 and/or single-chain integrin Integrin αDβ2, which can specifically target inflamed vascular endothelial cells and glomerular podocytes, providing effective tools and directions for the research of corresponding disease models, the development of drugs for clinically corresponding diseases, and the exploration of gene therapy methods.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Plasmid map of the mini-circle hRluc (1919 bp) of the present invention;

Fig. 2: pRP-EGFP (3657 bp) vector map of the present invention;

Figure 3: Cytation5 of the present invention photographs the expression of EGFP after exosomes enter cells;

Fig. 4: Plasmid map of pRP-Nanoluc (4929 bp) of the present invention;

FIG5 : NanoLuc enzyme activity is detected in the cell supernatant after incubation of the DNA-loaded exosomes of the present invention with cells;

Figure 6: Plasmid map of pSGLs-Nanoluc-pp-Fc (8929 bp) of the present invention;

FIG7 : Nanoluc enzyme activity was detected in the cell supernatant after incubation of the DNA-loaded exosomes of the present invention with cells;

FIG8 : Nanoluc enzyme activity was detected in the cell supernatant after incubation of the DNA-loaded exosomes of the present invention with cells;

FIG. 9 : Nanoluc enzyme activity in the cell supernatant of the present invention;

FIG10 : Immunofluorescence detection of exosome targeting effect of the present invention;

FIG. 11 : Targeted exosome-treated cells of the present invention express nanoluc.

Detailed ways

The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1: Loading small molecular weight plasmid mini-circle hRluc DNA (1919 bp) into milk exosomes, and comparing the effects of different polycations such as poly-lysine, poly-arginine, protamine and polyethyleneimine (PEI) on DNA loading.

1. Methods:

(1) DNA loading: The loading DNA plasmid was mini-circle hRluc DNA (plasmid vector, target fragment was hRluc, plasmid size was 1919 bp), and according to the ratio of DNA: exosome = 10:1 (copy number: number of particles), 7.5E+12 milk exosome particles were adjusted to pH 10.0 with 0.1 M Na2CO3/NaHCO3, 100 μg DNA was added and mixed, and the mixture was allowed to stand at 25°C for 10 min, and then protamine (or poly-lysine, poly-arginine, polyethyleneimine) was added. (PEI)), polycation: DNA = 5: 1 (w/w), after mixing, the sample was ultrasonically treated (10℃, 1200w, ultrasonic for 2h), and then the sample pH was adjusted to 8.0, and ultrasonication was continued for 1h; after ultrasonication, heparin (protamine: heparin = 1: 3w/w) was added and mixed, and high salt nuclease with a final concentration of 5U/mL was added and treated at 37℃ for 1h to digest free nucleic acids; all samples were purified by Capto Core700 chromatography column to remove free protamine-heparin-high salt nuclease, etc., and purified by anion exchange chromatography column for further analysis of different sample components (balance solution 20mM Tris pH 8.0, 100mM NaCl, elution balance solution 20mM Tris pH 8.0, 1M NaCl, gradient elution method).

(2) Loading verification: qPCR verification of loading effect.

2. Results:

As shown in Figure 1 and Table 1, the anion exchange chromatography column-low salt elution fraction contained the exosome peak after loading, and about 16-63 DNA plasmid copies were loaded per 100 exosome particles. The protamine group had the best loading effect, and no mini-circle hRluc DNA loading was detected in the experimental group without polycations.

Table 1. qPCR results and loading amounts

Example 2: Loading small molecular weight plasmid mini-circle hRuc DNA (1919bp) into milk exosomes to verify whether protamine is the key reagent for DNA loading.

1. Methods:

(1) DNA loading: The loaded DNA plasmid is mini-circle hRluc DNA (plasmid vector, target fragment is hRluc, plasmid size is 1919 bp), and the ratio of DNA: exosome = 10:1 (copy number: particle number) is 7.5E+12 milk exosome particles were adjusted to pH 10.0 with 0.1M Na2CO3/NaHCO3, 100μg DNA was added and mixed, and the mixture was allowed to stand at 25℃ for 10 min. Protamine was then added, protamine: DNA = 5:1 (w/w), and the sample was sonicated (10℃, 1200w, sonication for 2h). The pH of the sample was adjusted to 8.0, and sonication was continued for 1h. After sonication, heparin (protamine: heparin = 1:3w/w) was added and mixed, and high salt nuclease at a final concentration of 5U/mL was added and treated at 37℃ for 1h to digest the free nucleic acid, and the nucleic acid fragments were purified and removed using Capto Core700 chromatography column and anion exchange chromatography column (equilibrium solution 20mM Tris pH 8.0, 100mM NaCl, elution solution equilibration solution 20mM Tris pH 8.0, 1M NaCl, gradient elution method).

(2) Loading verification: qPCR verification of loading effect.

2. Results:

As shown in Table 2 , after DNA was compressed by protamine, about 59 DNA plasmid copies were loaded per 100 exosome particles, and no DNA loading was detected in the experimental group without protamine.

Table 2. qPCR results and loading amounts

Example 3: Compare the effects of centrifugation or non-centrifugation during the loading process on the efficiency of exosome loading with mini-circle hRluc DNA.

Protamine is positively charged and can bind to negatively charged DNA through electrostatic interaction, thereby compressing the DNA. The loading efficiency of exosomes for DNA can be changed by controlling the loading method. Since obvious precipitate will be generated during the loading process, heparin is added to dissolve the precipitate with or without centrifugation, and nuclease is added to digest the free nucleic acid. After removing the free protamine, heparin, and nucleic acid fragments in the system through Capto Core700, the effect of centrifugation on the DNA loading effect can be compared.

