Nanometer vesicle based on umbilical cord mesenchymal stem cell vesicle loaded type I photosensitive prodrug as well as preparation method and application of nanometer vesicle

Abstract

The invention discloses a nano-vesicle based on umbilical cord mesenchymal stem cell vesicle-loaded I-type photosensitive prodrug as well as a preparation method and application thereof. The nano-vesicle comprises a vesicle derived from human umbilical cord mesenchymal stem cells and the I-type photosensitive prodrug loaded on the vesicle. The nano vesicle has the advantages that the I-type photosensitive prodrug of the nano vesicle EVs (at) NBS-PB can respond to high-level H2O2 in a diabetic chronic wound microenvironment to release the I-type photosensitizer, and then a PDT effect is generated after laser excitation to release active oxygen to achieve the antibacterial purpose, so that the targeted anti-infection effect on the diabetic chronic wound is realized; the vesicles derived from human umbilical mesenchymal stem cells are combined to regulate the wound microenvironment so as to synergistically treat the chronic wound surface; the stem cell vesicles not only can improve the biocompatibility of the photosensitive prodrug, but also have the effects of promoting cell proliferation, migration and angiogenesis; the hydrogel microneedle patch loading administration mode not only has high drug delivery efficiency and minimally invasive property, but also can improve the compliance of a patient.

Description

Technical Field

[0001] The invention relates to the technical field of diabetic wound healing drugs, in particular to drugs of stem cells and photosensitizers. Background Art

[0002] The high incidence of diabetic wounds has placed an increasing burden on families and societies around the world. The hyperglycemic microenvironment of diabetic wounds increases susceptibility to bacterial infection. More importantly, bacterial infection often aggravates persistent chronic inflammation, exacerbates wound hypoxia and angiogenesis disorders, and further delays wound healing. Therefore, effective antibacterial treatment and regulation of the wound microenvironment for chronic diabetic wounds are ideal treatment strategies. In antimicrobial therapy, antibiotics have been widely used to eliminate pathogenic microorganisms in chronic wounds. However, the limitations of bacterial resistance and antibiotic abuse make the development of new alternative strategies an urgent need today.

[0003] Antimicrobial photodynamic therapy (aPDT) is considered a promising alternative due to its broad-spectrum antimicrobial activity and low resistance to drug resistance. However, the lack of targeting of conventional photosensitizers leads to serious toxic side effects. In addition, the hypoxic microenvironment at the site of wound infection severely limits the photodynamic efficiency of oxygen-consuming photosensitizers. In addition, in clinical practice, accurate assessment of the extent of chronic wound infection helps patients develop timely and effective treatment strategies. To this end, various composite nanomaterials have been widely developed for PDT in the diagnosis and targeting of infected wounds, but the uncertain toxicity and non-reproducibility of complex components limit their clinical application. Therefore, there is an urgent need to develop type I pro-photosensitizers with selective activation and oxygen-independence for accurate diagnosis and effective treatment of diabetic chronic wound infections. On the other hand, strategies to accelerate wound healing by regulating the wound microenvironment are also evolving. Summary of the invention

[0004] The purpose of the present invention is to provide a nanovesicle based on umbilical cord mesenchymal stem cell vesicles loaded with type I photosensitizer prodrug and its preparation method and application, so as to solve the problem of lack of effective antibacterial and healing-promoting drugs for diabetic wounds in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A type I photosensitizer prodrug, the structural formula of which is shown below:

[0007]

[0008] The present invention also provides a method for preparing the type I photosensitizer prodrug, which comprises: adding sodium bicarbonate and sodium dithionite to an N-benzophenothiazine solution, stirring and mixing, removing water from the mixture after the mixture turns yellow, adding the mixture to triethylamine, adding triphosgene solution, stirring, and then adding 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl chloroformate and triethylamine, fully mixing and reacting, and purifying to obtain the type I photosensitizer prodrug NBS-PB.

[0009] Furthermore, the specific process includes: dissolving 390 mg of N-benzophenothiazine in a 50% deionized water / dichloromethane solution, adding 168 mg of sodium bicarbonate and 526 mg of sodium dithionite and stirring for 2 hours, after the mixture turns yellow, removing water from the mixture, adding 170 μL of triethylamine; slowly adding 120 mg of triphosgene to 1 mL of dichloromethane, and adding it to the mixed system obtained in the previous step, stirring at room temperature for 0.5 hours; adding 296 mg of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl chloroformate and 140 μL of triethylamine to the above solution, collecting the mixture after one night of reaction, and purifying to obtain type I photosensitizer prodrug NBS-PB.

