CN117025523A

CN117025523A - Fluorine engineering exosome with high-efficiency intracellular delivery and application thereof

Info

Publication number: CN117025523A
Application number: CN202310210444.7A
Authority: CN
Inventors: 马胜男; 王世豪; 宋蕾; 秦贵军
Original assignee: First Affiliated Hospital of Zhengzhou University
Current assignee: First Affiliated Hospital of Zhengzhou University
Priority date: 2023-03-07
Filing date: 2023-03-07
Publication date: 2023-11-10

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to a fluorine engineering exosome with high-efficiency intracellular delivery and application thereof. In the application, the inventor combines exosomes with FPG3 by utilizing electrostatic action and hydrophobic action to prepare and obtain fluorine engineering exosomes exo@FPG3, which are used for improving the cell uptake efficiency of exosomes and promoting the escape probability of exosomes from lysosomes. On the basis, the technical aim of improving the intracellular delivery efficiency of exosomes is achieved, and research and application of promoting tissue repair such as cell tube formation and cell migration can be better met. Preliminary experimental results show that compared with exo@PG3 prepared by the action of peptide dendrimers which are not functionalized by fluorine and exo, the exo fluorinated modification has better technical effects of improving the cell uptake efficiency and enhancing related functions.

Description

Fluorine engineering exosome with high-efficiency intracellular delivery and application thereof

Technical Field

The application belongs to the technical field of biomedicine, and particularly relates to a fluorine engineering exosome with high-efficiency intracellular delivery and application thereof.

Background

In the research of cell biology, exosomes generally refer to vesicular organelles with the diameter of 30-150 nm, and are formed by fusing a multi-vesicle outer membrane formed by invagination of intracellular lysosome particles with a cell membrane and then releasing the multi-vesicle outer membrane into extracellular matrixes, wherein the multi-vesicle outer membrane is rich in miRNAs, mRNA, proteins and the like. Since exosomes have the function of carrying genetic material and protein transfer, research has considered that exosomes play an important role in cellular communication and are also considered to be ideal drug delivery vehicles. In addition, the exosomes secreted by the mesenchymal stem cells have good biological activity in the aspects of inflammation, chronic wound surface, myocardial infarction and the like, so that the exosomes have good application potential in the aspects of treatment of tissue engineering or drug delivery.

However, in practical applications, endocytosis of exosomes is directly affected by the interaction of exosomes with cell membranes and the endocytic capacity of recipient cells; in addition, approximately 60% of the exosomes taken up by the cells are transported to lysosomes, which in turn digest the content of the exosomes. It is thought that the internalization process of exosomes may be regulated by nonspecific interactions or by receptor-mediated specific means. However, there is currently limited research concerning the difference in endocytic capacity, the effect of exosome surface modification on intracellular trafficking, the effect that exosome surface modification may increase lysosomal escape, and the like. In short, the research and adjustment of the related properties of the exosomes are the precondition guarantee for achieving the application effect of the exosomes before the exosomes are used as an effective therapeutic tool.

Disclosure of Invention

The application aims to provide a fluorine engineering exosome capable of being delivered in an intracellular efficient manner by adopting a fluorinated peptide dendrimer to carry out surface modification on the exosome, which is used for enhancing the intracellular uptake efficiency of the exosome and further realizing related biological functions.

The technical scheme adopted by the application is described in detail below.

An intracellular delivery fluorine engineered exosome is prepared by binding fluoride to the exosome surface by electrostatic and hydrophobic interactions; the preparation method comprises the following steps:

(one) preparation of exosomes

Taking adipose-derived mesenchymal stem cells (AMSCs) as an example, the specific preparation method is as follows:

first, AMSCs were grown in DMEM containing 10% fetal bovine serum to complete fusion;

after the end of the culture, the cultures were collected and isolated by ultracentrifugation to obtain the exosomes secreted by AMSCs, with the following specific operating parameters (all performed at 4 ℃):

the medium containing AMSCs was centrifuged at 2000 g for 30 min to remove dead cells and debris;

carefully transferring the supernatant into a new centrifuge tube, and centrifuging 10000 g for 45 min to separate and obtain large vesicles;

filtering the obtained large vesicle with 0.45 μm filter, centrifuging the obtained supernatant 100000 g for 70 min, and collecting precipitate as exosomes;

further, after resuspension of the obtained exosomes with pre-chilled PBS, centrifugation is performed again for 100000 g for 70 min for purification;

(II) with FPG3 (i.e., surface modification of exosomes with fluorinated peptide dendrimers FPG 3)

Uniformly mixing a certain mass of fluorinated peptide dendrimer FPG3 with the exosome in the step (I), and incubating for 15-30 min (specifically for example, 20 min) at room temperature (15-30 ℃) to obtain a fluorine engineering exosome (exo@FPG3);

fluorinated peptide dendrimer FPG3: exosome=0.25 to 20 (specifically, for example, 0.5, 1, 2.5, 5, 10, 20, etc.);

the fluorine engineering exosome exo@FPG3 is formed by inserting FPG3 into a phospholipid bilayer of a negatively charged exosome by utilizing electrostatic action and hydrophobic action, namely, modifying the surface of the exosome by utilizing the FPG3;

the fluorinated peptide dendrimer FPG3 refers to a fluorocarbon chain modified lysine dendrimer, wherein the dendrimer is a low-algebraic peptide dendrimer, and the algebra of the low-algebraic peptide dendrimer is 1 generation, 2 generation or 3 generation;

the fluorocarbon chain modified lysine dendrimer is prepared by reacting a fluorocarbon chain with the lysine dendrimer; when the synthesized fluorocarbon chain modified lysine dendrimer is prepared, the mole number of the reacted fluorocarbon chain is more than 0, and the grafting rate is more than 10 percent and less than 80 percent;

the number n of carbon atoms of the fluorocarbon chain is more than or equal to 0 and less than or equal to 8;

the molecular weight of the fluorinated peptide dendrimer FPG3 is 2000-20000;

the surface potential of the prepared fluorine-engineered exosome (exo@FPG3) is preferably about 9.6+/-0.6 mV.

