Novel drug-loaded soluble microneedle for promoting healing of diabetic ulcer and preparation method of novel drug-loaded soluble microneedle

Abstract

The invention discloses a novel drug-loaded soluble microneedle for promoting healing of diabetic ulcer and a preparation method of the novel drug-loaded soluble microneedle. The microneedle comprises effective component extracts in lipidosome, hyaluronic acid, frankincense and myrrh. The carried medicine is a classical medicine pair with tissue regeneration promoting and sore astringing, namely frankincense-myrrh, and the key active ingredients for treating the diabetic ulcer are screened out by adopting artificial intelligence-based molecular docking and network target technologies, and biological verification is carried out on the action mechanism of the key active ingredients; by adopting the carrier system and the existing components lipidosome and hyaluronic acid in the organism, the material has extremely high biocompatibility and no toxic or side effect, and shows good stability in the subsequent test, so that the time and difficulty of clinical medicine replacement are greatly reduced; the preparation process of the drug-loaded microneedle is simple, conventional equipment is used, raw materials are easy to obtain, the drug-loaded microneedle is safe, pollution-free and low in cost, economic and mental burdens of patients are relieved, and the drug-loaded microneedle has good popularization and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a novel drug-loaded soluble microneedle for promoting healing of diabetic ulcers and a preparation method thereof. Background Art

[0002] Diabetic foot ulcer is one of the most serious and common complications of diabetes. It has the characteristics of high incidence, high recurrence rate, high disability rate, high economic cost and low treatment efficiency, so it has become the focus and difficulty of clinical treatment, seriously threatening the health and property safety of the people. Different from the orderly healing process of hemostasis, inflammation, proliferation and remodeling of ordinary wounds, diabetic foot patients suffer from metabolic immune dysfunction, vascular nerve degeneration, and dysfunction of cells and tissue factors involved in wound healing due to long-term hyperglycemia, which makes the local wound stagnate in the inflammatory stage for a long time and cannot progress to the proliferation and remodeling stage, resulting in persistent ulcers. Although governments and families around the world have invested far more money and energy than ordinary cancers in the research and development of mechanisms and drugs, and there are more and more drug formulations and options on the market, the clinical efficacy is not satisfactory. Therefore, actively exploring new strategies for wound healing of diabetic foot ulcers is crucial to improve the efficacy of diabetic foot ulcers, improve prognosis and quality of life of patients, and reduce the economic burden on society and families.

[0003] China has a history of more than 2,000 years of using traditional Chinese medicine to treat ulcers, including diabetic foot, with rich content and diverse methods. Long-term medical practice has screened out a group of low-cost and effective topical drugs, such as frankincense and myrrh. As early as in the Compendium of Materia Medica, it was recorded that the two can treat ulcers and promote healing, and they are often used as a pair of drugs. Modern pharmacological research has also confirmed that the combination of the two drugs is not just a simple addition of two pharmacological effects, but enhances the efficacy through synergistic effects. The combination of the two can resist inflammation and analgesia, promote cell proliferation and angiogenesis, thereby accelerating tissue repair and wound healing. It is a star drug with great market potential and transformation value in the treatment of diabetic foot ulcers. Unfortunately, the following three problems cannot be solved: 1) synergistic enhancement mechanism; 2) the mechanism of the combination of the two against diabetic foot wound ulcers; 3) inconvenience in use: Both are resin drugs, which are extremely inconvenient to use clinically, and there is no suitable drug delivery system, which seriously limits the development and application of the drug.

[0004] To solve the above problems, we have taken the following measures:

[0005] 1) Use molecular docking technology to predict the mechanism of synergistic enhancement of the two. Molecular docking technology is a technology that simulates the combination of receptors and ligands through computers. It can measure the binding energy and interaction relationship between them, and thus infer the target site of the active ingredient. P-glycoprotein (P-gp) is an important transport protein on the cell membrane. It has a wide range of substrate specificity and can recognize and bind to drugs and toxic substances of various structures. It pumps these molecules out of the cell through active transport to reduce their accumulation in the cell. It is closely related to drug synergistic enhancement and drug resistance. Therefore, we checked the effective active ingredients in frankincense and myrrh, and used molecular docking technology to screen out the monomers with the strongest competitiveness with P-gp to clarify the nature of synergistic enhancement.

[0006] 2) Use network pharmacology to predict the mechanism of the active ingredients of frankincense and myrrh in promoting diabetic foot wound healing. Network pharmacology (NP) provides a new methodological perspective for understanding traditional Chinese medicine from a holistic perspective, and has spawned cutting-edge disciplines such as network pharmacology of traditional Chinese medicine (TCM-NP). By constructing biological networks related to diseases or syndromes and deeply analyzing the interactions and regulatory relationships between these network elements or modules, we can systematically identify the mechanism of action of traditional Chinese medicine prescriptions or ingredients in intervening in diseases or syndromes, which also answers the second question.

