A spatially partitioned double-layer intelligent hydrogel as well as a preparation method and application thereof

Abstract

The present application belongs to the technical field of biomedical materials, and particularly relates to a spatially partitioned double-layer intelligent hydrogel and a preparation method and application thereof, which spatially separates infection monitoring function and drug treatment function, monitors temperature changes generated in the process of wound infection in real time through a temperature sensing layer, then introduces engineered microspheres with different functions in a functional treatment layer, and loads anti-inflammatory drugs and pro-angiogenic drugs in different microspheres, on the one hand, according to temperature changes, anti-inflammatory drugs loaded in the first microspheres in the functional treatment layer are released on demand, and on the other hand, the second microspheres in the functional treatment layer loaded with vascular endothelial growth factor are used to continuously release the vascular endothelial growth factor, thereby providing continuous support for wound repair. Through spatial isolation of different microspheres, the interaction between drugs and between drugs and the hydrogel matrix is reduced, thereby realizing independent regulation of the release behaviors of different therapeutic drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a spatially partitioned bilayer smart hydrogel, its preparation method, and its applications. Background Technology

[0002] With the development of smart biomaterials and wound healing technologies, hydrogel materials with environmental responsiveness and functional regulation capabilities are increasingly being applied to the treatment of chronic and diabetic wounds. Diabetic wounds are typically accompanied by pathological features such as persistent inflammation, impaired angiogenesis, and increased risk of infection. The wound microenvironment undergoes dynamic changes at different healing stages, thus requiring smart wound materials that can sense the wound condition and regulate treatment based on changes in the local microenvironment.

[0003] In existing technologies, smart hydrogels typically achieve wound microenvironment sensing by introducing stimulus-responsive materials such as temperature, pH, reactive oxygen species, and glucose, and further promote wound repair by loading therapeutic substances such as anti-inflammatory drugs, antibacterial drugs, or growth factors. Some studies have further combined sensing functions with drug delivery functions, enabling hydrogels to monitor wound conditions and achieve a certain degree of responsive treatment.

[0004] However, in existing technologies, the sensing module and the treatment module are often functionally coupled. The sensing materials and structures may affect drug delivery performance, while the structures used for drug delivery may in turn affect sensing performance. Multiple therapeutic drugs are often co-loaded in the same hydrogel matrix and passively released, which can easily lead to mutual influence between the release behaviors of different therapeutic components. This makes it difficult to independently and stage-wise regulate therapeutic effects such as inflammation control and angiogenesis according to different healing stages of diabetic wounds.

[0005] Therefore, there is still room for improvement in the existing smart wound hydrogels in terms of real-time wound condition sensing, responsive treatment, and synergistic regulation of multiple treatment functions. Summary of the Invention

[0006] To address the problems raised in the background art, this invention provides a spatially partitioned bilayer smart hydrogel, its preparation method, and its applications.

[0007] The technical solution of the present invention is as follows: The present invention provides a spatially partitioned bilayer smart hydrogel, comprising a temperature sensing layer as the bottom layer and a functional therapeutic layer disposed on the upper surface of the temperature sensing layer. The functional therapeutic layer contains a first microsphere loaded with anti-inflammatory drugs and a second microsphere loaded with vascular endothelial growth factor.

[0008] Based on the spatially partitioned bilayer intelligent hydrogel described above, the temperature sensing layer is an ion-conductive hydrogel, and the functional therapeutic layer is a gelatin-chitosan oligosaccharide composite hydrogel.

[0009] Based on the spatially partitioned bilayer smart hydrogel described above, the first microsphere is obtained by crosslinking gelatin methacrylate with poly(N-isopropylacrylamide), and the minimum critical dissolution temperature of the first microsphere is 40-50℃.

[0010] Based on the spatially partitioned bilayer smart hydrogel described above, the second microsphere is obtained by grafting heparin onto a gelatin methacrylate network via an EDC / HOBt-mediated coupling reaction; the loading efficiency of the second microsphere for vascular endothelial growth factor is higher than 83%.

