Dressing for treating diabetic foot ulcer, preparation method and application

Abstract

The invention belongs to the technical field of medicine, and discloses a dressing for treating diabetic foot ulcer, a preparation method and application. The dressing is characterized in that PLGA nanoparticles loaded with deferasirox are fixed on the inner walls of meshes of a silicone mesh stent through a polydopamine adhesion layer; the sodium alginate microspheres encapsulating the WAY 316606 are embedded into the carboxymethyl cellulose matrix, and the surface of the deferasirox-loaded PLGA nanoparticle layer is coated with the sodium alginate microspheres; and after the poloxamer 407 temperature-sensitive hydrogel containing rhPOSTN protein is molded by casting, the poloxamer 407 temperature-sensitive hydrogel containing rhPOSTN protein is coated with the sodium alginate microsphere layer for encapsulating WAY 316606. The thought that skin POSTN + fibroblasts regulate and control diabetic foot skin injury re-healing through iron metabolism regulation and interaction of the skin POSTN + fibroblasts and endothelial cells is proved, a molecular mechanism is disclosed, and a powerful basis is provided for later research.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, and in particular relates to a dressing for the treatment of diabetic foot ulcers, its preparation method and application. Background Technology

[0002] Diabetic foot ulcer (DFU) is a disease state characterized by ulceration and gangrene in the lower extremities due to severe peripheral neuropathy in diabetic patients, or insufficient perfusion caused by arterial disease. It has an extremely high rate of disability and mortality. Diabetic foot ulcers are chronic and prone to infection. Its pathogenesis is not fully understood. Collagen, which is crucial for DFU wound healing, is secreted by skin fibroblasts. Fibroblast dysfunction is one of the important reasons for delayed healing in DFU. Elucidating its pathogenesis and treatment remains a major global medical challenge. In DFU patients, multiple pathways are involved in disease progression and treatment, such as the PI3K / Akt / mTOR signaling pathway, the Wnt / β-catenin signaling pathway, the Notch signaling pathway, the JAK-STAT signaling pathway, and the NF-κB signaling pathway. These signaling pathways cross-regulate with each other, and this complex regulatory network offers endless possibilities for research. The Wnt / β-catenin signaling pathway is involved in angiogenesis, cell proliferation, and differentiation. Upregulation of the Wnt / β-catenin signaling pathway enhances endothelial cell proliferation, differentiation, and migration, accelerating ulcer healing. Downregulation of this pathway suppresses the biological activity of ulcerated skin cells and the expression of cytokines, leading to immune dysfunction, poor granulation tissue development, and thus delaying wound healing. Furthermore, some studies have indicated that Wnt5a and Wnt2 may be potential targets for controlling diabetic complications. DFU (digestive fusion) is commonly used for debridement of ulcers, and the use of bioactive molecules for specific treatment at the cellular level has received widespread attention in recent years. [5] DFU healing is a multicellular, multi-signaling pathway process, and treatment strategies and pathogenesis still require further exploration. Limitations of existing therapies: Debridement: cannot address dysfunctional subpopulations (such as POSTN). + Fibroblast deficiency); growth factor therapy (such as rhVEGF): poor heterogeneous response to plantar / dorsolateral foot ulcers, with an efficacy rate of only 40-60%; stem cell therapy: high cost, survival rate <10%.

[0003] Breakthroughs in molecular mechanism research further highlight the shortcomings of treatment strategies: Ferrapreosis, as a novel type of programmed cell death, leads to the accumulation of free iron in DFU tissues through NCOA4-mediated ferritin degradation, subsequently triggering lipid peroxidation. However, clinically used iron chelators have low clearance rates of intracellular iron pools due to their inability to penetrate cell membranes. More seriously, the anatomical heterogeneity of DFU has long been overlooked: plantar ulcers require enhanced keratinocyte barrier function, while the core challenge of dorsum-of-the-foot ulcers lies in POSTN. + Fibroblast ferroptosis occurs, but current technologies all adopt a "one-size-fits-all" approach, resulting in a lack of personalized treatment.

[0004] Patent analysis confirms technological gaps: while iron-chelating hydrogels reduce total iron content in tissues, they fail to address the key intracellular ferroptosis target NCOA4; SFRP1 inhibitor microneedle patches were discontinued in clinical trials due to low biomembrane permeability. These failures reflect the inability of existing technologies to synergistically address the cascade pathology of ferroptosis-signal interruption-cell subset imbalance. Therefore, developing a technology that can target and regulate POSTN is crucial. + Fibroblast ferroptosis, repair of Wnt-FZD-mediated cell interactions, and smart dressings adapted to the anatomical heterogeneity of the foot have become scientific challenges and clinical needs to be addressed in the field of DFU treatment.

[0005] Current technologies, particularly growth factor therapy, a representative of DFU adjuvant therapy, rely on the local application of exogenous recombinant growth factors to activate downstream Ras / MAPK and PI3K / Akt signaling pathways, ultimately promoting fibroblast proliferation, collagen synthesis, and endothelial cell angiogenesis. For example, using 0.01% rhPDGF-BB gel, the standard procedure is to apply the gel evenly to the ulcer surface after daily debridement, cover with a breathable dressing, and continue treatment for up to 20 weeks. This approach is based on two key studies: in vitro experiments confirmed that rhPDGF-BB at a concentration of 10 ng / mL significantly increases the proliferation rate of normal fibroblasts; and in animal models, in a STZ-induced diabetic rat model, the rhPDGF-BB treatment group showed significantly faster wound healing than the control group. However, this approach suffers from a fundamental design flaw in the diabetic pathological microenvironment: its mechanism of action depends on the sustained high expression of growth factor receptors on the target cell surface. Fibroblasts are key effector cells involved in extracellular matrix secretion and tissue healing.

[0006] The fundamental flaw of existing growth factor therapies lies in the irreconcilable contradiction between their linear action model and the pathological complexity of DFU.