1. Methods:

(1) DNA loading: The DNA plasmid loaded was mini-circle hRluc DNA (plasmid vector, target fragment was hRluc, plasmid size was 1919 bp), and the loading was performed according to DNA: exosome = 20:1 (copy number: particle number). First, 200 μL of OMEM + protamine was mixed and allowed to stand at 25°C for 1 min, and 200 μL of OMEM + 25 μg of DNA was mixed and allowed to stand at 25°C for 1 min; then OMEM-DNA was added to OMEM + protamine (protamine: DNA = 7:1, w/w), then vortexed for 1 min, and allowed to stand at 25°C for 10 min. The OMEM-DNA-protamine mixture was added to the milk exosomes, mixed while adding, and then the sample was placed in a metal bath at 37°C, 120 rpm, for 30 min, and then immediately placed in an ice-water mixture (4°C) and allowed to stand for 10 min. This was one cycle; the heat shock was repeated 10 times. After the sample was heat-shocked, it was divided into two equal parts: one part was directly added with heparin, mixed evenly, and then DNaseI enzyme was added to digest at 37℃ overnight; the other part was centrifuged at 12000rpm for 20min, and the precipitate and supernatant were collected respectively. The precipitate was washed 3 times with 1*PBS, redissolved with heparin (protamine: heparin = 1: 3w/w) and fixed to 500μL with 1*PBS, and DNaseI enzyme was added to digest at 37℃ overnight. The supernatant was directly added with heparin + DNaseI enzyme to digest at 37℃ overnight. All samples were purified by Capto Core700 column to remove free protamine, heparin and DNaseI.

(2) qPCR verification of loading effect.

2. Results: After 37-0℃ heat shock cycle treatment, the Cq value was not significantly affected by sample centrifugation or non-centrifugation, indicating that centrifugation is not necessary during the loading process, thereby reducing the number of operation steps and time.

Table 3. qPCR results and loading amount

Embodiment 4:

Purpose of the experiment: To load mini-circle hRluc DNA into milk exosomes and verify the effects of different ratios of protamine to DNA and the number of heat shock cycles on the loading efficiency.

(1) DNA loading: The DNA plasmid loaded was mini-circle hRluc DNA (plasmid vector, target fragment was hRluc, plasmid size was 1919 bp), and the DNA: exosome ratio was 20:1 (copy number: particle number). First, 200 μL of OMEM + protamine was mixed and allowed to stand for 1 min, and 200 μL of OMEM + 10 μg of DNA was mixed and allowed to stand for 1 min; then OMEM-DNA was added to OMEM + protamine (protamine: DNA mass ratio was 2:1, 5:1, 7:1, 10:1), then vortexed for 1 min, and allowed to stand at 25°C for 10 min. The OMEM-DNA-protamine mixture was added to 2.5E+11 milk exosomes, mixed and incubated at 37°C, 120 rpm, for 30 min. Then, the sample was immediately placed in an ice-water bath (0°C) and allowed to stand for 10 minutes (sample group, 37°C-ice-water bath standing step, repeated 1-3 times respectively). After centrifugation at 12000rpm for 20 minutes, the precipitate was collected. After the precipitate was washed twice with 1*PBS, the precipitate was redissolved with heparin (protamine: heparin = 1: 3w/w). The dissolved samples were all fixed to 500μL with 1*PBS. All samples were passed through Capto Core700 to remove free protamine-heparin.

(2) Loading verification: qPCR verification of loading effect.

(3) Expression verification: The exosomes with the highest loading efficiency (DNA+protamine+milk exosomes (DNA and protamine mass ratio 1:10 group)+heparin-incubated for 2 cycles) were injected into mice via the tail vein. Heart, liver, spleen, lung, and kidney tissues were obtained 4 hours after administration, ground, and centrifuged to obtain the supernatant, and the Rluc enzyme activity was detected.

2. Results

As shown in Tables 4-5, different ratios of DNA-protamine can promote the loading of nucleic acids by exosomes, and the loading effects are relatively good at ratios of 1:2 to 1:10, especially at 1:7. Increasing the number of heat shock cycles can increase the loading capacity of exosomes, and the loading efficiency of 3 cycles is significantly higher than that of 1 cycle. In vivo experiments clearly detected the expression of hRluc and exerted enzymatic activity, proving that exosomes loaded with Rluc gene plasmids can transfect cells in vivo and achieve gene expression.

Table 4. qPCR results and loading amount

name of association		Repeat 1	Repeat 2	Repeat 3	Mean	SD
Heart	201	230	214	215	14.5
liver	641	660	639	647	11.6
spleen	189	193	176	186	8.9
lung	200	209	189	199	10.0
kidney	231	240	242	238	5.9
Heart	1004	1109	1120	1078	64.0

Table 5. Results of RLuc enzyme activity detection in tissues after exosome injection in vivo

Example 5: The medium molecular weight plasmid pRP-EGFP (3657 bp) was loaded into cells and exosomes, the ratio of protamine to DNA was fixed, and the difference in loading efficiency between cell exosomes and milk exosomes was verified.

1. Methods:

(1) DNA loading: The DNA plasmid to be loaded is pRP-EGFP DNA (expression vector, target fragment is EGFP, plasmid size is 3657 bp), and the loading ratio is DNA: exosome = 10:1 (copy number: number of particles). Take 2 mL of OMEM medium and mix it with protamine, and mix 2 mL of OMEM with DNA; then add OMEM-DNA to OMEM-protamine (protamine: DNA = 7:1, w/w), vortex for 1 min, and let stand at 25°C for 10 min. Add the above mixture to cell exosomes or milk exosomes and mix well. Place the sample in an ice-water bath, let stand for 30 min, then heat shock at 42°C for 90 s, and then let stand on ice (0°C) for 3 min. This is one cycle. Repeat 10 cycles of heat shock and add heparin to all samples (protamine: heparin = 1:3 w/w). Each sample was then divided into two equal parts, one of which was digested with nuclease DNaseI (final concentration 0.1 mg/mL) at 37°C overnight and then purified by Capto Core 700, and the other was directly purified by Capto Core 700 without enzyme digestion.

(2) NanoFCM detection of sample particles after loading;

(3) qPCR detection of loading effect;

(4) Verification of the cellular entry effect of exosomes after loading: The loaded samples were incubated with HepG2 cells. After 72 h of cell culture, fluorescent cells were photographed using Cytation5 (Biotek) to detect protein expression after the plasmid entered the cells.

