A hydrogel for exosome delivery and its preparation and application

Abstract

The present application relates to the technical field of hydrogel, in particular to a hydrogel for exosome delivery and preparation and application thereof.The hydrogel for exosome delivery prepared by the present application integrates multiple functions, has good biocompatibility, and has the characteristics of injectability, self-healing, tissue adhesion and exosome binding, which is conducive to the maintenance of the integrity of the hydrogel scaffold and the integration of the hydrogel and the host tissue; the exosome-hydrogel system prepared further is conducive to the retention and long-term controlled release of exosomes at the affected area, and is conducive to the continuous exertion of the therapeutic effect, and improves the quality and efficiency of tissue regeneration.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, and in particular to a hydrogel for exosome delivery and its preparation and application. Background Art

[0002] Clinically, there is a huge demand for repairing critical-size tissue defects, but its treatment still faces severe challenges. Existing therapies, such as autologous tissue transplantation and cell-based therapies, have many limitations, including limited donor tissue, potential immune rejection, the risk of tumorigenesis, high costs, and ethical issues. In recent years, therapeutic strategies based on extracellular vesicles, especially exosomes, have been proposed as new methods to promote tissue regeneration. This is because they contain rich components such as proteins, nucleic acids, and lipids, have the potential to serve as effective drug delivery vehicles, and have many advantages, such as low immunogenicity, easy cross-biological barriers, and easy engineering modification to obtain special functions.

[0003] In existing exosome-based and engineered exosome-based therapies, exosomes are typically mixed with phosphate-buffered saline and injected into the desired treatment area via a syringe and needle. Disadvantages of this approach include rapid diffusion of exosomes through body fluids, difficulty accumulating at the affected site, low bioavailability, and the need for multiple dosing. There are also reports on the use of hydrogels to encapsulate exosomes in therapeutics. Hydrogels are hydrophilic, porous, and flexible materials composed of three-dimensional polymer networks with high water content that closely mimic the extracellular matrix. Over the past few decades, hydrogels have attracted significant research attention due to their promising applications in tissue engineering. However, existing hydrogels typically suffer from low mechanical strength and are prone to rupture when subjected to external tension. They also lack tissue adhesion, hindering good tissue integration and prone to migration from the defect area. Furthermore, due to a lack of exosome binding sites, exosomes are released too quickly, making sustained therapeutic efficacy difficult. There is an urgent need to develop functionally optimized hydrogels for exosome delivery. Summary of the Invention

[0004] To address the above-mentioned issues, the present invention aims to provide a hydrogel for exosome delivery, and its preparation and application. The hydrogel for exosome delivery prepared by the present invention integrates multiple functions, exhibits excellent biocompatibility, and exhibits injectable, self-healing, tissue-adhesive, and exosome-binding properties, which facilitate the maintenance of the integrity of the hydrogel scaffold and the integration of the hydrogel with the host tissue. Furthermore, the resulting exosome-hydrogel system facilitates the retention of exosomes in the affected area and their long-term sustained release, facilitating the sustained therapeutic effect and improving the quality and efficiency of tissue regeneration.

[0005] The hydrogel for exosome delivery of the present invention is composed of specific hydrazide-grafted hyaluronic acid HA-A and aldehyde-modified hyaluronic acid HA-C, and has self-healing, tissue adhesion and exosome binding properties to meet the expected requirements of exosome delivery and tissue defect regeneration and repair treatment in clinical settings.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The first object of the present invention is to provide a hydrogel for exosome delivery, wherein the hydrogel for exosome delivery is a HA-AC hydrogel, which is obtained by mixing hydrazide-grafted hyaluronic acid (abbreviated as "HA-A") and aldehyde-modified hyaluronic acid (abbreviated as "HA-A").

[0008] In one embodiment of the present invention, the molar ratio of the hydrazide-grafted hyaluronic acid to the aldehyde-modified hyaluronic acid is 1-2:1-3.

[0009] A second object of the present invention is to provide a method for preparing a hydrogel for exosome delivery, comprising the following steps:

[0010] The hydrazide-grafted hyaluronic acid and the aldehyde-modified hyaluronic acid were mixed and reacted to prepare the HA-AC hydrogel.

