Chlorogenic acid-loaded dressing based on polypeptide self-assembled composite hydrogel as well as preparation method and application of chlorogenic acid-loaded dressing

Abstract

The invention discloses a chlorogenic acid-loaded dressing based on polypeptide self-assembled composite hydrogel as well as a preparation method and application of the chlorogenic acid-loaded dressing, and belongs to the technical field of dressings. According to the dressing based on the polypeptide self-assembled composite hydrogel loaded chlorogenic acid disclosed by the invention, the chlorogenic acid can be encapsulated in the hydrogel through a physical or chemical means, so that the stability of the chlorogenic acid is improved, and the release rate of the chlorogenic acid is controlled. The acting time of the medicine on the wound can be prolonged, so that a better treatment effect is realized, and the chronic wound is effectively treated.

Description

Technical Field

[0001] The present invention relates to the technical field of dressings, and more particularly to a dressing based on polypeptide self-assembled composite hydrogel loaded with chlorogenic acid, and a preparation method and application thereof. Background Art

[0002] Diabetes is a clinical metabolic disease. After the onset of the disease, the blood sugar level in the patient's body rises. With the development of society, people's living standards have gradually improved, and the incidence of diabetes has continued to rise. The number of diabetes patients in the world and China has increased year by year, and the diabetes diagnosis and treatment market has great potential. Clinically, the treatment of chronic wound infection in diabetes is relatively complicated, and the treatment cycle of this disease is long. The wound is difficult to heal, and the patient is prone to repeated infections, forming a vicious cycle. So far, there is still a lack of drugs that can effectively cure such diseases.

[0003] Adding antibiotics, anti-inflammatory drugs and growth factors to drugs is currently the main way to eliminate inflammation in chronic diabetic wounds and promote cell proliferation. However, the problems of these bioactive drugs being easily inactivated, prone to drug resistance and toxic side effects cannot be ignored. Chlorogenic acid, as a water-soluble phenolic compound, is widely distributed in the plant kingdom and exhibits strong biological activity and multifaceted pharmacological effects. When studying the effects of chlorogenic acid on diabetic wound healing in rats, the relevant team found that chlorogenic acid can accelerate wound healing. However, there are still great deficiencies in the researchers' exploration of the molecular mechanism of chlorogenic acid and target verification. How to accurately and targetedly deliver chlorogenic acid and related therapeutic drugs to inflammatory wounds and perform sustained-release drugs is also an issue that needs to be urgently addressed in future research.

[0004] In recent years, the research on diabetic chronic wound dressings has become a long-standing problem in the clinical treatment of diabetes. Traditional wound dressings lack corresponding biological functions and are difficult to replace. They cannot effectively protect wounds, and are prone to local oxidative stress, vascular damage and secondary infection in wounds, leading to prolonged healing. A small number of studies have reported the use of dressing materials themselves to regulate the inflammatory microenvironment of the wound surface. However, most of these dressing materials can only play a role in one reason for wound failure to heal, and the overall healing effect is not ideal. Nanogels have attracted widespread attention in the field of medical dressings due to their unique advantages, while the exploration of peptide / sugar composite hydrogels as potential biomaterials for chronic diabetic wound healing is still limited. At present, teams at home and abroad have conducted relevant research on the construction of nanogel systems loaded with active substances for diabetic wound healing. However, there are few basic data for the research, the construction of carriers is scarce, and the exploration of the effects of chlorogenic acid is relatively simple.

[0005] Therefore, providing a dressing based on polypeptide self-assembled composite hydrogel loaded with chlorogenic acid and a preparation method and application thereof is an issue that needs to be urgently addressed by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention aims to solve the problems of the prior art such as lack of effective drugs and difficulty in targeted relief, lack of new wound dressings with mechanical functions similar to skin tissue and multiple biological functions, and provides a dressing based on polypeptide self-assembled composite hydrogel loaded with chlorogenic acid and its preparation method and application, constructs a naphthalene-modified diphenylalanine-hyaluronic acid gel carrier, loads the natural product active substance chlorogenic acid, innovatively utilizes the polypeptide self-assembled composite hydrogel loaded with chlorogenic acid for sustained drug release, promotes the healing of chronic diabetic wounds, and forms a hydrogel complex that can be used for the treatment of chronic diabetic wounds.

