Preparation method and application of mesenchymal stem cell source exosome entrapped valproic acid compound

Abstract

The invention belongs to the field of biological medicine, and discloses a preparation method and application of a mesenchymal stem cell-derived exosome entrapped valproic acid compound. According to the invention, the mesenchymal stem cell source exosome is used as a carrier, valproic acid is entrapped, and the compound of VPA entrapped by the exosome of MSCs is prepared by an ultrasonic method, so that the VPA-EXO prepared by the invention is proved to be capable of promoting faster repair of the wound surface by inhibiting inflammation, promoting collagen formation, accelerating angiogenesis and reepithelization of the wound surface. The valproic acid-loaded human umbilical cord mesenchymal stem cell exosome prepared by the invention is VPA-EXO prepared by an ultrasonic method, has no obvious toxicity to cells, and has obvious advantages.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, specifically to the technical field of exosome drug delivery, and more specifically to a preparation method and application of a mesenchymal stem cell-derived exosome-encapsulated valproic acid complex. Background Art

[0002] The self-repair ability of skin tissue is limited. During the wound healing process, the wound may be difficult to repair and take a long time to heal, which may lead to serious consequences such as damaged skin barrier function, tissue infection and necrosis, and severe local or systemic dysfunction. When the skin is over-repaired, pathological scars will be generated, which will affect the beauty of the skin and also have an impact on the patient's psychology. Traditional wound treatments include wound dressings, debridement of damaged tissues, and autologous skin transplantation.

[0003] Emerging biomedical research technologies include smart wound dressings, bioactive biomaterial matrices, stem cell therapy, gene therapy, targeted drug / growth factor delivery, and bioengineered skin transplants, which can restore the original function of the skin after healing and reconstruct damaged skin tissue. Therefore, it is an urgent problem to develop carrier materials that can achieve the delivery of drug molecules, rapidly regenerate the skin's natural protective structure, and reduce the risk of infection. Summary of the invention

[0004] The present invention uses mesenchymal stem cell-derived exosomes (MSC EXO ) was used as a carrier to encapsulate valproic acid (VPA), and a complex (VPA-EXO) composed of MSCs exosomes encapsulating VPA was prepared by ultrasound. It was confirmed that the VPA-EXO prepared by the present invention can promote faster wound repair by inhibiting inflammation, promoting collagen formation, accelerating angiogenesis and re-epithelialization of the wound.

[0005] The present invention provides a mesenchymal stem cell-derived exosome-loaded valproic acid complex, and a preparation method and application thereof.

[0006] The present invention first provides a method for preparing a mesenchymal stem cell-derived exosome-encapsulated valproic acid complex, which comprises the following steps: using mesenchymal stem cell-derived exosomes MSC as a carrier to encapsulate valproic acid VPA, and preparing the exosome MSC-encapsulated valproic acid VPA complex MSC-Exo by an ultrasonic method.

[0007] Specifically, the preparation method of mesenchymal stem cell-derived exosomes MSC is as follows:

[0008] S1: washing human umbilical cord mesenchymal stem cells with a fusion rate of 80-90% with a buffer, adding serum-free medium for continued culture, and collecting the cell culture supernatant;

[0009] S2: After the collected stem cell culture supernatant is centrifuged to remove impurities, ECS reagent is added, the mixture is mixed by vortex oscillator, and then placed at 0-4°C for stabilization;

[0010] S3 centrifuge and discard the supernatant, add buffer to resuspend;

[0011] S4 centrifuges and retains the supernatant, which is transferred to the upper chamber of the EPF column to obtain the crude exosome particles;

[0012] S5 centrifugation to collect the liquid at the bottom of the EPF column tube, which is the purified exosome MSC particles;

[0013] Optionally, store it in aliquots at -80°C.

[0014] Specifically, the buffer is PBS buffer.