[0011] This invention also provides a method for preparing the aforementioned spatially partitioned bilayer smart hydrogel, comprising: Step (1): Dissolve acrylamide, photoinitiator, crosslinking agent, gelatin, polyvinyl alcohol and NaCl in deionized water, mix evenly and then photocrosslink to form the first chemical crosslinking network, and then form the physical crosslinking network through borax treatment and freeze-thaw cycle to obtain the temperature sensing layer. Step (2): Methacrylamide gelatin, N-isopropylacrylamide, crosslinking agent and photoinitiator are mixed in proportion and microdroplets are prepared by droplet microfluidics. The first microspheres are obtained by photocrosslinking. The first microspheres are freeze-dried and incubated in an anti-inflammatory drug solution to obtain the first microsphere loaded with anti-inflammatory drugs. Step (3): Heparin was grafted onto the gelatin methacrylic acid network using an EDC / HOBt-mediated coupling reaction to obtain the second microspheres. The second microspheres were then freeze-dried and incubated in a vascular endothelial growth factor solution to obtain the second microspheres loaded with vascular endothelial growth factor. Step (4): The first microsphere loaded with anti-inflammatory drugs and the second microsphere loaded with vascular endothelial growth factor are mixed and dispersed in the gelatin-chitosan composite hydrogel precursor solution to obtain the functional therapeutic layer precursor solution. Step (5): The functional therapeutic layer precursor liquid is deposited on the upper surface of the temperature sensing layer by 3D printing and then photocrosslinked.

[0012] Based on the above-described method for preparing spatially partitioned bilayer smart hydrogels, in step (4), the first microsphere loaded with anti-inflammatory drugs and the second microsphere loaded with vascular endothelial growth factor are mixed at a mass ratio of 1:3-3:1.

[0013] Based on the above-described method for preparing spatially partitioned bilayer intelligent hydrogels, in step (4), the total concentration of the first microsphere loaded with anti-inflammatory drugs and the second microsphere loaded with vascular endothelial growth factor in the gelatin-chitosan composite hydrogel precursor solution is 10-100 mg / mL.

[0014] The present invention also provides an application of the aforementioned spatially partitioned bilayer smart hydrogel in the preparation of chronic wound repair materials.

[0015] Furthermore, the chronic wound is a diabetic wound.

[0016] Specifically, in application, the functional therapeutic layer of the spatially partitioned double-layer smart hydrogel is attached to the wound surface, while the temperature sensing layer faces the air side.

[0017] Beneficial effects This invention constructs a smart hydrogel with a functionally partitioned structure, spatially separating infection monitoring and drug treatment functions. A temperature sensing layer monitors temperature changes during wound infection in real time, converting these changes into detectable electrical signals, thus enabling dynamic monitoring of the wound infection status. Then, engineered microspheres with different functions are introduced into the functional treatment layer, loading anti-inflammatory drugs and pro-angiogenic drugs onto separate microspheres. On one hand, the anti-inflammatory drugs loaded on the first microsphere in the functional treatment layer are released as needed based on temperature changes; on the other hand, a second microsphere loaded with vascular endothelial growth factor (VEGF) is used to continuously release VEGF, providing sustained support for wound repair. The spatial isolation of the different microspheres reduces interactions between drugs and between drugs and the hydrogel matrix, thereby achieving independent regulation of the release behavior of different therapeutic drugs. Attached Figure Description

[0018] Figure 1 In Figure a, it is a schematic diagram of the preparation process of the functional therapeutic layer; in Figure b, it is a schematic diagram of the preparation process of the temperature sensing layer; and in Figure c, it is a schematic diagram of the application mechanism of the spatially partitioned bilayer smart hydrogel.

[0019] Figure 2 This is a schematic diagram of the preparation process of the temperature sensing layer in Example 1: PPG-NaCl ion-conductive hydrogel.

[0020] Figure 3 In Example 2, a represents the TCR value of PPG-NaCl ion-conducting hydrogels with different NaCl contents; b represents the ΔR / R0 response curve of PPG-1.0NaCl hydrogels in the range of 35-42℃; and c represents the change in ionic conductivity of PPG-1.0NaCl hydrogels in the range of 35-42℃.