[0007] 3) Finally, in order to deliver drugs to achieve the maximum therapeutic effect and for more convenient clinical use, we designed a hyaluronic acid drug-loaded microneedle (MN) patch with ultra-high biosafety. In addition to the remarkable characteristics of low invasiveness and good patient compliance, compared with other types, it can be degraded by hyaluronidase in the body and completely absorbed by the skin to take effect quickly, while releasing the embedded drug for a long time during the dissolution process, so as to continue to work. In addition, HA's strong water absorption helps to increase skin moisture, promote wound healing, and can be combined with a variety of drugs to improve the stability and release characteristics of the drug, thereby enhancing the efficacy.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The purpose of the present invention is to solve the shortcomings of the prior art, and to propose a novel drug-loaded soluble microneedle for promoting the healing of diabetic ulcers and a preparation method thereof, in order to solve the problem of repeated debridement and dressing changes in the current clinical treatment of diabetic ulcers, which increases the pain of patients and consumes huge time and energy for medical staff. The Chinese medicine pair of frankincense and myrrh has a history of more than 2,000 years in promoting ulcer healing, but its treatment mechanism and composition are unknown, and the unique physical and chemical properties of resin drugs, such as volatility, stickiness and difficulty in grinding, have hindered the clinical expansion of applications.

[0010] The present invention firstly determines 3-O-Acetyl-β-boswellic acid (AKBA) in frankincense and β-elemene (β-elemene) in myrrh as key active ingredients through molecular docking and network pharmacology screening. The combination of the two can improve intracellular drug concentration and bioavailability, and can also significantly inhibit the activation of PI3K / AKT / NF-κB signaling pathway, reduce the inflammatory response mediated by M1 macrophages, and then promote cell proliferation, angiogenesis and accelerate wound healing. Then, the hydrophilic β-elemene is wrapped in the core liposome, and then made into a microneedle patch with AKBA and hyaluronic acid.

[0011] Thus, a new dual drug-loaded soluble microneedle (E-Lipo@A-HMN) patch and preparation method are provided. The active ingredients of frankincense and myrrh are synergistically enhanced by regulating P-gp. The microneedle patch can penetrate the hard gangrenous layer on the surface of diabetic ulcers, achieve precise drug delivery and lasting therapeutic effect, and provide a new, efficient and safe solution for the treatment of diabetic ulcers, showing broad clinical application prospects.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] A novel drug-loaded soluble microneedle for promoting the healing of diabetic ulcers. The microneedle comprises liposomes, hyaluronic acid, and extracts of effective ingredients in frankincense and myrrh.

[0014] Preferably, the active ingredient extract in frankincense is 3-O-Acetyl-β-boswellic acid (AKBA).

[0015] Preferably, the active ingredient extract in the myrrh is β-elemene.

[0016] Preferably, the carrier of β-elemene is a liposome capable of encapsulating a hydrophilic drug in the core.

[0017] Preferably, the microneedles are liposomes loaded with β-elemene and hyaluronic acid loaded with AKBA.

[0018] A method for preparing a novel drug-loaded soluble microneedle for promoting healing of diabetic ulcers, the method comprising the following steps:

[0019] Step 1: Review the literature on the active pharmacological components of the frankincense and myrrh drug pair, and use molecular docking to identify the key components that can enhance the synergy between the two.

[0020] Step 2: Use network target technology to screen the active pharmacological components of frankincense and myrrh for intervention in diabetic ulcer mechanism;

[0021] Step 3: Prepare β-elemene-loaded liposomes (ELE-Lipo) by thin film hydration method. The average particle size of blank liposomes (Blank-Lipo) is 76.3 nm, while the particle size of β-elemene-loaded ELE-Lipo increases to 94.36 nm;

[0022] Step 4: The above-mentioned ELE-Lipo, AKBA and hyaluronic acid are mixed and injected into a mold for drying to prepare a drug-loaded microneedle patch (E-Lipo@A-HMN);

[0023] Step 5: In vitro and in vivo experiments verify the biological mechanism of action of the lactucin extract in promoting the healing of diabetic ulcers;

[0024] Step 6: In vivo experiments verify the synergistic effect of the two.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The drugs contained in the drug are a classic drug pair for promoting tissue regeneration and healing ulcers: frankincense-myrrh. The key active ingredients and mechanisms of action for the treatment of diabetic ulcers were screened out using molecular docking and network target technology based on artificial intelligence for biological verification;

[0027] 2. The carrier system uses liposomes and hyaluronic acid, which are existing components in the organism. It has extremely high biocompatibility and no toxic side effects. In subsequent tests, the material showed good stability, which will greatly reduce the time and difficulty of changing drugs in clinical practice;

[0028] 3. The preparation process of the drug-loaded microneedles of the present invention is simple, conventional equipment is used, raw materials are easily available, safe and pollution-free, low-cost, and reduces the economic and mental burden of patients, and has good prospects for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more specifically and intuitively 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 are briefly introduced below.