[0015] In a specific implementation,

[0016] Continue culturing in S1 for 18-28 hours;

[0017] In S2, impurities are removed by centrifugation at 2000-40000 g for 5-15 min at 0-4°C; the standing is standing at 0-4°C for 8-20 h;

[0018] In S3, the samples were centrifuged at 8000-12000 g for 45-75 min at 0-4 °C;

[0019] In S4, the cells were centrifuged at 10,000-15,000 g for 1-4 min at 0-4 °C;

[0020] In S5, the samples were centrifuged at 2000-40000 g for 5-15 min at 0-4 °C;

[0021] Preferably,

[0022] Continue culturing in S1 for 23-25 ​​hours, for example, 24 hours;

[0023] In S2, impurities are removed by centrifugation at 3000 g for 10 min at 4°C; the standing is standing at 4°C for 14 h;

[0024] In S3, the samples were centrifuged at 10,000 g for 60 min at 4 °C;

[0025] In S4, the samples were centrifuged at 12,000 g for 2 min at 4 °C;

[0026] In S5, the cells were centrifuged at 3000 g for 10 min at 4°C.

[0027] In a preferred embodiment, the ultrasonic method is performed as follows:

[0028] The exosome MSC and valproic acid VPA are mixed in a mass ratio of 1-20:10-1, and treated with an ultrasonic cell disruptor; after ultrasonic treatment, the mixed solution is incubated to restore the exosome membrane.

[0029] More specifically, the ultrasonic parameters were set as follows: 20% amplitude, 30s on / off, 6 cycles in total, with a 2-min cooling time between each cycle; the mixed solution was incubated at 35-38°C for 45-75 min. According to experimental testing, when the mass ratio of VPA to exosomes was 5:1, the drug loading rate could reach 11.54%.

[0030] In addition, the method further includes the step of separating free valproic acid VPA, specifically using an ultrafiltration tube to centrifuge at a speed of 4000-6000×g for 5-15 minutes to separate the free drug and obtain drug-loaded exosomes.

[0031] The present invention provides a complex MSC-Exo of exosome MSC encapsulating valproic acid (VPA) obtained according to the preparation method.

[0032] The present invention further provides the use of the exosome MSC-Exo complex containing valproic acid (VPA) in the preparation of an agent for promoting skin wound healing.

[0033] Preferably, the concentration of the exosome MSC-Exo complex containing valproic acid (VPA) is 100 μg / mL when administered; the dosage form suitable for injection or addition to wound dressings to promote wound healing can be used.

[0034] The human umbilical cord mesenchymal stem cell exosomes loaded with valproic acid prepared by the present invention have good therapeutic effects and potential mechanisms of action in wound healing. Compared with the prior art, the invention of the present invention is that the VPA-EXO prepared by the ultrasonic method of the present invention has no obvious toxicity to cells. The VPA-EXO prepared by the present invention has obvious effects of promoting cell proliferation and migration. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 、Characterization of UC-MSC-Exo. Among them, (A) characteristic markers of exosomes; (B) transmission electron microscopy morphology of exosomes; (C) nanoparticle tracking analysis of exosomes; (D) nanoparticle diameter distribution of exosomes.

[0036] Figure 2, characterization of VPA-EXO. Among them, (A) transmission electron microscopy morphology of VPA-EXO; (B) nanoparticle diameter distribution of VPA-EXO; (C) characteristic markers of VPA-EXO.

[0037] Figure 3 , PKH26-labeled VPA-EXO was taken up by HSF cells (A) and HUVEC cells (B).

[0038] Figure 4 , optical images and quantitative results of cell scratch assay. Among them, (A) HSF cell scratch assay; (B) quantitative results of HSF cell scratch assay; (C) HUVEC cell scratch assay; (D) quantitative results of HUVEC cell scratch assay.

[0039] Figure 5 , Optical images and quantitative results of cell migration Transwell experiment. Among them, (A) HSF cell Transwell experiment; (B) HUVEC cell Transwell experiment; (C) Quantitative results of HSF cell Transwell experiment; (D) Quantitative results of HUVEC cell Transwell experiment.

[0040] Figure 6 , optical images and quantitative results of in vitro angiogenesis of endothelial cells. Among them, (A) optical image of tube formation experiment; (B) fluorescent staining tube formation experiment; (C) quantitative results of tube formation experiment.

[0041] Figure 7 , ELISA method was used to detect the expression of cytokines. Among them, (A) IL-1β; (B) IL-8; (C) TNF-α; (D) MMP-9; (E) PG-E2; (F) VEGF.