[0007] At the molecular level: receptor downregulation and SFRP1 overexpression constitute a dual signaling barrier, which inactivates the vast majority of exogenous growth factors;

[0008] Cellular level: Ignoring POSTN + Iron death in fibroblasts leads to the collapse of ECM synthesis;

[0009] Anatomical level: The high-pressure environment of the sole of the foot reduces drug penetration rate to below the critical value. Summary of the Invention

[0010] To overcome the problems existing in related technologies, the present invention discloses an embodiment of a dressing for the treatment of diabetic foot ulcers, a preparation method thereof, and its application. To solve the above problems, the present invention provides a method for targeted drug delivery through a specific fibroblast subset.

[0011] The technical solution is as follows: A dressing for the treatment of diabetic foot ulcers, the dressing comprising, from the contact side of the wound to the dorsal side:

[0012] Contact layer, silicone mesh support;

[0013] Anti-ferrode layer, PLGA nanoparticles loaded with larosse;

[0014] Signal activation layer, encapsulating sodium alginate microspheres of WAY ​​316606;

[0015] Repair-promoting layer: poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein;

[0016] Specifically, PLGA nanoparticles loaded with delarose are fixed to the inner wall of the silicone mesh scaffold through a polydopamine adhesion layer; sodium alginate microspheres encapsulating WAY 316606 are embedded in a carboxymethyl cellulose matrix and coated on the surface of the PLGA nanoparticle layer loaded with delarose; and poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein is cast and molded to encapsulate the sodium alginate microsphere layer encapsulating WAY 316606.

[0017] Furthermore, the silicone mesh scaffold has a thickness of 0.5 mm and a pore size of 50 μm;

[0018] The particle size of the PLGA nanoparticles loaded with larosx is 152±9 nm.

[0019] The sodium alginate microspheres encapsulating WAY 316606 have a particle size of 20-50 μm;

[0020] The concentration of poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein was 25% w / v.

[0021] In the iron-death layer, the mass ratio of derarosilicate to PLGA is 1:2;

[0022] Drug loading 20–35%;

[0023] In the signal activation layer, the concentration of WAY ​​316606 in the microspheres was 5 mg / mL, with a drug loading rate of 12.5% ​​w / w;

[0024] At pH 5.5, the sodium alginate microspheres encapsulating WAY 316606 cumulatively released 82.3±4.7% over 24 hours.

[0025] In the repair-promoting layer, the concentration of rhPOSTN was 10–100 μg / mL;

[0026] The repair-promoting layer includes a foot-type thermosensitive hydrogel without added potassium titanate phosphate (KTP) peptides;

[0027] And to add potassium titanate phosphate KTP peptide to a foot-type thermosensitive hydrogel, with a KTP peptide concentration of 0.5–2 mM.

[0028] The dorsum-of-the-foot thermosensitive hydrogel is made of 25% poloxamer 407 plus 50 μg / mL rhPOSTN.

[0029] The plantar thermosensitive hydrogel is composed of 25% poloxamer 407, 50 μg / mL rhPOSTN, and 1 mM KTP peptide.

[0030] The anti-iron death layer uses chitosan-citric acid nanoparticles;

[0031] The signal activation layer uses gelatin-oxidized dextran.

[0032] The dressing for treating diabetic foot ulcers, from the wound contact side to the dorsal side, consists of:

[0033] Contact layer, silicone mesh support;

[0034] A composite layer of anti-ferrode death layer and signal activation layer, including PLGA / WAY 316606 / delarosus co-loaded nanoparticles;

[0035] Repair-promoting layer: poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein.

[0036] Another object of the present invention is to provide a method for preparing a dressing for the treatment of diabetic foot ulcers, the method comprising:

[0037] S1, PLGA nanoparticles loaded with delarose as an anti-ferrode layer were prepared; the prepared PLGA nanoparticles loaded with delarose were fixed to the inner wall of the mesh of a silicone mesh scaffold through a polydopamine adhesion layer.

[0038] S2, as a signal activation layer, is a pH-responsive sodium alginate microsphere encapsulated with WAY 316606. The prepared sodium alginate microspheres encapsulated with WAY 316606 are embedded in a carboxymethyl cellulose matrix and coated on the surface of a PLGA nanoparticle layer loaded with delarose.

[0039] S3, a layer of sodium alginate microspheres from WAY 316606, is encapsulated after the poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein is cast and molded.

[0040] Step S1, the preparation of PLGA nanoparticles loaded with delarose includes:

[0041] Oil phase preparation: Weigh out derarosi and PLGA, dissolve in dichloromethane, and vortex until clear;

[0042] Aqueous phase emulsification: The oil phase is injected into a 2% PVA solution and ultrasonically treated with a probe;

[0043] Solvent evaporation: Pour the emulsion into a 0.5% PVA solution and stir magnetically to evaporate the organic solvent;

[0044] Antibody conjugation: Nanoparticles were collected by centrifugation, resuspended in pH 7.4 phosphate buffered saline, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution were added. The reaction was carried out with shaking at room temperature.

[0045] Purification and freeze-drying: Wash with phosphate-buffered saline, add 5% mannitol, pre-freeze and then freeze-dry under vacuum;

[0046] Step S2, the preparation of pH-responsive encapsulated sodium alginate microspheres of WAY ​​316606 includes:

[0047] Drug mixing: WAY 316606 is dissolved in a 2% sodium alginate solution;

[0048] Microsphere molding: Microfluidic chip is injected into a 1.5% CaCl2 crosslinking bath;

[0049] Shell coating: Microspheres are impregnated with 0.5% chitosan-acetic acid solution and stirred;

[0050] Curing and washing: Rinse with physiological saline, pre-freeze and then vacuum freeze-dry;

[0051] In step S3, the sodium alginate microsphere layer encapsulating WAY316606 after the poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein is cast and molded specifically includes:

[0052] Hydrogel casting includes:

[0053] The foot-shaped thermosensitive hydrogel was cast with 25% poloxamer 407 and 50 μg / mL rhPOSTN, solidified at 4℃, and then sterilized by γ-irradiation.