[0010] The present invention also provides an application of the type I photosensitizer prodrug in preparing a medicine for treating diabetic wounds.

[0011] The present invention also provides a nanovesicle using the type I photosensitizer prodrug, which includes a vesicle derived from human umbilical mesenchymal stem cells and the type I photosensitizer prodrug loaded thereon, namely, the nanovesicle EVs@NBS-PB.

[0012] The present invention also provides a method for preparing the nanovesicles using the type I photosensitizer prodrug, the process comprising: lysing human umbilical mesenchymal stem cells, repeatedly grinding and centrifuging to obtain the supernatant, repeatedly extruding the supernatant through 800nm ​​and 400nm polycarbonate membranes in a liposome extruder to obtain cell vesicles, adding type I photosensitizer prodrug to the cell vesicle solution, and repeatedly extruding with a 200nm polycarbonate membrane of the liposome extruder to obtain nanovesicles EVs@NBS-PB.

[0013] Further, the detailed process is as follows: 10 7Human umbilical cord mesenchymal stem cells (HUMSCs) were lysed with 1 ml hypotonic lysis buffer at 4°C overnight, the lysate was repeatedly ground 20 times, centrifuged at 3200 g and 4°C for 5 min, and the supernatant was taken. The supernatant was repeatedly extruded 20 times through 800 nm and 400 nm polycarbonate membranes in a liposome extruder to obtain cell vesicles UC-EVs. 10 μl of 100 mM type I photosensitizer prodrug NBS-PB was added to 1 ml UC-EVs solution and vortexed to mix, and then repeatedly extruded 20 times through a 200 nm polycarbonate membrane of a liposome extruder to obtain nanovesicles EVs@NBS-PB.

[0014] The present invention also provides an application of the nanovesicles using the type I photosensitizer prodrug in preparing a medicine for treating diabetic wounds.

[0015] The present invention also provides an application of the nanovesicles using the type I photosensitizer prodrug in preparing a hydrogel microneedle patch. In clinical applications, the umbilical cord mesenchymal stem cell vesicles loaded with type I photosensitizer prodrug can be loaded with a hydrogel microneedle patch and then applied to the wound surface for treatment with near-infrared laser irradiation.

[0016] The advantages of the present invention are: Figure 1 As shown, the type I photosensitizer prodrug of the nanovesicle EVs@NBS-PB can respond to the high level of H2O2 in the microenvironment of chronic diabetic wounds to release type I photosensitizer, and then produce PDT effect after laser excitation to release reactive oxygen to achieve the antibacterial purpose, thereby realizing targeted anti-infection effects on chronic diabetic wounds, and combining with vesicles derived from human umbilical mesenchymal stem cells to regulate the wound microenvironment to synergistically treat chronic wounds; stem cell vesicles can not only improve the biocompatibility of photosensitizer prodrugs, but also promote cell proliferation, migration and angiogenesis; the method of loading and administering drugs with hydrogel microneedle patches not only has high drug delivery efficiency and minimal invasiveness, but also can improve patient compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of the present application, and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 Schematic diagram of H2O2-responsive EVs@NBS-PB MNs synergizing oxygen-independent phototherapy and microenvironment regulation in hypoxic diabetic wounds;

[0019] Figure 2 The synthetic route of type I photosensitizing prodrug NBS-PB;

[0020] Figure 3 This is the H NMR spectrum of type I photosensitizer prodrug NBS-PB;

[0021] Figure 4 is the inductively coupled plasma mass spectrum of type I photosensitizer prodrug NBS-PB;

[0022] Figure 5 The absorption spectra of type I photosensitizer prodrug NBS-PB in different concentrations of H2O2;

[0023] Figure 6 The fluorescence spectra of type I photosensitizer prodrug NBS-PB in different concentrations of H2O2;

[0024] Figure 7 SOSG detection after different treatments 1 Fluorescence intensity result diagram of O2;

[0025] Figure 8 DLS results of nanovesicles EVs@NBS-PB, with insert: magnified TEM image of EVs@NBS-PB;