[0007] The present invention provides a more convenient, efficient, new and safe treatment method for chronic diabetic wound healing; explores newer, efficient, convenient and accurate treatment methods for chronic diabetic wound healing; and paves the way for studying the application of new composite hydrogels as potential sustained-release drug carriers. The dressing prepared by the present invention utilizes the pharmacological effects of chlorogenic acid and its combination to improve the characteristics of sustained-release drugs to further promote wound healing, paving the way for subsequent research on the application effect of chlorogenic acid on wound healing.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A method for preparing a dressing based on polypeptide self-assembled composite hydrogel loaded with chlorogenic acid, the specific steps are as follows:

[0010] (1) adding 1 ml of DMSO to the lyophilized peptide to obtain a peptide solution with a concentration of 100.0 mg / mL; the peptide is R-FF-OH, R = naphthalene (N));

[0011] (2) HA powder was dissolved in deionized water to obtain HA solution I with a concentration of 10.0 mg / mL;

[0012] (3) Add 400 μl of HA solution to a round-bottom Eppendorf tube, and then add 80 μl of ddH2O to obtain HA solution II;

[0013] (4) Add chlorogenic acid at a concentration of 100 mg / ml to a conical bottom Eppendorf tube, then add 10 μl of the peptide solution to the tube and mix well to obtain a chlorogenic acid-peptide mixture solution;

[0014] (5) Add the chlorogenic acid-peptide mixture solution to the HA solution II and mix immediately to obtain a dressing with a chlorogenic acid concentration of 0.01-0.2 wt %.

[0015] Furthermore, the method described above prepares a dressing based on a polypeptide self-assembled composite hydrogel loaded with chlorogenic acid.

[0016] Furthermore, the polypeptide self-assembled composite hydrogel-loaded chlorogenic acid dressing is used in the preparation of dressings for treating diabetic chronic wounds.

[0017] The dressing is used in the field of wound healing.

[0018] The present invention has the following technical advantages:

[0019] (1) Synthetic bionanomaterials for drug delivery and wound healing

[0020] The present invention aims to explore the future prospects of synthetic bio-nanomaterials in drug delivery and wound healing, and further explore the application of peptide / sugar composite hydrogels as potential chronic diabetic wound healing biomaterials, providing new ideas for wound repair in the treatment of chronic diabetes, and as a new treatment option, verifying that the controlled release of bioactive factors using composite hydrogels is a new and efficient strategy for repairing chronic wounds.

[0021] (2) Effect of using chlorogenic acid to promote wound healing

[0022] Chlorogenic acid has shown a series of pharmacological effects in current research, such as antioxidant, anti-inflammatory, antimicrobial, anti-diabetic and lipid-lowering functions; however, there are few specific in vivo experimental data. The present invention attempts to explore the therapeutic potential of chlorogenic acid in diabetic wound repair and regeneration, paving the way for subsequent research on the application effect of chlorogenic acid on wound healing.

[0023] (3) Comprehensive integration of various disciplines for integrated innovation

[0024] Chronic wounds are a multifactorial disease that often depends on the healing mechanisms of damaged patients, microorganisms from different sources of wounds, and environmental determinants around the wound (moisture, temperature, pH, etc.). Strategies to address this widespread health-damaging problem will need to focus on all aspects, not just one aspect as in the past. Use a multidisciplinary, holistic approach to address this complex health problem. This project is an integrated innovation that integrates drug delivery technology, nanomolecular technology, new biomaterial technology, etc. It is a multidisciplinary intersection. During the experimental exploration process, the drug-loaded composite hydrogel was further optimized to lay a scientific theoretical foundation for the treatment of chronic diabetic wounds.

[0025] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a dressing based on polypeptide self-assembly composite hydrogel loaded with chlorogenic acid and its preparation method and application, which has the following characteristics:

[0026] Beneficial effects:

[0027] (1) Higher biocompatibility, which is beneficial for wound cleaning.