[0054] Foot-type thermosensitive hydrogel casting: 25% poloxamer 407, 50 μg / mL rhPOSTN, and 1 mM KTP peptide; solidified at 4°C and then sterilized by γ-irradiation.

[0055] Before step S1, a bottom layer treatment is performed: plasma activation of the silicone mesh scaffold;

[0056] In step S3, the sol-gel phase transition temperature of the poloxamer 407 hydrogel in the repair-promoting layer is 32°C.

[0057] Another object of the present invention is to provide the use of a dressing for the treatment of diabetic foot ulcers in the preparation of a medicament for treating and reducing inflammation of diabetic foot ulcer tissue.

[0058] Combining all the above technical solutions, the beneficial effects of this invention are as follows:

[0059] First, addressing the issue that existing technologies rely on growth factor-receptor binding (e.g., PDGF-BB→PDGFRβ) to activate signaling pathways, but multiple pathways function in the diabetic microenvironment, this invention deepens the understanding of iron metabolism disorders and POSTN. + The correlation between fibroblast subgroups and the Wnt pathway was proposed and demonstrated, and skin POSTN was proposed and demonstrated. + The concept of fibroblasts regulating the rehearing of diabetic foot skin lesions through iron metabolism and their interaction with endothelial cells, along with the elucidation of the molecular mechanisms, provides strong evidence for future research. This invention, through targeted iron metabolism and intervention with Wnt2 and Wnt5a, effectively reduces the occurrence of inflammation in diabetic foot ulcer tissue, demonstrating its potential to effectively intervene in the progression of diabetic foot ulcers.

[0060] Secondly, diabetic foot ulcer treatment is one of the largest and fastest-growing sub-segments. The composite dressing of this invention, as a high-end medical device, has clear commercial value: compared to existing recombinant growth factor gels with limited efficacy (such as... (Since single-drug treatments are expensive and the treatment course is long), the dressing of this invention, through multi-target synergistic treatment, is expected to increase the 12-week healing rate of moderate to severe DFU from less than 40% to more than 70%, significantly shortening the treatment course and reducing the total treatment cost. It is extremely attractive to the medical insurance payment system and individual patients, and has the market potential to quickly replace traditional therapies.

[0061] Third, this invention provides a local therapeutic approach targeting ferroptosis in fibroblast subsets. Existing technologies (such as patent WO2021167892A1) only focus on iron overload at the tissue level, using ordinary iron chelating hydrogels, which cannot solve the core problem of intracellular ferroptosis. This invention's pioneering CD44 antibody-modified PLGA nanoparticles achieve targeted treatment of POSTN… +This invention represents a pioneering achievement both domestically and internationally in the active targeted drug delivery to fibroblasts, precisely intervening in their ferroptosis process. It also presents a mature product targeting the SFRP1-Wnt5a-FZD axis for local intervention. While the role of the Wnt pathway in wound healing is recognized, its complex regulation and high systemic toxicity have hindered the development of safe and effective local intervention strategies. This invention utilizes pH-responsive microsphere technology to locally deliver the SFRP1 inhibitor WAY 316606, specifically reactivating the non-canonical Wnt5a signaling pathway crucial for healing, avoiding the carcinogenic risks of β-catenin activation, and filling the gap in local therapeutic products targeting this pathway.

[0062] Fourth, this invention addresses the issue that previous research often focused on "supplementing stimulating factors" (such as growth factors) to promote cell function, neglecting the fact that cells themselves may be "on the verge of death" or severely impaired due to novel damage mechanisms such as ferroptosis in a high-glucose microenvironment. This invention is the first to explicitly point out POSTN + Fibroblast ferroptosis is one of the core reasons for the poor healing of DFU (diabetic wound healing) and provides an effective rescue strategy, fundamentally solving the problem of cell dysfunction. Given the bias that "poor wound healing in diabetes should be attributed to cell function suppression caused by high glucose levels, rather than cell death," research has consistently focused on how to "activate" or "stimulate" cells (e.g., using growth factors). This invention breaks through this mindset, and through rigorous single-cell sequencing and molecular biological evidence, it is the first to confirm the efficacy of POSTN. + In a high-glucose environment, fibroblast subsets undergo programmed cell death, represented by ferroptosis, rather than simply functional inhibition. Therefore, the treatment strategy must shift from "stimulation" to "survival salvage," representing a fundamental paradigm shift. Attached Figure Description

[0063] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure;

[0064] Figure 1 This is a flowchart of a dressing preparation method for the treatment of diabetic foot ulcers provided in an embodiment of the present invention;

[0065] Figure 2 This is a schematic diagram of the dressing for the treatment of diabetic foot ulcers provided in an embodiment of the present invention;

[0066] Figure 3 This is a verification diagram of the fibroblast cell line stably overexpressing Wnt5a and SFRP1 provided in the embodiments of the present invention;

[0067] Figure 4 This figure shows the cell proliferation of endothelial cells 24 hours after the invention demonstrated that the Wnt5a pathway significantly promotes eNOS activity in fibroblast cell lines.

[0068] Figure 5 This is a schematic diagram illustrating the eNOS activity in the Wnt5a pathway of the present invention, which significantly promotes eNOS activity in fibroblast cell lines.

[0069] Figure 6 This is a diagram showing the cell proliferation of endothelial cells 24 hours after WAY 316606 of the present invention can significantly restore the eNOS activity of fibroblasts.

[0070] Figure 7 This is a schematic diagram illustrating the eNOS activity in WAY 316606 of the present invention, which can significantly restore eNOS activity in fibroblasts.

[0071] Figure 8 The invention shows that inhibiting inflammation levels can significantly promote angiogenesis.

[0072] Figure 9 This invention significantly promotes skin damage healing by inhibiting inflammation levels.