[0026] Fig. 9 A is the absorbance result of EVs@NBS-PB without / with H2O2;

[0027] Fig. 9 B is the fluorescence spectrum result of EVs@NBS-PB without / with H2O2;

[0028] Fig.10 A is the scratch micrograph of HUVECs under different treatments, scale bar: 100 μm;

[0029] Fig.10 B is the quantitative histogram of micro scratches;

[0030] Fig.11 A is the migration image of HUVECs stained with crystal violet, scale bar: 100 μm;

[0031] Fig.11 B is the result diagram of HUVECs mobility;

[0032] Fig.12 A is the image of HUVECs tube formation under different treatments under optical microscope, scale: 100 μm;

[0033] Fig.12 B is the statistical result of the number of HUVECs nodes under different treatments under optical microscope;

[0034] Fig.12 C is the statistical result of the total length of HUVECs branches under different treatments under optical microscope;

[0035] Fig.13 A is the in vivo fluorescence image of wounds with and without bacterial infection after EVs@NBS-PB MNs treatment;

[0036] Fig.13 B is the fluorescence intensity analysis result of mouse wound surface after EVs@NBS-PB MNs treatment;

[0037] Fig.14 A is a typical wound image;

[0038] Fig.14 B is a diagram of simulated in vivo wound closure traces under different treatments, corresponding to the five groups of PBS, EVs MNs, NBS-PB MNs+light, EVs@NBS-PB MNs, and EVs@NBS-PB MNs+light from top to bottom;

[0039] Fig.14 C is the image of Staphylococcus aureus colonies in wound tissue on day 3;

[0040] Fig.14 D is the statistical analysis result of wound area;

[0041] Fig.14 E is the corresponding quantitative results of bacterial colony growth on the wound surface of different treatment groups;

[0042] Fig.15 A is a statistical graph of IL-6 levels on day 3 of mice infected with Staphylococcus aureus under different treatments;

[0043] Fig.15 B is a statistical graph of IL-10 levels on day 9 in mice infected with Staphylococcus aureus under different treatments;

[0044] Fig.15 C is a statistical graph of VEGF levels on day 9 in mice infected with Staphylococcus aureus under different treatments;

[0045] Fig.15 D is the heat map of IL-6, IL-10, and VEGF expression detected by QPCR. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0047] Example 1 Preparation of Type I Photosensitizer Prodrug NBS-PB

[0048] The specific synthesis steps of the type I photosensitizer prodrug provided by the present invention are as follows: Figure 2 As shown, the specific steps are as follows:

[0049] N-Benzophenothiazine (390 mg, 1 mmol) was dissolved in a solution of deionized water / dichloromethane (50%), sodium bicarbonate (168 mg, 2 mmol) and sodium dithionite (526 mg, 1.5 mmol) were added and stirred for 2 h. After the mixture turned yellow, the water in the mixture was removed and added to a round-bottom flask containing triethylamine (TEA, 170 μL, 1.2 mmol). Triphosgene (TPG, 120 mg, 0.32 mmol) was slowly added to 1 mL of dichloromethane and added to the mixed system obtained in the previous step and stirred at room temperature for 0.5 h. 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl chloroformate (296 mg, 1 mmol) and triethylamine (140 μL, 1 mmol) were added to the above solution. After reacting overnight, the mixture was collected and evaporated under reduced pressure. The precipitate was purified by chromatography and eluted with ethyl acetate / petroleum ether (20-50%) to obtain type I photosensitizer prodrug NBS-PB (82 mg, green solid, yield 12.6%).

[0050] N-Benzophenethiazine, the nitrogen atom of the benzophenothiazine rings, hereinafter referred to as NBS, is a BP that was reported in "Totally Caged Type I Pro-Photosensitizer for Oxygen-Independent Synergistic Phototherapy of Hypoxic Tumors", which reported the synthesis route of BP (i.e., NBS).

[0051] 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylchloroformate, abbreviated as PB, is originally called 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylchloroformate. Its synthesis route has been reported in "H2O2-responsive biodegradable nanomedicine for cancer-selective dualmodal imaging guided precise photodynamic therapy".

