A PD-168077-based double-layer programmed release hydrogel dressing, and a preparation method and application thereof

Abstract

This invention provides a bilayer programmed release hydrogel dressing based on PD-168077. The hydrogel dressing is formed by photocrosslinking a lower fast-release layer and an upper slow-release layer. The fast-release layer is formed by photocrosslinking and curing a first pregel solution containing a first concentration of methacrylamide hyaluronic acid, methacrylamide collagen, a photoinitiator, and PD-168077. The slow-release layer is formed by photocrosslinking and curing a second pregel solution containing a second concentration of methacrylamide hyaluronic acid, methacrylamide collagen, a photoinitiator, and PD-168077. This invention also provides a method for preparing the dressing and its application. This invention constructs a biomimetic dermal triple network structure through a photopolymerization strategy of "single formulation, dual concentration, dual curing," achieving programmed release of PD-168077 with a fast-then-slow release. This synergistic effect of the drug, including anti-inflammatory, antioxidant, and angiogenesis-promoting properties, along with matrix bioactivity, effectively promotes the healing of chronic diabetic wounds.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedical materials, and in particular relates to a bilayer hydrogel dressing loaded with dopamine D4 receptor agonist PD-168077, having a biomimetic dermal structure and programmed drug release function, as well as its preparation method and application. Background Technology

[0002] Diabetic chronic wounds (DCWs) are among the most serious and costly complications of diabetes, characterized by a healing process stalled in the inflammatory phase, failing to enter the normal proliferative and remodeling stages. Their complex pathological microenvironment involves a persistent state of high inflammation, excessive oxidative stress, impaired angiogenesis, and an imbalance between the synthesis and degradation of the extracellular matrix (ECM). While systemic glycemic control is fundamental, targeted interventions against the abnormal local microenvironment of the wound are crucial to breaking the healing stagnation.

[0003] Dopamine D4 receptor (DRD4) belongs to the D2-like receptor family. Previous basic research has shown that DRD4 activation has anti-inflammatory, antioxidant, and endothelial protective effects in the cardiovascular system. Its selective agonist, PD-168077, can exert its effects by regulating signaling pathways such as PI3K / Akt / eNOS. However, current research mainly focuses on neurological and cardiovascular applications, and there are no reports of PD-168077 being used to treat chronic diabetic wounds. Meanwhile, how to stably and effectively deliver such small molecules with multi-pathway regulatory potential to the dynamically changing wound microenvironment, and achieve spatiotemporal control that matches the wound healing process, remains a key challenge for clinical translation. Traditional monophasic release dressings, due to their singular release kinetics, cannot simultaneously meet the differentiated needs of early wound inflammation regulation and subsequent continuous tissue repair.

[0004] In recent years, biomimetic hydrogels based on natural ECM components (such as hyaluronic acid and collagen) have attracted widespread attention due to their excellent biocompatibility and ability to mimic the cellular microenvironment. Achieving programmed or sequential drug release through engineered design is a cutting-edge direction for improving the therapeutic performance of dressings. However, existing bilayer or multilayer dressing preparation technologies often involve complex chemical reaction steps, phase separation, or electrospinning processes, resulting in problems such as cumbersome preparation, weak interlayer bonding, or difficulty in loading bioactive molecules. Summary of the Invention

[0005] One objective of this invention is to provide a double-layer programmed release hydrogel dressing based on PD-168077. Another objective of this invention is to provide a method for preparing the double-layer programmed release hydrogel dressing based on PD-168077. A third objective of this invention is to provide the application of the double-layer programmed release hydrogel dressing in the preparation of drugs or dressings for treating chronic diabetic wounds.

[0006] To achieve the first objective of this invention, the following technical solution is adopted:

[0007] A bilayer programmed release hydrogel dressing (PCHMA) based on PD-168077, wherein PD-168077 is screened and determined by the following steps:

[0008] T1. By comparing and analyzing the differential transcriptome gene expression characteristics of skin samples with different healing outcomes related to diabetic chronic wounds, and combining the Cmap database, PD-168077 was screened and identified as a potential candidate drug for reversing the non-healing state of diabetic chronic wounds.

[0009] T2. By conducting local administration experiments in a diabetic chronic wound animal model, and combining in vitro functional tests of fibroblasts, keratinocytes and vascular endothelial cells, we verified that PD-168077 has the effect of promoting wound healing, and further determined its preferred concentration range in promoting cell proliferation, migration and angiogenesis related to wound repair.

[0010] T3. Within the preferred concentration range determined in step T2, transcriptome sequencing analysis showed that PD-168077 can significantly regulate inflammation-related signaling pathways, remodel the functional state of macrophages under inflammatory stimulation, and is associated with inhibiting excessive inflammatory response and promoting macrophage polarization towards a regenerative phenotype.

[0011] In this invention, the preferred effective concentration range of PD-168077 can be obtained through the above screening and determination steps.

[0012] Specifically:

[0013] Step T1 involved screening small molecule compounds based on a computational model of gene expression association, and PD-168077 was identified as a potential candidate drug that could reverse the non-healing state of chronic diabetic wounds.

[0014] Step T2, based on PD-168077 as a candidate agonist, systematically evaluated the effects of different concentrations of PD-168077 on the healing of chronic diabetic wounds through in vitro and in vivo experiments, and assessed its regulatory effects on the viability and function of keratinocytes, fibroblasts, and vascular endothelial cells. Ultimately, an optimal effective concentration range was determined, which effectively improves cell function while maintaining cell viability, providing a precise pharmacological window for subsequent mechanistic studies.

[0015] Step T3, based on the screening in step T1 and the concentration optimization in step T2, involved performing whole transcriptome sequencing analysis on macrophages treated with the optimized effective concentration of PD-168077. The results showed that PD-168077 could significantly affect the gene expression profile of macrophages and suggested that the intervention of PD-168077 was related to the polarization process of macrophages toward specific functional phenotypes, thus further confirming the application value of PD-168077 in regulating the immune microenvironment.

[0016] In summary, through an integrated strategy of "computational model screening → in vivo and in vitro functional verification and concentration optimization → transcriptome mechanism exploration", PD-168077 was identified as a potential drug for the healing of chronic diabetic wounds, and its preferred effective concentration range was provided. This provides a new treatment option for chronic, refractory diabetic wounds and lays the foundation for subsequent research.