[0073] Figure 10 This is a representative fluorescence image showing how the WNT5a pathway can affect cytoskeleton formation, as described in this invention.

[0074] Figure 11 This is a graph showing the quantitative results of F-actin fluorescence intensity in the WNT5a pathway affecting cytoskeleton formation, as presented in this invention.

[0075] Figure 12 The graph shows the changes in blood glucose levels in rats during the detection of DFO and WAY 316606 significantly promoting the healing of skin ulcers in rats with type II diabetes.

[0076] Figure 13 The graph shows the change in rat body weight over time in which DFO and WAY 316606 of this invention significantly promote the healing of skin ulcers in rats with type II diabetes.

[0077] Figure 14 The images show the histopathological (HE) and collagen (Masson) content in different groups of rat models (n=8 per group).

[0078] Figure 15 The graph shows the expression of collagen and biomarker-related genes in different rat models (n=8 for each group). Detailed Implementation

[0079] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0080] The innovation of this invention lies in proposing and validating a novel targeted synergistic therapy strategy: targeting and inhibiting POSTN through deirarosi nanoparticles. + Fibroblast ferroptosis was addressed by reversing SFRP1 inhibition of the Wnt5a pathway through WAY 316606 microspheres to restart angiogenesis signaling, and the damaged cellular function was directly repaired via the rhPOSTN protein. This approach is the first to reveal the effects of POSTN at the single-cell level. + A novel mechanism by which fibroblasts cause poor healing of diabetic foot ulcers through iron metabolism imbalance and impaired interaction with endothelial cells has been discovered. The feasibility and significant efficacy of targeted intervention in iron metabolism and the non-classical Wnt pathway (Wnt5a) have been demonstrated, providing a breakthrough solution for the precision treatment of diabetic foot ulcers.

[0081] Targeted synergistic mechanism: This invention is the first to propose "ferroptosis inhibition (deferrasirox) → Wnt pathway activation (WAY 316606) → POSTN + Cellular function repair (rhPOSTN) employs a three-tiered regulatory strategy, overcoming the limitations of single-target approaches. Intelligent release control: nanoparticle sustained release (>6 days), PLGA degradation matching the iron metabolism cycle; first-ever validation of skin ASPN. + Fibroblasts regulate the mechanism of skin re-healing in diabetic foot lesions through iron metabolism regulation and their interaction with endothelial cells. This study is the first to demonstrate the feasibility of intervention targeting iron metabolism or the Wnt2 and Wnt5a pathways.

[0082] Example 1: The dressing for treating diabetic foot ulcers provided in this embodiment of the invention has a four-layer composite structure, which consists of the following layers from the wound contact side to the dorsal side:

[0083] Contact layer, silicone mesh support (0.5 mm thick, 50 μm pore size);

[0084] Anti-ferrode layer, loaded with PLGA (Poly(lactic-co-glycolic acid) nanoparticles (particle size 152±9nm) of larosli.

[0085] Signal activation layer, encapsulating sodium alginate microspheres (particle size 20-50 μm) of WAY ​​316606 (2-({3-(2-Fluorophenyl)-4-oxo-3H,4H-thieno[3,2-d]pyrimidin-2-yl}sulfanyl)acetic acid ethyl ester, an SFRP1 protein inhibitor);

[0086] Repair-promoting layer, containing recombinant human POSTN + Poloxamer 407 thermosensitive hydrogel of protein (rhPOSTN (Recombinant Human Periostin)) (concentration 25% w / v).

[0087] In this process, PLGA nanoparticles loaded with delamination of laseros are fixed to the inner wall of the silicone mesh scaffold via a polydopamine adhesion layer; sodium alginate microspheres encapsulating WAY 316606 are embedded in a carboxymethyl cellulose matrix (310) and coated on the surface of the PLGA nanoparticle layer loaded with laseros; recombinant human POSTN... + After the poloxamer 407 thermosensitive hydrogel of protein (rhPOSTN) was cast and molded, it encapsulated the sodium alginate microsphere layer of WAY ​​316606. The sol-gel phase transition temperature of the poloxamer 407 hydrogel in the repair layer 400 was 32℃ (gelation at body temperature to prevent loss).

[0088] An example is an anti-ferrode layer (PLGA nanoparticles loaded with delarose).

[0089] Drug loading and encapsulation efficiency: The mass ratio of deferasirox to PLGA was 1:2 (50mg:100mg).

[0090] HPLC analysis revealed the following:

[0091] Drug loading = (actual encapsulated drug amount / total mass of nanoparticles) × 100%, which can reach 33.3 ± 2.1%;

[0092] Encapsulation efficiency = (encapsulated drug amount / initial drug amount) × 100%, which can reach 89.5 ± 3.4%;

[0093] Preferably, the drug loading is limited to 20–35%.

[0094] For example, the signal activation layer (sodium alginate microspheres, i.e., sodium alginate-Ca²⁺ encapsulating WAY 316606) +Microspheres); Drug loading parameters: The concentration of WAY ​​316606 in the microspheres was 5 mg / mL (20 mg / 4 mL sodium alginate solution), with a drug loading rate of 12.5% ​​w / w. Release characteristics: At pH 5.5 (simulated wound environment), the cumulative release over 24 h was 82.3 ± 4.7% (vs. <20% at pH 7.4).

[0095] For example, a repair-promoting layer (thermosensitive hydrogel, i.e., poloxamer 407 thermosensitive hydrogel containing recombinant human POSTN⁺ protein (rhPOSTN)); rhPOSTN concentration: effective range 10–100 μg / mL (optimal 50 μg / mL), below 10 μg / mL has no proliferative effect, above 100 μg / mL induces apoptosis (Biomaterials. 2022;281:121357). The repair-promoting layer may not contain potassium titanyl phosphate KTP peptide, and is a dorsum-of-the-foot type; the repair-promoting layer may contain potassium titanyl phosphate KTP peptide (sole-of-the-foot type), with a KTP peptide concentration of 0.5–2 mM (optimal 1 mM), enhancing the keratinocyte barrier (Claudin-1 expression higher than 3.2-fold). Potassium titanyl phosphate (KTiOPO4, abbreviated as KTP) crystal is an excellent nonlinear optical crystal.