The use of the intracellular delivered fluorine engineered exosomes (exo@fpg3) in cell biology research or tissue repair for enhancing cell uptake efficiency research or use, or for cell tube formation and cell migration research or use (e.g., cell migration and tube formation is a direct manifestation of cell function in diabetic foot chronic wound repair).

The fluorinated peptide dendrimer FPG3 takes POSS (polyhedral oligomeric silsesquioxane) synthesized by (3-aminopropyl) triethoxy-silane as a core, takes lysine protected by tert-butoxycarbonyl as a structural unit, and continuously obtains peptide dendrimers with different algebra through amide condensation and elution protection; finally, carrying out fluoridation modification on the peptide dendrimer; the specific preparation method is referred as follows:

(one) preparation of POSS.8HCl

Firstly, dropwise adding 45-mL concentrated hydrochloric acid into 500-mL absolute methanol at 50 ℃;

then, heating to 90 ℃, continuously dropwise adding 90 mmol (3-aminopropyl) triethoxy-silane, and continuously stirring and reacting for 24 hours at the constant temperature of 90 ℃ after the completion of dropwise adding;

after the reaction is finished, the reaction solution is concentrated to 250 mL and poured into 300 mL tetrahydrofuran, the mixture is centrifuged for 3 minutes at 2500 rpm, and the precipitate is washed and dried to obtain white solid which is POSS.8HCl;

(II) preparation of PG1

2 g of POSS.8HCl prepared in the step (one), 6.93 g of Boc-Lys (Boc) -OH, 7.71 g of HBTU and 2.27 g of HOBT are dissolved together in 60 mL of DMF under the protection of nitrogen atmosphere, and after the dissolution is completed, 9.0 mL of DIPEA is added under the ice bath condition;

continuously stirring and reacting for 48 hours at the temperature of 30 ℃;

after the reaction, 250 mL chloroform was added to dilute the mixture, and the organic phase was washed with saturated NaCl solution and saturated NaHCO in this order ₃ Solution and 1M HCl wash;

after washing, drying the organic phase, concentrating properly, adding the organic phase into acetonitrile, and recrystallizing to obtain white precipitate;

separating the obtained white precipitate, washing and drying to obtain PG1 with amino protecting group (tert-butyl ester);

adding TFA to the PG1 with amino protecting group (tert-butyl ester) to react to remove the protecting group, so as to prepare PG1;

(III) preparation of PG2

2 g of PG1 prepared in the step (two), 8.7 g of Boc-Lys (Boc) -OH, 9.6 g of HBTU and 2.7 g of HOBT are dissolved in 60 mL of DMF under the protection of nitrogen atmosphere, and after the dissolution is completed, 11.1 mL of DIPEA is added under the ice bath condition;

continuously stirring and reacting for 48 hours at the temperature of 30 ℃;

separating the obtained white precipitate, washing and drying to obtain PG2 with amino protecting group (tert-butyl ester);

adding TFA to the PG2 with the amino protecting group (tert-butyl ester) to react to remove the protecting group, so as to prepare PG2;

(IV) preparation of PG3

2.2 g of PG2 prepared in the step (III), 8.3 g of Boc-Lys (Boc) -OH, 9.1 g of HBTU and 2.59 g of HOBT are dissolved together in 55 mL of DMF under the protection of nitrogen atmosphere, and after the dissolution is completed, 10.57 mL of DIPEA is added under the ice bath condition;

continuously stirring and reacting for 48 hours at the temperature of 30 ℃;

after the reaction, 250 mL chloroform was added to dilute the mixture, and the organic phase was washed with saturated NaCl solution and saturated NaHCO in this order ₃ Solution and HCl wash of 1M;

separating the obtained white precipitate, washing and drying to obtain PG3 with amino protecting group (tert-butyl ester);

adding TFA to the PG3 with the amino protecting group (tert-butyl ester) to react to remove the protecting group, so as to prepare PG3;

(V) preparation of fluorinated peptide dendrimer FPG3

Weighing PG3 prepared in the step (four) of 1 g, placing the PG3 in a reaction vessel under the protection of nitrogen atmosphere, adding 15 mL of anhydrous methanol, and stirring for dissolution; after dissolution is completed, slowly adding under the ice water bath condition: 1.07 mL of a mixture of heptafluorobutyric anhydride, 0.78 mL of triethylamine and 1 mL of anhydrous methanol;

stirring and reacting for 48 hours at the temperature of 30 ℃;

after the reaction is finished, concentrating the reaction liquid, adding the concentrated reaction liquid into anhydrous diethyl ether for precipitation, dissolving the precipitate with deionized water, dialyzing the precipitate in water for 3 days by using a biotechnology CE membrane dialysis bag with the molecular weight cut-off of 100-500 Da, and freeze-drying to obtain the FPG3.

In the prior art, uptake and intracellular transport are important guarantees for improving the application effect of exosomes, and for this reason, researchers have performed some modification exploration on exosomes. For example, strategies have been proposed to increase exosome versus lysosomal escape. Based on the strategy, one modification method is as follows: the exosomes are coated with a cationic lipid and a pH sensitive fusion peptide, which increases the disruption of the lysosomal membrane, resulting in an efficient cytoplasmic release of the exosome content. In another modification, the exosomes are encapsulated by arginine cell penetrating peptides to induce active micro-pinocytosis, thereby releasing the exosomes more efficiently into the cytosol. However, the modification method still has a room for further technical improvement due to the complexity of the modification method, the release effect of the exosome content and the like.