[0021] Figure 4 This is a schematic diagram of the preparation process of the first microsphere loaded with anti-inflammatory drugs in Example 3.

[0022] Figure 5In Figure 1, a represents the temperature-dependent rheological behavior of PNIPAM / GelMA hydrogels with different mass ratios; b represents the differential scanning calorimetry (DSC) analysis of PNIPAM / GelMA hydrogels with different GelMA contents; c represents the compressive stress-strain curve of PG / GH microgels; d represents the compressive modulus of PG / GH microgels; e represents the DS loading of PNIPAM / GelMA microspheres; and f represents the DS release curve of PG11MGs and its relationship with temperature.

[0023] Figure 6 This is a schematic diagram of the preparation process of the second microsphere loaded with vascular endothelial growth factor in Example 5.

[0024] Figure 7 In the figure, a represents the VEGF encapsulation efficiency in GMs and GHMs, b represents the VEGF release curves of GMGs and GHMGs, and c represents the cumulative VEGF release of GMGs and GHMGs.

[0025] Figure 8 In Figure a, DS release curve is shown in the spatially partitioned bilayer smart hydrogel; VEGF release curve is shown in the spatially partitioned bilayer smart hydrogel; and DS release curve and relative resistance change (ΔR / R0) are shown in Figure c, which are temperature-dependent cumulative DS release and relative resistance change (ΔR / R0) over 24 hours at 35, 37, 39 and 42 °C.

[0026] Figure 9 Photographs showing the application of spatially partitioned double-layer smart hydrogel in wound monitoring.

[0027] Figure 10 Typical wound images and wound trajectory simulations at 0, 3, 7, 10, and 12 days. Detailed Implementation

[0028] The following examples are intended to illustrate the present invention, and not to further limit the invention.

[0029] The present invention provides a spatially partitioned bilayer smart hydrogel, comprising a temperature sensing layer as the bottom layer and a functional therapeutic layer disposed on the upper surface of the temperature sensing layer. The functional therapeutic layer contains a first microsphere loaded with anti-inflammatory drugs and a second microsphere loaded with vascular endothelial growth factor.

[0030] To address the limitations of traditional wound dressings and existing hydrogel systems in real-time monitoring of wound conditions and proactive regulation based on changes in the wound microenvironment, this invention constructs a smart hydrogel with a functional partition structure. This structure spatially separates infection monitoring and drug therapy functions. A temperature sensing layer monitors temperature changes during wound infection in real time and converts these changes into detectable electrical signals, enabling dynamic monitoring of the wound infection status. Furthermore, engineered microspheres with different functions are introduced into the functional therapy layer. Anti-inflammatory and angiogenic drugs are loaded into different microspheres, and the spatial isolation between these microspheres reduces interactions between drugs and between drugs and the hydrogel matrix, thereby achieving independent regulation of the release behavior of different therapeutic drugs.

[0031] Through the above spatial partitioning design, the wound condition perception function and the treatment function are respectively undertaken by different functional layers, thus forming a double-layer intelligent hydrogel with real-time perception and treatment response capabilities.

[0032] Specifically, the temperature sensing layer is an ion-conducting hydrogel, and the functional therapeutic layer is a gelatin-chitosan oligosaccharide composite hydrogel.

[0033] Among them, the ion-conductive hydrogel is a dual-network structure composed of chemical cross-linking and physical cross-linking, which is used to sense local temperature changes in the wound and realize real-time monitoring of wound temperature through the resistance changes caused by temperature changes.

[0034] The methacrylic acid gelatin-chitosan oligosaccharide composite hydrogel is used to fix and support two types of functional microspheres.

[0035] Regarding the engineered microspheres with different functions introduced into the functional therapeutic layer, the first microsphere is obtained by crosslinking gelatin methacrylate with poly(N-isopropylacrylamide), and the minimum critical dissolution temperature of the first microsphere is 40-50℃. The first microsphere utilizes temperature-responsive characteristics to achieve temperature-responsive release of anti-inflammatory drugs.