[0042] Figure 8 , mRNA expression levels of wound healing related factors. Among them, (A) TNF-α; (B) IL-1β; (C) TGF-β1; (D) Fibronectin; (E) Col-Ⅰ; (F) Ang-1; (G) Tie2; (H) VEGFR-2; (I) VEGFA; (J) HIF-1α.

[0043] Fig. 9 , Western blotting results. (A) Relative protein expression of VEGF and PEDF; (B) Quantitative analysis of relative protein expression. DETAILED DESCRIPTION

[0044] Example 1: Isolation and identification of MSC-Exo

[0045] 1. Isolation of MSC-Exo

[0046] Take out the human umbilical cord mesenchymal stem cells with a fusion rate of 80-90% from the incubator, discard the liquid, add PBS to wash 2-3 times, and add serum-free medium to continue culturing. Collect the cell culture supernatant after 24 hours. Transfer the collected stem cell culture supernatant sample to a centrifuge tube, centrifuge at 3000g for 10 minutes at 4°C, add Exosome Concentration Solution (ECS reagent) in proportion to the supernatant after removing impurities, shake and mix on a vortex oscillator for 1 minute, and then place it at 4°C for 14 hours. Centrifuge at 10000g for 60 minutes at 4°C, discard the supernatant, and resuspend in PBS. Centrifuge at 12000g for 2 min at 4°C, retain the supernatant, and transfer it to the upper chamber of Exosome Purification Filter (EPF column) to obtain crude exosome particles. Then centrifuge at 3000g for 10 min at 4°C. Collect the liquid at the bottom of the EPF column after centrifugation. This liquid is the purified exosome particles, which are stored at -80°C after aliquoting.

[0047] 2. Identification of MSC-Exo

[0048] 2.1 Morphology of exosomes

[0049] Drop the exosome sample onto the hydrophilized side of the copper mesh facing up. After incubation for 1 minute, use filter paper to absorb excess liquid from the side. Use tweezers to wash the sample side of the copper mesh with ultrafiltered water for 5-10 seconds, and use filter paper to absorb excess water. Then, place the copper mesh sample side facing up, add uranyl acetate (UA) dye solution to stain for 30 seconds, and dry naturally. Place the sample under a transmission electron microscope and observe the morphology and size of the exosomes.

[0050] 2.2 Exosome Particle Size and Concentration

[0051] The particle size and concentration of exosomes were detected using a nanoparticle tracking analyzer (NanoSightNS500, Zetaview, UK). Calibration was performed before use and the samples were washed three times with PBS. The exosome samples diluted with PBS were added to the nanopores to measure and analyze the particle size and concentration of the exosomes. The principle is to track and analyze the Brownian motion of each particle, and then calculate the hydrodynamic diameter and concentration of the nanoparticles using the equation.

[0052] 2.3 Protein markers of exosomes

[0053] WB protein immunoblotting method: add lysis buffer to the exosome sample for lysis, the ratio of lysis buffer to PMSF is 100:1, place on ice for 3-5 minutes, and shake repeatedly during the period. Centrifuge at 12000×g for 5 minutes at 4℃, remove the supernatant and divide it into sterile EP tubes. Take a part of the lysed protein, add the loading buffer according to the proportion, and heat it in a metal bath (100℃, 10min) for denaturation for use. Put the gel caster into the electrophoresis tank and add enough electrophoresis fluid, add the sample and then electrophoresed. The voltage of the concentrated gel is 75V, and the voltage of the separation gel is 120V. When the bromophenol blue is about 1cm away from the bottom, the electrophoresis can be stopped, and then the membrane is transferred. Activate the PVDF membrane with methanol for 2 minutes, clamp the transfer clips together in the order of (white board) sponge, filter paper, separation gel, PVDF membrane, filter paper, sponge (black board), and transfer the membrane at 300mA constant current for half an hour. During the transfer process, ice bags should be placed around the transfer equipment to cool down. Add TBST to wash the membrane, then add skim milk and block at room temperature for 30 minutes; after washing, add the prepared primary antibodies CD63 (1:1000) and CD81 (1:1000) respectively and incubate slowly at 4°C overnight. Wash the membrane with TBST for 5 minutes each time, three times; add the secondary antibody diluted in proportion to the incubation tank and incubate at room temperature on a shaker for 2 hours. Then add TBST to wash the membrane again, 5 minutes each time, three times. Mix equal volumes of ECLA solution and ECLB solution in a dark room, add the mixed ECL solution to fully react, and after 1-2 minutes, take the band image through the chemiluminescence image analysis system.