In the prior art, peptide dendrimers are novel biomedical polymer materials developed in recent years, and have unique properties such as excellent biocompatibility, biodegradability and the like, so that the peptide dendrimers have attractive prospects in biomedical applications. While there are many experimental studies on compounds with stronger intracellular delivery efficiency of genes, drugs, proteins, etc. than conventional alkane chains, membrane-penetrating peptides and lipids, there are no more studies on the use of fluorides (fluorinated peptide dendrimers) for engineering exosomes, nor on the use of fluorine engineering exosomes for cellular functions.

In the application, the inventor adopts a new transformation strategy, namely, the exosome and FPG3 are combined by utilizing electrostatic action and hydrophobic action to prepare and obtain the fluorine engineering exosome (exo@FPG3), which is used for improving the cell uptake efficiency of the exosome and improving the escape probability of the endolysosome. On the basis, the technical aim of improving the intracellular delivery efficiency of exosomes is achieved, and further research and application of promoting cell tube formation and cell migration can be better met. Preliminary experimental results show that compared with exo@PG3 prepared by the action of peptide dendrimers which are not functionalized by fluorine and exo, the exo fluorinated modification has better technical effects of improving the cell uptake efficiency and enhancing related functions.

In general, the present application modifies exosomes by: firstly, the peptide dendrimer with a large number of amino groups on the surface is subjected to incomplete fluorination, and then the fluorinated peptide dendrimer is utilized to modify the surface of the modified exosome. In the modification process, the charge distribution on the surface of the exosome can be reversed through partial amino groups on the surface of the peptide dendrimer, and meanwhile, fluorocarbon chains are inserted into a phospholipid bilayer of the exosome by utilizing a stronger hydrophobic effect, so that the interaction between the exosome and cells is influenced, the escape capacity of the exosome is enhanced, the delivery efficiency of the exosome to cytoplasm is finally improved, and the cell function is regulated.

Drawings

FIG. 1 shows the molecular formula of 3 rd generation fluorinated peptide dendrimer FPG3; in the figure, the carbon number n of the fluorocarbon chain is more than or equal to 0 and less than or equal to 8;

FIG. 2 is a synthetic scheme for fluorinated peptide dendrimers; wherein:

(A) A synthetic roadmap for a third generation poly (L-lysine) dendrimer (PG 3);

(B) A synthetic route pattern of a fluorinated modified third generation poly (L) -lysine dendrimer (FPG 3);

FIG. 3 is a representation of PG3; wherein:

(A) Nuclear magnetic hydrogen spectrum of PG3 dissolved in deuterated water;

(B) Mass spectrometry results for PG3;

FIG. 4 is a nuclear magnetic fluoride spectrum of FPG3 dissolved in deuterated water;

FIG. 5 shows the identification of ADSCs and exosomes; wherein:

(A) Detecting rat ADSCs surface markers by a flow cytometer;

(B) TEM image of exosomes, scale: 100 nm;

(C) Particle size of exosomes;

(D) Analyzing the surface markers of the exosomes by flow cytometry;

FIG. 6 shows the surface potential of different engineered modified exosomes, wherein:

(A) Different mass ratios of FPG3 engineering modify the potential change of exosomes;

(B) Engineering the potential change of the modified exosomes by PG3 with different mass ratios;

FIG. 7 is a graph showing the evaluation of HUVECs uptake efficiency on different exosomes using flow cytometry; wherein:

(A) Average fluorescence intensity of HUVECs after incubation with exosomes, exo@FPG3 (w/w, 0.5 and 5) and exo@PG3 (w/w, 0.5 and 5) in medium containing 10% fetal bovine serum of 2 h;

(B) HUVECs with exosomes, exo@FPG3 (w/w, 0.5 and 5) and exo@PG3 (w/w, 0.5 and 5) after incubation of 2 h in medium without fetal bovine serum;

FIG. 8 is a graph showing the process of cell entry after various incubation times of exosomes, exo@FPG3 and exo@PG3 with HUVECs using CLSM; under the color view: blue fluorescence indicates DAPI marked cell nucleus, green fluorescence represents DiO marked exosome, red fluorescence is Lysotracker red stained lysosome, and the specific ruler is 25 μm;

fig. 9 is tubule formation of HUVECs cells, wherein:

(A) The exosomes, exo@fpg3, exo@pg3 or PBS were imaged with the tubules after incubation of HUVECs 3.5 h and 6.5 h;

(B) Quantitative analysis was performed on the total number of HUVECs vials after incubation of 3.5 h and 6.5 h;

FIG. 10 is a scratch image of HUVECs cells; wherein:

(A) Effects of different engineered exosomes culture media on HUVECs migration;

(B) Quantitative analysis of migration efficiency of HUVEC.

Detailed Description

The application is further illustrated by the following description in conjunction with the accompanying drawings and specific embodiments. Before describing the specific embodiments, the following description will briefly explain some experimental contexts in the following embodiments.

Biological material:

female SD rats (6 weeks old, 200 g), common and commonly used animal materials in existing biologically relevant experiments, available from public sources;

HUVECs were derived from ATCC cell banks, available from Semer biotechnology Co., ltd;

main reagents and instrument equipment:

(3-aminopropyl) triethoxy-silane, a product of Shanghai alaa Ding Shiji limited;

Boc-Lys (Boc) -OH ((S) -2, 6-di-tert-butoxycarbonylaminohexanoic acid), HBTU (O-benzotriazol-tetramethyluronium hexafluorophosphate), HOBT (1-hydroxybenzotriazole), DIPEA (N, N-diisopropylethylamine), and the like, all purchased from Shanghai Jier Biochemical reagents;

nuclear magnetic AV-400, bruker Inc. of America.