[0052] like Figure 3-4 Here are the characterization data of the product NBS-PB:

[0053] 1H NMR (600MHz, CDCl3) δ7.73(d,J=6Hz,2H),7.71(M,J=6.0Hz,1H),7.52(dd,J=6.0Hz,1H),7.36(m,J=24.0Hz,1H),7.27(d,J=6.0Hz,1H),7.08(m,J=12.0Hz ,1H),7.00(d,J=6.0Hz,2H),6.98(s,1H),6.85(dd,J=12.0Hz,2H),3.39(q,J =24.0Hz,8H),2.74(s,2H),1.30(m,J=102.0Hz,12H),1.18(t,J=18.0Hz,12H)

[0054] MS Calcd for C 38 H 46 BN3O4S[MH] - :651.3,found:651.3.

[0055] Example 2 Absorption spectra of type I photosensitizer prodrug NBS-PB in different concentrations of H2O2

[0056] Absorption spectrum: Take the type I photosensitizer NBS-PB obtained in Example 1, prepare a type I photosensitizer solution and a hydrogen peroxide aqueous solution, mix the type I photosensitizer solution and the hydrogen peroxide aqueous solution to form a series of reaction systems with different concentrations of hydrogen peroxide, in which the concentration of the type I photosensitizer in the reaction system with different concentrations of hydrogen peroxide is 10 μM and the concentration of hydrogen peroxide is 0 to 50 μM. After the type I photosensitizer solution and the hydrogen peroxide aqueous solution are mixed for 30 minutes, the absorption thereof is measured; the absorption spectrum is as follows Figure 5 The five blue lines above represent the reaction systems containing 10, 20, 30, 40, and 50 μM hydrogen peroxide, respectively. Figure 5 It can be seen that in the same system, the absorption coefficient of type I photosensitizer prodrug is the highest at 675nm. As the concentration of hydrogen peroxide increases, the solution changes from colorless to cyan, and the absorption coefficient of type I photosensitizer prodrug gradually increases, especially at 675nm.

[0057] Example 3 Fluorescence spectra of type I photosensitizer prodrug NBS-PB in different H2O2 concentrations

[0058] Fluorescence spectrum: A series of reaction systems with different concentrations of hydrogen peroxide were prepared according to the method of Example 2. In the reaction systems with different concentrations of hydrogen peroxide, the concentration of type I photosensitizer prodrug was 10 μM and the concentration of hydrogen peroxide was 0-50 μM. The fluorescence of type I photosensitizer prodrug solution was measured after mixing with hydrogen peroxide aqueous solution for 30 minutes. The fluorescence spectrum is shown in FIG. Figure 6The five red lines above represent the reaction systems containing 10, 20, 30, 40, and 50 μM hydrogen peroxide from bottom to top. Figure 6 It can be seen that in the same system, the fluorescence intensity of type I photosensitizer prodrug is the strongest at 705 nm. As the concentration of hydrogen peroxide increases, the fluorescence intensity of type I photosensitizer prodrug gradually increases, especially at 705 nm.

[0059] Example 4 SOSG (singlet oxygen sensor green) detection after different treatments 1 Fluorescence intensity of O2

[0060] Detection 1 O2 generation: 10 μM type I photosensitizer prodrug was prepared in 2 groups. The first group was treated with 680nm light, and the second group was treated with hydrogen peroxide solution and 680nm light. Singlet oxygen detection reagent SOSG (singlet oxygen detection reagent purchased from Thermo Fisher Scientific (China); added in an amount of 5 μM) was added to the two groups of solutions, and the fluorescence of SOSG at 545nm in the two groups of solutions was detected. The results are shown in Figure 7 As shown, after the type I photosensitive prodrug reacts with hydrogen peroxide solution and is irradiated by laser, it can continuously generate singlet oxygen 1 O2. This indicates that type I photosensitizer prodrug has a good photodynamic effect under the conditions of hydrogen peroxide and light.

[0061] Example 5 Preparation and particle size characterization of nanovesicles EVs@NBS-PB

[0062] Preparation of nanovesicles EVs@NBS-PB: 10 7 Human umbilical cord mesenchymal stem cells were lysed with 1 ml hypotonic lysis buffer at 4 ° C overnight, and the lysate was repeatedly ground 20 times, centrifuged at 3200g and 4 ° C for 5 minutes, and the supernatant was taken. The supernatant was repeatedly extruded 20 times through 800nm ​​and 400nm polycarbonate membranes in a liposome extruder to obtain cell vesicles UC-EVs (hereinafter referred to as EVs). 10μl 100mM type I photosensitive prodrug NBS-PB was added to 1ml UC-EVs solution and vortexed to mix, and then repeatedly extruded 20 times with a 200nm polycarbonate membrane of a liposome extruder to obtain nanovesicles EVs@NBS-PB.