[0054] Results: TEM results showed that the extract had a typical cup-shaped morphology of MSC-Exo. NTA results showed that the particle size of most extracts was in the range of 30-150nm, which was consistent with the expected characteristics of exosomes. WB experiments showed that the membrane of the exosomes in the extracts was rich in the tetraspanin family (CD63, CD81 and CD9) involved in exosome transport, the heat shock protein family (HSP60, HSP70, HSPA5, CCT2 and HSP90) and some cell-specific proteins. Through WB experiments, the exosome markers CD63 and CD81 were detected in the exosome samples, see for details. Figure 1 .

[0055] Example 2: Preparation and identification of VPA-EXO

[0056] 1. Preparation of VPA-EXO

[0057] The study used ultrasonic treatment for drug loading. The exosomes prepared in Example 1 and VPA were mixed in a mass ratio of 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, and 20:1, and the mixture of exosomes and VPA was treated with drug loading using an ultrasonic cell disruptor. The ultrasonic parameters were set as follows: 20% amplitude, 30s on / off, 6 cycles in total, and 2 minutes of cooling time between each cycle. After ultrasonic treatment, the mixed solution was incubated at 37°C for 60 minutes to restore the exosome membrane. The free drug was separated using an ultrafiltration tube at a speed of 5000×g for 10 minutes to obtain drug-loaded exosomes.

[0058] The drug loading rate was detected by HPLC.

[0059] 2. Identification of VPA-EXO

[0060] 2.1 Determination of exosome drug loading efficiency by liquid chromatography

[0061] Mobile phase A was acetonitrile-0.1 mol / L sodium acetate (4.2 g of anhydrous sodium acetate, 970 mL of water to dissolve 30 mL of acetonitrile) solution (3:97) (pH 6.5 adjusted with acetic acid). Mobile phase B was acetonitrile-water (4:1). The chromatographic column was Aglient C18 (4.6×250 mm×5 μm); the flow rate was 1.0 mL per minute; the column temperature was 40°C; the detection wavelength was 210 nm, and the elution gradient was shown in Table 1.

[0062] Table 1 Mobile phase conditions

[0063]

[0064] 2.2TEM morphology observation: the operation is the same as above.

[0065] 2.3NTA identification of particle size and concentration: the operation is the same as above.

[0066] 2.4WB detection of protein markers: the operation is the same as above.

[0067] 2.5 Statistical analysis

[0068] GraphPad Prism 9 was used to analyze the differences among the groups using one-way ANOVA. p < 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0069] result:

[0070] (1) Liquid chromatography to determine the drug loading efficiency of exosomes

[0071] When VPA and MSCEXO When the mass ratio increased from 1:10 to 20:1, the encapsulation efficiency showed a downward trend overall, and the drug loading showed an increasing trend. When the mass ratio increased from 1:10 to 2:1, the encapsulation efficiency gradually decreased, and the drug loading slowly increased. When the mass ratio increased from 2:1 to 5:1, the drug loading increased significantly, and the encapsulation efficiency also increased to 11.58%; when the mass ratio increased from 5:1 to 20:1, the drug loading increased, but the encapsulation efficiency gradually decreased. When the mass ratio was 1:10, the encapsulation efficiency was the highest, about 24.26%, but its drug loading was the lowest. VPA and MSC EXO When the mass ratio was 5:1, the drug loading effect was the best and the encapsulation efficiency could reach 11.54%. This ratio was used for subsequent experiments.

[0072] (2) Identification of drug-loaded exosomes

[0073] TEM results showed that VPA-EXO had a typical cup-shaped morphology of exosomes. NTA results showed that the particle size of VPA-EXO was mostly in the range of 30-150nm, which was consistent with the expected characteristics of exosomes. WB experiments detected exosome markers CD9 and TSG101 in the samples, which were consistent with the characteristics of exosomes. Figure 2 (Take VPA-EXO prepared with a mass ratio of exosomes to VPA of 5:1 as an example).