[0096] For example, the principle of a dressing for the treatment of diabetic foot ulcers is as follows: Figure 2 As shown.

[0097] In Example 2, in the anti-ferrode death layer carrier system, the PLGA nanoparticles loaded with delaros can be chitosan-citric acid nanoparticles (pH-responsive degradation); in the signal activation layer of the microsphere delivery system, the sodium alginate microspheres encapsulating WAY 316606 can be gelatin-oxidized dextran (thermosensitive sol-gel transition).

[0098] For example, the three-layer variant may employ a combined anti-ferrode layer and a signal activation layer, namely PLGA / WAY 316606 / delarose co-loaded nanoparticles.

[0099] By way of example, the present invention provides the use of the dressing for the treatment of diabetic foot ulcers in the preparation of a medicament for treating and reducing inflammation of diabetic foot ulcer tissue.

[0100] Example 3, as Figure 1 The method for preparing a dressing for the treatment of diabetic foot ulcers provided in this embodiment of the invention includes:

[0101] S1, PLGA nanoparticles loaded with delarose as an anti-ferrode layer were prepared; the prepared PLGA nanoparticles loaded with delarose were fixed to the inner wall of the mesh of a silicone mesh scaffold through a polydopamine adhesion layer.

[0102] S2, as a signal activation layer, is a pH-responsive sodium alginate microsphere encapsulated with WAY 316606. The prepared sodium alginate microspheres encapsulated with WAY 316606 are embedded in a carboxymethyl cellulose matrix and coated on the surface of a PLGA nanoparticle layer loaded with delarose.

[0103] S3, a layer of sodium alginate microspheres from WAY 316606, is encapsulated after the poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein is cast and molded.

[0104] Contains recombinant human POSTN + After the poloxamer 407 thermosensitive hydrogel of protein (rhPOSTN) was cast and molded, it encapsulated the sodium alginate microsphere layer of WAY ​​316606. The sol-gel phase transition temperature of the poloxamer 407 hydrogel in the repair layer (400) was 32℃ (gelation at body temperature to prevent loss).

[0105] For example, step S1, the preparation of PLGA nanoparticles loaded with delarose includes:

[0106] 1.1 Materials and Methods;

[0107] Raw materials: Deferasirox (MCE HY-B2175), PLGA (LA:GA=75:25, Mw 38 kDa, Sigma 739944), PLGA (LA:GA=75:25, molecular weight 38 kDa, Sigma 739944), CD44 antibody (clone number IM7, Abcam ab196415);

[0108] Equipment: Ultrasonic breaker (Sonics VCX750), freeze dryer (Christ Alpha 1-4 LDplus);

[0109] 1.2 Preparation steps:

[0110] Oil phase preparation: Accurately weigh 50 mg deferasirox and 100 mg PLGA, dissolve in 5 mL dichloromethane (HPLC grade), and vortex until clear; Aqueous phase emulsification: Inject the oil phase into 20 mL of 2% PVA solution (containing 0.1 mg / mL CD44 antibody), and sonicate with a probe (200 W, 70% amplitude, pulse 2 s on / 1 s off) for 2 min; Solvent evaporation: Pour 0.5% of the emulsion into the solvent. PVA solution, magnetically stirred (800 rpm) for 6 h to evaporate organic solvent; antibody conjugation: collect nanoparticles by centrifugation (15,000 g × 30 min), resuspend in pH 7.4 phosphate buffered saline, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution (50 mM) and N-hydroxysuccinimide solution (25 mM), and react with shaking at room temperature for 2 h; purification and lyophilization: wash 3 times with phosphate buffered saline, add 5% mannitol, pre-freeze at -80℃ for 24 h and then lyophilize under vacuum.

[0111] For example, step S2, the preparation of pH-responsive encapsulated sodium alginate microspheres of WAY ​​316606, includes:

[0112] 2.1 Materials and Methods; Raw materials: WAY 316606 (MCE HY-10858), sodium alginate, chitosan;

[0113] 2.2 Preparation steps: Drug mixing: 20 mg WAY 316606 was dissolved in 4 mL of 2% sodium alginate solution (0.1 M MES buffer, pH 6.0); Microsphere molding: Microfluidic chip (channel width 200 μm) was injected into a 1.5% CaCl2 crosslinking bath at a rate of 100 μL / min; Shell coating: Microspheres were immersed in 0.5% chitosan acetic acid solution (pH 5.0) and gently stirred for 10 min; Curing and washing: Rinse 3 times with 0.9% physiological saline, pre-freeze at -80℃ for 24 h and then freeze-dry under vacuum.

[0114] For example, step S3, containing recombinant human POSTN + The poloxamer 407 thermosensitive hydrogel of protein (rhPOSTN) encapsulates the sodium alginate microspheres of WAY ​​316606 after casting. Specifically, the encapsulation layer includes:

[0115] Hydrogel casting includes: dorsal foot type thermosensitive hydrogel casting, 25% poloxamer 407 plus 50 μg / mL rhPOSTN, solidified at 4℃ and then sterilized by γ-irradiation (25 kGy); plantar foot type thermosensitive hydrogel casting, 25% poloxamer 407 plus 50 μg / mL rhPOSTN plus 1 mM KTP peptide; solidified at 4℃ and then sterilized by γ-irradiation (25 kGy).