The sample composition is shown in the table:

sample name	pRP-EGFP DNA (μg)	Protamine (μg)	Exosomes	Heparin (μg)	DNaseI
Exosomes	152.6	1068.2	2.27E+12	3205	-
Exosomes+DNaseI	152.6	1068.2	2.27E+12	3205	0.1mg/mL
Milk exosomes	30	210	1.05E+12	630	-
Milk exosomes + DNaseI	30	210	1.05E+12	630	0.1mg/mL

2. Results:

As shown in Figure 2-3 and Table 6, on average, about 20 plasmid copies were loaded per 100 exosome particles, and the loading efficiency of cell exosomes was higher. Due to the action of syncytin, the cell exosomes loaded with DNA were released into the cell after the fusion of the exosome membrane and the cell membrane, avoiding decomposition by lysosomes. After entering the nucleus for transcription and then translating EGFP, obvious green fluorescence was observed. No obvious green fluorescence was observed in milk exosomes loaded with the same amount of DNA, which may be because most of the DNA was decomposed by lysosomes after entering the cell.

Example 6: Loading the medium molecular weight plasmid pRP-Nanoluc (4929 bp) into cell or milk exosomes, and comparing the effects of different heat shock methods on the loading efficiency of exosomes from different sources during the loading process.

1. Methods:

(1) DNA loading: The DNA plasmid to be loaded is pRP-Nanoluc DNA (expression vector, target fragment is Nanoluc, plasmid size is 4929 bp), and it is loaded at a ratio of DNA: exosome = 3:1 (copy number: particle number). For each group of samples, 2 mL of OMEM + protamine was mixed and allowed to stand for 1 min, and 2 mL of OMEM + 3 μg of DNA was mixed and allowed to stand at 25°C for 1 min; then OMEM-DNA was added to OMEM + protamine (protamine: DNA = 7:1, w/w), then vortexed for 1 min, and allowed to stand at 25°C for 10 min. The OMEM-DNA-protamine mixture was added to the cell exosomes or milk exosomes, and mixed while adding. Two heat shock methods: samples treated at 60℃ were shaken at 120rpm for 1h in a 60℃ shaker, then placed in an ice bath (0℃) for 5min until the samples were completely cooled. This was one cycle and repeated 3 times. Samples treated at 42℃ were first placed in an ice-water bath (0℃) and allowed to stand for 30min, then heat-shocked at 42℃ for 90s, then placed in an ice-water bath (0℃) for 3min. This was one cycle and repeated 10 times. After heat shock, all samples were added with heparin and vortexed at 25℃ for 1min. After loading, all samples were added with heparin (protamine: heparin = 1: 3w/w). Each sample was then divided into two equal parts, one of which was digested with nuclease DNaseI at 37℃ overnight and purified by Capto Core 700, and the other was purified directly by Capto Core 700 without enzyme digestion.

(2) NanoFCM detection of sample particle count after loading;

(3) qPCR detection of loading effect;

(4) Verify the cellular entry effect of loaded exosomes: The loaded samples were incubated with HepG2 cells, and the Nanoluc enzyme activity in the cell supernatant was detected after 72 h of cell culture.

The sample composition is shown in the table:

sample name	Nanoluc-pp-Fc DNA (μg)	Protamine (μg)	Exosomes	Heparin (μg)	Benzonase
Exosomes 60℃	2.4	16.8	1.2E+11	50.4	-
Exosomes+DNaseI 60℃	2.4	16.8	1.2E+11	50.4	5U/mL
Exosomes 42℃	0.5	3.5	2.5E+10	10.5	-
Exosomes+DNaseI 42℃	0.5	3.5	2.5E+10	10.5	5U/mL
Milk exosomes 60℃	40	280	2.0E+12	840	-
Milk exosomes + DNaseI 60℃	40	280	2.0E+12	840	5U/mL
Milk exosomes 42℃	0.5	3.5	2.5E+10	10.5	-
Milk exosomes + DNaseI 42℃	0.5	3.5	2.5E+10	10.5	5U/mL

2. Results:

As shown in Figures 4-5 and Tables 7-8, on average, every 100 exosome particles can be loaded with up to 50 plasmid copies. Under 60°C heat shock conditions, the loading capacity of cell exosomes for pRP-Nanoluc plasmid is relatively high, while under 42°C heat shock conditions, the loading capacity of milk exosomes for pRP-Nanoluc plasmid is relatively high. After DNA enters the cells, Nanoluc protein with enzymatic activity is successfully expressed and secreted, and Nanoluc enzyme activity can be detected in the cell supernatant.

Table 7. Sample related data and loading amount after loading

sample name		Repeat 1	Repeat 2	Repeat 3	Mean	SD
Exosomes 60℃	68928	68821	69128	68959	156
Exosomes+DNaseI 60℃	20437	20929	20480	20615	272
Exosomes 42℃	13232	13230	13199	13220	19
Exosomes+DNaseI 42℃	2694	2737	2753	2728	31
Exosomes-Blank	281	272	260	271	11
Milk exosomes 60℃	768	774	783	775	8
Milk exosomes + DNaseI 60℃	569	582	550	567	16
Milk exosomes 42℃	39286	38216	39168	38890	587
Milk exosomes + DNaseI 42℃	13768	13689	13682	13713	48
Milk Exosomes-Blank	244	255	258	252	7

Table 8. Nanoluc enzyme activity data detected in the cell supernatant after incubation of DNA-loaded exosomes with cells

Example 7: The 42°C heat shock method was used to determine whether milk exosomes could be successfully loaded with the high molecular weight plasmid pSGLs-Nanoluc-pp-Fc (8929 bp).