[0030] Figure 1 . Amino acid site map of the interaction between the active ingredients of frankincense and myrrh and P-glycoprotein;

[0031] Figure 2 .Network pharmacology analysis results of active ingredients of frankincense and myrrh and diabetic ulcer.A) Venn diagram of the intersection of diabetic ulcer disease genes and AKBA and β-elemene target genes;B) KEGG enrichment analysis pathway diagram;C) Schematic diagram of AKBA+β-elemene inhibiting PI3K / AKT / NF-kB signaling pathway;D) GO enrichment analysis diagram;

[0032] Figure 3 Preparation and characterization of ELE-Lipo and E-Lipo@A-HMN. A) Schematic diagram of the preparation process of E-Lipo@A-HMN; B) TME image of Blank-Lipo; C) TEM image of ELE-Lipo; D) Force and displacement curves of E-Lipo@A-HMN; EJ) Morphological exploration of E-Lipo@A-HMN; K) UV absorption spectrum and standard curve of AKBA; E) Standard curve of β-elemene; M)

[0033] Drug release profile of E-Lipo@A-HMN; N) Micropores formed after E-Lipo@A-HMN penetrated into pig skin; O) Solubility of E-Lipo@A-HMN at different times after insertion into the skin;

[0034] Figure 4 .In vivo performance of E-Lipo@A-HMN on DW healing. A) Diagram of in vivo infected diabetic wound model; B, C) Photos and wound area statistics of wounds with different treatments on days 1, 3, 7 and 12; D) Mouse weight statistics; E) Mouse blood glucose statistics; F) H&E staining of wounds on day 7 (yellow arrows are lymphocytes, blue arrows are skin stratum corneum, green arrows are damaged hair follicles, black arrows are new hair follicles, and red arrows are new blood vessels); G, H) Masson staining and quantitative analysis statistics of wounds on day 12; IK) The expression levels of TNF-α, IL-1β, and IL-6 in wound tissues of each group on day 7 were determined by enzyme-linked immunosorbent assay (ELISA); LP) Immunohistochemical staining and quantitative analysis statistics of TNF-α and IL-6 in wound tissues on day 7 and MMP2 and VEGF in wound tissues on day 12. (Data are expressed as mean ± SD, n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001);

[0035] Figure 5 .E-Lipo@A-HMN can inhibit the PI3K / AKT / NF-κB signaling pathway. A) WB detection of the effect of E-Lipo@A-HMN on the expression levels of the main target proteins of the PI3K / AKT / NF-κB pathway; BE) Immunohistochemical staining and quantitative analysis of AKT, PI3K, and IκBα in wound tissue on the 12th day; FM) WB quantitative analysis of the main target proteins of the PI3K / AKT / NF-κB pathway. (Data are expressed as mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001);

[0036] Figure 6.E-Lipo@A-HMN inhibits the activity of P-glycoprotein. A, D) Immunohistochemical staining and quantitative analysis of P-gp in wound tissue on day 12; B, C) WB experiment and quantitative analysis of P-gp in wound tissue on day 12. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0038] Reference Figure 1-5 A novel drug-loaded soluble microneedle for promoting the healing of diabetic ulcers and a preparation method thereof, the preparation method comprising the following steps:

[0039] Step 1: Review the literature on the active pharmacological components of the frankincense and myrrh drug pair and use molecular docking to identify the key components of the synergistic effect of the two. We selected 14 main monomers in frankincense and myrrh as ligands and used P-gp as the receptor for molecular docking (Table 1).

[0040] The results showed that the binding energy of AKBA to P-gp was -8.9 kcal / mol, which was lower than the threshold of -7.9 kcal / mol, indicating that it had a high affinity with P-gp. In addition, AKBA and β-elemene had three common competitive amino acid sites (PH239A, PHE994A and LEU65A) when binding to P-gp, and the binding energy of the two was significantly different.

[0041] This indicates that AKBA can preferentially bind to P-gp, thereby inhibiting its activity, thereby increasing the intracellular concentration of β-elemene and enhancing its bioavailability. Therefore, we selected AKBA and β-elemene as representative monomers of frankincense and myrrh for subsequent research.