[0036] The second microsphere was obtained by grafting heparin onto a gelatin methacrylate network via an EDC / HOBt-mediated coupling reaction; the loading efficiency of the second microsphere for vascular endothelial growth factor was higher than 83%.

[0037] Addressing the issues of fixed drug release mechanisms and difficulty in distributing drugs according to the inflammatory state of wounds using traditional wound hydrogels, this invention provides a spatially partitioned, dual-layer intelligent hydrogel. This hydrogel can monitor temperature changes in real time to perceive the wound state and release anti-inflammatory drugs loaded on the first microsphere in the functional therapeutic layer according to temperature changes. Furthermore, it can utilize the second microsphere loaded with vascular endothelial growth factor (VEGF) in the functional therapeutic layer to continuously release VEGF, providing continuous support for wound repair.

[0038] This invention also provides a method for preparing the aforementioned spatially partitioned bilayer smart hydrogel, comprising: Step (1): Acrylamide, photoinitiator, crosslinking agent, gelatin, polyvinyl alcohol, and NaCl are dissolved in deionized water to obtain a PPG-NaCl precursor solution. After uniform mixing, the solution is crosslinked under 405nm blue light to form a first chemical crosslinking network. Then, it undergoes borax treatment and freeze-thaw cycles to form a physical crosslinking network, resulting in a temperature sensing layer. (See...) Figure 1 b.

[0039] In the PPG-NaCl precursor solution, the mass concentrations of acrylamide, polyvinyl alcohol (PVA), gelatin, crosslinking agent MBA (N,N'-methylenebisacrylamide), photoinitiator LAP, and NaCl are 0.5-3 wt%, preferably 1 wt%.

[0040] 405nm blue light intensity 20-100mW / cm 2 50mW / cm is preferred 2 .

[0041] The hydrogel obtained from the first chemical cross-linking network is immersed in a 0.5-1.5 wt% borax solution. Reversible borate ester bonds are formed through the dynamic complexation between borate and PVA segments. Combined with 1-5 freeze-thaw cycles, stable physical cross-linking regions are formed in the PVA segments. Finally, a PPG-NaCl dual-network ion-conductive hydrogel composed of both chemical and physical cross-linking networks is obtained, which is the temperature sensing layer.

[0042] Step (2): Methacrylamide gelatin and poly(N-isopropylacrylamide) were mixed in a certain proportion to obtain PNIPAM-GelMA precursor solution. Microdroplets were then prepared using a droplet microfluidic method. The microdroplets were cross-linked using 405nm blue light to obtain the first microspheres. After freeze-drying, the first microspheres were incubated in an anti-inflammatory drug solution at 4℃ for 12-24 hours to allow the anti-inflammatory drug to diffuse into the interior of the microspheres, thus obtaining the first microspheres loaded with anti-inflammatory drugs. See [link to relevant documentation]. Figure 1 a.

[0043] The mass ratio of poly(N-isopropylacrylamide) to methacrylamide gelatin (GelMA) is 1:0-2:1, preferably 1:1.

[0044] Then, using the obtained PNIPAM-GelMA precursor liquid as the dispersed phase and mineral oil containing Span 80 as the continuous phase, uniformly sized composite microdroplets were prepared using a droplet microfluidic chip. During the microfluidic process, the microsphere size was controlled by adjusting the flow rates of the dispersed and continuous phases, with the dispersed phase flow rate being 10-100 μL / min and the continuous phase flow rate being 50-500 μL / min, preferably a flow rate ratio of 200 / 35 (continuous phase / dispersed phase) μL / min.

[0045] Subsequently, the formed droplets were subjected to photocrosslinking under 405 nm blue light to form a crosslinked network structure between GelMA and NIPAM, yielding the first PNIPAM-GelMA microspheres. After crosslinking, the microspheres were repeatedly washed to remove mineral oil, Span 80, and unreacted substances. The microspheres were then mixed with ethyl acetate at a volume ratio of 1:1, and subjected to low-temperature pre-freezing and lyophilization to obtain the first PNIPAM-GelMA microspheres in a dry state.