[0011] In one embodiment of the present invention, the preparation of hydrazide-grafted hyaluronic acid comprises the following steps:

[0012] After dissolving hyaluronic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 1-hydroxy-benzotriazole are added and mixed for reaction, and then ADH is added, mixed for reaction, and then post-processed to obtain hydrazide-grafted hyaluronic acid.

[0013] In one embodiment of the present invention, the molar ratio of hyaluronic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 1-hydroxy-benzotriazole and ADH is 1:2-6:2-6:10-30.

[0014] In one embodiment of the present invention, during the reaction, the temperature is room temperature and the reaction time is 12 to 36 hours.

[0015] In one embodiment of the present invention, the post-treatment is deep dialysis using deionized water (molecular cut-off of 10,000 MWCO) to purify the reaction product.

[0016] In one embodiment of the present invention, the preparation of aldehyde-modified hyaluronic acid comprises the following steps:

[0017] HA is dissolved and reacted with sodium periodate. After the reaction is completed, the reaction is stopped and post-processed to obtain aldehyde-modified hyaluronic acid.

[0018] In one embodiment of the present invention, the molar ratio of HA to sodium periodate is 0.5-1.5:0.5-1.5; during the reaction, the temperature is room temperature and the reaction time is 4-8 hours.

[0019] In one embodiment of the present invention, the post-treatment is deep dialysis using deionized water (molecular cut-off of 10,000 MWCO) to purify the reaction product.

[0020] The third object of the present invention is to provide a use of a hydrogel for exosome delivery in the preparation of an exosome-hydrogel system.

[0021] The fourth object of the present invention is to provide an exosome-hydrogel system obtained by mixing exosomes, hydrazide-grafted hyaluronic acid and aldehyde-modified hyaluronic acid.

[0022] A fifth object of the present invention is to provide a method for preparing an exosome-hydrogel system, comprising the following steps:

[0023] The exosomes are mixed with hydrazide-grafted hyaluronic acid, and then mixed with aldehyde-modified hyaluronic acid and reacted to obtain an exosome-hydrogel system.

[0024] In one embodiment of the present invention, the usage ratio of exosomes, hydrazide-grafted hyaluronic acid, and aldehyde-modified hyaluronic acid is 10-100 μg:0.5 mL:0.5 mL.

[0025] The sixth object of the present invention is to provide an application of an exosome-hydrogel system in the preparation of a bone defect repair drug.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The exosome delivery hydrogel of the present invention exhibits excellent biocompatibility and possesses multiple practical functions that meet clinical needs, including injectability, self-healing, tissue adhesion, and exosome binding properties. It is of great significance for the regeneration and repair of lacunar, anatomically complex, and irregular tissue defects. The exosome delivery hydrogel prepared using the present invention improves the efficiency of encapsulating, retaining, delivering, and releasing exosomes to target tissue sites, optimizing the hydrogel's potential for tissue engineering applications.

[0028] (1) The hydrogel for exosome delivery of the present invention can be directly gelled in situ at the tissue defect site, the reaction is mild and rapid, no additional cross-linking reagents need to be introduced, and it is non-toxic and harmless.

[0029] (2) Exosomes can be premixed with hydrazide-grafted hyaluronic acid before use, which is convenient for preoperative preparation. The hydrogel precursor solution has a certain fluidity and is easy to fill and store, which facilitates industrial production.

[0030] (3) The hydrogel for exosome delivery of the present invention is injectable, easy to operate, and conforms to the concept of minimally invasive treatment.

[0031] (4) The initial hydrogel solution can fill defects of any shape and can fit tightly with the defective tissue, which is of great significance for filling defects of complex morphology tissues.

[0032] (5) The hydrogel for exosome delivery of the present invention can adhere to the tissue while forming a gel, preventing the hydrogel from dislocating, and seamlessly connects with the host tissue to form a perfect integration.

[0033] (6) The hydrogel for exosome delivery of the present invention can heal itself after being damaged by external forces, which is beneficial to maintaining the integrity of the hydrogel scaffold.