[0116] Example 3, as another embodiment of the present invention, provides a method for preparing a dressing for the treatment of diabetic foot ulcers, comprising:

[0117] Substrate treatment: Silicone mesh scaffold (2×2cm) plasma activation (29.6W, 5min); PLGA nanoparticle coating loaded with deraxis: PLGA-deraxis nanoparticles (30mg / mL) sprayed (drug loading 1.5mg / cm²); Sodium alginate microsphere layer fixation: Sodium alginate microspheres dispersed in 1% CMC solution and coated (wet weight 0.8mg / cm²); Thermosensitive hydrogel casting: Dorsal type: 25% poloxamer 407 + 50μg / mL rhPOSTN; Sole type: 25% poloxamer 407 + 50μg / mL rhPOSTN + 1mM KTP peptide; Final product: Sterilized by γ-irradiation after coagulation at 4℃ (25kGy).

[0118] For example, after preparing a dressing for the treatment of diabetic foot ulcers, an in vitro co-culture experiment was conducted, specifically including:

[0119] 1. Dressing assembly:

[0120] Substrate treatment: Silicone mesh scaffold (2×2cm) plasma activation (29.6W, 5min); Nanoparticle coating: PLGA-deferrasiro nanoparticles (30mg / mL) sprayed (drug loading 1.5mg / cm²); Microsphere layer fixation: Sodium alginate microspheres dispersed in 1% CMC solution and coated (wet weight 0.8mg / cm²).

[0121] Hydrogel casting: Dorsal type: 25% poloxamer 407 + 50 μg / mL rhPOSTN; Sole type: 25% poloxamer 407 + 50 μg / mL rhPOSTN + 1 mM KTP peptide; Final product: Sterilized by γ-irradiation (25 kGy) after solidification at 4℃.

[0122] 2. Preparation of dressing extracts; Sampling: Take 1 cm² of each type of dressing (blank / single-drug / composite dressing), aseptically cut into small pieces; Extraction: Immerse the fragments in 10 mL of phosphate-buffered saline (pH 7.4), and incubate at 37°C with shaking for 24 h; Sterilization: Filter through a 0.22 μm filter membrane, aliquot, and store at -80°C. Concentration determination: Deferrasiro extract: HPLC concentration 15 ± 1.2 μg / mL (corresponding to a dressing drug loading of 1.5 mg / cm²); WAY 316606 extract: LC-MS concentration 2.0 ± 0.3 μg / mL (corresponding to a microsphere drug loading rate of 12.5%); Composite extract: Contains both of the above + rhPOSTN (50 μg / mL);

[0123] 3. Establishment of co-culture model; Cell seeding: Lower layer: Human umbilical vein endothelial cells (HUVECs), HUVECs at a density of 2×10⁶ cells / year. 4Cells / wells were seeded in 24-well plates (coated with Matrigel to simulate the basement membrane); top layer: human dermal fibroblasts (HDF, high-glucose culture), HDF cells were seeded at a density of 5 × 10⁶ cells / well. 4 Cells / inserts were seeded into Transwell chambers;

[0124] 4: Animal pharmacodynamic evaluation (foot dorsum ulcer model);

[0125] 4.1 Model establishment: Animals: SPF grade SD rats (200±20g, n=50); Diabetes induction: fed with high-fat diet (45% fat) for 4 weeks → STZ intraperitoneal injection (50mg / kg); Wound preparation: full-thickness skin defects (deep to myofascia layer) were created using an 8mm biopsy puncturist.

[0126] 4.2 Grouping and processing: See Table 1.

[0127] Table 1 Grouping and Processing Group Solution Dressing size Blank control group Blank dressing (without medication) 2×2cm Positive control group 0.01% rhPDGF-BB gel 0.01% rhPDGF-BB gel, equivalent to wound surface Experimental group 1 Deferox monotherapy dressing 2×2cm Experimental group 2 WAY 316606 Single-drug dressing 2×2cm Invention Group Composite dressing (dorsum of the foot type) 2×2cm

[0128] Diabetes model and ulcer modeling; Diabetes induction: After 1 week of adaptive feeding, rats were fed a high-fat diet for 4 weeks; STZ (40 mg / kg) was injected intraperitoneally (dissolved in 0.1 M citrate buffer, pH 4.5); fasting blood glucose ≥16.7 mmol / L was considered successful modeling on day 1, day 7, and day 14 (n=50). Ulcer wound preparation: Anesthesia: 2% isoflurane inhalation anesthesia; Skin preparation: shaving the back and disinfecting with iodine; Wound: 8 mm diameter full-thickness skin defect (biopsy perforator, depth reaching the myofascia layer).

[0129] 3. Experimental grouping and treatment schemes are shown in Table 2.

[0130] Table 2 Experimental Grouping and Treatment Scheme Group Handling method Dressing size Sample size Blank control group Drug-free silicone dressing (scaffold only + blank hydrogel) 2×2cm n=8 Positive control group 0.01% rhPDGF-BB gel (apply daily after wound cleaning) Cover the wound n=8 Experimental group 1 Deferasirox monotherapy dressing (drug loading 1.5 mg / cm²) 2×2cm n=8 Experimental group 2 WAY 316606 Single-drug dressing (drug loading 0.5 mg / cm²) 2×2cm n=8 Invention Group Composite dressing (dorsum of foot type: containing rhPOSTN 50μg / mL) 2×2cm n=8

[0131] Operating procedure: After dressing is applied, it is secured with a breathable adhesive membrane (Tegaderm™); change every 48 hours (after anesthesia, wound cleaning and rinsing with saline); the positive control group has rhPDGF-BB gel applied daily (simulating clinical protocol).

[0132] 4.3 Evaluation of therapeutic effect:

[0133] 1) Wound healing rate:

[0134] 2) Histological analysis (day 14), see Table 3.

[0135] Table 3. Histological analysis of changes between the present invention group and the control group. index Detection methods <![CDATA[POSTN + Cell density Immunofluorescence Number of new blood vessels CD31 IHC CD31 IHC Collagen deposition rate Masson staining Iron deposition in tissues Prussian blue staining

[0136] 3) qPCR and Western Blot were used to detect the expression of Wnt5a, Wnt2, SFRP1, FZDs receptor, iron metabolism disorder markers (TFR1, FPN, GPX4, NCOA4, Ferritin), and angiogenesis markers (TGF-1β, PDGF-C, VEGF-A, ANGPT2) in each group.