[0046] Step (3): Heparin was grafted onto a gelatin methacrylate network using an EDC / HOBt-mediated coupling reaction to obtain a second microsphere. The second microsphere was then freeze-dried and incubated in a vascular endothelial growth factor (VEGF) solution to load VEGF into the microspheres, resulting in a second microsphere loaded with VEGF. (See attached image.) Figure 1 a.

[0047] In a preferred embodiment, 0.001-0.05 mmol EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 0.001-0.05 mmol HOBt (1-hydroxybenzotriazole) are used to activate the carboxyl group of heparin, and 1-50 mg of heparin sodium is added to carry out the coupling reaction for 12-48 h.

[0048] In a preferred embodiment, 100 mg of the second microspheres are added to 3 mL of a vascular endothelial growth factor solution with a concentration of 50-100 ng / mL, and incubated at 4°C for 12-24 h. After incubation, the microspheres are collected by centrifugation to obtain the second microspheres loaded with vascular endothelial growth factor.

[0049] Step (4): The first microsphere loaded with anti-inflammatory drugs and the second microsphere loaded with vascular endothelial growth factor were mixed and dispersed in a gelatin-chitosan composite hydrogel precursor solution to obtain the functional therapeutic layer precursor solution. Figure 1 a.

[0050] Preferably, the first microsphere loaded with anti-inflammatory drugs and the second microsphere loaded with vascular endothelial growth factor are mixed in a mass ratio of 1:3 to 3:1, preferably 1:1.

[0051] The total concentration of the first microsphere loaded with anti-inflammatory drugs and the second microsphere loaded with vascular endothelial growth factor in the gelatin-chitosan composite hydrogel precursor solution is 10-100 mg / mL, preferably 60 mg / mL.

[0052] The precursor solution for the gelatin-chitosan oligosaccharide composite hydrogel is composed of methacrylated gelatin (GelMA), chitosan oligosaccharide (COS), and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).

[0053] The concentration of GelMA is 5-20% (w / v), preferably 10% (w / v); the concentration of COS is 1-20 mg / mL, preferably 10 mg / mL; and the concentration of LAP is 0.05-1%, preferably 0.3%.

[0054] Step (5): The functional therapeutic layer precursor liquid is deposited on the upper surface of the temperature sensing layer by 3D printing and photocrosslinked by 405nm blue light.

[0055] The 3D printing parameters can be adjusted according to actual needs. The preferred printing speed is 0.1-5mm / s, the printing line spacing is 200-1000μm, and the single-layer printing thickness is 50-500μm.

[0056] This invention utilizes a spatial partitioning design to integrate temperature sensing and drug delivery functions into different hydrogel layers, ensuring the PPG-NaCl ion-conducting network and functional drug microspheres are independent and avoiding interference between conductive components, drug carriers, and bioactive factors. Specifically, the upper functional therapeutic layer is 3D printed to form a porous network with a regular pore structure, improving the uniformity of the functional microsphere distribution and increasing drug diffusion channels, which is beneficial for promoting the release of anti-inflammatory drugs and vascular endothelial growth factor.

[0057] The present invention also provides an application of the aforementioned spatially partitioned bilayer smart hydrogel in the preparation of chronic wound repair materials.

[0058] Preferably, the chronic wound is a diabetic wound.

[0059] Unlike ordinary chronic wounds, diabetic chronic wounds are characterized by persistent inflammation and difficulty in timely monitoring of elevated local temperatures. Utilizing the spatially partitioned bilayer intelligent hydrogel of this invention, the synergistic effect of real-time infection monitoring via a temperature-sensing layer, temperature-responsive anti-inflammatory drug release, and continuous release of vascular endothelial growth factor allows the intelligent hydrogel to be dynamically adjusted according to the needs of different healing stages of diabetic wounds. This enables anti-inflammatory intervention in the early inflammatory stage of the wound and continuous promotion of angiogenesis in the subsequent tissue repair stage, thereby achieving a synergistic treatment of "infection monitoring - on-demand anti-inflammatory - continuous angiogenesis promotion." Figure 1 c.