[0034] (7) The hydrogel for exosome delivery of the present invention has exosome binding sites, which is beneficial to the retention of exosomes in the affected area and long-term sustained release, and is beneficial to the continued exertion of therapeutic effects.

[0035] (8) The hydrogel for exosome delivery of the present invention has good biocompatibility and strong adjustability, and the hydrogel preparation and application parameters can be customized for different tissue application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a diagram showing the working principle of the hydrogel for exosome delivery of the present invention;

[0037] Figure 2 is the NMR spectrum of HA-A;

[0038] Figure 3 is the FITR map of HA-C;

[0039] Figure 4 Schematic diagram of the storage modulus and loss modulus of HA-AC hydrogels with different concentrations;

[0040] Figure 5 Schematic diagram of the gelation time of HA-AC hydrogels with different concentrations;

[0041] Figure 6 Schematic diagram of the swelling rate of HA-AC hydrogels with different concentrations;

[0042] Figure 7 Schematic diagram of HA-AC hydrogel injected into PBS solution;

[0043] Figure 8Schematic diagram of rheological test of self-healing performance of HA-AC hydrogel;

[0044] Figure 9 Schematic diagram of the macroscopic image of the self-healing performance of HA-AC hydrogel;

[0045] Figure 10 This is a scanning electron micrograph of HA-AC hydrogel tissue adhesion;

[0046] Figure 11 Schematic diagram of HA-AC hydrogel tissue adhesion mechanics test;

[0047] Figure 12 Schematic diagram of cell activity in co-culture of HA-AC hydrogel and stem cells;

[0048] Figure 13 Schematic diagram of cell apoptosis levels in co-culture of HA-AC hydrogel and stem cells;

[0049] Figure 14 Schematic diagram of the distribution of exosomes in HA-AC hydrogel;

[0050] Figure 15 Schematic diagram of the release curve of exosomes in HA-AC hydrogel;

[0051] Figure 16 Schematic diagram of the exosome-hydrogel system promoting regeneration and repair of bone defects. DETAILED DESCRIPTION

[0052] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0053] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0054] In the following embodiments, the exosomes can be exosomes or engineered exosomes derived from any cell with regenerative properties.

[0055] Example 1

[0056] This embodiment provides a hydrogel for exosome delivery, an exosome-hydrogel system, and a preparation method thereof.

[0057] (S1) Preparation of hydrazide-grafted hyaluronic acid (HA-A): Hyaluronic acid (HA) was dissolved in MES buffer (pH = 5.3), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBT) were added to activate HA for 2 hours. Next, ADH was added to the mixed solution, and the pH was adjusted to 6.8 every 30 minutes for 4 hours. The reaction was then continued for 24 hours without pH adjustment. After completion of the reaction, the reaction product was purified by deep dialysis against deionized water (molecular weight cutoff 10,000 MWCO) to obtain hydrazide-grafted hyaluronic acid.

[0058] Among them, HA:EDC:HOBT:ADH=1:4:4:20 (molar ratio).

[0059] Aldehydated hyaluronic acid (HA-C) was prepared by dissolving HA in deionized water and reacting it with sodium periodate (NaIO4) in the dark for 6 hours at a molar ratio of HA:NaIO4 = 1:1. The reaction was then terminated by the addition of 1% (final concentration) of ethylene glycol. Following completion of the reaction, the product was thoroughly purified by dialysis against deionized water (molecular weight cutoff 10,000 MWCO) in the dark to yield aldehydated hyaluronic acid.