[0042] Table 1 The docking results of the active ingredients of Myrrh and P-gp as molecules

[0043]

[0044]

[0045] Step 2: The network target technology was used to predict the targets of the screened active ingredients AKBA and β-elemene, and their potential mechanisms of action in diabetic ulcer effect targets were explored through GO and KEGG enrichment analysis. The results showed that there were 33 common genes between the diabetic ulcer target genes and the 78 targets of AKBA and β-element, which were centered around pathways such as inflammation, apoptosis, and oxidative stress. Through the visualization of KEGG pathway analysis and GO enrichment analysis, it was found that AKBA and β-elemene can regulate reactive oxygen metabolism, participate in biological processes such as lipopolysaccharide-induced inflammatory response and collagen metabolism, and are enriched in 8 signal pathways.

[0046] These results indicate that the therapeutic mechanism of AKBA and β-elemene on diabetic ulcer is closely related to inflammatory response. Considering that diabetic ulcer is a complication of diabetes, we selected the AGE / RAGE signaling pathway as the research object and verified PI3K / AKT / NF-κB as the downstream signaling pathway.

[0047] Step 3: First, the optimal drug loading of AKBA and β-elemene was determined by cytotoxicity and Griess experiments, which were 20μM and 10μM, respectively. The thin film hydration method was used to prepare β-elemene-loaded liposomes (ELE-Lipo). The average particle size of blank liposomes (Blank-Lipo) was 76.3nm, while the particle size of ELE-Lipo loaded with β-elemene increased to 94.36nm. The polydispersity index (PDI) of all samples was less than 0.3, showing a good size distribution. Both blank Lipo and ELE-Lipo showed positive charge, with average zeta potentials of 56mV and 57.47mV, respectively.

[0048] Table 2. Particle size, zeta potential, and encapsulation efficiency (EE) of Blank-Lipo and ELE-Lipo

[0049]

[0050] Step 4: The above-mentioned ELE-Lipo, AKBA and hyaluronic acid were mixed and injected into the mold for drying to prepare a drug-loaded microneedle patch (E-Lipo@A-HMN). The microneedle patch is shown as a circle with a diameter of 2.3 cm in digital camera and super-depth microscope images, light milky white, composed of 112 (7*8*2) needles, arranged in a 7*8 staggered pattern. The microneedle consists of a quadrangular prism-shaped needle body and a quadrangular pyramid-shaped tip. Compared with traditional pyramid-shaped microneedles, it has a larger drug loading space and can improve drug loading efficiency. Scanning electron microscopy (SEM) and inverted fluorescence microscopy images show that MNs are uniform in size and sharp in tip. Their height, width and needle distance are 1000μm, 300μm and 1.2mm, respectively. In addition, the mechanical strength of E-Lipo@A-HMN is a key factor in its ability to penetrate the skin. The force-displacement curve shows that the addition of liposomes and drugs effectively enhances the strength of the microneedles.

[0051] In addition, we inserted E-Lipo@A-HMN into pig skin and observed its dissolution behavior. We found that E-Lipo@A-HMN could penetrate the skin and leave a needle hole, and completely dissolved after 10 minutes of insertion into the skin, indicating that it has excellent skin penetration and solubility. Sustained drug release from microneedles is an important parameter of drug delivery systems. E-Lipo@A-HMN was placed in a dialysis bag to simulate the in vivo drug release behavior. The in vitro drug release curve showed that the AKBA release rate of E-Lipo@A-HMN within 2 hours was 50.55%, and it reached 90.65% in 6 hours, indicating that the drug release rate of AKBA was very high. The release rate of E-Lipo@A-HMN in the first hour was 30.04%, and the curve was close to a straight line, indicating that the drug was released suddenly in the first hour. This part of the drug may come from drug molecules that are not encapsulated, adhered to the surface of the liposome, or embedded shallowly, resulting in a short-term rapid release of the drug. After 1 hour, the drug encapsulated in the liposome is slow, thereby achieving the sustained release of the liposome, and releasing 60.23% within 24 hours. The above results indicate that ELE-Lipo has a sustained-release effect.

[0052] Therefore, the microneedle patch has a well-defined drug loading and release profile, which facilitates subsequent in vitro and in vivo studies.