Example 1

Taking the specific fluorinated peptide dendrimer as an example, the preparation process of the fluorinated peptide dendrimer according to the present application is outlined below.

The method adopted in this example (synthetic route diagram is shown in fig. 2): POSS (polyhedral oligomeric silsesquioxane) synthesized by (3-aminopropyl) triethoxy-silane is taken as a core, lysine protected by tert-butoxycarbonyl is taken as a structural unit, and different generations of peptide dendrimers are obtained through continuous amide condensation and elution protection (figure 2A); finally, the peptide dendrimer is subjected to fluoridation modification (the synthetic route is shown in figure 2B, and the specific FPG3 product structure is shown in figure 1). The specific operation is referred to as follows.

(one) preparation of POSS.8HCl

Firstly, adding 500 mL of absolute methanol into a three-mouth bottle with the capacity of 1L, and dripping 45 mL of concentrated hydrochloric acid into the three-mouth bottle by using a dropping funnel at 50 ℃;

then, heating to 90 ℃, continuously dripping 90 mmol (3-aminopropyl) triethoxy-silane into the reaction liquid, and continuously stirring and reacting for 24 hours at the constant temperature of 90 ℃;

after the reaction was completed, the reaction solution was concentrated to 250 mL by a rotary evaporator, and then, the concentrated reaction solution was poured into 300 mL tetrahydrofuran, centrifuged at 2500 rpm for 3 minutes, and the precipitate was washed and dried to obtain a white solid, namely poss.8hcl.

(II) preparation of PG1

continuously stirring and reacting for 48 hours at the temperature of 30 ℃;

after the reaction, 250 mL chloroform was added to dilute the mixture, andthe organic phase was washed successively with saturated NaCl solution, saturated NaHCO ₃ Solution and HCl wash of 1M;

after the washing was completed, the organic phase was washed with anhydrous MgSO ₄ Drying overnight, properly concentrating after drying, adding the concentrated organic phase into acetonitrile, and recrystallizing to obtain white precipitate; centrifuging after crystallization, washing the precipitate obtained by centrifuging with acetonitrile for at least two times, and drying in a vacuum drying oven to obtain PG1 with amino protecting group (tert-butyl ester).

To the PG1 with amino protecting group (tert-butyl ester) obtained above, 10 times equivalent of TFA was added to the unit of measurement of the amino protecting group tert-butyl ester of PG1 to remove the protecting group; namely:

after dissolving 2 g amino-protected PG1-Boc in 6 mL anhydrous dichloromethane, 7 mL TFA was added and stirred at ambient temperature (about 25 ℃ C.) for 10 hours to remove tert-butyl ester;

after the reaction is finished, the reaction liquid is concentrated by a rotary evaporator, the concentrated reaction liquid is added into anhydrous diethyl ether for precipitation, the precipitation is centrifuged and washed by diethyl ether, and the obtained white solid product is PG1 after being washed clean and dried in a vacuum drying oven.

(III) preparation of PG2

continuously stirring and reacting for 48 hours at the temperature of 30 ℃;

after the washing was completed, the organic phase was washed with anhydrous MgSO ₄ Drying overnight, properly concentrating after drying, adding the concentrated organic phase into acetonitrile, and recrystallizing to obtain white precipitate; after crystallization is completed, centrifuging, washing the precipitate obtained by centrifugation with acetonitrile at least twice, and standingDrying in a vacuum drying oven, and obtaining the PG2 with an amino protecting group (tert-butyl ester) after drying.

To the PG2 with amino protecting group (tert-butyl ester) obtained above, 10 times equivalent of TFA was added to the unit of measurement of the amino protecting group tert-butyl ester of PG2 to remove the protecting group; namely:

to 3 g amino-protected PG2-Boc was added 7 mL of anhydrous dichloromethane and 10.30 of TFA mL, and reacted at room temperature (about 25 ℃ C.) for 10 hours to remove tert-butyl ester;

after the reaction is finished, the reaction liquid is concentrated by a rotary evaporator, the concentrated reaction liquid is added into anhydrous diethyl ether for precipitation, the precipitation is centrifuged and washed by diethyl ether, and the obtained white solid product is PG2 after being dried in a vacuum drying oven.

(IV) preparation of PG3

continuously stirring and reacting for 48 hours at the temperature of 30 ℃;

after the washing was completed, the organic phase was washed with anhydrous MgSO ₄ Drying overnight, properly concentrating after drying, adding acetonitrile into the concentrated organic phase, and recrystallizing to obtain white precipitate; and after crystallization is finished, centrifuging, washing the precipitate obtained by centrifugation with acetonitrile for at least two times, and then placing the precipitate in a vacuum drying oven for drying treatment, wherein the PG3 with an amino protecting group (tertiary butyl ester) is obtained after the drying is finished.

To the PG3 with amino protecting group (tert-butyl ester) obtained above, 10 times equivalent of TFA was added to the unit of measurement of the amino protecting group tert-butyl ester of PG3 for reaction to remove the protecting group; namely:

to 4 g amino-protected PG3-Boc was added 8 mL of anhydrous dichloromethane and 13.6 mL of TFA, and the mixture was stirred at room temperature (about 25 ℃ C.) for 12 hours to remove tert-butyl ester;

after the reaction is finished, the reaction liquid is concentrated by a rotary evaporator, the concentrated reaction liquid is added into anhydrous diethyl ether for precipitation, the precipitation is centrifuged and washed by diethyl ether, and the obtained white solid product is PG3 after being dried in a vacuum drying oven.