[0060] Exosome preparation: Bone marrow mesenchymal stem cells were expanded in a conventional culture medium and then cultured for 48 hours in an exosome-free culture medium (37°C, 5% CO2 incubator). The cell culture medium was collected and exosomes were isolated from the culture medium by continuous centrifugation. The continuous centrifugation steps are as follows:

[0061] 1) Centrifuge the collected liquid at 300g for 10 minutes at 4°C, discard the precipitate, and collect the supernatant;

[0062] 2) The collected supernatant was centrifuged at 2000 g for 20 minutes at 4°C, the precipitate was discarded, and the supernatant was collected;

[0063] 3) The collected supernatant was centrifuged at 10,000 g for 30 minutes at 4°C, the precipitate was discarded, and the supernatant was collected;

[0064] 4) Filter the supernatant using a 0.22 μm filter, then centrifuge at 100,000 × g for 90 minutes at 4°C in an ultracentrifuge, discard the supernatant, and retain the precipitate;

[0065] 5) The precipitate was washed with PBS and ultracentrifuged again at 100,000 g for 90 minutes. The resulting precipitate was resuspended in a small amount of PBS to obtain exosomes.

[0066] (S2) The hydrazide-grafted hyaluronic acid prepared in step (S1) is mixed with aldehyde-modified hyaluronic acid at a volume ratio of 1:1 to obtain a hydrogel for exosome delivery: HA-A-C hydrogel.

[0067] Further, 50 μg of exosomes are mixed with 0.5 mL of hydrazide-grafted hyaluronic acid (2% by mass), and then mixed with 0.5 mL of aldehyde-modified hyaluronic acid (2% by mass), and after mixing, cross-linking is performed to obtain an exosome-hydrogel system.

[0068] Performance analysis:

[0069] HA-A-C reaction principle: As shown in Figure 1 , HA-A and HA-C can be self-assembled and cross-linked by the interaction of ADH and aldehyde groups in a mild and rapid reaction to form a dynamic and reversible C=N covalent bond, thereby realizing self-healing performance; in addition, aldehyde groups can be combined with amine groups on tissues or exosomes.

[0070] Characterization of HA-A: Purified HA-A is dissolved using D2O and detected by nuclear magnetic resonance hydrogen spectrum (NMR). As shown in Figure 2 , the appearance of the ADH characteristic peak indicates the successful synthesis of HA-A.

[0071] Characterization of HA-C: Purified HA-C is characterized by Fourier transform infrared spectroscopy (FTIR). As shown in Figure 3 , the characteristic peak at 1716 cm -1 indicates the successful synthesis of HA-C.

[0072] Storage modulus and energy dissipation modulus detection of HA-A-C hydrogel: Time scanning oscillation test of different concentrations of HA-A-C hydrogel is carried out in a rotational rheometer under CD mode at 1 Hz and 1% strain, as shown in Figure 4 , the storage modulus of HA-A-C hydrogel increases with the increase of solid content, and the solid content of 3% can exceed 10000 Pa, indicating that the HA-A-C hydrogel has excellent mechanical properties and stability.

[0073] Gelation time detection of HA-A-C hydrogel: The gelation time of different concentrations of HA-A-C hydrogel is tested using the small bottle tilting method. Specifically, the HA-A-C hydrogel is added to a glass bottle and left to stand at 37°C for a period of time. When the HA-A-C hydrogel solution stops flowing after the glass bottle is tilted, the time required for standing is recorded. As shown in Figure 5 , the gelation time of HA-A-C hydrogel is inversely proportional to its concentration, and the concentration changes from 0.5% to 3%, and the gelation time of HA-A-C hydrogel decreases from more than 25 minutes to less than 30 seconds.

[0074] HA-AC hydrogel swelling rate test: HA-AC hydrogels with different concentrations were immersed in DPBS (pH = 7.4) at 37°C for 24 hours. The initial weight of the sample (W0) and the weight of the sample after swelling (W1) were recorded to calculate the swelling rate using the following equation: Swelling rate (%) = (W1 / W0-1) * 100%. Figure 6 As shown, the swelling ratio of HA-AC hydrogels is proportional to their concentration. Hydrogels with higher solid content exhibited greater swelling ratios. When the concentration varied from 0.5% to 3%, the swelling ratios ranged from approximately 7% to approximately 60%. Considering the mechanical properties, gelation time, and swelling ratio, a 2% concentration was selected for subsequent experiments.

[0075] Injectability of HA-AC hydrogel: HA-AC hydrogel was injected into PBS solution and it was found that HA-AC hydrogel had excellent injectability (e.g. Figure 7 shown).