[0053] Step 5: In vivo experiments verified that the extract of Lactobacillus niger promoted the healing of diabetic ulcers. A spontaneous type 2 diabetes mouse model (DB mouse) was used to study its effect in the treatment of diabetic ulcers in vivo. After one week of adaptive feeding, a full-thickness skin wound model was established on the back of DB mice, and a control group (untreated injury group) and Lipo@HMN, A-HMN, and E-Lipo@HMN patches were set up as control groups to evaluate the effect of E-Lipo@A-HMN on wound healing. In order to evaluate the therapeutic effect of each group, digital photographs of the wounds on days 1, 3, 7, and 12 were recorded, and ImageJ software was used to calculate the wound area and closure rate for effective observation. The results showed that compared with the control group, the other four groups showed varying degrees of accelerated wound healing during the treatment process. On the 12th day, the wounds in the E-Lipo@A-HMN group were basically healed, with only 6.75±1.48% of the wound area remaining, while those in the A-HMN group and the E-Lipo@HMN group were 15.48±0.67% and 19.49±4.09%, respectively, indicating that E-Lipo@A-HMN is effective in promoting wound healing.

[0054] In addition, blood glucose and body weight of each group remained relatively stable throughout the treatment period, indicating that E-Lipo@A-HMN exhibited good biocompatibility in vivo. To further verify the anti-inflammatory, cell proliferation and angiogenesis effects of E-Lipo@A-HMN, we performed enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) experiments on wound tissues.

[0055] The ELISA results showed that the expression levels of TNF-α, IL-1β and IL-6 in the E-Lipo@A-HMN group were lower than those in the other groups (see Figure 4IK part in the middle). IHC results showed that the proinflammatory factors TNF-α and IL-6 were significantly downregulated in the E-Lipo@A-HMN group, and the downregulation effect was better than that in the A-HMN group and the ELE-Lipo@HMN group. In order to observe the effect of drug-loaded microneedles on the PI3K / AKT / NF-κB signaling pathway that causes delayed healing of diabetic ulcers, we performed Western blotting (WB) and IHC experiments to verify several key targets in the pathway, including AKT, p-AKT, PI3K, p-PI3K, IκBα, p-IκBα, P65, and p-P65. WB results showed that the expression levels of target proteins such as AKT, PI3K and IκBα in the wound tissue of the AE group (E-Lipo@A-HMN group) were significantly lower than those of the other four groups; IHC experiments also further verified that the expression levels of AKT, PI3K and IκBα were the lowest in the E-Lipo@A-HMN group, which indicates that the PI3K / AKT / NF-κB signaling pathway was inhibited after E-Lipo@A-HMN treatment, thereby alleviating the inflammatory response in diabetic wounds and promoting the wound from the inflammatory stage to the cell proliferation stage to accelerate wound healing.

[0056] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A novel drug-loaded soluble microneedle for promoting healing of diabetic ulcers, characterized in that: The microneedle comprises liposomes, hyaluronic acid, and extracts of effective ingredients in frankincense and myrrh.

2. A novel drug-loaded soluble microneedle for promoting healing of diabetic ulcers according to claim 1, characterized in that: The active ingredient extract in the frankincense is 3-O-Acetyl-β-boswellic acid, namely AKBA.

3. A novel drug-loaded soluble microneedle for promoting healing of diabetic ulcers according to claim 2, characterized in that: The effective ingredient extract in the myrrh is β-elemene.

4. A novel drug-loaded soluble microneedle for promoting healing of diabetic ulcers according to claim 3, characterized in that: The carrier of the β-elemene is a liposome capable of encapsulating a hydrophilic drug in the core.

5. A novel drug-loaded soluble microneedle for promoting healing of diabetic ulcers according to claim 4, characterized in that: The microneedles are liposomes loaded with β-elemene and hyaluronic acid loaded with AKBA.

6. A method for preparing a novel drug-loaded soluble microneedle for promoting healing of diabetic ulcers as claimed in claim 5, characterized in that: The preparation method comprises the following steps: Step 1: Review the literature on the active pharmacological components of the frankincense and myrrh drug pair, and use molecular docking to identify the key components that can enhance the synergy between the two. Step 2: Use network target technology to screen the active pharmacological components of frankincense and myrrh for intervention in diabetic ulcer mechanism; Step 3: Prepare β-elemene-loaded liposomes (ELE-Lipo) by thin film hydration method. The average particle size of blank liposomes (Blank-Lipo) is 76.3 nm, while the particle size of β-elemene-loaded ELE-Lipo increases to 94.36 nm; Step 4: The above-mentioned ELE-Lipo, AKBA and hyaluronic acid are mixed and injected into a mold for drying to prepare a drug-loaded microneedle patch (E-Lipo@A-HMN); Step 5: In vitro and in vivo experiments verify the biological mechanism of action of the lactucin extract in promoting the healing of diabetic ulcers; Step 6: In vivo experiments verify the synergistic effect of the two.

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