[0074] Example 3: Effect of VPA-EXO on promoting skin wound healing

[0075] 3.1 Uptake of VPA-EXO by HSF and HUVEC cells

[0076] By co-culturing PKH26-labeled VPA-EXO (prepared with a mass ratio of exosomes to VPA of 5:1, the same below) with HSF and HUVEC, the exosomes, cytoskeleton and cell nucleus were dyed red, green and blue, respectively, and the absorption of VPA-EXO by HSF and HUVEC was verified by observing under an inverted fluorescence microscope to test whether the cells can take up the drug-loaded exosomes. If red can be observed in the cells, it means that HSF and HUVEC cells can take up VPA-EXO.

[0077] 3.2 VPA-EXO promotes the proliferation and migration of HSF and HUVEC cells in vitro

[0078] HSF and HUVEC cells were subjected to scratch test and Transwell migration assay, respectively. VPA-EXO, EXO, VPA prepared in DMEM at a concentration of 100 μg / mL and NC group with only DMEM were used on HSF and HUVEC cells, respectively, to observe the effect of VPA-EXO on cell proliferation and migration, so as to evaluate the ability of VPA-EXO to promote wound healing. In the scratch experiment, photography was performed under a microscope at 0h, 8h, and 16h, and the distance of cell migration was counted and the scar area was calculated. In the Transwell migration experiment, the cells were fixed and stained before being photographed.

[0079] 3.3 VPA-EXO promotes the tube formation of endothelial cells in vitro

[0080] Different drugs in the VPA-EXO, EXO, VPA, and NC groups were co-cultured with HUVEC cells, and the tubule formation in each group was observed under an inverted microscope.

[0081] 3.4 VPA-EXO regulates the expression of wound healing-related factors

[0082] ELISA was used to detect the secretion of related factors such as IL-1β, TNF-α, VEGF and other cytokines in vascular endothelial cells and fibroblasts.

[0083] 3.5VPA-EXO regulates the expression of genes related to wound healing

[0084] The expression levels of several angiogenesis-related genes were detected at the mRNA level using qRT-PCR technology to explore the effect of VPA-EXO on the expression of wound healing-related genes such as TNF-α, IL-1β, Ang-1 and other cytokine genes.

[0085] 3.6 VPA-EXO regulates the expression of VEGF and PEDF proteins in HUVEC

[0086] The Western blot method was used to detect the expression of related proteins involved in regulating angiogenesis (such as VEGF and PEDF).

[0087] result:

[0088] (1) In the microscope images, PKH26-labeled drug-loaded exosomes (red) were localized in HSF and HUVEC cells, confirming that the drug-loaded exosomes were internalized by the cells and were able to enter the cells. Figure 3 .

[0089] (2) Optical microscopy confirmed that VPA-EXO promoted the proliferation of endothelial cells and fibroblasts at 12 hours and 24 hours in the scratch test. Transwell assay further proved that VPA-EXO enhanced the migration ability of cells. Figure 4 and Figure 5 .

[0090] (3) Compared with the EXO group and the VPA group, more tubule structures were observed in HUVECs treated with VPA-EXO, followed by the EXO group, and the vascular network structure formation in the NC group was the worst. The tubule length of each group was quantitatively analyzed using ImageJ software. The results showed that the tubule length of the VPA-EXO group was significantly higher than that of the NC group (p<0.01), indicating that the tube formation ability of HUVEC cells was enhanced after VPA-EXO treatment. For details, see Figure 6 .

[0091] ELISA results showed that compared with the NC group, VPA-EXO, EXO, and VPA were able to reduce the expression of IL-1β, IL-8, TNF-α, MMP-9, and PG-E2, and promote the expression of VEGF. The expression of IL-1β, IL-8, TNF-α, MMP-9, and PG-E2 in the VPA-EXO group was significantly lower than that in the NC group, with the best inhibitory effect, and the expression of VEGF was significantly increased. This shows that VPA-EXO has the effect of inhibiting pro-inflammatory factors and promoting angiogenic factors, and has a certain promoting effect on wound healing. For details, see Figure 7 .

[0092] (4) qRT-PCR results showed that the expression of TNF-α and IL-1β mRNA in HSF cells treated with VPA-EXO was lower than that in the NC group, and the expression of TGF-β1 and Fibronectin was significantly increased. The expression of angiogenesis-related factors such as Ang-1, Tie2, and HIF-1α in HUVEC was significantly increased, while the expression of VEGFR-2 and VEGFA was not significantly increased. The results showed that VPA-EXO could inhibit inflammation and promote angiogenesis after acting on vascular endothelial cells and fibroblasts. Figure 8 .