[0017] Preferably, the concentration of PD-168077 in both the rapid-release layer and the slow-release layer is 50-200 nM. Within this concentration range, it can promote keratinocyte migration, fibroblast proliferation and vascular endothelial cell lumen formation, and accelerate wound healing in animal models, while avoiding high concentrations that inhibit cell activity.

[0018] In this invention, PD-168077 at this concentration significantly promotes the formation of tubular structures in human umbilical vein endothelial cells (HUVECs), the migration of HaCaT keratinocytes, and the proliferation of NIH3T3 fibroblasts, while avoiding the cell activity inhibition that may occur at higher concentrations. In vivo animal experiments further confirmed that topical application of PD-168077 at this concentration range effectively accelerates wound healing in diabetic mouse models.

[0019] The hydrogel dressing is formed by photocrosslinking a lower fast-release layer PCHMA-1 and an upper slow-release layer PCHMA-2. The fast-release layer is formed by crosslinking and curing a first pregel solution containing a first concentration of methacryloyl hyaluronic acid (HAMA), a first concentration of methacryloyl collagen (ColMA), a photoinitiator, and a therapeutically effective amount of PD-168077 through a first round of light irradiation. The polymer network crosslinking density of the fast-release layer is relatively low, and its structure is relatively loose.

[0020] The slow-release layer is formed by cross-linking and curing a second pregel solution containing a second concentration of methacrylamide hyaluronic acid, a second concentration of methacrylamide collagen, a photoinitiator, and a therapeutically effective amount of PD-168077 on the surface of the fast-release layer through a second round of light irradiation, and chemically bonding it with the underlying layer.

[0021] Wherein, the second concentration is greater than the first concentration, so that the crosslinking density of the slow-release layer is higher than that of the fast-release layer, thereby forming a bilayer hydrogel with different release characteristics in structure.

[0022] The methacrylamide collagen is not a single component, but a mixture obtained by methacrylamide modification of type I collagen and type III collagen respectively.

[0023] Type I collagen provides mechanical strength, while Type III collagen is associated with tissue flexibility and early repair. The combination of the two better mimics the composition of the natural dermal matrix. The fact that the second concentration is greater than the first concentration is the physical basis for the difference in release kinetics. The concentration difference between the two leads to significant differences in swelling ratio, degradation rate, and network pore size between the two hydrogel layers, thereby achieving a programmed mode of rapid initial release of PD-168077 from the lower layer and continuous slow release from the upper layer.

[0024] Preferably, in the first concentration, the mass-volume concentration of methacrylated hyaluronic acid is 2-4%, and the mass-volume concentration of methacrylated collagen is 8-12%; in the second concentration, the mass-volume concentration of methacrylated hyaluronic acid is 5-7%, and the mass-volume concentration of methacrylated collagen is 18-22%.

[0025] In this invention, the concentration range of each component in the first and second concentrations takes into account both the operability of the pregel solution and the mechanical properties, swelling behavior and degradation rate of the photocured hydrogel. Within this range, the lower layer (fast-release layer) and the upper layer (slow-release layer) can form significantly different pore sizes and cross-linking densities, which is the structural basis for realizing programmed drug release, while ensuring that the two layers can form a stable integrated bond through photocross-linking.

[0026] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate.

[0027] In this invention, lithium phenyl-2,4,6-trimethylbenzoyl phosphate exhibits high initiation efficiency and reaction rate under visible light excitation at a wavelength of 405 nm. Its reaction conditions are mild and do not cause significant damage to the loaded bioactive molecule PD-168077, making it suitable for constructing a drug-loaded biomedical photocrosslinked hydrogel system.

[0028] To achieve the second objective of this invention, the following technical solution is adopted:

[0029] A method for preparing a bilayer programmed release hydrogel dressing based on PD-168077 as described above includes the following steps:

[0030] S1. Preparation of the first pregel solution: Dissolve the first concentration of methacrylamide hyaluronic acid, the first concentration of methacrylamide collagen, PD-168077 and photoinitiator in phosphate buffer solution, stir in the dark until completely dissolved, and obtain the first pregel solution.

[0031] S2. Preparation of the second pregel solution: Dissolve the second concentration of methacrylamide hyaluronic acid, the second concentration of methacrylamide collagen, PD-168077 and photoinitiator in phosphate buffer solution, and stir in the dark until completely dissolved to obtain the second pregel solution; the second concentration is greater than the first concentration;

[0032] S3. Preparation of the bilayer hydrogel: The first pregel solution is injected into a mold and subjected to a first round of light irradiation to crosslink and solidify it to form a fast-release layer; the second pregel solution is added to the surface of the fast-release layer and subjected to a second round of light irradiation to crosslink and solidify it and combine it with the lower layer to form a slow-release layer, thus obtaining the bilayer programmed release hydrogel dressing.

[0033] Preferably, in S1 and S2, the method for preparing the methacrylamide collagen includes: dissolving a mixture of type I collagen and type III collagen in phosphate buffer, adding methacrylic anhydride, reacting at pH 7.4-7.8 and 50°C for 2-4 hours, and after the reaction is completed, dialyzing and lyophilizing to obtain the methacrylamide collagen.

[0034] Preferably, the wavelength of the light irradiation is 405 nm.

[0035] To achieve the third objective of this invention, this invention also provides an application scenario for the dual-layer programmed release hydrogel dressing based on PD-168077 described above.

[0036] Specifically, the application of the PD-168077-based dual-layer programmed release hydrogel dressing as a wound dressing to promote wound healing.

[0037] Preferably, the wound is a diabetic chronic non-healing wound, i.e., DCWs.

[0038] In this invention, PD-168077 can be used as a drug to promote wound healing. A bilayer programmed release hydrogel dressing based on PD-168077 can be used as a wound dressing for wound healing.