[0060] When applying, the functional therapeutic layer of the spatially partitioned double-layer smart hydrogel is attached to the wound surface, with the temperature sensing layer facing the air side.

[0061] The temperature sensing layer converts changes in wound temperature into a resistance signal, which is recorded in real time via external electrodes, enabling non-invasive monitoring of wound temperature. Furthermore, utilizing the physical properties of the first microsphere within the functional treatment layer, anti-inflammatory drugs are released on demand based on temperature changes, while continuously released microspheres within the functional treatment layer continuously release loaded vascular endothelial growth factor.

[0062] Although existing technologies include smart dressings that utilize temperature, electrochemistry, or other sensing elements to monitor wound conditions, and wound repair materials that utilize temperature-sensitive materials or microsphere carriers to achieve drug response release, the monitoring and treatment functions in existing technologies are usually integrated into the same hydrogel. The monitoring results are mainly used for wound condition assessment, while the treatment process still relies on manual intervention, making it difficult to achieve coordinated regulation between wound microenvironment perception and treatment response.

[0063] In this invention, both the temperature sensing layer and the functional treatment layer utilize local temperature changes at the wound site as external information sources related to the wound's condition. The former converts temperature changes into recordable resistance signals to perceive the wound's condition, while the latter uses temperature changes to induce a response in the first microsphere, thereby regulating the release of anti-inflammatory drugs. This allows the wound condition monitoring function and the temperature-responsive treatment function to form a functional association based on shared microenvironmental stimulation within the same system. Furthermore, the temperature-sensitive anti-inflammatory drug microspheres and vascular endothelial growth factor-loaded microspheres in the functional treatment layer work together to address the treatment needs at different stages of diabetic wound repair. The former primarily regulates the release of anti-inflammatory drugs through temperature response, while the latter continuously releases the drugs to meet subsequent angiogenesis requirements. The two types of microspheres are independent in function and release mechanism but complementary in treatment timing, thus achieving a synergistic effect of different therapeutic factors.

[0064] This invention integrates real-time wound monitoring, drug response release, and independent multi-drug delivery into a single intelligent hydrogel system. It addresses the problems in existing technologies, such as the incompatibility between wound monitoring and treatment functions, the potential for mutual interference and release coupling when multiple drugs are co-loaded, and the difficulty in providing precise treatment based on different healing stages of diabetic wounds. This improves the controllability and adaptability of drug delivery during chronic wound treatment, providing an intelligent method for real-time monitoring, inflammation regulation, angiogenesis, and tissue regeneration of diabetic chronic wounds.

[0065] Example 1 This embodiment provides the preparation process of the temperature sensing layer: a polypropylene glycol-sodium chloride (PPG-NaCl) ion-conducting hydrogel, see [link to documentation]. Figure 2 .

[0066] Acrylamide (AM), photoinitiator LAP, crosslinking agent MBA, gelatin, and polyvinyl alcohol (PVA) were dissolved in deionized water, and NaCl was added to prepare a PPG-NaCl precursor solution. The mass concentrations of acrylamide (5-15 wt%), PVA (5-15 wt%), gelatin (1-10 wt%), MBA (0.05-1 wt%), LAP (0.05-1 wt%), and NaCl (0.5-3 wt%) were all specified.

[0067] Subsequently, at 405nm blue light (light intensity 50mW / cm²) 2 Photocrosslinking is performed under certain conditions to form the first chemical crosslinking network of acrylamide.

[0068] The obtained hydrogel was further immersed in a 0.5-1.5 wt% borax solution to form reversible borate ester bonds through dynamic complexation between borate and PVA segments. Combined with three freeze-thaw cycles, the PVA segments were promoted to form stable physical cross-linked regions, and finally a PPG-NaCl dual-network ion-conductive hydrogel composed of chemical cross-linked network and physical cross-linked network was obtained.

[0069] Example 2 To optimize the temperature sensing performance of the PPG-NaCl ion-conductive hydrogel, based on Example 1, this example further adjusted the NaCl addition ratio, using acrylamide (8%), PVA (10%), gelatin (1.25%), MBA (0.3%), and LAP (0.35%) as the base concentrations.