[0028] (2) Targeted drug delivery: The constructed composite hydrogel system can encapsulate chlorogenic acid inside the hydrogel by physical or chemical means, improve the stability of chlorogenic acid, and control its release rate. It can prolong its action time at the wound site to achieve better therapeutic effects, thereby effectively treating chronic wounds.

[0029] (3) The effectiveness of chlorogenic acid as a drug: It has a broad-spectrum antifungal and antibacterial effect. Compared with other bioactive dressings, chlorogenic acid-hydrogel dressings have the initiative and directness to heal wounds, rather than just indirectly regulating the wound microbial environment to achieve the purpose of wound treatment.

[0030] (4) Focus on the field of high-end medical dressings: Currently, China's high-end medical dressings are mostly imported. Due to price factors, the current usage rate of high-end medical dressings in the Chinese market is only 10%. The present invention also studies high-end dressings, which can not only help wounds heal faster, but also have the advantages of high aesthetics, less scarring, and no cross infection.

[0031] (5) The present invention is an integrated innovation that integrates drug delivery technology, nanomolecular technology, new biomaterial technology, etc. It is a multidisciplinary cross and opens up a new path for project production in the dressing field. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0033] Figure 1 The accompanying drawing is a flow chart of the preparation process of the composite hydrogel of the present invention;

[0034] Figure 2 The accompanying drawing is the structural formula of R-FF-OH (R = naphthalene (N)) of the present invention;

[0035] Wherein, A: R-FF-OH; B: R; C: structural formula of peptide;

[0036] Figure 3 The accompanying drawings show the physical properties of the HA hydrogel according to the present invention;

[0037] Figure 4 The accompanying drawings show the characteristics of the self-assembled hydrogel of the present invention;

[0038] A: From left to right, transmission electron micrographs of HA hydrogel, R-FF-OH / HA, and hydrogel samples loaded with 0.2% CGA; B: scanning electron micrographs of hydrogel samples loaded with 0.2% CGA, the three images are from three different fields of view; N-FF in the figure represents R-FF-OH;

[0039] Figure 5 The attached figure shows the evaluation results of the R-FF-OH / HA loaded with CGA on diabetic skin wound healing of the present invention; NF in the figure represents R-FF-OH;

[0040] A: Full-thickness defects of diabetic rats in each group were photographed on days 0, 6, and 12 after treatment; B: Wound area of ​​each group; values ​​are mean ± standard deviation, n = 5; C: Body weight of mice in each group, n = 5; D: Biochemical indicators of mice in each group, n = 5. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Example 1

[0043] A preparation method of a dressing based on polypeptide self-assembled composite hydrogel loaded with chlorogenic acid, the flow chart is shown in Figure 1 , the specific steps are as follows:

[0044] Step 1 Materials and Preliminary Preparation

[0045] Lyophilized peptide R-FF-OH (R = naphthalene (N)) (structural formula see Figure 2 ) was purchased from GL Biochemical Co., Ltd. (Shanghai, China); hyaluronic acid (HA) was purchased from Theines; dimethyl sulfoxide (DMSO) was purchased from Solebol; streptozotocin (STZ) was purchased from Sigma-Aldrich (St. Louis, MO); phosphate buffered saline (PBS) was purchased from Gibco; and chlorogenic acid (CGA) was purchased from Macklin.

[0046] Step 2 Preparation of hyaluronic acid hydrogel

[0047] HA powder was dissolved in deionized water to obtain HA solution (10.0 mg / mL), 400 μl of which was pipetted into a round-bottom Eppendorf tube, and then 80 μl of ddH2O and 20 μl of DMSO were added to obtain hyaluronic acid hydrogel.