[0039] The PD-168077-based bilayer programmed release hydrogel dressing of this invention exhibits a multimodal mechanism for promoting healing in application, with beneficial effects manifested in at least the following aspects: ① Effectively removes excessive reactive oxygen species (ROS) in the wound tissue, reducing oxidative stress damage; ② Regulates the transformation of macrophages in the wound from the pro-inflammatory M1 phenotype to the pro-repair M2 phenotype; ③ Downregulates the expression levels of key pro-inflammatory cytokines IL-1β and IL-6 in the wound microenvironment, breaking the chronic inflammatory cycle; ④ Promotes the formation of tubular structures by vascular endothelial cells, accelerating wound vascularization; ⑤ Stimulates the proliferation and migration of fibroblasts, promoting granulation tissue formation and ECM secretion; ⑥ Accelerates the migration of keratinocytes, promoting the re-epithelialization process; ⑦ Optimizes ECM remodeling, manifested as increased total collagen deposition and a higher proportion of type III collagen in the wound tissue, contributing to a repair outcome closer to regeneration.

[0040] The beneficial effects of this invention are:

[0041] The PD-168077-based bilayer programmed release hydrogel dressing of the present invention achieves programmed drug release through a bilayer structure. The lower layer (rapid release layer) has a loose structure, which can rapidly release sufficient amount of PD-168077 in the early stage of wound healing to inhibit excessive inflammation and oxidative damage in the early stage. The upper layer (slow release layer) has a dense structure, which can provide long-term sustained release of the drug and continuously promote angiogenesis and tissue repair in the middle and late stages of healing. This release mode is consistent with the natural process of wound healing.

[0042] The PD-168077-based bilayer programmed release hydrogel dressing matrix of this invention is composed of hyaluronic acid and type I and type III collagen, mimicking the core components of the skin's extracellular matrix. This matrix supports cell migration and proliferation, and its degradation products can activate cell repair behavior. Simultaneously, PD-168077, by acting on the DRD4 receptor, downregulates inflammatory factors such as IL-1β and IL-6, promoting the transformation of macrophages to a repair phenotype and activating pathways such as PI3K / Akt / eNOS to promote angiogenesis. The microenvironment provided by the matrix synergistically enhances the healing of chronic diabetic wounds by reducing oxidative stress, regulating immunity, promoting vascularization, and facilitating cell migration.

[0043] The preparation process of the PD-168077-based bilayer programmed release hydrogel dressing of the present invention is based on visible light curing technology. By sequentially curing two precursor solutions of different concentrations, a firmly bonded bilayer structure is directly formed. The preparation method of the present invention is mild, simple, easy to repeat, and suitable for large-scale production.

[0044] This invention utilizes a photopolymerization strategy of "single formulation, dual concentration, and dual curing" to construct a biomimetic dermal triple network structure, achieving programmed release of PD-168077 with a rapid initial release followed by a slower release. This synergistic effect of the drug, including anti-inflammatory, antioxidant, and angiogenesis-promoting properties, along with matrix bioactivity, effectively promotes the healing of chronic diabetic wounds. Attached Figure Description

[0045] Figure 1 A schematic diagram of PD-168077 screening, CHMA hydrogel preparation, and drug-loaded bilayer hydrogel PCHMA for DCW therapy;

[0046] Figure 2 Screening and in vitro / in vivo concentration optimization of the DRD4 agonist PD-168077; among which, Figure 2 (a) Volcano plot of differentially expressed genes (DEGs) between self-healing and non-self-healing DCW patient samples (GSE143735); Figure 2 (b) A diagram of the protein-protein interaction (PPI) network constructed from DEG; Figure 2 (c) is a GO enrichment analysis plot of DEG; Figure 2 (d) shows the binding posture of PD-168077 (orange) within the active site of the DRD4 receptor (gray) as displayed in the docking simulation. Figure 2 (e) is a 2D ligand-receptor interaction diagram of PD-168077 and DRD4; Figure 2 (f) shows a representative image of the wound healing process in db / db mice; Figure 2 (g) is Figure 2 (f) A quantitative graph of wound healing rate; Figure 2 (h) is a statistical graph of the activity of NIH3T3 fibroblasts after treatment with various concentrations of PD-168077; Figure 2 (i) is a statistical graph of HaCaT keratinocyte activity after treatment with various concentrations of PD-168077; Figure 2 (j) is a statistical graph of the viability of HUVEC endothelial cells after treatment with various concentrations of PD-168077; Figure 2 (k) is a statistical graph of the healing rate of HaCaT cells in the scratch test; Figure 2 (l) Representative images of HaCaT cell migration scratch assay at 0, 24 and 48 hours; Figure 2 (m) is a statistical graph of the healing rate of HUVEC cells in the scratch test; Figure 2 (n) is a representative image of the HUVEC cell tube formation assay; Figure 2 (o) is a statistical graph of the average mesh size formed by HUVEC cell tubes; Figure 2(p) is a statistical graph of the total tube length in the HUVEC cell tube formation assay; Figure 2 (q) is a statistical graph of the number of connection points in the HUVEC cell tube formation assay; Figure 2 (r) is a statistical diagram of the number of meshes in the HUVEC cell tube formation assay; where, Figure 2 (o) to Figure 2 The data in (r) are expressed as mean ± SD (n=3), p < 0.05, p < 0.01, p < 0.001. ;

[0047] Figure 3 This is a transcriptome analysis of the effect of PD-168077 on LPS-induced macrophage polarization; among which, Figure 3 (a) A graph showing the number of differentially expressed genes (DEGs) upregulated and downregulated in PD-treated LPS-induced macrophages relative to the control. Figure 3 (b) is a volcano diagram of DEGs; Figure 3 (c) Visualization of DEGs expression patterns and corresponding Reactome function annotation diagrams; Figure 3 (d) is a bar chart of GO function annotations; Figure 3 (e) is a bar chart annotating the KEGG pathway; Figure 3 (f) is a KEGG chord diagram showing the relationship between DEG and enrichment pathways; Figure 2 (g) is a bubble chart of GO enrichment; Figure 3 (h) is a bubble chart of KEGG enrichment;