[0063] Particle size analysis: Take 10 μl of nanovesicle EVs@NBS-PB and add it to 1 ml of PBS solution. After ultrasonic vibration for 10 minutes, put it into the quartz sample cell of the particle size analyzer to measure the hydrated particle size of nanovesicle EVs@NBS-PB. The results are as follows Figure 8 As shown, the hydrated particle size of the nanovesicle EVs@NBS-PB is about 176 nm and is spherical.

[0064] Example 6 Characterization of the response performance of nanovesicles EVs@NBS-PB

[0065] Absorption spectrum of nanovesicle EVs@NBS-PB: Prepare nanovesicle EVs@NBS-PB solution and divide it into two groups. The first group is not treated, and the second group is added with hydrogen peroxide solution. The absorption of the second group is measured thirty minutes after mixing evenly. The concentration of EVs@NBS-PB in the second group reaction system is 10μM, and the hydrogen peroxide solution is 100μM. The concentration of EVs@NBS-PB in the first and second groups is the same. The absorption spectrum is shown in Fig. 9 As shown in A, after EVs@NBS-PB responded to hydrogen peroxide solution, its absorption coefficient at 680nm was significantly enhanced.

[0066] Fluorescence spectrum of nanovesicle EVs@NBS-PB: Nanovesicle EVs@NBS-PB solution was prepared and divided into two groups. The first group was not treated, and the second group was added with hydrogen peroxide solution. The fluorescence of the second group was measured thirty minutes after mixing evenly; the concentration of EVs@NBS-PB in the reaction system of the second group was 10μM, and the hydrogen peroxide solution was 100μM. The concentration of EVs@NBS-PB in the first and second groups was the same. Fluorescence spectrum is shown in Fig. 9 As shown in B, EVs@NBS-PB responded to hydrogen peroxide solution, and its fluorescence intensity was significantly enhanced around 700 nm.

[0067] The above shows that EVs@NBS-PB can release type I photosensitizer NBS under hydrogen peroxide conditions.

[0068] Example 7 Verification of the ability of nanovesicles EVs@NBS-PB to promote cell migration

[0069] Cell scratch experiment: HUVEC cells (human umbilical vein endothelial cells) were cultured in complete medium and plated on 6-well plates and cultured overnight. Use a pipette tip to draw a line along the midline of the well plate and then wash twice with PBS. Prepare EVs@NBS-PB solution and EVs solution, and set up three groups. The first group added PBS, the second group added EVs solution, and the third group added EVs@NBS-PB solution. Among them, the concentrations of EVs and EVs@NBS-PB nanovesicle proteins were both 0.4 mg / ml. The reagents that needed to be added to the three groups were mixed with the culture medium (1% fetal bovine serum, 1% double antibody) and incubated with the cells in the 6-well plate. The migration of the cells was observed at 0, 12, and 24 hours. The results are as follows: Fig.10 As shown in A, there is a significant migration phenomenon in both the EVs group and the EVs@NBS-PB group over time, and referring to the statistical situation Fig.10 As shown in B, the migration-promoting efficiency of the EVs@NBS-PB group was the most obvious.

[0070] Example 8 Transwell verification of nanovesicles EVs@NBS-PB

[0071] Transwell assay: 200 μL of 5 × 10 5 cells / mL HUVEC cell suspension was inoculated into the transwell chamber, 500 μL complete medium was added to the lower chamber, and PBS, EVs, and EVs@NBS-PB were added to the upper chamber. After incubation at 37°C and 5% CO2 for 12 hours, the transwell chamber was removed. The transwell chamber was repeatedly washed 3 times with PBS, and then the cells on the upper surface of the membrane were wiped with a cotton swab. The lower surface was stained with crystal violet for 20 minutes, washed 3 times with PBS, and observed under a microscope. The results are shown in Fig.11 As shown in A, compared with the Control group, the purple color of the EVs group and the EVs@NBS-PB group was more obvious, indicating that the vesicles had an excellent effect on promoting cell migration. Fig.11 B also verified this conclusion.