[0048] Step 3: Preparation of hyaluronic acid-peptide hydrogel

[0049] Add 1 ml of DMSO to the lyophilized peptide R-FF-OH (R = naphthalene (N)) to obtain a peptide solution with a concentration of 100.0 mg / mL. Dissolve HA powder in deionized water to obtain HA solution I (10.0 mg / mL). Take a 2 ml round-bottom Eppendorf tube, add 400 μl of HA solution to the tube, and then add 80 μl of ddH2O to obtain HA solution II for use. In a 1.5 ml pointed-bottom Eppendorf tube, add 10 μl of DMSO and 10 μl of peptide solution (100 mg / ml), mix to obtain a DMSO-peptide mixed solution. Add 20 μl of the DMSO-peptide mixed solution to HA solution II, shake to mix, and invert the reaction tube. If the liquid gels and does not flow, the hyaluronic acid-peptide hydrogel (R-FF-OH / HA) can be obtained.

[0050] Step 4 Preparation of chlorogenic acid loaded hydrogel

[0051] Add 1 ml of DMSO to the lyophilized peptide R-FF-OH (R = naphthalene (N)) to obtain a peptide solution with a concentration of 100.0 mg / mL. Dissolve HA powder in deionized water to obtain HA solution I (10.0 mg / mL). Take 5 2 ml round-bottom Eppendorf tubes, add 400 μl of HA solution to each tube, and then add 80 μl of ddH2O to obtain HA solution II for use. Take 5 1.5 ml conical-bottom Eppendorf tubes, add different volumes of 100 mg / ml chlorogenic acid (10 μl, 5 μl, 2.5 μl, 1 μl, 0.5 μl, and the remaining 4 are filled to 10 μl with DMSO), and then add 10 μl of peptide solution (100.0 mg / ml) to each tube and mix well. Add 20 μl of the chlorogenic acid-peptide mixture solution to HA solution II and mix immediately. When the flowing liquid is gelled, the hydrogel R-FF-OH / HA-CGA (0.2wt%, 0.1wt%, 0.05wt%, 0.02wt%, 0.01wt%) containing different concentrations of chlorogenic acid can be obtained.

[0052] Test Example 1 Tube Reversal Method

[0053] First add 400μl HA with a concentration of 10mg / ml to a round-bottom Ep tube, and then add 80μl ddH2O. Take another pointed-bottom Ep tube, and add CGA and DMSO respectively according to Table 1 to form a mixed solution. Then add the CGA-DMSO mixed solution to the previously prepared HA-ddH2O solution to prepare the hydrogel CGA-HA loaded with different concentrations of chlorogenic acid (0.2%, 0.1%, 0.05%, 0.01%); at the same time, set up a simple HA control group, add 20μl DMSO to the HA-ddH2O solution, and shake to mix. Invert all the prepared tubes in a water bath and gradually heat them. During this period, record the highest temperature of the hydrogel formed in the absence of liquid flow, and set it as the gel-solution transition temperature. The results are shown in Figure 3 . Figure 3 The left picture shows that when the hydrogel containing peptides reaches a high temperature of more than 100°C, it can still maintain a gel state. The right picture shows a hydrogel without peptides, which has turned into a solution state when heated to 30°C. This shows that this R-FF-OH / HA composite hydrogel has good stability under high temperature conditions, and loading CGA does not affect its gelation performance.

[0054] Table 1

[0055]

[0056]

[0057] Experimental Example 2 Morphological Characterization of Drug-Loaded Composite Hydrogel

[0058] The prepared HA hydrogel, R-FF-OH / HA, and 0.2% CGA-loaded hydrogel samples were diluted 3 times with ddH2O to form a flowable liquid. Droplets of the hydrogel liquid were placed on a copper grid and dyed with phosphotungstic acid dye (3.0wt%) after drying. The filter paper absorbed the excess solution and the sample was dried overnight to obtain a transmission electron microscopy sample. The transmission electron microscopy results showed that the hyaluronic acid gel alone exhibited a porous structure. The fiber network in the N-FF / HA gel with added peptides was physically cross-linked by entangled soft nanofibers. Even if 0.2% CGA was loaded in the N-FF / HA, the fiber network structure was not destroyed, which was conducive to the long-term release of the drug ( Figure 4 A).