[0048] Figure 4 The diagram shows the preparation process and physicochemical characterization results of the PD-168077-based bilayer programmed release hydrogel dressing of the present invention; wherein, Figure 4 (a) is a schematic diagram of the photocrosslinking of ColMA and HAMA to form CHMA; Figure 4 (b) FTIR spectra of ColMA, blank CHMA hydrogel and drug-loaded PCHMA hydrogel; Figure 4 (c) shows the gelation time of different hydrogels; Figure 4 (d) shows the cross-linking and curing diagram of PCHMA-2 hydrogel; Figure 4 (e) is a rheological frequency scan plot showing G′ and G″; Figure 4 (f) is a diagram of the average energy storage modulus at 1 Hz; Figure 4 (g) shows the compressive stress-strain curves of different hydrogels under strains of 0-80%; Figure 4 (h) shows the compressive stress-strain curves of different hydrogels under 10-20% strain; Figure 4(i) is a diagram of the compressive modulus derived from the stress-strain curve; Figure 4 (j) is the tensile stress-strain curve; Figure 4 (k) is a representative figure of the tensile test of PCHMA-2 hydrogel; Figure 4 (l) Images of hydrogels solidified into various shapes; Figure 4 (m) is a graph showing the swelling rate of the hydrogel in PBS over 24 hours; Figure 4 (n) is a graph showing the swelling rate of the hydrogel in PBS over 1 hour; Figure 4 (o) shows the degradation of the hydrogel in PBS; Figure 4 (p) shows the degradation of the hydrogel in collagenase solution; Figure 4 (q) is a quantitative diagram of the hydrogel pore size; Figure 4 (r) is a quantitative diagram of the porosity of the hydrogel; Figure 4 (s) is the force-displacement curve of the tissue adhesion test; Figure 4 (t) is a quantification plot of adhesion strength; Figure 4 (u) is a schematic diagram of the bilayer hydrogel and a representative cross-sectional SEM image of the bilayer structure; Figure 4 (v) is a comparison of the degradation rates of monolayer (CHMA-1) and bilayer hydrogel (CHMA-1 + CHMA-2) in collagenase;

[0049] Figure 5 This is a diagram showing the in vitro antioxidant and macrophage regulation results of the PCHMA hydrogel in Example 4; where, Figure 5 (a) A representative fluorescence image of intracellular ROS levels in RAW264.7 macrophages stained with DCFH-DA (H2O2 induced); Figure 5 (b) is Figure 5 (a) Quantitative map of DCF positive regions; Figure 5 (c) is a quantitative map of the DCF-positive region in LPS-induced macrophages; Figure 5 (d) is a flow cytometry analysis of DCF fluorescence intensity; Figure 5 (e) is a representative diagram of DPPH free radical scavenging activity; Figure 5 (f) is a quantitative diagram of DPPH free radical scavenging activity; Figure 5 (g) is a representative graph of NBT (superoxide anion) scavenging activity; Figure 5 (h) is a quantification diagram of NBT (superoxide anion) scavenging activity; Figure 5 (i) Immunofluorescence staining of M1 marker CD86 (red) and M2 marker CD206 (green) in RAW264.7 macrophages; Figure 5 (j) is Figure 5 (i) is a fluorescence intensity quantization plot of CD86; Figure 5 (k) is Figure 5 (i) Quantitative fluorescence intensity diagram of CD206;

[0050] Figure 6 This is a diagram showing the in vitro evaluation results of PCHMA's healing-promoting function and biocompatibility in Example 4; where, Figure 6 (a) is Figure 6 (b) Quantization plot; Figure 6 (b) is a representative immunofluorescence staining image of α-SMA expression (green) in NIH3T3 fibroblasts; Figure 6 (c) is a statistical graph of the cell proliferation assay of NIH3T3 fibroblasts; Figure 6 (d) is a representative diagram of the HUVEC cell tube formation assay; Figure 6 (e) is a statistical graph of the common tube length formed by HUVEC cells; Figure 6 (f) is a statistical graph showing the number of branching points formed by HUVEC cells; Figure 6 (g) is a statistical graph showing the number of tubes formed by HUVEC cells; Figure 6 (h) is a statistical graph of HaCaT cell migration scratch assay; Figure 6 (i) is a representative image of the HaCaT cell migration scratch assay; Figure 6 (j) is a graph quantifying the hemolysis rate; Figure 6 (k) is a representative graph of the hemolysis test, NC (negative control, PBS), PC (positive control, deionized water); Figure 6 (l) is a representative image of live / dead cell staining of NIH3T3, HaCaT, RAW264.7 and HUVEC cells after PCHMA-2 treatment; Figure 6 (m) is a quantitative map of NIH3T3 cell viability; Figure 6 (n) is a quantitative graph of HaCaT cell viability; Figure 6 (o) is a quantitative graph of RAW264.7 cell viability; Figure 6 (p) is a quantitative map of HUVEC cell viability;

[0051] Figure 7 The in vivo efficacy evaluation diagram for the wound-healing function of PCHMA hydrogel in implementation 5 is shown; among them, Figure 7 (a) is a schematic diagram of the in vivo experimental design; Figure 7 (b) Gross wound representations of the control group, CHMA group, PD group and PCHMA group on days 0, 3, 7 and 14; Figure 7 (c) is a quantitative graph of wound healing rate; Figure 7 (d) Histological quantitative images of wound length on day 14 for the control group, CHMA group, PD group and PCHMA group; Figure 7 (e) is a histological quantitative map of epidermal thickness on day 14 for the control group, CHMA group, PD group and PCHMA group; Figure 7 (f) is a histological quantification of collagen volume fraction on day 14 in the control group, CHMA group, PD group and PCHMA group; Figure 7 (g) is a representative image of H&E staining of wound tissue on day 14; Figure 7 (h) is a representative image of Masson's trichrome staining (collagen appears in blue);

[0052] Figure 8 This is a diagram showing the collagen composition, inflammation, vascularization, and myofibroblast staining analysis results of the wound tissue on day 14 after modeling in Example 5; where, Figure 8 (a) is a representative image of picric acid-Sirius red staining observed under polarized light, showing type I collagen (red / orange) and type III collagen (green). Figure 8 (b) is Figure 8 (a) Quantitative graph of the proportion of type III collagen (green area / total collagen area); Figure 8 (c) is a representative image of IL-1β immunohistochemical (IHC) staining; Figure 8 (d) is Figure 8 (c) Quantitative map of the IL-1β positive region; Figure 8 (e) is a representative image of IHC staining for IL-6; Figure 8 (f) is Figure 8 (e) Quantitative map of IL-6 positive regions; Figure 8 (g) is a representative immunofluorescence co-staining image of macrophage markers CD86 (red, M1 type) and CD206 (green, M2 type); Figure 8 (h) is Figure 8 (g) Quantitative statistical graph of CD86 / CD206 fluorescence intensity ratio; Figure 8 (i) A representative IHC staining image of the endothelial marker CD31 (marking blood vessels); Figure 8 (j) is Figure 8 (i) is a statistical chart of the number of blood vessels per field of view; Figure 8 (k) is a representative IHC staining image of the myofibroblast marker α-SMA; Figure 8 (l) is Figure 8 Quantitative statistical plot of α-SMA positive regions in (k). Detailed Implementation

[0053] The invention can be further understood through the specific embodiments given below, but they are not intended to limit the invention.