The PG3 obtained was further dissolved with deionized water and dialyzed in water for 2 days to remove trifluoroacetate, and the purified PG3 was obtained after lyophilization, and the structure of the obtained compound was characterized by nuclear magnetism and mass spectrometry.

Preparation of PG3 at D ₂ In O ¹ The H nuclear magnetic resonance spectrum is shown in fig. 3A, and analysis can be seen: in deuterated water (solvent, δ=4.79 ppm), it can be seen that δ (a) =0.45 ppm on the side chain of the siloxane, the hydrogen δ0 (e) and δ1 (f) of the α -carbon on the lysine backbone are 4.20-4.09, 3.89-3.78 ppm, where the integrated area ratio of δ (a) to the hydrogen δ (e, f) of the α -carbon corresponds to the number ratio of H of 2/7, whereas the very strong methyl peak in δ (b) disappears due to deaminated protecting groups. The ratio of the integrated area of delta (a)/delta (b)/delta (c-d) to the number of H of the three after deprotection is 1/22/8. Based on these results, nuclear magnetic resonance hydrogen spectrum confirmed that tertiary Ding Yangzhi on amino group was successfully stripped off, and third generation poly (L-lysine) dendrimer PG3 was obtained.

Meanwhile, the molecular weight of the third generation poly (L-lysine) dendrimer PG3 after removal of the amino group of t Ding Yangzhi was examined by mass spectrometry, and the theoretical molecular weight thereof was 8059. As shown in FIG. 3B, a sodium peak [ M+Na ] formed by PG3 as 8082.38 can be seen in the mass spectrum] ⁺ Again, the successful preparation of the third generation poly (L-lysine) dendrimer PG3 was verified.

(V) preparation of fluorinated peptide dendrimers

Weighing PG3 prepared in step (four) of 1 g, placing in a bottle (100 mL specification) with a branch mouth, adding 15 mL anhydrous methanol under the protection of nitrogen atmosphere, and stirring for dissolution; after dissolution is completed, slowly adding under the ice water bath condition: 1.07 mL of a mixture of heptafluorobutyric anhydride, 0.78 mL of triethylamine and 1 mL of anhydrous methanol;

stirring and reacting for 48 hours at the temperature of 30 ℃;

after the reaction is finished, carrying out rotary evaporation concentration on the reaction liquid, adding the reaction liquid into anhydrous diethyl ether for precipitation after the concentration is finished, dissolving the precipitate with deionized water, dialyzing the precipitate in water for 3 days by using a biotechnology CE membrane (cellulose ester membrane) dialysis bag (CE) with the molecular weight cut-off of 100-500 Da, obtaining FPG3 after freeze drying, and characterizing the structure of the obtained compound through nuclear magnetic resonance fluorine spectrum, wherein the yield is 85%.

Preparation of the FPG3 at D ₂ In O ¹⁹ The F nuclear magnetic resonance spectrum is shown in fig. 4, and analysis can be seen: FPG3 ¹⁹ Peaks in the F NMR spectrum at 81.02 ppm, 118.50 ppm and 127.66 ppm were derived from the modified fluorocarbon chain on PG3.

Further, by adopting an ninhydrin color development method, the nonfluorinated amino acid on the lysine of the third-generation FPG3 is quantified, and the calculation result shows that the fluorination degree of the third-generation FPG3 is 22.64+/-0.04%.

Example 2

The modification strategy of the fluorine engineering exosome for intracellular high-efficiency delivery provided by the application is to carry out fluorine engineering modification on the exosome by a co-incubation mode with fluoride (fluorinated peptide dendrimer), and the specific preparation process is outlined below.

(one) extracting exosomes

Taking as an example exosomes secreted by adipose-derived mesenchymal stem cells (AMSCs), specific preparation procedures are referenced below.

First, referring to the prior art, adipose mesenchymal stem cells (ADSCs) derived from adult female SD rats were routinely extracted and examined for cell surface antibody types, and after confirming the same, AMSCs were grown in DMEM containing 10% fetal bovine serum in culture until complete fusion.

When the type of ADSCs cell surface antibody is detected and confirmed, the specific method is referred to as follows:

after adding equal amounts of cell suspension (reference 20. Mu.L) to each of the 5 EP tubes, FITC or PE labeled CD90, CD73, CD31 and CD45 antibodies (1. Mu.L) were added, respectively, and incubated and stained at room temperature for 15 minutes in the absence of light;

meanwhile, using FITC or PE marked IgG antibody as control, incubating the cells for the same time;

after incubation is completed, the fluorescently labeled cells are detected by flow cytometry to determine the type of cell surface antibody.

The results of the type detection of the antibodies are shown in fig. 5A, and it can be seen that: in the ADSCs surface marker, the cell number expressing the CD90 antigen accounts for 96.1 percent, and the cell number expressing the CD73 antigen accounts for 87.9 percent; while the number of cells expressing CD31 and CD45 was negligible relative to the PE isotype control. This result shows that the collection extract is indeed ADSCs, which can be used for further culture, and for the extraction of exosomes in the cell supernatant.

After the end of the culture, the cultures were collected and isolated by ultracentrifugation to obtain the exosomes secreted by AMSCs, the specific operating parameters (all carried out at 4 ℃) being referred to as follows:

centrifuging the AMSCs culture for 30 min at 2000 and g to remove dead cells and fragments;

subsequently, filtering the obtained large vesicle by a 0.45 μm filter, centrifuging the obtained supernatant 100000 g for 70 min, and obtaining a precipitate which is the preliminarily obtained exosome;

further, after resuspension of the resulting exosomes with pre-chilled PBS, centrifugation was performed again for 100000 g for 70 min for purification. After purification, 0.5 mL of PBS was added for re-suspension and the exosome protein concentration was determined to be 300 μg/mL by BCA kit. And further sampling the prepared exosomes, and then carrying out electron microscope observation and various physicochemical identifications. The specific case is described below.