1. Methods:

(1) DNA loading: The DNA plasmid was pSGLs-Nanoluc-pp-Fc DNA (expression vector, target fragment was Nanoluc, plasmid size was 8929 bp), and the DNA: exosomes were loaded at a ratio of 10:1 (copy number: particle number). For each sample group, 1 mL of OMEM medium was mixed with protamine, and 1 mL of OMEM medium was mixed with 8 μg of DNA. Then, OMEM-DNA was added to OMEM-protamine (protamine: DNA = 7:1, w/w), vortexed for 1 min, and allowed to stand at 25°C for 10 min. The OMEM-DNA-protamine mixture was added to 8.80E+10 cell exosomes. Then, heat shock was performed: 42°C, 120 rpm, heat shock for 1 h, and then the sample was placed in an ice water bath (0°C) and allowed to stand for 5 min until the sample was completely cooled. This was one cycle, and the cycle was repeated 3 times. After heat shock, all samples were added with heparin and vortexed for 1 min. After loading, all samples were added with heparin protamine: heparin = 1: 3 (w/w). Each sample was then divided into two parts, one of which was digested with Benzonase nuclease at 37°C overnight and purified by Capto Core 700, and the other was directly purified by Capto Core 700 without enzyme digestion;

(2) NanoFCM detection of sample particle count after loading;

(3) qPCR detection of loading effect;

(4) Verify the cellular entry effect of loaded exosomes: The loaded samples were incubated with HepG2 cells, and the Nanoluc enzyme activity in the cell supernatant was detected after 72 h of cell culture.

2. Results:

As shown in Figure 6 and Table 9, under the conditions of heat shock at 42°C for 1 h, followed by ice bath at 0°C for 5 min, and repeated heat shock three times, approximately 20 plasmid copies were loaded per 100 exosome particles, and the addition of nuclease could remove free DNA attached to the surface of exosomes.

Table 9. Sample data and loading amount after loading

Example 8: Loading high molecular weight plasmid pSGLs-Nanoluc-pp-Fc into cell- and milk-derived exosomes, and comparing the effects of different heat shock methods on the efficiency of DNA loading in exosomes from different sources during the loading process.

1. Methods:

(1) DNA loading: The DNA plasmid was pSGLs-Nanoluc-pp-Fc DNA (expression vector, target fragment was Nanoluc, plasmid size was 8929 bp), and the loading ratio was DNA: exosome = 10:1 (copy number: particle number). For each group of samples, 2 mL of OMEM medium was mixed with protamine, and 2 mL of OMEM medium was mixed with 6 μg of DNA; then OMEM-DNA was added to OMEM-protamine (protamine: DNA = 7:1, w/w), vortexed for 1 min, and allowed to stand at 25°C for 10 min. The OMEM-DNA-protamine mixture was added to 2.0E+11 cell exosomes or 2.0E+11 milk exosomes. The control group samples included all other samples except exosomes. Two heat shock methods: For samples treated at 60°C, oscillate at 120 rpm for 1 hour in a 60°C shaker, then place in an ice bath (0°C) for 5 minutes until the sample is completely cooled. This is one cycle and is repeated three times. For samples treated at 42°C, first place the sample in an ice-water mixed bath (0°C), let it stand for 30 minutes, then heat shock at 42°C for 90 seconds, then place in an ice-water mixed bath (0°C) for 3 minutes. This is one cycle and is repeated 10 times. After heat shock, all samples were vortexed with heparin for 1 minute. After loading, all samples were added with heparin protamine: heparin = 1:3 (w/w). Each sample was then divided into two equal parts, one of which was digested with Benzonase nuclease overnight at 37°C and purified by Capto Core 700, and the other was directly purified by Capto Core 700 without enzyme digestion.

(2) NanoFCM detection of sample particle count after loading;

(3) qPCR detection of loading effect;

(4) Verify the cellular entry effect of loaded exosomes: The loaded samples were incubated with HepG2 cells, and the Nanoluc enzyme activity in the cell supernatant was detected after 72 h of cell culture.

2. Results:

As shown in Figure 7 and Tables 10-11, approximately 2 to 40 plasmid copies were loaded per 100 exosome particles. The loading efficiency of cell exosomes was higher than that of milk exosomes, and the loading efficiency of cell exosomes at 60°C was better than that at 42°C.

sample name	Number of particles	qPCR Cq value (Mean±SD)	Plasmid copy number per 100 particles (Mean±SD)
Exosomes are not digested at 60℃	6.46E+10	10.9±1.45	38±7
Digestion of exosomes at 60°C	9.50E+10	10.98±1.15	28±3
Exosomes 60℃Blank	1.35E+11	22.57±0.21	0±2
Exosomes are not digested at 42℃	6.05E+10	17.65±0.87	3±6
Digestion of exosomes at 42°C	8.91E+10	19.9±0.30	0±5
Exosomes 42℃B1ank	5.70E+10	23.23±0.70	0±7
Milk exosomes undigested at 60℃	4.79E+10	16.57±0.42	7±3
Digestion of milk exosomes at 60°C	3.98E+10	16.77±0.70	2±6
Milk Exosomes 60℃Blank	4.68E+10	21.80±0.22	0±1
Milk exosomes undigested at 42°C	2.13E+10	18.21±0.19	4±2
Digestion of milk exosomes at 42°C	2.91E+10	15.66±0.72	5±5
Milk Exosomes 42℃Blank	1.04E+10	23.17±0.05	0±1

Table 10. Sample data and loading amount after loading

sample name		Repeat 1	Repeat 2	Repeat 3	Mean	SD
Exosomes are not digested at 60℃	3256	3606	3580	3481	195
Digestion of exosomes at 60°C	668	614	657	646	29
Exosomes 60℃Blank	twenty three	26	19	twenty three	4
Exosomes are not digested at 42℃	707	719	722	716	8
Digestion of exosomes at 42°C	239	239	281	253	twenty four
Exosomes 42℃Blank	15	18	16	16	2
Milk exosomes undigested at 60℃	137	129	148	138	10
Digestion of milk exosomes at 60°C	56	54	63	58	5
Milk Exosomes 60℃Blank	twenty one	twenty one	25	twenty two	2
Milk exosomes undigested at 42°C	98	88	96	94	5
Digestion of milk exosomes at 42°C	94	102	98	98	4
Milk Exosomes 42℃Blank	18	16	20	18	2

Table 11. Nanoluc enzyme activity data detected in the cell supernatant after incubation of DNA-loaded exosomes with cells

Example 9: In order to improve the efficiency of loading the high molecular weight plasmid pSGLs-Nanoluc-pp-Fc into exosomes and to express Nanoluc with enzymatic activity after entry into the cells, different heat shock temperatures and times during the loading process were compared.