[0072] Example 9: Verification of the angiogenesis-promoting ability of nanovesicles EVs@NBS-PB

[0073] Tube formation experiment: 50 μL Matrigel (purchased from Corning) was added to each well of a 96-well plate and dried at 37°C for 30 minutes. 50 μL of HUVEC cell suspension (2×10^4 / cells) was added to each well, and then PBS, EVs, and EVs@NBS-PB were added respectively. After incubation at 37°C for 4 hours, the capillary structure was observed under a light microscope (Nikon). Image-J software was used to analyze the total number of tubes and nodes (n=3). The results are shown in Figure 2. Fig.12 As shown in A, there was no significant change in cell morphology in the PBS group, while the EVs group and EVs@NBS-PB group induced HUVECs to form obvious and interconnected tubule structures. The number of nodes and total number of tubes were also counted. Fig.12 As shown in B and 12C, after HUVECs were treated with EVs@NBS-PB, the number of nodes was approximately 62 and the total branch length was approximately 1885, and both numbers increased significantly.

[0074] Example 10 In vivo imaging verification of EVs@NBS-PB MNs

[0075] A hydrogel microneedle patch loaded with EVs@NBS-PB was prepared and named EVs@NBS-PB MNs.

[0076] Diabetic mouse modeling: Male C57BL / 6 mice (3-4 weeks old) were fed for 1 week. After preparing STZ solution with sodium citrate buffer to a concentration of 10 mg / mL STZ solution, 200 μL was injected intraperitoneally into each mouse. Inject once every other day for a total of three injections. On the 7th day, the blood glucose of the mice was tested with a blood glucose meter. If it reached 16.7 mmol / L, the diabetic mouse model was successfully established.

[0077] Diabetic full-thickness skin wound model: After removing the hair on the back of the mouse, a full-thickness wound (6 mm × 6 mm) was made on the back of the mouse using a skin remover. Then, the infection group was inoculated with 10 μL of Staphylococcus aureus (1×10 8 CFU / mL) were used to infect the wound for 12 h, and the normal group was not treated with infection.

[0078] In vivo imaging of EVs@NBS-PB: EVs@NBS-PB MNs were applied to the wounds of mice in the infection group and the normal group, and the mice were anesthetized with isoflurane. The fluorescence intensity of the wounds of mice at different time points (0, 2, 6, 12, 24 h) was observed using an animal imaging system (AniView 600, BLT). The fluorescence intensity statistics of the wounds were calculated using the software related to the animal imaging system (n = 3). The results are shown in Figure 2. Fig.13 A Fluorescence image and Fig.13 As shown in the fluorescence statistics chart B, with the increase of time, the fluorescence of the wound surface in the normal group did not change, while the wound surface in the infected group showed obvious fluorescence at 2 hours and the fluorescence intensity also increased with the increase of time.

[0079] Example 11 Verification of the therapeutic effect of EVs@NBS-PB MNs

[0080] Prepare a hydrogel microneedle patch loaded with EVs, which is named EVs MNs;

[0081] The NBS-PB loaded hydrogel microneedle patch was prepared and named NBS-PB MNs;

[0082] A hydrogel microneedle patch loaded with EVs@NBS-PB was prepared and named EVs@NBS-PB MNs.

[0083] Treatment of chronic diabetic wounds: Skin contraction has a huge impact on the wound surface, so 3M tape is used during treatment to prevent skin contraction caused by external factors. The diabetic full-thickness skin wound model mice were divided into different groups (n=5): PBS, EVs MNs, NBS-PB MNs+light, EVs@NBS-PB MNs, EVs@NBS-PB MNs+light. After the wounds of mice in all groups were infected with Staphylococcus aureus, the wounds were treated with corresponding drugs and incubated for 6 hours, and then laser (680nm, 0.5W / cm 2,15min) group, and the treatment lasted for three days. On days 0, 3, 5, 7, and 9, the wounds were photographed under the same conditions. Image J was used to simulate the wound shape, calculate the wound area, and calculate the healing rate. On day 3, 20μL PBS was used to collect bacteria from the wounds of each group and spread on LB agar plates.