[0054] This invention provides a bilayer programmed release hydrogel dressing based on PD-168077 and its preparation method, and establishes PD-168077 as a promising new candidate drug for DCW treatment. Figure 1The flowchart provides a new strategy for the study of the mechanisms and treatment of DCWs. Figure 1 The screening of PD-168077, preparation of CHMA dressings, and preparation of PCHMA, a bilayer programmed release hydrogel dressing for DCWs treatment, were demonstrated.

[0055] Example 1

[0056] The synthesis of methacrylamide collagen (ColMA) includes the following steps:

[0057] Type I collagen and Type III collagen powders were mixed at a mass ratio of 1:1. The mixture was weighed and dissolved in phosphate buffer (PBS, pH 7.4) preheated to 50°C to prepare a mixed collagen solution with a total mass volume concentration of 10% (w / v).

[0058] Under continuous stirring and in the dark, methacrylic anhydride was slowly added dropwise to the solution. The amount of methacrylic anhydride added was 10:1 based on the molar ratio of free amino groups in the collagen molecules. The reaction temperature was maintained at 50°C, and the pH of the system was stabilized between 7.4 and 7.8 by adding 1M NaOH solution. After the reaction continued for 3 hours, 4 times the volume of the reaction solution was added and PBS preheated to 37°C was added to terminate the reaction.

[0059] The reaction mixture was transferred to a dialysis bag with a molecular weight cutoff of 12-14 kDa and dialyzed in deionized water at 40°C for 7 days, with the external dialysis fluid replaced every 8-12 hours during the period.

[0060] After dialysis, the liquid was pre-frozen at -80°C and then freeze-dried (48-72 h) to obtain a white, loose methacrylamide collagen solid.

[0061] Example 2

[0062] The preparation of PCHMA, a bilayer programmed release hydrogel dressing based on PD-168077, includes the following steps:

[0063] S1. Preparation of the first pregel solution: Methacrylamide hyaluronic acid (final concentration 3% w / v), methacrylamide collagen prepared in Example 1 (final concentration 10% w / v), PD-168077 (final concentration 100 nM), and phenyl-2,4,6-trimethylbenzoyl lithium phosphate (final concentration 0.5% w / v) were dissolved in phosphate buffer (pH 7.4) and stirred at 37°C in the dark until completely dissolved. The solution was then filtered through a 0.22 μm sterile filter membrane to obtain the first pregel solution, i.e., the rapid release layer pregel solution. The rapid release layer pregel solution was stored at 4°C in the dark.

[0064] S2. Preparation of the second pregel solution: Methacrylamide hyaluronic acid (final concentration 6% w / v), methacrylamide collagen prepared in Example 1 (final concentration 20% w / v), PD-168077 (final concentration 100 nM), and phenyl-2,4,6-trimethylbenzoyl lithium phosphate (final concentration 0.5% w / v) were dissolved in phosphate buffer (pH 7.4) and stirred at 37°C in the dark until completely dissolved. The solution was then filtered through a 0.22 μm sterile filter membrane to obtain the second pregel solution, namely the slow-release layer pregel solution. The slow-release layer pregel solution was stored at 4°C in the dark.

[0065] Preparation of S3 and PCHMA bilayer hydrogel: A cylindrical polydimethylsiloxane mold with a diameter of 10 mm and a depth of 2 mm was sterilized by ultraviolet light. 100 μL of fast-release layer pregel liquid was injected into the mold, and the mold was irradiated for the first time with an LED light source with a wavelength of 405 nm and a light intensity of 15 mW / cm² for 30 s to crosslink and solidify it to form the lower fast-release layer. 100 μL of slow-release layer pregel liquid was added to the surface of the solidified lower fast-release layer, and the mold was irradiated for the second time with an LED light source with a wavelength of 405 nm and a light intensity of 15 mW / cm² for 60 s to crosslink and solidify the slow-release layer pregel liquid and combine it with the lower layer to form the upper slow-release layer. After demolding, the PD-168077-loaded bilayer hydrogel dressing was obtained and labeled as PCHMA.

[0066] Furthermore, PD-168077 in this embodiment 2 is screened and determined according to the following steps:

[0067] T1. By comparing and analyzing the differential transcriptome gene expression characteristics of skin samples with different healing outcomes related to diabetic chronic wounds, and combining the Cmap database, PD-168077 was screened and identified as a potential candidate drug for regulating the non-healing state of diabetic chronic wounds.

[0068] T2. By conducting local drug administration experiments in a diabetic chronic wound animal model, and combining in vitro functional tests of fibroblasts, keratinocytes and vascular endothelial cells, we verified the regulatory effect of PD-168077 on the behavior of wound repair-related cells and further determined its preferred concentration range.

[0069] T3. Within the preferred concentration range determined in step T2, the effects of PD-168077 on macrophage gene expression profiles were analyzed by transcriptome sequencing. The results showed that PD-168077 can regulate inflammation-related signaling pathways and is associated with the regulation of inflammatory responses and changes in macrophage functional status.

[0070] The results of PD-168077 screening and determination are as follows: Figure 2((a)-(r)) and Figure 3 As shown in (a)-(h)). Figure 2 This is used to illustrate the overall technical roadmap of PD-168077 from candidate screening to parameter determination. Figure 3 The experimental results used to illustrate the effects of PD-168077 on inflammation-related signaling pathways and immune regulation under preferred conditions are used to support the technical effects described in this embodiment.