[0093] (5) After VPA-EXO treatment, the expression of VEGF was significantly increased (P<0.01), and the expression of PDEF was significantly decreased (P<0.01). Pigment epithelium-derived factor (PEDF) is an endogenous inhibitor of angiogenesis, which has differentiation and neuroprotective activities as well as anti-angiogenic activities. It can inhibit the migration of endothelial cells in vitro in a dose-dependent manner. For details, see Fig. 9 .

Claims

1. A method for preparing a mesenchymal stem cell-derived exosome-loaded valproic acid complex, characterized in that: The method comprises the following steps: using exosomes MSC derived from mesenchymal stem cells as a carrier to encapsulate valproic acid VPA, and preparing a complex MSC-Exo of exosomes MSC encapsulating valproic acid VPA by ultrasonic method.

2. The preparation method according to claim 1, characterized in that The preparation method of mesenchymal stem cell-derived exosomes MSC is as follows: S1: washing human umbilical cord mesenchymal stem cells with a fusion rate of 80-90% with a buffer, adding serum-free medium for continued culture, and collecting the cell culture supernatant; S2: After the collected stem cell culture supernatant is centrifuged to remove impurities, ECS reagent is added, the mixture is mixed by vortex oscillator, and then placed at 0-4°C for stabilization; S3 centrifuge and discard the supernatant, add buffer to resuspend; S4 centrifuges and retains the supernatant, which is transferred to the upper chamber of the EPF column to obtain the crude exosome particles; S5 centrifugation to collect the liquid at the bottom of the EPF column tube, which is the purified exosome MSC particles; Optionally, store it in aliquots at -80°C.

3. The preparation method according to claim 2, characterized in that: The buffer is PBS buffer.

4. The preparation method according to claim 2, characterized in that: Continue culturing in S1 for 18-28 hours; In S2, impurities are removed by centrifugation at 2000-40000 g for 5-15 min at 0-4°C; the standing is standing at 0-4°C for 8-20 h; In S3, the samples were centrifuged at 8000-12000 g for 45-75 min at 0-4 °C; In S4, the cells were centrifuged at 10,000-15,000 g for 1-4 min at 0-4 °C; In S5, the samples were centrifuged at 2000-40000 g for 5-15 min at 0-4 °C; Preferably, Continue culturing in S1 for 23-25 ​​hours, for example, 24 hours; In S2, impurities are removed by centrifugation at 3000 g for 10 min at 4°C; the standing is standing at 4°C for 14 h; In S3, the samples were centrifuged at 10,000 g for 60 min at 4 °C; In S4, the samples were centrifuged at 12,000 g for 2 min at 4 °C; In S5, the cells were centrifuged at 3000 g for 10 min at 4°C.

5. The preparation method according to claim 1, characterized in that: The ultrasonic method is operated as follows: The exosome MSC and valproic acid VPA are mixed in a mass ratio of 1-20:10-1, and treated with an ultrasonic cell disruptor; after ultrasonic treatment, the mixed solution is incubated to restore the exosome membrane.

6. The preparation method according to claim 5, characterized in that: The ultrasonic parameters were set as follows: 20% amplitude, 30s on / off, 6 cycles in total, with a 2-min cooling time between each cycle; the mixed solution was incubated at 35-38° C. for 45-75 min.

7. The preparation method according to claim 6, characterized in that: The method further includes the step of separating free valproic acid VPA, specifically using an ultrafiltration tube to centrifuge at a speed of 4000-6000×g for 5-15 min to separate the free drug and obtain drug-loaded exosomes.

8. The exosome MSC-Exo complex containing valproic acid (VPA) obtained according to the preparation method according to any one of claims 1 to 7.

9. Use of the exosome MSC-Exo complex containing valproic acid (VPA) as claimed in claim 8 in the preparation of an agent for promoting skin wound healing.

10. The use according to claim 9, characterized in that The concentration of the exosome MSC-Exo complex containing valproic acid (VPA) is 100 μg / mL when administered; the dosage form can be used for injection or added to wound dressings to promote wound healing.

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