[0137] 4) H&E staining analysis of tissue inflammation in mice in each group.

[0138] 5) ELISA detection of IL-1β and IL-6 inflammatory factors.

[0139] 6) Immunohistochemical detection: The slides were incubated with 5% normal goat serum for 1 hour, and then incubated overnight with primary antibodies against the expression and proliferation markers of VEGF, VE-cadherin, CD31 and PCNA.

[0140] Stable overexpression cell lines were constructed using a lentiviral packaging system. Both Wnt5a and SFRP1 were conjugated to mCherry fluorescence. After puromycin drug screening, cell fluorescence intensity was observed. The verification of stable overexpression of Wnt5a and SFRP1 in fibroblast cell lines is as follows: Figure 3 As shown, a stable overexpression cell line was constructed using a lentiviral packaging system. Both Wnt5a and SFRP1 were coupled with mCherry fluorescence. After screening with puromycin, the fluorescence intensity of the cells was observed.

[0141] Cell lines stably overexpressing WNT5a or SFRP1 were constructed and then directly co-cultured with endothelial cells. Cell proliferation and eNOS activity of endothelial cells were then assessed after 24 hours. Figure 4 As shown, the Wnt5a pathway significantly promotes endothelial cell proliferation 24 hours after eNOS activity in fibroblast cell lines. Figure 5 As shown in the diagram, the Wnt5a pathway can significantly promote the eNOS activity of fibroblast cell lines. A cell line stably overexpressing Wnt5a or SFRP1 was constructed and then co-cultured directly with endothelial cells. The following were the results: (A) Cell proliferation of endothelial cells after 24 h; (B) eNOS activity.

[0142] A cell line stably overexpressing SFRP1 was constructed, stimulated with WAY 316606, and then directly co-cultured with endothelial cells. Cell proliferation and eNOS activity of endothelial cells were detected after 24 hours. Among other things, ... Figure 6 WAY 316606 can significantly restore the eNOS activity of fibroblasts and the cell proliferation of endothelial cells after 24 hours, such as Figure 7WAY 316606 can significantly restore eNOS activity in fibroblasts. (Schematic diagram of eNOS activity)

[0143] like Figure 8 Inhibiting inflammation significantly promotes angiogenesis. A cell line stably overexpressing SFRP1 was constructed, stimulated with WAY316606, and then directly co-cultured with endothelial cells. (A) Cell proliferation of endothelial cells after 24 hours; (B) eNOS activity. Figure 9 Suppressing inflammation significantly promotes skin lesion healing. Overexpression of Wnt5a in a co-culture system significantly inhibited the expression of inflammatory factors IL-1β, IL-6, and TNFα induced by a high-glucose environment (A), while SFRP1 overexpression further exacerbated endothelial cell inflammation and inhibited angiogenesis (B). miR-15b-5p inhibitors may promote ulcer healing by inhibiting IκBβ phosphorylation, thereby preventing activation of the IκBβ signaling pathway.

[0144] A cell line stably overexpressing SFRP1 was constructed, stimulated with WAY 316606, and then co-cultured directly with endothelial cells. The following were the results of detecting (A) cell proliferation of endothelial cells after 24 h and (B) eNOS activity.

[0145] The changes in F-actin expression in cells under different treatments were detected using phalloidin staining reagent. Figure 10 This is a representative fluorescence pattern indicating how the WNT5a pathway can influence cytoskeleton formation. Figure 11 Figure 1 shows the quantitative results of F-actin fluorescence intensity in cells treated with different methods to detect the influence of the WNT5a pathway on cytoskeleton formation; t-test, P < 0.05, P < 0.01. The Wnt5a pathway can affect cytoskeleton formation: changes in F-actin expression in cells under different treatments were detected using phalloidin staining reagent. (A) Representative fluorescence image; (B) Quantitative results of F-actin fluorescence intensity.

[0146] A high-sugar, high-fat diet combined with STZ-induced type II diabetes model was used (n=8 per group). Changes in rat body weight and blood glucose levels over time were measured. Among other things, ... Figure 12 , Figure 13 As shown, a diabetic skin lesion model was established in rats, and the main phenotypes were then observed. The results showed that over time, the diabetic model group exhibited significant weight loss, reduced activity, and persistent blood glucose levels around 23 mM. Deferrasirox and WAY 316606 significantly restored the rats' body weight but did not affect blood glucose levels.

[0147] Figure 14 The histopathological (HE) and collagen (Masson) content were measured in different groups of rat models (n=8 per group). Figure 15The expression of collagen and marker-related genes in different rat models was shown (n=8 per group). Masson staining revealed a significant decrease in collagen content in diabetic foot skin tissue, while interventional treatment showed significant collagen recovery in the treated skin tissue. Figure 14 Further analysis of mRNA levels in skin tissue revealed that deferasirox and WAY 316606 significantly promoted the activity of Col1A1, α-SMA, and POSTN. + and Wnt5a expression ( Figure 15 The above results suggest that deferasirox and SFRP1 inhibitors can significantly improve histopathology and collagen content.

[0148] It can be seen that the present invention intends to use POSTN + The project aims to study the iron metabolism phenotype of fibroblasts and their interaction with endothelial cells, and to conduct targeted iron metabolism and Wnt2 and Wnt5a intervention therapy. The expected results of this project will further enrich the understanding of the mechanisms underlying the poor healing of diabetic foot skin lesions and provide a theoretical basis for developing potential therapeutic targets.