[0071] The preparation principle and process of the PD-168077-based bilayer programmed release hydrogel dressing in Example 2 are as follows: Figure 4 As shown in (a, cd), the dressing has a double-layer structure as follows: Figure 4 As shown in (u).

[0072] Non-drug-loaded dressing control group

[0073] The non-drug-loaded control dressing CHMA was prepared according to the methods of Examples 1 and 2. The difference between CHMA and the dressing labeled PCHMA is that PD-168077 was not added to the non-drug-loaded control dressing, while the rest were the same. The resulting non-drug-loaded control dressing was labeled CHMA.

[0074] Example 3

[0075] Physicochemical characterization of dressings

[0076] The physicochemical characterization of the PD-168077-based bilayer programmed release hydrogel dressing (PCHMA) and the drug-free control dressing (CHMA) prepared in Example 2 is as follows:

[0077] (1) Spectral characterization

[0078] The material was characterized by Fourier transform infrared spectroscopy.

[0079] like Figure 4 As shown in (b), the spectrum of methacrylamide collagen is at approximately 1450 cm⁻¹. -1 and 1330cm -1 The presence of a characteristic -CH3 bending vibration absorption peak at the 1015 cm⁻¹, attributed to the methacrylate group, indicates successful methacrylation. The spectrum of the unloaded CHMA hydrogel shows characteristic CO and COC vibrational peaks of the hyaluronic acid polysaccharide backbone. After loading PD-168077, the PCHMA spectrum shows a peak at approximately 10¹⁵ cm⁻¹. -1 and 950cm -1 A new absorption peak belonging to the heterocyclic structure of the PD molecule appeared, confirming that the drug was successfully encapsulated in the hydrogel matrix.

[0080] (2) Mechanical properties and rheological characterization

[0081] The viscoelasticity of the hydrogel was determined using a rotational rheometer.

[0082] like Figure 4 (e) and Figure 4 As shown in (f), during the frequency scan, the storage modulus (G′) of CHMA-2 is significantly higher than that of CHMA-1, while the incorporation of PD-168077 has no significant effect on the modulus.

[0083] Compression test ( Figure 4 (gh) shows that the compressive modulus of CHMA-2 ( Figure 4 (i) The modulus of PCHMA-2 was higher than that of CHMA-1, while there was no significant difference between PCHMA-2 and unloaded CHMA-2;

[0084] Tensile test ( Figure 4 (j) indicates that CHMA-2 has a lower maximum elongation than CHMA-1 due to its higher crosslinking density; such as Figure 4 As shown in (k), the cured hydrogel has good mechanical integrity.

[0085] (3) Molding properties and swelling behavior

[0086] The pregel solution has good plasticity and can be injected into a mold and photocured to form the desired shape. Figure 4 (l)).

[0087] Swelling experiment such as Figure 4 As shown in (mn), after soaking in phosphate buffer, the swelling rate of CHMA-1 was significantly higher than that of CHMA-2.

[0088] (4) Degradation behavior

[0089] Degradation experiments such as Figure 4 As shown in (op), in phosphate buffer containing collagenase, the degradation rate of CHMA-1 is significantly faster than that of CHMA-2;

[0090] Figure 4 (v) further shows that the degradation curve of the PCHMA bilayer hydrogel exhibits a two-stage pattern different from that of the monolayer hydrogel, which is consistent with its bilayer structure design.

[0091] (5) Microstructure

[0092] Scanning electron microscopy observation showed ( Figure 4 (qr)), the internal structure of the sustained-release hydrogel CHMA-2 is more compact, with smaller pore size and lower porosity than that of the immediate-release hydrogel CHMA-1. The incorporation of PD-168077 did not significantly change the microstructure of the hydrogel.

[0093] (6) Adhesion performance

[0094] The adhesion strength between the hydrogel and biological tissue was evaluated using tissue adhesion testing. Figure 4 (st). The results showed that the adhesion strength of the sustained-release layer CHMA-2 was higher than that of the immediate-release layer CHMA-1, and the loading of PD-168077 had no significant effect on the adhesion strength.

[0095] (7) Verification of the double-layer structure

[0096] like Figure 4 As shown in (u), the cross-sectional scanning electron microscope image clearly shows the different structural morphologies of the upper and lower layers in the bilayer hydrogel, confirming the successful construction of the bilayer structure.

[0097] Example 4

[0098] In vitro biological function and biocompatibility evaluation

[0099] (1) In vitro antioxidant and macrophage regulation

[0100] Intracellular reactive oxygen species (ROS) levels were assessed using the DCFH-DA fluorescent probe method.

[0101] like Figure 5 As shown in (ac), after H2O2 stimulation, the intracellular fluorescence intensity of RAW264.7 cells in the PCHMA hydrogel treatment group was significantly lower than that in the PBS control group and the CHMA hydrogel group, and the effect was comparable to that in the free PD-168077 solution group. Flow cytometry analysis ( Figure 5 (d) further confirms this result.

[0102] DPPH free radical scavenging experiment ( Figure 5 (ef)) and NBT method were used to detect superoxide anion scavenging ability. The results showed that PCHMA hydrogel itself has free radical scavenging activity (ef) Figure 5 (gh)).

[0103] Macrophage polarization markers were analyzed using immunofluorescence staining. For example... Figure 5 As shown in (ik), in the lipopolysaccharide-induced M1 polarized macrophage model, PCHMA hydrogel treatment can reduce the expression of the M1 marker CD86 in RAW264.7 cells, while upregulating the expression of the M2 marker CD206.

[0104] (2) In vitro healing-promoting function

[0105] Immunofluorescence staining was used to detect α-smooth muscle actin expression to assess fibroblast activation. For example... Figure 6 As shown in (ab), PCHMA hydrogel can promote the expression of α-SMA in NIH3T3 cells. Cell proliferation was detected by CCK-8 assay. Figure 6(c) shows that PCHMA hydrogel can significantly enhance the proliferation activity of NIH3T3 fibroblasts.