[0076] Rheological characterization of the self-healing behavior of HA-AC hydrogel: HA-AC hydrogel was subjected to oscillation sweep test in the strain range of 1% to 10000% in a rotational rheometer. The damaged HA-AC hydrogel was then allowed to self-heal for 15 minutes, and then the self-healing effect was characterized by time oscillation sweep test. Figure 8 As shown in the figure, the hydrogel structure was destroyed with the increase of strain, which was manifested as G" being greater than G'. After allowing the destroyed HA-AC hydrogel to self-heal for 15 minutes, the time oscillation scanning test showed that the destroyed HA-AC hydrogel recovered the initial G' / G", showing significant self-healing performance.

[0077] Macroscopic observation of the self-healing ability of HA-AC hydrogel: HA-AC hydrogel that was gelled into sheets and dyed with different colors was cut into two parts and then attempted to reconnect after exchanging them. Figure 9 Figure 2 shows a macroscopic image of the self-healing properties of HA-AC. A HA-AC hydrogel cut into two pieces attempts to reassemble after exchange. The boundaries of the assembled hydrogel sheet blur at the healing interface, re-forming a new, complete sheet.

[0078] Scanning electron microscopy observation of the microstructure of HA-AC hydrogel: The HA-AC hydrogel was freeze-dried and broken, and then the surface was sprayed with gold. Next, the sample was photographed by scanning electron microscopy (Zeiss, Germany). In order to evaluate the integration of HA-AC hydrogel and tissue, HA-AC hydrogel was applied to the tissue and gelled in situ. After freeze-drying and gold spraying, the sample was observed by scanning electron microscopy. Figure 10To evaluate the excellent tissue adhesion and integration ability of HA-A-C hydrogel, the adhesion interface of hydrogel to tissue was observed using scanning electron microscopy analysis. The seamless contact between HA-A-C hydrogel and tissue confirmed their firm adhesion and integration.

[0079] HA-A-C hydrogel tissue adhesion test: The tissue adhesion was tested using a lap shear test, and fresh skin (10 mm x 10 mm) was adhered to a glass slide using cyanoacrylate glue. The HA-A-C hydrogel was evenly coated on the surface of the skin. Another skin-pasted glass slide was placed on the first glass slide in a skin-to-skin manner. After in situ gelation at 37°C, the bonded test sample was subjected to tensile mechanical testing by a universal testing machine. The tissue adhesion strength of the HA-A-C hydrogel was determined according to the maximum tensile stress divided by the adhesion area. As shown in Figure 11 The results show that the HA-A-C hydrogel exhibits higher adhesion strength than the HAMA hydrogel and commercial fibrin glue. After loading the exosomes (multifunctional exosome delivery hydrogel) into the HA-A-C hydrogel, the adhesion strength decreased slightly, which may be related to the occupation of some aldehyde groups by exosomes. However, the multifunctional exosome delivery hydrogel still has stronger adhesion than HAMA and fibrin glue.

[0080] HA-A-C hydrogel co-cultured with stem cells cell activity test: MSCs were incubated with HA-A and HA-C and the formed HA-A-C hydrogel using a transwell model to evaluate the cell compatibility of the HA-A-C hydrogel. Cell viability was detected using a CCK-8 kit at set time points 1 day, 3 days, 5 days. As shown in Figure 12 The results show that the hydrogel precursors HA-A and HA-C and the formed HA-A-C hydrogel do not affect cell viability.

[0081] HA-A-C hydrogel co-cultured with stem cells cell apoptosis level test: MSCs were cultured on the surface of HA-A-C hydrogel, and cells seeded in the well plate were used as a control. After 3 days, the apoptosis level was detected using a flow cytometer. As shown in Figure 13 The results show that culturing stem cells on the surface of the formed HA-A-C hydrogel does not significantly change the cell apoptosis rate compared to the control group.