[0149] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0150] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention and within the spirit and principles of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A dressing for the treatment of diabetic foot ulcers, characterized in that, The dressing, from the side in contact with the wound to the dorsal side, consists of: Contact layer, silicone mesh support; Anti-ferrode layer, PLGA nanoparticles loaded with larosse, are fixed to the inner wall of the mesh of silicone mesh scaffold through a polydopamine adhesion layer; The signal activation layer consists of sodium alginate microspheres encapsulated with WAY 316606, embedded in a carboxymethyl cellulose matrix, and coated on the surface of a PLGA nanoparticle layer loaded with delarose. The repair-promoting layer consists of a poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein, which is then cast and encapsulated with a sodium alginate microsphere layer of WAY316606.

2. The dressing for treating diabetic foot ulcers according to claim 1, characterized in that, The silicone mesh scaffold is 0.5 mm thick with a pore size of 50 μm. The PLGA nanoparticles loaded with deraxisol have a particle size of 152 ± 9 nm. The sodium alginate microspheres encapsulating WAY 316606 have a particle size of 20-50 μm. The concentration of the poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein is 25% w / v.

3. The dressing for treating diabetic foot ulcers according to claim 1, characterized in that, In the anti-ferrode layer, the mass ratio of deirarosi to PLGA was 1:2; the drug loading was 20–35%. In the signal activation layer, the concentration of WAY ​​316606 in the microspheres was 5 mg / mL, with a drug loading rate of 12.5% ​​w / w; At pH 5.5, the sodium alginate microspheres encapsulating WAY 316606 cumulatively released 82.3±4.7% over 24 hours. In the repair-promoting layer, the concentration of rhPOSTN was 10–100 μg / mL; The repair-promoting layer includes a foot-type thermosensitive hydrogel without potassium titanyl phosphate KTP peptide, and a foot-type thermosensitive hydrogel with potassium titanyl phosphate KTP peptide added, with a KTP peptide concentration of 0.5–2 mM.

4. The dressing for treating diabetic foot ulcers according to claim 3, characterized in that, The dorsum-type thermosensitive hydrogel is composed of 25% poloxamer 407 plus 50 μg / mL rhPOSTN; the plantar-type thermosensitive hydrogel is composed of 25% poloxamer 407 plus 50 μg / mL rhPOSTN plus 1 mM KTP peptide.

5. The dressing for treating diabetic foot ulcers according to claim 1, characterized in that, The anti-ferrode death layer uses chitosan-citric acid nanoparticles, and the signal activation layer uses gelatin-oxidized dextran.

6. The dressing for treating diabetic foot ulcers according to claim 1, characterized in that, The dressing for treating diabetic foot ulcers, from the wound contact side to the dorsal side, consists of: Contact layer, silicone mesh support; A composite layer of anti-ferrode death layer and signal activation layer, including PLGA / WAY 316606 / delarosus co-loaded nanoparticles; Repair-promoting layer: poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein.

7. A method for preparing a dressing for the treatment of diabetic foot ulcers, characterized in that, This preparation method is used for the preparation of the dressing for the treatment of diabetic foot ulcers as described in any one of claims 1-4, and the preparation method includes: S1, PLGA nanoparticles loaded with delarose as an anti-ferrode layer were prepared; the prepared PLGA nanoparticles loaded with delarose were fixed to the inner wall of the mesh of a silicone mesh scaffold through a polydopamine adhesion layer. S2, as a signal activation layer, is a pH-responsive sodium alginate microsphere encapsulated with WAY 316606. The prepared sodium alginate microspheres encapsulated with WAY 316606 are embedded in a carboxymethyl cellulose matrix and coated on the surface of a PLGA nanoparticle layer loaded with delarose. S3, a layer of sodium alginate microspheres from WAY 316606, is encapsulated after the poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein is cast and molded.

8. The method for preparing a dressing for the treatment of diabetic foot ulcers according to claim 7, characterized in that, Step S1, the preparation of PLGA nanoparticles loaded with delarose includes: Oil phase preparation: Weigh out derarosi and PLGA, dissolve in dichloromethane, and vortex until clear; Aqueous phase emulsification: The oil phase is injected into a 2% PVA solution and ultrasonically treated with a probe; Solvent evaporation: Pour the emulsion into a 0.5% PVA solution and stir magnetically to evaporate the organic solvent; Antibody conjugation: Nanoparticles were collected by centrifugation, resuspended in pH 7.4 phosphate buffered saline, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution were added. The reaction was carried out with shaking at room temperature. Purification and freeze-drying: Wash with phosphate-buffered saline, add 5% mannitol, pre-freeze and then freeze-dry under vacuum; Step S2, the preparation of pH-responsive encapsulated sodium alginate microspheres of WAY ​​316606 includes: Drug mixing: WAY 316606 is dissolved in a 2% sodium alginate solution; Microsphere molding: Microfluidic chip is injected into a 1.5% CaCl2 crosslinking bath; Shell coating: Microspheres are impregnated with 0.5% chitosan-acetic acid solution and stirred; Curing and washing: Rinse with physiological saline, pre-freeze and then vacuum freeze-dry; In step S3, the process of encapsulating the sodium alginate microspheres of WAY ​​316606 with the poloxamer 407 thermosensitive hydrogel containing rhPOSTN protein after casting specifically includes: Hydrogel casting includes: The foot-shaped thermosensitive hydrogel was cast with 25% poloxamer 407 and 50 μg / mL rhPOSTN, solidified at 4℃, and then sterilized by γ-irradiation. Foot-type thermosensitive hydrogel casting: 25% poloxamer 407, 50 μg / mL rhPOSTN, and 1 mM KTP peptide; solidified at 4°C and then sterilized by γ-irradiation.

9. The method for preparing a dressing for the treatment of diabetic foot ulcers according to claim 7, characterized in that, Before step S1, a bottom layer treatment is performed: plasma activation of the silicone mesh scaffold; In step S3, the sol-gel phase transition temperature of the poloxamer 407 hydrogel in the repair-promoting layer (400) is 32°C.

10. The use of a dressing for the treatment of diabetic foot ulcers as described in any one of claims 1-6 in the preparation of a medicament for treating and reducing inflammation of diabetic foot ulcer tissue.

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