[0106] The ability of vascular endothelial cells to form new blood vessels is assessed through tube formation experiments. For example... Figure 6 As shown in (dg), cells treated with PCHMA hydrogel formed tubular structures that were significantly superior to the PBS control group and the CHMA hydrogel group in terms of total tube length, number of branch points, and number of pores. The migration ability of keratinocytes was evaluated using a scratch assay. Figure 6 As shown in (hi), the scratch closure rate of HaCaT cells in the PCHMA hydrogel treatment group was significantly higher than that in the control group at 24 hours and 48 hours.

[0107] (3) Biocompatibility evaluation: Blood compatibility is evaluated through hemolysis tests. For example... Figure 6 As shown in (jk), the hemolysis rate of PCHMA material is less than 1%.

[0108] The cell compatibility of the hydrogel was evaluated using a live / dead cell staining method. For example... Figure 6 As shown in (lp), the survival rates of NIH3T3, HaCaT, RAW264.7 and human umbilical vein endothelial cells co-cultured with PCHMA hydrogel were all higher than 95%.

[0109] Example 5

[0110] The evaluation of in vivo chronic wound healing in diabetic patients was approved by the ethics committee for animal experiments (approval number: 20251107161553).

[0111] (1) Animal model establishment and experimental grouping

[0112] like Figure 7 As shown in (a), two full-thickness skin defects with a diameter of 8 mm were prepared on the backs of 8-week-old male db / db mice. The animals were randomly divided into four groups (n=6 per group): PBS control group, CHMA dressing group (non-drug-loaded bilayer hydrogel), free PD-168077 solution group (PD, 100 nM), and PCHMA dressing group (PD-168077-loaded bilayer hydrogel).

[0113] (2) Observation of wound healing process

[0114] The wound was photographed and documented on days 0, 3, 7, and 14 post-injury. For example... Figure 7 As shown in (b), the wound closure was slow in the PBS control group. Wound healing was improved in the free PD group. The PCHMA group showed the fastest wound closure and significantly better gross morphology than the other groups. Figure 7As shown in (c), the healing rate of the PCHMA group was significantly higher than that of the other three groups on days 3, 7 and 14.

[0115] (3) Histopathological analysis of the wound tissue The animal was euthanized on the 14th day after the injury, and full-thickness wound tissue was taken for paraffin sectioning and staining analysis.

[0116] Hematoxylin-eosin staining: such as Figure 7 As shown in (g), the PCHMA group exhibited the most complete reepithelialization of the wound, with continuous new epidermis; the granulation tissue was also the most abundant, rich in cells and newly formed microvessels. Quantitative analysis showed that ( Figure 8 (de)), the PCHMA group had the shortest wound length and the best epidermal thickness recovery.

[0117] Masson trichrome staining: such as Figure 7 As shown in (h), a direct comparison reveals that collagen deposition (blue) was significantly higher in all treatment groups than in the PBS control group. Quantitative analysis ( Figure 7 (f) shows that the collagen volume fraction of the CHMA group and the PD group increased significantly, while the collagen fraction of the PCHMA group was comparable to that of the PBS control group. However, combined with its superior macroscopic healing and the observed new hair follicle structure, it indicates that its healing tends to be more regenerative rather than simply fibrotic filling.

[0118] (4) Analysis of collagen composition, inflammation, vascularization and myofibroblasts in wound tissue

[0119] Sirius red polarized light staining: such as Figure 8 As shown in (ab), the distribution of type I collagen (red / orange) and type III collagen (green) was observed under polarized light. The proportion of green (type III collagen) signal in the PCHMA group and CHMA group was significantly higher than that in the PBS control group and the free PD group.

[0120] Immunohistochemical staining:

[0121] Pro-inflammatory cytokines: such as Figure 8 (cf) shows that IL-1β in the wound tissue of the PCHMA group ( Figure 8 (cd)) and IL-6 ( Figure 8 The positive staining intensity and area of ​​(ef) were significantly lower than those of the other groups.

[0122] Endothelial markers: such as Figure 8 As shown in (ij), the number of CD31-positive microvessels in the wound tissue of all treatment groups was greater than that in the PBS control group, with the PCHMA group having the highest number.

[0123] Myofibroblast markers: such as Figure 8As shown in (kl), during the later healing stage (day 14), the CHMA group and PD group had larger positive areas of α-SMA, while the PCHMA group had relatively lower α-SMA expression levels.

[0124] (5) Macrophage phenotype analysis

[0125] The phenotype of macrophages in the wound was detected by immunofluorescence co-staining. For example... Figure 8 As shown in (gh), the fluorescence intensity of CD86 (red), a marker of pro-inflammatory M1 macrophages, was relatively decreased in the PCHMA group wound tissue, while the fluorescence intensity of CD206 (green), a marker of pro-repair M2 macrophages, was increased, and the CD86 / CD206 fluorescence intensity ratio decreased.

[0126] As can be seen from the results of Examples 1-5 above, the present invention provides a treatment solution for chronic diabetic wounds by combining specific small molecule drugs with biomimetic double-layer hydrogel dressings.

[0127] The technical solution of this invention first demonstrates a complete and controllable preparation process through Examples 1 and 2, from the methacrylylation modification of collagen raw materials to the construction of a bilayer hydrogel dressing, forming a stable and repeatable preparation process. This process is based on visible light-induced polymerization. Under the same chemical system, by adjusting the concentration of the polymer precursor and performing stepwise photocuring, a simple molding of an integrated bilayer hydrogel dressing with a clearly defined upper and lower layer structure is achieved. The physicochemical performance test results in Example 3 confirm that the structural characteristics of the obtained bilayer dressing conform to the initial design: the lower layer (immediate-release layer) composed of a lower concentration of polymer exhibits a higher swelling rate and faster degradation behavior, with a relatively loose network structure; while the upper layer (sustained-release layer) composed of a higher concentration of polymer exhibits a lower swelling rate, a slower degradation rate, and a denser network structure. A stable bond is formed between the two layers through chemical bonding, and the loaded PD-168077 drug is successfully encapsulated within the hydrogel matrix.