[0059] The prepared 0.2% CGA-loaded hydrogel samples were placed in a freeze dryer for freeze drying overnight, and then placed on the workbench using black double-sided tape. Before imaging, the surface of the hydrogel samples was coated with gold for a few seconds under argon. Scanning electron microscopy results showed that the CGA-loaded N-FF / HA gel had a microporous structure, which may be the reason for the prolonged drug release ( Figure 4 B).

[0060] Experimental Example 3 In vivo experiment

[0061] BALB / c mice (male, 4-6 weeks old, 18-22 g) were purchased from the Experimental Animal Center of Guangdong Medical University. After all mice were adapted for 12 days, a single injection of STZ (120 mg / kg) in citrate buffer solution (0.1 M, pH 4.5) was administered intraperitoneally to begin inducing type I diabetes. Blood glucose concentration was monitored by tail blood using an Accu-Chek blood glucose meter (Roche, Switzerland). After the blood glucose level reached 11.1 mM, the mice were selected and divided into 5 groups, with 5 mice in each group, for the next experiment. Before surgery, a 50 mg / kg dose of sodium pentobarbital (Sigma-Aldrich) injection was administered intraperitoneally, and the dorsal hair of the mice was removed using a depilatory cream. A standardized full-thickness skin wound of 10×10 mm (approximately 100 mm) was created in the dorsal thoracic and lumbar region of the mice. 2 ). Then, Histoacryl was used to stick the silicone ring to the edge of the wound to prevent wound contraction. 50 μL of the prepared saline, HA hydrogel, R-FF-OH / HA hydrogel, R-FF-OH / HA-CGA hydrogel, and free chlorogenic acid solution (100 mg / ml) were applied to the wounds of each group of mice, respectively, and then covered and protected with 3M Tegaderm. All mice were dosed once every 3 days for a total of 5 treatments, and the weight changes of all mice were monitored every 6 days. The wound area was photographed on days 0, 6, and 12 after surgery and measured using Image J software. On the 12th day after surgery, three animals in each group were killed and serum was collected for further analysis. The biochemical indices were tested using urea nitrogen (BUN), creatinine (CREA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (CHO), total bilirubin (TBIL), alkaline phosphatase (ALP) (all kits were from Nanjing Jiancheng), and urea assay kit (UREA) (purchased from Solebao). The body weight of the remaining animals was observed until 18 days. The results are shown in Figure 5 .

[0062] The results showed that compared with the blank control group (NS, normal saline), N-FF / HA-CGA hydrogel could promote the healing of mouse wounds, and the healing effect was better than that of the free chlorogenic acid group. This showed that N-FF / HA-CGA hydrogel had a good effect in healing wounds, and the biochemical indicators of the experimental group were not much different from those of the NS-treated control group, indicating that the hydrogel had good biocompatibility.

[0063] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a dressing based on polypeptide self-assembled composite hydrogel loaded with chlorogenic acid, characterized in that: The specific steps are as follows: (1) adding 1 ml of DMSO to the lyophilized peptide to obtain a peptide solution with a concentration of 100.0 mg / mL; the peptide is R-FF-OH, R = naphthalene (N); (2) HA powder was dissolved in deionized water to obtain HA solution I with a concentration of 10.0 mg / mL; (3) Add 400 μl of HA solution to a round-bottom Eppendorf tube, and then add 80 μl of ddH2O to obtain HA solution II; (4) Add chlorogenic acid at a concentration of 100 mg / ml to a conical bottom Eppendorf tube, then add 10 μl of the peptide solution to the tube and mix well to obtain a chlorogenic acid-peptide mixture solution; (5) Add the chlorogenic acid-peptide mixture solution to the HA solution II and mix immediately to obtain a dressing with a chlorogenic acid concentration of 0.01-0.2 wt %.

2. A dressing based on polypeptide self-assembled composite hydrogel loaded with chlorogenic acid prepared according to the method of claim 1.

3. Use of the polypeptide self-assembled composite hydrogel loaded with chlorogenic acid as claimed in claim 2 in the preparation of dressings for treating chronic diabetic wounds.

Images