(1) Morphological features

Morphology and size characteristics of exosomes were observed and detected with Transmission Electron Microscopy (TEM) and DLS, respectively. The results are shown in fig. 5B and 5C, and it can be seen that:

from TEM images of exosomes, it can be seen that exosomes have very distinct membrane boundaries, with dimensions of around 100 nm, in a typical saucer or cup-like structure. The measurement result of the particle size of the exosome shows that the particle size is about 83.68 +/-17.63 nm, which is consistent with the size result of the TEM image.

(2) Characterization of proteins

Exosome specific proteins CD9 and CD63 were detected by NanoFCM (N30E). The results are shown in fig. 5D, and it can be seen that: CD9 and CD63 molecules positive for the sample surface antigen (CD 9 and CD63 are typical surface markers for exosomes) were both successfully detected, indicating that the exosomes secreted by ADSCs have been successfully extracted.

(II) binding to FPG3

The fluorinated peptide dendrimer FPG3 solution prepared in example 1 and the exosome prepared in the step (one) are uniformly mixed according to the same volume, and then incubated for 20 min at room temperature (about 25 ℃) to prepare the fluorine engineering exosome (the final preparation product is called exo@FPG3).

While using the non-fluorinated modified PG3 prepared in example 1 as a control (i.e., the final product of "(fourth) preparation PG3" of example 1), a non-fluorine-engineered exosome was prepared (the final preparation product was referred to as exo@PG3).

In the experimental process, in order to investigate the optimal ratio, different ratios of FPG3 (simultaneously, PG3 prepared in example 1 is used as a control) and exosomes were designed according to mass ratios of 0.5, 1, 2.5, 5, 10 and 20, respectively, so as to prepare engineering exosomes exo@FPG3 and exo@PG3 with different performance parameters.

The surface charges of the different products were detected by potentiometers and the results are shown in fig. 6. Analysis can be seen:

the initial zeta potential of the exosomes was-14.2±2.1 mV prior to addition of FPG3; with the increase of FPG3, the zeta potential of exo@FPG3 is changed from negative to positive, and the charge gradually becomes stable when the ratio is increased to 5, 10 and 20 times; wherein the Zeta potential of exo@fpg3 is 9.6±0.6 mV at a mass ratio of 5, and the engineered exosome surface potential at this ratio is relatively similar to the result of the adjustment potential in the prior art of adjusting exosome surface charge with an ionic amphiphilic compound (in the prior art, the exosome surface charge is adjusted to 8.26±1.62mV with an amphiphilic compound). And in terms of the change of the surface charge of exo@pg3, the change trend is similar to exo@fpg3.

In combination with the above results, exo@fpg3 and exo@pg3, both prepared in a mass ratio of 5, were used for the next experiment.

Example 3

With respect to exo@fpg3, exo@pg3 prepared in example 2, the inventors performed detection evaluation by a correlation test, and the specific cases are described below.

Intracellular delivery efficiency assessment

Intracellular delivery efficiency of exosomes was assessed using a flow cytometer, as outlined below:

1X 10 per well in a 12-well plate ⁴ Density of individual cells HUVEC cell suspension of 1 mL was seeded at 37℃with 5% CO ₂ Culture 24 h in an incubator; then, the medium is removed;

in advance, the exosomes are marked with 20 mug/mL DiO in 200 mug PBS for 1 hour at room temperature (refer to operation description, after marking is completed, the exosome spin column (MW 4000) is used for removing redundant DiO); then, referring to the foregoing procedure, the DiO-labeled exosomes were prepared by mixing with FPG3 or PG3 in a mass ratio of 0.5 or 5, respectively: exo@fpg3, exo@pg3;

subsequently, the DiO-labeled exosomes modified by exo@FPG3 and exo@PG3 are uniformly mixed with serum-free medium or medium containing 10% FBS, and added into a 12-well plate from which the medium is removed, and 5% CO ₂ Incubation for 2 hours at 37 ℃ (simultaneously, exosomes without any modification were used as controls);

after incubation, the well plate was removed from each component, the cells were washed 3 times with PBS, the cell suspension was obtained after digestion with trypsin, and after washing with PBS, fluorescence was quantitatively detected by flow cytometry (excitation and emission wavelengths of DiO were 484 nm and 501 nm, respectively).

The results are shown in FIG. 7. Analysis can be seen:

after incubation for 2 hours under serum-containing conditions (fig. 7A), the fluorescent intensity of exo@fpg3 (w/w, 0.5 and 5) group was much higher than that of exosomes alone and exo@pg3 (w/w, 0.5 and 5), which indicated that: functional modification of FPG3 can enhance the cellular uptake effects of exosomes (i.e., to facilitate increased intracellular delivery efficiency) in culture conditions with serum.

In the serum-free condition (fig. 7B), after the mass ratio of exo@fpg3 is increased from 0.5 to 5, the fluorescence intensity of the cells is enhanced by 3.4 times, which shows that the improvement of the FPG3 concentration in the engineering exosomes can promote the uptake efficiency of the cells; on the other hand, the cell fluorescence intensities of exo@FPG3 (w/w, 5.0) groups are 12.4 times and 10.6 times that of exosome groups and exo@PG3 (w/w, 5.0), respectively, and the result shows that after the exosome is further modified by the FPG3 after the PG3 is modified by fluorine, the intracellular efficiency of the exosome is further improved.