[0084] Results such as wound healing Fig.14 As shown in A, compared with the Control group, the wound healing effect of the EVs@NBS-PB MNs+light group was obvious over time, and the wound simulation shape Fig.14 B also proved this conclusion. And the wound bacterial culture on the third day was as follows Fig.14 As shown in C, the EVs@NBS-PB MNs+light group had a better antibacterial effect than other groups. Fig.14 Colony counts on day D and day 3 Fig.14 E also showed that the EVs@NBS-PB MNs group had an excellent healing effect.

[0085] Example 12 Changes in cytokine levels in EVs@NBS-PB MNs

[0086] Cytokine detection: The skin tissues of the wound sites of the mice in the five experimental groups of Example 11 on the 3rd and 9th days were stored in RNA stabilizing solution, and the expression of the corresponding cytokines was detected by RT-QPCR. Fig.15 As shown in AD, during the treatment process, the EVs@NBS-PB MNs+light group had the lowest expression level of inflammatory factor IL-6 on the 3rd day compared with other groups, and the highest expression levels of anti-inflammatory factor IL-10 and vascular endothelial growth factor VEGF on the 9th day.

[0087] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A type I photosensitizer prodrug, characterized in that: The structural formula is as follows:

2. A method for preparing the type I photosensitizer prodrug according to claim 1, characterized in that: The process includes: adding sodium bicarbonate and sodium dithionite to an N-benzophenothiazine solution and stirring and mixing, removing water from the mixture after the mixture turns yellow, then adding it to triethylamine, adding triphosgene solution and stirring, then adding 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl chloroformate and triethylamine, fully mixing and reacting, and purifying to obtain type I photosensitizer prodrug NBS-PB.

3. A method for preparing a type I photosensitizer prodrug according to claim 1 or 2, characterized in that: The specific process includes: dissolving 390 mg of N-benzophenothiazine in a 50% deionized water / dichloromethane solution, adding 168 mg of sodium bicarbonate and 526 mg of sodium dithionite and stirring for 2 hours, removing water from the mixture after the mixture turns yellow, and adding 170 μL of triethylamine; slowly adding 120 mg of triphosgene to 1 mL of dichloromethane, and adding it to the mixed system obtained in the previous step, stirring at room temperature for 0.5 hours; adding 296 mg of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl chloroformate and 140 μL of triethylamine to the above solution, collecting the mixture after reacting overnight, and purifying to obtain type I photosensitizer prodrug NBS-PB.

4. Use of the type I photosensitizer prodrug according to claim 1 in the preparation of a drug for treating diabetic wounds.

5. A nanovesicle using the type I photosensitizer prodrug according to claim 1, characterized in that: The invention comprises vesicles derived from human umbilical mesenchymal stem cells and the type I photosensitizer prodrug loaded thereon, which is a nanovesicle EVs@NBS-PB.

6. A method for preparing nanovesicles using the type I photosensitizer prodrug as claimed in claim 5, characterized in that: The process includes: lysing human umbilical cord mesenchymal stem cells, repeatedly grinding and centrifuging to obtain the supernatant, repeatedly extruding the supernatant through 800nm ​​and 400nm polycarbonate membranes in a liposome extruder to obtain cell vesicles, adding type I photosensitizer prodrug to the cell vesicle solution, and then repeatedly extruding it through a 200nm polycarbonate membrane of a liposome extruder to obtain nanovesicles EVs@NBS-PB.

7. The method for preparing nanovesicles using the type I photosensitizer prodrug according to claim 6, characterized in that: The detailed process is as follows: 7 Human umbilical cord mesenchymal stem cells (HUMSCs) were lysed with 1 ml hypotonic lysis buffer at 4°C overnight, the lysate was repeatedly ground 20 times, centrifuged at 3200 g and 4°C for 5 min, and the supernatant was taken. The supernatant was repeatedly extruded 20 times through 800 nm and 400 nm polycarbonate membranes in a liposome extruder to obtain cell vesicles UC-EVs. 10 μl of 100 mM type I photosensitizer prodrug NBS-PB was added to 1 ml UC-EVs solution and vortexed to mix, and then repeatedly extruded 20 times through a 200 nm polycarbonate membrane of a liposome extruder to obtain nanovesicles EVs@NBS-PB.

8. Use of the nanovesicles comprising the type I photosensitizer prodrug as claimed in claim 5 in the preparation of a drug for treating diabetic wounds.

9. Use of the nanovesicles containing the type I photosensitizer prodrug as claimed in claim 8 in preparing a hydrogel microneedle patch.

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