[0082] Exosome distribution in multifunctional exosome delivery hydrogel: Exosomes were first mixed in the HA-A hydrogel solution, and then mixed with an equal amount of HA-C solution to gelate. Subsequently, the sample was scanned and reconstructed using a confocal laser scanning microscope. As shown in Figure 14 The results show that the exosomes labeled with red fluorescence are uniformly distributed in the HA-A-C hydrogel.

[0083] Exosome release profile of multifunctional exosome delivery hydrogel: Equal amounts of exosomes were added to the HA-AC hydrogel precursor solution and the HAMA hydrogel precursor solution, respectively. After gelation, the samples were immersed in PBS buffer. 10 μL of PBS was collected at different examination time points and supplemented with equal amounts of fresh PBS. The release of exosomes was detected by BCA protein detection kit. Figure 15 As shown in Figure 3, the release profile of exosomes encapsulated in HA-AC hydrogel (multifunctional exosome delivery hydrogel) was flatter and lasted longer compared with HAMA, which may be attributed to the binding of exosomes to the aldehyde groups in the hydrogel.

[0084] Micro-CT evaluation of the regenerative and repair effects of a hydrogel / exosome system (multifunctional exosome-delivering hydrogel) on diabetic bone defects: Four-week-old male Sprague Dawley rats were fed a high-fat diet for four weeks and then injected intraperitoneally with streptozotocin at a dose of 30 mg / kg. One week later, rats with blood glucose levels consistently above 16.7 mM were considered diabetic. Subsequently, the diabetic rats were randomly divided into three groups: 1) Balnk group, 2) Gel group, and 3) Gel-Exo group. Under general anesthesia, the surgical site was shaved and disinfected. A 3 mm diameter and 2.5 mm deep bone defect was created near the medial epicondyle of each rat using a drill. The defects were then fully filled with the different hydrogels, and the surgical site was sutured layer by layer. Four weeks later, the rats were sacrificed, and the femurs were removed and fixed in 10% neutral formalin. The samples were scanned and analyzed using a μCT50 (Scanco Medical, Switzerland) at 70 kVp, 114 μA, and 15 μm resolution. The defect area was selected as the region of interest (ROI) for quantitative analysis of bone volume / tissue volume (BV / TV) and bone mineral density (BMD). Figure 16 As shown in the results, compared with the control group (Blank), the HA-AC hydrogel group (Gel) formed more new bone, and the multifunctional exosome delivery hydrogel group (Gel-Exo) showed the best bone regeneration-promoting effect, indicating that the prepared multifunctional exosome delivery hydrogel has good potential for tissue engineering applications.

[0085] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the explanations of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An application of an exosome-hydrogel system in the preparation of a bone defect repair drug, characterized in that: The exosome-hydrogel system was prepared by the following method: The exosomes are mixed with hydrazide-grafted hyaluronic acid, and then mixed with aldehyde-modified hyaluronic acid and reacted to obtain an exosome-hydrogel system; The method for preparing exosomes is as follows: Bone marrow mesenchymal stem cells were expanded through conventional culture and cultured in exosome-free culture medium for 48 hours. The cell culture medium was collected and exosomes were isolated from it by continuous centrifugation. The preparation of hydrazide-grafted hyaluronic acid comprises the following steps: After dissolving hyaluronic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 1-hydroxy-benzotriazole were added and mixed and reacted for 2 hours; ADH was then added, mixed and reacted, and then post-treated to obtain hydrazide-grafted hyaluronic acid; the molar ratio of hyaluronic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 1-hydroxy-benzotriazole and ADH was 1:4:4:20, the reaction temperature was room temperature, and the reaction time was 24 hours; The preparation of aldehyde-modified hyaluronic acid comprises the following steps: HA is dissolved and reacted with sodium periodate. After the reaction is completed, the reaction is stopped and post-processed to obtain aldehyde-modified hyaluronic acid. The molar ratio of HA to sodium periodate is 1:

1. The reaction temperature is room temperature and the reaction time is 6 hours.

2. The use according to claim 1, characterized in that The dosage ratio of exosomes, hydrazide-grafted hyaluronic acid, and aldehyde-modified hyaluronic acid is 10-100 μg:0.5 mL:0.5 mL.

Images