1. Methods:

(1) DNA loading: The DNA plasmid to be loaded is pSGLs-Nanoluc-pp-Fc DNA (expression vector, target fragment is Nanoluc, plasmid size is 8929 bp), and the loading is performed according to DNA: exosome = 10:1 (copy number: number of particles). Take 1 mL of OMEM medium and mix it with protamine, then add DNA and mix well; then add DNA-protamine to the prepared pH 8.0 milk exosomes while mixing (protamine: DNA = 7:1, w/w). Preheat the water bath and shaker at different temperatures half an hour in advance. For samples that were heat-shocked once at different temperatures, the samples were placed on ice for 10 minutes, heated at the corresponding temperature for 9 minutes, and then placed on a preheated shaker at the corresponding temperature for 175 minutes at 120 rpm, and then placed on ice for 5 minutes; for samples that were heat-shocked 5 times at different temperatures, the samples were placed on ice for 10 minutes, heated at the corresponding temperature for 9 minutes, and then placed on a preheated shaker at the corresponding temperature for 31 minutes at 120 rpm, and then placed on ice for 5 minutes, and the heat shock was repeated 5 times; for samples that were heat-shocked 10 times at different temperatures, the samples were placed on ice for 10 minutes, heated at the corresponding temperature for 9 minutes, and then placed on a preheated shaker at the corresponding temperature for 13 minutes at 120 rpm, and then placed on ice for 5 minutes, and the heat shock was repeated 10 times; for samples that were heat-shocked 20 times at different temperatures, the samples were placed on ice for 10 minutes, heated at the corresponding temperature for 9 minutes, and then placed on ice for 5 minutes, and the heat shock was repeated 20 times. After the heat shock was completed, heparin was added to all samples and mixed by inversion. For all samples, 1 mM MgCl2 and a final concentration of 5 U/mL Benzonase were added and digested at 37°C overnight. The digested samples were treated with Capto Core700, and the flow-through was collected to remove free protamine-heparin-Benzonase.

The sample composition is shown in the table:

(2) NanoFCM detection of sample particle count after loading

(3) qPCR detection of loading effect

(4) Detect enzyme activity with an ELISA instrument to verify the expression of Rluc after entry into cells.

2. Results:

As shown in Table 12, heat shock of milk exosomes at 60°C for 20 times can increase the loading capacity of large molecular weight DNA.

sample name	Number of particles	qPCR Cq value (Mean±SD)	Plasmid copy number per 100 particles (Mean±SD)
25℃-heat shock once	1.15E+11	15.71±0.17	48±2
25℃-heat shock 5 times	1.24E+11	17.02±0.73	18±10
25℃-heat shock 10 times	7.86E+10	18.81±0.17	8±2
25℃-heat shock 20 times	8.49E+10	17.62±0.41	17±4
37℃-heat shock once	6.99E+10	16.97±0.20	33±3
37℃-heat shock 5 times	7.44E+10	17.35±0.13	23±1
37℃-heat shock 10 times	5.07E+10	16.96±0.27	45±3
37℃-heat shock 20 times	6.2E+10	16.94±0.24	37±3
42℃-heat shock once	7.91E+10	17.65±0.37	18±4
42℃-heat shock 5 times	6.39E+10	17.42±0.33	26±4
42℃-heat shock 10 times	8.27E+10	16.51±0.59	38±6
42℃-heat shock 20 times	4.92E+10	17.94±0.13	23±1
42℃-heat shock 3 times digestion	8.42E+10	17.84±0.48	15±4
60℃-heat shock once	8.57E+10	17.38±0.21	20±2
60℃-heat shock 5 times	6.48E+10	16.34±0.21	55±2
60℃-heat shock 10 times	8.00E+10	16.40±1.05	43±1
60℃-heat shock 20 times	7.38E+10	15.79±1.36	71±16

Table 12. qPCR results and loading amounts

Example 10: SGLs-Nanoluc-pp-Fc plasmid was loaded into cell exosomes to verify whether the addition of different auxiliary molecules (including ionizable lipid molecules or CaCl2) during the loading process could promote the loading of DNA by exosomes, and the effect of different ratios of protamine to DNA on the loading efficiency of exosomes under the action of the addition of auxiliary molecules.

1. Methods:

(1) DNA loading: The DNA plasmid was pSGLs-Nanoluc-pp-Fc DNA (expression vector, target fragment was Nanoluc, plasmid size was 8929 bp), and the ratio of DNA: exosomes = 20:1 (copy number: particle number) was used for loading, SM102/DLin-MC3-DMA: protamine: DNA = 3:1:1 (w/w) or 6:2:1 (w/w), and the pH of the cell exosomes was adjusted to 5.5 before loading. For the SM102 and DLin-MC3-DMA sample groups, 1 mL of OMEM medium was mixed with protamine and SM102/DLin-MC3-DMA in each group, and DNA was added to the mixture of OMEM medium-protamine-SM102/DLin-MC3-DMA, and then the mixture was added to the cell exosomes and mixed. That is, the sample group includes OMEM culture medium, protamine, SM102/DLin-MC3-DMA, DNA and cell exosomes, and the control group includes all other samples except exosomes. For the CaCl2 sample group, 1 mL of OMEM was mixed with protamine, and 2 mL of OMEM was mixed with 20 μg of DNA; then the DNA was added to OMEM+protamine (protamine: DNA = 7: 1, w/w). The above mixture was mixed with 1E+11 cell exosomes and then added with a final concentration of 20 mM CaCl ₂ . That is, the sample group includes OMEM culture medium, protamine, CaCl ₂ , DNA and cell exosomes, and the control group includes all other samples except exosomes. All samples were shaken at 120 rpm for 1 h in a 42°C shaker, and then ice-bathed (0°C) for 5 min until the samples were completely cooled. This was one cycle. After repeating the cycle 3 times, the pH of the solution was adjusted to neutral. Heparin (protamine: heparin = 1: 3 w/w) was added to all samples after loading. Each sample was then divided into two parts, one of which was digested with Benzonase nuclease at 37°C overnight and then purified by Capto Core700, and the other was directly purified by Capto Core700 without enzyme digestion.