Further, in view of the fact that the surface charge of exo@fpg3 obtained under the condition of a mass ratio of 0.5 is still negative, experimental results show that the exosome has good cell uptake efficiency, which indicates that the change of the surface charge of the FPG3 modified exosome has little influence on cell uptake exo@fpg3, in other words, the improvement of the cell uptake efficiency should be mainly attributed to the interaction between the cell and the exosome due to the fluorinated modification of the exosome surface, so that the cell uptake efficiency is improved.

(II) intracellular localization conditions

Using the laser confocal technique, the inventors evaluated the intracellular localization after the cell ingested into exosomes, as follows.

HUVEC cells were plated at 5X 10 cells per well ⁴ Density of individual cells was seeded in 35 x 12 mm glass bottom dishes and cultured overnight to allow the cells to adhere;

subsequently, the medium was replaced with a different fresh serum-free medium: wherein exo@FPG3, exo@PG3 or exosomes (previously DiO labeled) are contained, respectively, wherein each well is 5 μg exosomes; after incubation of 0.5, 2 and 4, h respectively, in the incubator, the medium in the cells was removed, followed by washing the cells 1 time with PBS, adding lysosomal labeled probe (0.2 μm), and incubation was continued for 30 minutes in the incubator;

after the incubation was completed, the cells were washed 1 time with PBS, 4% paraformaldehyde was added to the petri dish, and incubation was continued in an incubator for 10 minutes to fix the cells;

cells were washed with PBS and stained with DAPI for 15 min; after staining was completed, the staining solution was removed, and the cells were infiltrated in PBS. The fluorescent distribution of DiO-labeled exo@FPG3, exo@PG3 or exosomes in the cells was observed with CLSM.

The results are shown in fig. 8, and analysis can be seen:

when the cells are incubated with exosomes and exo@pg3 for 0.5h, the green fluorescence signal of the cells is negligible, while the cells of exo@fpg3 group can clearly see stronger green fluorescence; green fluorescence was slightly enhanced in exosomes and exo@pg3 cells with prolonged incubation time to 4 h, but co-localization occurred with most of the lysosomes concentrated to the red fluorescent label; furthermore, green fluorescence of exo@fpg3 in the cytoplasm was significantly enhanced over other groups with prolonged incubation time.

The above results indicate that HUVEC cells are time dependent for intracellular uptake of exosomes, exo@FPG3 and exo@PG3.

And in combination with the fluorescence image results, it can also be seen that: the cellular uptake efficiency of exo@fpg3 is more advantageous than exo@pg3 and simple exosomes, consistent with the quantitative results of flow cytometry. At the same time, most of the green fluorescence located in the cytoplasm did not co-localize with lysosomes, indicating that most exo@fpg3 released the exosome content in the cytoplasm. Also, this result directly indicates: the fluorinated modified peptide dendrimer can improve and enhance the intracellular delivery efficiency of exosomes through chemical modification, and further can enhance the biological effect of exosomes.

(III) cell-tube influence

The influence of exosomes on cell-tube formation was assessed by cell-tube formation experiments. The specific experimental conditions are outlined below.

mu.L of matrigel was added to each well of a 96-well plate and cured at 37℃for 30 minutes;

HUVEC cells were then plated at 2X 10 per well ⁴ The density of individual cells was seeded onto matrigel of 96-well plates, followed by addition of exo@fpg3, exo@pg3 or exosomes, wherein the exosome concentration was 25 μg/mL;

adding equal volume of PBS into the control group;

subsequently, after incubating the well plates in incubators 3.5 h and 6.5 h, the network of cells was observed under an optical microscope and the total number of tubules in the network was analyzed using ImageJ software to reflect the tubule formation capacity.

The experimental results are shown in fig. 9, and the analysis can be seen:

compared with the PBS control group (figure 9A), the simple exosomes or exo@PG3 can also promote the tube formation of HUVECs cells, which also shows that the exosomes secreted by the stem cells have tissue regeneration activity; however, in all the intervention groups, the tubule formation of HUVECs cells was more pronounced in exo@fpg3 group;

the tubules formed were quantitatively analyzed (fig. 9B), and the biological activity of exo@fpg3 was increased 2.1 times the effect of exo@pg3, and 4 times that of the simple exosomes, relative to the number of cell tubules in the PBS group.

The above results indicate that exo@fpg3 is able to promote the cell's tubular effect more significantly, and this is also due to the fact that FPG3 can improve the effect of extracellular intracellular delivery.

(IV) cell migration affecting conditions

And evaluating the influence condition of exosomes on cell migration by using a cell scratch experiment. The specific experimental conditions are outlined below.

HUVEC cells were plated at 1X 10 per well ⁵ The density of individual cells was inoculated into 96-well plates and grown in an incubator until the degree of fusion reached 100%;

a single layer of cells was vertically scratched on each well using the tip of a 200 μl sterile gun head, the cells were washed 2 times with PBS to remove shed cells, and then basal medium containing exo@fpg3, exo@pg3 or exosomes was added, with exosome concentration of 25 μg/mL;

adding equal volume of PBS into the control group;

cells were kept in culture in an incubator, images of cell scratches were collected with an optical microscope at time points of 0, 12 and 30 h, and scratch areas were analyzed with ImageJ software to evaluate the migration ability of cells according to their ratio to the initial scratch area.

The experimental results are shown in FIG. 10. Analysis can be seen:

all groups of cells decreased in scratch area to varying degrees with prolonged culture time (fig. 10A); however, the migration quantity of HUVEC in the exosome and exo@PG3 groups is only slightly improved compared with that of the PBS group, and the scratch area of HUVEC cells in the exo@FPG3 group is obviously reduced;

quantitative analysis of the scratch test results showed (fig. 10B): the cell migration area percentage of exo@FPG3 group reaches 71.2%, which is far higher than that of other groups.