[0128] Of particular importance is that, as a known dopamine D4 receptor agonist, the application of PD-168077 in the repair of chronic diabetic wounds lacks a mature or well-defined technical solution in the existing technology. This invention introduces PD-168077 into a local treatment system for chronic diabetic wounds and achieves programmed delivery to the wound site through a double-layer hydrogel dressing, thereby giving the drug new application value in this indication and expanding its application scope in the field of tissue repair.

[0129] In vitro functional evaluation results showed that the PCHMA hydrogel dressing, while maintaining the bioactivity of PD-168077 itself, further amplified its comprehensive effects in wound repair-related processes. Specifically, the dressing not only exhibited effects such as scavenging reactive oxygen species, regulating macrophage function, promoting the formation of tubular structures in vascular endothelial cells, and enhancing the migration and proliferation of keratinocytes and fibroblasts, but also showed a more significant synergistic promoting effect compared to simple non-drug-loaded hydrogel matrices or free drug forms. Simultaneously, the material demonstrated good blood compatibility and cell compatibility, providing a safety basis for its subsequent in vivo application.

[0130] Animal experiments further confirmed that, in a diabetic chronic wound model, treatment with the PCHMA dressing described in this invention significantly improved wound healing speed, reepithelialization, granulation tissue formation quality, and collagen deposition compared to the PBS buffer control group, the drug solution group alone, and the non-drug-loaded hydrogel dressing group. Further histological analysis revealed that this dressing effectively inhibited the expression of key pro-inflammatory factors IL-1β and IL-6 in the wound area, promoted the infiltration of reparative M2 macrophages, increased the density of newly formed microvessels, and moderately regulated the activation of myofibroblasts in the later stages of healing. These effects collectively propelled the wound microenvironment from a state of persistent inflammation to an active regenerative repair phase.

[0131] Comprehensive analysis reveals that the core design of this invention lies in combining the pharmacological effects of the dopamine D4 receptor agonist PD-168077 with the bioactivity and controllable release characteristics of a biomimetic matrix. By constructing a bilayer structure with a concentration gradient, an organic combination of rapid early drug release and sustained later drug supply is achieved, thereby synergistically regulating inflammatory responses, angiogenesis, and tissue regeneration processes at both temporal and spatial levels. This provides a novel technical solution for the effective intervention of diabetic chronic wounds.

[0132] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the concept and technical principles of the present invention should be considered to fall within the scope of protection of the present invention.

Claims

1. A bilayer programmed release hydrogel dressing based on PD-168077, characterized in that, The hydrogel dressing is formed by integrating a lower fast-release layer and an upper slow-release layer through photocrosslinking. The rapid release layer is formed by photocrosslinking and curing a first pregel solution containing a first concentration of methacrylamide hyaluronic acid, a first concentration of methacrylamide collagen, a photoinitiator, and PD-168077. The slow-release layer is formed by photocrosslinking and curing a second pregel solution containing a second concentration of methacrylamide hyaluronic acid, a second concentration of methacrylamide collagen, a photoinitiator, and PD-168077. Wherein, the second concentration is higher than the first concentration, so that the crosslinking density of the slow release layer is higher than that of the fast release layer, thereby forming a bilayer hydrogel with different structural properties; The methacrylamide collagen is a mixture of type I methacrylamide collagen and type III methacrylamide collagen.

2. The PD-168077-based bilayer programmed release hydrogel dressing according to claim 1, characterized in that, In the first concentration, the mass-volume concentration of methacrylated hyaluronic acid is 2-4%, and the mass-volume concentration of methacrylated collagen is 8-12%; in the second concentration, the mass-volume concentration of methacrylated hyaluronic acid is 5-7%, and the mass-volume concentration of methacrylated collagen is 18-22%.

3. The PD-168077-based bilayer programmed release hydrogel dressing according to claim 1, characterized in that, PD-168077 was selected and identified as a candidate drug for reversing the non-healing state of chronic diabetic wounds through screening and identification by "transcriptome differential gene analysis combined with Cmap calculation screening → in vivo and in vitro functional verification and concentration optimization → transcriptome gene sequencing mechanism confirmation".

4. The PD-168077-based bilayer programmed release hydrogel dressing according to claim 1 or 2, characterized in that, The concentration of PD-168077 in both the fast-release layer and the slow-release layer is 50–200 nM.

5. The dual-layer programmed release hydrogel dressing based on PD-168077 according to claim 1, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate.

6. A method for preparing a bilayer programmed release hydrogel dressing based on PD-168077 as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Preparation of the first pregel solution: Dissolve the first concentration of methacrylamide hyaluronic acid, the first concentration of methacrylamide collagen, PD-168077 and photoinitiator in phosphate buffer solution, stir in the dark until completely dissolved, and obtain the first pregel solution. S2. Preparation of the second pregel solution: Dissolve the second concentration of methacrylamide hyaluronic acid, the second concentration of methacrylamide collagen, PD-168077 and photoinitiator in phosphate buffer solution, and stir in the dark until completely dissolved to obtain the second pregel solution; the second concentration is greater than the first concentration; S3. Preparation of the bilayer hydrogel: The first pregel solution is injected into a mold and subjected to a first round of light irradiation to crosslink and solidify it to form a fast-release layer; the second pregel solution is added to the surface of the fast-release layer and subjected to a second round of light irradiation to crosslink and solidify it and combine it with the lower layer to form a slow-release layer, thus obtaining the bilayer programmed release hydrogel dressing.

7. The method for preparing a bilayer programmed release hydrogel dressing based on PD-168077 according to claim 6, characterized in that, In S1 and S2, the preparation method of the methacrylamide collagen includes: dissolving a mixture of type I collagen and type III collagen in phosphate buffer, adding methacrylic anhydride, reacting at pH 7.4-7.8 and 50°C for 2-4 hours, and after the reaction is completed, dialyzing and lyophilizing to obtain the methacrylamide collagen.

8. The method for preparing a bilayer programmed release hydrogel dressing based on PD-168077 according to claim 6, characterized in that, The wavelength of the light irradiation is 405 nm.

9. The application of a PD-168077-based bilayer programmed release hydrogel dressing as described in any one of claims 1-5 for promoting wound healing.

10. The application of the PD-168077-based double-layer programmed release hydrogel dressing according to claim 9 for promoting wound healing, characterized in that, The wound in question is a chronic, slow-healing wound caused by diabetes.

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