(2) NanoFCM detects the number of sample particles after loading.

(3) qPCR detection of loading effect.

(4) Verify the cellular entry effect of loaded exosomes: The loaded samples were incubated with HepG2 cells, and the Nanoluc enzyme activity in the cell supernatant was detected after 72 h of cell culture.

2. Results:

As shown in Figure 8 and Tables 13-14, about 1 to 60 plasmid copies were loaded per 100 exosome particles, and the loading effect of the CaCl2 group was poor. Nanoluc enzyme activity was clearly detected in the cell supernatant of the SM102 and DLin-MC3-DMA groups. When the ratio of DNA to protamine was 1:2, the loading amount of the DLin-MC3-DMA group was the highest.

sample name	Number of particles	qPCR Cq value (Mean±SD)	Plasmid copy number per 100 particles (Mean±SD)
SM102 1∶1 experimental group before enzyme digestion	6.20E+09	13.93±0.2	171±13
SM102 1∶1 experimental group after enzyme digestion	1.65E+10	15.68±0.21	62±10

SM102 1∶1 control group before enzyme digestion	ND	22.66±0.11	ND
SM102 1∶1 control group after enzyme digestion	ND	20.44±0.23	ND
DLin-MC3-DMA 1∶1 experimental group before enzyme digestion	8.00E+09	20.39±0.05	2±13
DLin-MC3-DMA 1∶1 experimental group after enzyme digestion	1.01E+i0	20.42±0.17	2±10
DLin-MC3-DMA 1∶1 control group before enzyme digestion	ND	26.10±0.21	ND
DLin-MC3-DMA 1∶1 control group after enzyme digestion	ND	27.40±0.30	ND
SM102 1∶2 experimental group before enzyme digestion	3.60E+09	15.25±0.30	50±8
SM102 1∶2 experimental group after enzyme digestion	3.98E+09	17.30±0.29	30±6
SM102 1∶2 control group before enzyme digestion	ND	23.95±0.03	ND
SM102 1∶2 control group after enzyme digestion	ND	27.50±0.05	ND
DLin-MC3-DMA 1∶2 experimental group before enzyme digestion	4.80E+09	13.05±0.13	72±4
DLin-MC3-DMA 1∶2 experimental group after enzyme digestion	5.34E+09	14.80±0.23	62±8
DLin-MC3-DMA 1∶2 control group before enzyme digestion	ND	20.12±0.02	ND
DLin-MC3-DMA 1∶2 control group after enzyme digestion	ND	28.30±0.05	ND
CaCl2 1∶2 experimental group before enzyme digestion	1.82E+10	17.88±0.08	86±10
After enzyme digestion in the CaCl2 1∶2 experimental group	2.34E+10	20.17±0.64	13±2
CaCl2 1∶2 control group before enzyme digestion	1.02+07	26.75±0.30	0±5
CaCl2 1∶2 control group after enzyme digestion	1.16+07	29.30±0.19	0±2

Table 13. Sample related data and loading amount after loading

sample name	Repeat 1	Repeat 2	Repeat 3	Mean	SD
SM102 1∶1 experimental group before enzyme digestion	5328	5481	5452	5420	81
SM102 1∶1 experimental group after enzyme digestion	179	169	185	178	8
SM102 1∶1 control group before enzyme digestion	39	35	39	38	2
SM102 1∶1 control group after enzyme digestion	43	42	44	43	1
DLin-MC3-DMA 1∶1 experimental group before enzyme digestion	44	41	33	39	6
DLin-MC3-DMA 1∶1 experimental group after enzyme digestion	63	53	55	57	5
DLin-MC3-DMA 1∶1 control group before enzyme digestion	46	42	35	41	6
DLin-MC3-DMA 1∶1 control group after enzyme digestion	41	38	46	42	4
SM102 1∶2 experimental group before enzyme digestion	1628	1635	1612	1625	12
SM102 1∶2 experimental group after enzyme digestion	79	69	85	78	8
SM102 1∶2 control group before enzyme digestion	38	27	twenty four	30	7
SM102 1∶2 control group after enzyme digestion	36	45	63	48	14
DLin-MC3-DMA 1∶2 experimental group before enzyme digestion	21838	22110	21773	21907	179
DLin-MC3-DMA 1∶2 experimental group after enzyme digestion	1725	1704	1735	1721	16
DLin-MC3-DMA 1∶2 control group before enzyme digestion	36	39	34	36	3
DLin-MC3-DMA 1∶2 control group after enzyme digestion	35	38	25	33	7
CaCl 2 1∶2 experimental group before enzyme digestion	40	34	42	39	4
CaCl 2 1∶2 experimental group after enzyme digestion	42	31	31	35	6
CaCl 2 1∶2 control group before enzyme digestion	39	41	37	39	2
CaCl 2 1∶2 control group after enzyme digestion	32	35	29	32	3

*The ratios in the experimental and control groups are the ratios of DNA to protamine

Table 14. Nanoluc enzyme activity data detected in the cell supernatant after incubation of DNA-loaded exosomes with cells

Example 11: SGLs-Nanoluc-pp-Fc plasmid DNA is loaded into cell exosomes, and after entry into the cell, Nanoluc with enzymatic activity can be expressed; verify whether the cell entry ability of exosomes engineered to express syncytin-1 is improved.