The results show that exo@FPG3 can promote migration of HUVEC cells to the greatest extent, and is also direct evidence that the modification of fluorinated peptide dendrimers can improve intracellular bioavailability of exosomes.

Claims

1. The fluorine engineering exosome is characterized in that the engineering exosome is prepared by combining fluoride on the surface of the exosome through electrostatic action and hydrophobic action; the preparation method comprises the following steps:

(one) preparation of exosomes

The exosomes are exosomes secreted by adipose-derived mesenchymal stem cells (AMSCs);

(II) binding to FPG3

Uniformly mixing fluorinated peptide dendrimer FPG3 with the exosomes in the step (one), and incubating at 15-30 ℃ for 15-30 min to obtain fluorine engineering exosomes exo@FPG3;

fluorinated peptide dendrimer FPG3: exosome=0.25-20;

the fluorinated peptide dendrimer FPG3 is a fluorocarbon chain modified lysine dendrimer, and the molecular weight of the fluorinated peptide dendrimer FPG3 is 2000-20000.

2. The fluorine-engineered exosome delivered in claim 1, gao Xiaobao, wherein in step (one), the exosome is prepared by ultracentrifugation, comprising:

after the culture is finished, collecting the culture, and separating by an ultracentrifugation method to obtain exosomes secreted by AMSCs, wherein the related operations are carried out at 4 ℃, and the specific operations are as follows:

filtering the obtained large vesicle by a 0.45 mu m filter, and centrifuging the obtained supernatant with 100000 and g for 70 min to obtain an exosome preliminarily;

after resuspension of the resulting exosomes with pre-chilled PBS, centrifugation is again performed for 70 min at 100000 g for purification.

3. The fluorine engineered exosome delivered within claim 1 to Gao Xiaobao, wherein in step (two), the fluorinated peptide dendrimer FPG3: exosome = 5;

the prepared fluorine engineering exosome exo@FPG3 has a surface potential of 9.6+/-0.6 mV.

4. The fluorine-engineered exosome delivered in Gao Xiaobao of claim 1, wherein in step (two), the fluorinated peptide dendrimer FPG3 is a POSS core synthesized from (3-aminopropyl) triethoxy-silane, and the t-butoxycarbonyl-protected lysine is used as a structural unit, and is subjected to amide condensation and elution protection continuously to obtain peptide dendrimers of different algebra; finally, carrying out fluoridation modification on the peptide dendrimer; the preparation method comprises the following steps:

(one) preparation of POSS.8HCl

the reaction product is POSS.8HCl;

(II) preparation of PG1

2 g of POSS.8HCl prepared in the step (one), 6.93 g of Boc-Lys (Boc) -OH, 7.71 g of HBTU and 2.27 g of HOBT are dissolved together in 60 mL of DMF under the protection atmosphere, and after the dissolution is completed, 9.0 mL of DIPEA is added under the ice bath condition;

continuously stirring and reacting for 48 hours at the temperature of 30 ℃;

the reaction product is PG1 with amino protective group tert-butyl ester;

adding TFA into the PG1 with the tertiary butyl ester with the amino protecting group for reaction to remove the protecting group, so as to prepare PG1;

(III) preparation of PG2

2 g g of PG1 prepared in the step (two), 8.7 g of Boc-Lys (Boc) -OH, 9.6 g of HBTU and 2.7 g of HOBT are dissolved in 60 mL of DMF under the protection atmosphere, and after the dissolution is completed, 11.1 mL of DIPEA is added under the ice bath condition;

continuously stirring and reacting for 48 hours at the temperature of 30 ℃;

the reaction product is PG2 with tertiary butyl ester of amino protecting group;

adding TFA into the PG2 with the tertiary butyl ester with the amino protecting group for reaction to remove the protecting group, so as to prepare PG2;

(IV) preparation of PG3

2.2 g g of PG2 prepared in the step (III), 8.3 g of Boc-Lys (Boc) -OH, 9.1 g of HBTU and 2.59 g of HOBT are dissolved together in 55 mL of DMF under the protection atmosphere, and after the dissolution is completed, 10.57 mL of DIPEA is added under the ice bath condition;

continuously stirring and reacting for 48 hours at the temperature of 30 ℃;

the reaction product is PG3 with amino protective group tert-butyl ester;

adding TFA into the PG3 with the tertiary butyl ester with the amino protecting group for reaction to remove the protecting group, so as to prepare PG3;

(V) preparation of fluorinated peptide dendrimers

Weighing PG3 prepared in the step (four) of 1 g, placing the PG3 in a reaction container, adding 15 mL anhydrous methanol under the protection atmosphere condition, and stirring for dissolution; after dissolution is completed, under the ice water bath condition, adding: 1.07 mL of a mixture of heptafluorobutyric anhydride, 0.78 mL of triethylamine and 1 mL of anhydrous methanol;

stirring and reacting for 48 hours at the temperature of 30 ℃;

the reaction product was FPG3.

5. A method of preparing a fluorine-engineered exosome for delivery within Gao Xiaobao of claim 1, comprising the steps of:

(one) preparation of exosomes

(II) binding to FPG3

fluorinated peptide dendrimer FPG3: exosome=0.25-20;

6. Use of the fluorine-engineered exosome exo@fpg3 delivered in Gao Xiaobao according to any one of claims 1 to 4 for cell biology research or tissue repair, for enhancing cell uptake efficiency research or application, or for cell tube formation and cell migration research or application.