1. Methods:

(1) DNA loading: The DNA plasmid to be loaded was pSGLs-Nanoluc-pp-Fc DNA (expression vector, target fragment was Nanoluc, plasmid size was 8929 bp), and the loading ratio was DNA: exosome = 10:1 (copy number: particle number). 293F exosomes and 293F-syncytium 1 exosomes were used for loading respectively. 2 mL of OMEM medium was mixed with protamine, and 2 mL of OMEM was mixed with 3 μg of DNA; then OMEM-DNA was added to OMEM-protamine (protamine: DNA = 7:1, w/w), vortexed for 1 min, and allowed to stand at 25°C for 10 min. The above mixture was added to 1E+11 293F exosomes or 1E+11 293F-syncytium 1 exosomes and mixed. The samples were placed at 42°C, 120 rpm for 1 hour, and then placed on ice (4°C) for 5 minutes. This was one cycle. After three cycles of heat shock, heparin was added to all samples, with protamine: heparin = 1:3 (w/w). Each sample was then divided into two parts, one of which was digested with nuclease DNaseI (final concentration 5U/mL) at 37°C overnight and then purified by Capto Core 700, and the other was directly purified by Capto Core 700 without enzyme digestion.

(2) NanoFCM detection of sample particle count after loading;

(3) qPCR detection of loading effect;

(4) qPCR verification of the cellular entry effect of exosomes after loading: The successfully loaded samples were incubated with HepG2 cells. After 6 h of culture, the culture medium was replaced to remove the free plasmids, and the cells were taken for qPCR.

(5) Detection of Nanoluc expression by microplate reader: Detect Nanoluc enzyme activity in the cell supernatant after 72 h of culture.

2. Results:

As shown in Figure 9 and Tables 15-17, there is basically no difference in the DNA loading capacity between engineered exosomes and 293F wild-type exosomes. The exosomes engineered to express Syncytin-1 have stronger cellular entry ability and higher Nanoluc expression level.

Table 15. Sample data and loading amount after loading

Table 16. Results of exosome cellular entry ability test after loading

sample name		Repeat 1	Repeat 2	Repeat 3	Mean		SD
293F exosomes + DNA + protamine + heparin	1766	1723	1760	1750	twenty three

293F exosomes + DNA + protamine + heparin + DNaseI	1201	1143	1107	1150	47
293F-syncytin 1 exosomes + DNA + protamine + heparin	3744	3720	3731	3732	12
293F-syncytin 1 exosomes + DNA + protamine + heparin + DNaseI	3266	3501	3602	3456	172
DNA	twenty one	15	17	18	3
Lipo3000	8433	8100	8814	8449	357

17. Nanoluc enzyme activity detection data in cell supernatant

Example 12: Engineered cell exosomes expressing single-chain integrin IntegrinαLβ2 (or single-chain IntegrinαDβ2) on the membrane surface can be loaded with pSGLs-Nanoluc-pp-Fc plasmid, specifically targeting inflamed vascular endothelial cells and glomerular podocytes, respectively.

1. Methods:

(1) DNA loading: The DNA plasmid was pSGLs-Nanoluc-pp-Fc DNA (expression vector, target fragment was Nanoluc, plasmid size was 8929 bp), and the DNA: exosome ratio was 10:1 (copy number: particle number). For each sample group, 2 mL of OMEM medium was mixed with protamine, and 2 mL of OMEM medium was mixed with 3 μg of DNA. Then, OMEM-DNA was added to OMEM+protamine (protamine: DNA = 7:1, w/w), vortexed for 1 min, and allowed to stand at 25°C for 10 min. The OMEM-DNA-protamine mixture was added to 1E+11 engineered cell exosomes. In the control group, DNA was loaded into wild-type 293F-exosomes; heat shock treatment was performed at 60°C, and the samples were shaken at 120 rpm for 1 h in a shaker at 60°C, and then placed in an ice bath (0°C) for 5 min until the samples were completely cooled. This was one cycle, and the cycles were repeated 3 times. After heat shock, all samples were added with heparin and vortexed for 1 min. After loading, all samples were added with heparin (protamine: heparin = 1: 3 w/w). Each sample was then digested with Benzonase nuclease at 37°C overnight, and the loaded exosomes were labeled with FITC-Integrinβ2 antibody and then purified by Capto Core700.

(2) NanoFCM detection of sample particle count after loading

(3) qPCR verification of loading effect

(4) Immunofluorescence verification of exosomes targeting ICAM-1 positive cells and glomerular podocytes: ICAM-1 positive cells and glomerular podocytes were inoculated at 2E5 cells per well in a 24-well plate with a slide placed in advance. After 24 hours, the slide was removed and gently washed 3 times with PBS. Fix with 500 μL paraformaldehyde for 10 minutes and wash 3 times with PBS for 5 minutes each time. Add 1 mL 5% BSA/PBS buffer to block at room temperature for 2 hours, wash 3 times with PBS for 5 minutes each time, and add unlabeled wild-type EVs at a cell to EV ratio of 1:30000 to continue blocking for 2 hours.

At the same time, take engineered EVs and wild-type EVs loaded with single-chain integrin IntegrinαLβ2 (or single-chain IntegrinαDβ2), adjust the concentration to about 4E10/ml, add an equal volume of PKH67 labeling buffer, mix well, add 10μ1 PKH67 dye, incubate at room temperature in the dark for 30 minutes, remove free dye through core700, add 5E9 PKH67-labeled engineered EVs and wild-type EVs loaded with single-chain integrin IntegrinαLβ2 (or single-chain IntegrinαDβ2) to the well of the slide, 1ml/slide, incubate overnight at 4℃ in the dark. Wash 3 times with PBST, 5min each time. Take out the slide and seal it with a sealing agent containing DAPI, and take pictures with a laser confocal microscope after the slide is dry.

(5) ICAM-1 positive cells and glomerular podocytes were seeded into 24-well plates at a density of 2E5 cells per well. After 24 h, wild-type EVs were added at a cell to EV ratio of 1:30,000 and blocked at room temperature for 2 h. 5E9 engineered EVs loaded with single-chain integrin IntegrinαLβ2 (or single-chain IntegrinαDβ2) and wild-type EVs (Blank and loaded with DNA) were added to the cells and incubated at 37°C for 72 h. The cells were then harvested for nanoluc enzyme activity detection.

Sequence information:

SEQ.1 (Syncytin1 (1-484aa) amino acid sequence):

SEQ.2 (Integrin αLβ2 amino acid sequence):

Sequence composition: SP(EWIF)-IntegrinαL(26-155)-(GGGGS)3-Integrinβ2(124-363)

-(GGGGS)3-IntegrinαL(338-612)-Fc-NPTN(Ig1-3)-TMD(EWIF)

SEQ.3 (Integrin αD β2 amino acid sequence):

Sequence composition: SP(EWIF)-αD(19-149)-(GGGGS)3-β2(124-363)

-(GGGGS)3-αD(338-612)-Fc-NPTN(Ig1-3)-TMD(EWIF)

2. Results:

As shown in Figures 10-11 and Tables 18-19, the amount of DNA loaded in engineered exosomes and wild-type exosomes was relatively consistent, with an average of 40-60 DNA copies per 100 exosome particles; immunofluorescence results showed that engineered exosomes loaded with single-chain IntegrinαLβ2 (or single-chain IntegrinαDβ2) (green fluorescence) could specifically bind to ICAM-1 positive cells and glomerular podocytes, and after further loading with pSGLs-Nanoluc-pp-Fc DNA plasmid, nanoluc enzyme activity detection results showed that targeted engineered EVs could bring more loaded plasmids into target cells and express more nanoLuc.

Table 18. Sample data and loading amount after loading

Table 19. Nanoluc enzyme activity detection data of cells

Claims (28)

1. A method for efficiently loading DNA into exosomes, wherein the exosomes are derived from mammalian cells or milk, characterized in that:

   The specific steps are:

   (1) thoroughly mixing the polycationic compacting agent with DNA;

   (2) adding the polycation-DNA complex to mammalian cell or milk-derived exosomes and mixing them evenly;

   (3) Complete DNA loading.
2. The method for efficient DNA loading according to claim 1 is characterized in that it can include step (4) of removing unloaded DNA and fragments.
3. The method for efficient DNA loading according to claim 2 is characterized in that: in the step (4), heparin is added to dissociate the unloaded DNA-protamine complex, and the method for removing the unloaded free DNA can be selected from: one or more of high-salt nuclease, DNAseI treatment, and Benzonase treatment, and the removal of nucleic acid fragments can be selected from anion exchange chromatography column purification and/or Capto Core700 chromatography column purification method.
4. The method for efficient DNA loading according to claim 1 is characterized in that it can include step (5) of verifying the loading effect using a qPCR method.
5. The method for efficient DNA loading according to claim 3 is characterized in that: in the step (1), the exosomes derived from mammalian cells or milk can be unmodified exosomes or engineered exosomes.
6. The method for efficient DNA loading according to claim 5, characterized in that: the engineered exosomes are exosomes modified with syncytin-1.
7. The method for efficient DNA loading according to claim 5 is characterized in that the engineered exosomes are exosomes modified with single-chain integrin Integrin αLβ2.
8. The method for efficient DNA loading according to claim 5 is characterized in that the engineered exosomes are exosomes modified with single-chain integrin Integrin αDβ2.
9. The method for efficiently loading DNA according to any one of claims 6 to 8, characterized in that in step (1), the ratio of DNA:exosomes is 3-20:1.
10. The method for efficiently loading DNA according to claim 1, characterized in that: in the step (2), the polycationic compression agent can be selected from one or more of protamine, polylysine, polyarginine, and polyethyleneimine.
11. The method for efficiently loading DNA according to claim 10 is characterized in that the polycationic compression agent can be protamine, and the ratio of protamine to DNA is 2-10:1.
12. The method for efficiently loading DNA according to claim 11 is characterized in that the polycationic compression agent can be protamine, and the ratio of protamine to DNA is 7:1.
13. The method for efficiently loading DNA according to claim 1, characterized in that: in the step (2), after the protamine is mixed with the exosomes and the DNA, the resulting precipitate can be separated from the supernatant by centrifugation.
14. The method for efficient DNA loading according to claim 1 is characterized in that: in the step (3), the DNA loading method can be an ultrasonic method and/or a heat shock/thermal cycling method.
15. The method for efficient DNA loading according to claim 12, characterized in that the number of cycles of the heat shock/thermal cycling loading method is 1-20 times.
16. The method for efficient DNA loading according to claim 15 is characterized in that the number of cycles of the heat shock/thermal cycling loading method is 10-20 times.
17. The method for efficient DNA loading according to claim 16 is characterized in that the heat shock temperature of the heat shock/thermal cycling loading method can be 25-60°C, and the cooling temperature can be 0°C or 4°C.
18. The method for efficiently loading DNA according to claim 17 is characterized in that the loading heat shock temperature of milk exosomes is 60°C, the cooling temperature is 0°C, the number of heat shocks is 20 times, and the loading heat shock temperature of cell exosomes is 60°C.
19. The method for efficient DNA loading according to claim 1 is characterized in that the exosomes are pretreated before DNA loading, such as adjusting the pH.
20. The method for efficient DNA loading according to claim 1 is characterized in that: in the step (3), the DNA loading into the exosome membrane is promoted by adding auxiliary molecules, and the auxiliary factors can be selected from one or both of SM102 and DLin-MC3-DMA.
21. The method for efficient DNA loading according to claim 20 is characterized in that the auxiliary molecule addition ratio is SM102/DLin-MC3-DMA: protamine: DNA = 3:1:1 (w/w) or 6:2:1 (w/w).
22. The method for efficiently loading DNA according to claim 21 is characterized in that when the auxiliary molecule is selected as DLin-MC3-DMA, the ratio of DNA to protamine is 1:2.
23. The method for efficiently loading DNA according to claim 1: the size of the loaded DNA is 1.9kbp-9kbp.
24. An exosome efficiently loaded with DNA prepared according to the method of claims 1-23, wherein the exosome is derived from mammalian cells or milk.
25. A use of the exosomes described in claim 24 in preparing a drug.
26. The use according to claim 25 is characterized in that the drug is a drug used as a non-viral vector for in vivo DNA delivery in gene therapy.
27. A medicine comprising the exosomes according to claim 24.
28. A gene therapy method comprising administering the drug according to claim 27.

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