[0070] NaCl, as an ion-conducting medium, can generate migratable Na+ in hydrogel networks. + and Cl - Ions exhibit altered migration behavior with temperature, thus converting temperature signals into resistance signals. Therefore, by optimizing the NaCl content, a balance can be achieved between ion migration ability and network stability.

[0071] When the NaCl concentration was 1 wt%, the resulting PPG-NaCl hydrogel exhibited the best overall sensing performance. Figure 3 (a) has a physiological temperature range of -2.88% within the range of 35-42℃. -1 Temperature coefficient (TCR), linear correlation coefficient R 2 Reaching 0.998 ( Figure 3(b) Further testing showed that the conductivity of this hydrogel increased with increasing temperature from 0.486 mS / cm at 35℃ to 0.607 mS / cm at 42℃. Figure 3 (c)

[0072] Finally, the PPG-NaCl hydrogel with the best overall sensing performance, i.e. the hydrogel obtained when the NaCl mass concentration is 1wt%, is designated as PPG-1.0NaCl hydrogel and used as the temperature sensing layer.

[0073] Example 3 This embodiment provides the preparation process of the first microsphere loaded with anti-inflammatory drugs, see [link to documentation]. Figure 4 .

[0074] A precursor solution was prepared by mixing poly(N-isopropylacrylamide) and methacrylamide gelatin (GelMA) at a mass ratio of 1:0 to 2:1. The resulting PNIPAM-GelMA precursor solution was used as the dispersed phase, and mineral oil containing Span 80 was used as the continuous phase. Uniform composite microdroplets were then fabricated using a droplet microfluidic chip. During the microfluidic process, the microsphere size was controlled by adjusting the flow rates of the dispersed and continuous phases, with the dispersed phase flow rate at 10-100 μL / min and the continuous phase flow rate at 50-500 μL / min.

[0075] Subsequently, the formed droplets were subjected to photocrosslinking under 405 nm blue light to form a crosslinked network structure between GelMA and NIPAM, yielding the first PNIPAM-GelMA microspheres. After crosslinking, the microspheres were repeatedly washed to remove mineral oil, Span 80, and unreacted substances. The microspheres were then mixed with ethyl acetate at a volume ratio of 1:1, and subjected to low-temperature pre-freezing and lyophilization to obtain the first PNIPAM-GelMA microspheres in a dry state.

[0076] Further, 100 mg of PNIPAM-GelMA first microspheres were added to 3 mL of diclofenac sodium (DS) solution and incubated at 4 °C for 12-24 h to allow DS molecules to diffuse into the microspheres through the concentration gradient, thus achieving drug loading. After incubation, the microspheres were collected by centrifugation to obtain PNIPAM-GelMA first microspheres loaded with the anti-inflammatory drug DS.

[0077] Example 4 To regulate the phase transition temperature and drug release behavior of the first microsphere, based on Example 3, this example uses a solution with GelMA concentration of 10% and LAP concentration of 0.3% in solution A, and NIPAM concentration of 10%, LAP concentration of 0.3%, and methylene acrylamide concentration of 0.5% in solution B. With solutions A and B mixed in a 1:1 volume ratio, the mass ratio of poly(N-isopropylacrylamide) to methacrylamide gelatin was optimized to prepare composite microspheres with mass ratios of 1:0, 2:1, 1:1, and 1:2 for poly(N-isopropylacrylamide) to methacrylamide gelatin.

[0078] Figure 4 The results showed that the introduction of GelMA could regulate the density of the poly(N-isopropylacrylamide) network structure and increase the phase transition temperature of the composite system, making it more suitable for drug-responsive release within the wound temperature range. With increasing GelMA ratio, the mechanical stability of the microspheres improved, while drug diffusion was somewhat restricted, thus achieving release rate regulation. Specifically, at a mass ratio of 1:1, the composite microspheres exhibited superior temperature response characteristics and drug release performance, with a minimum critical solution temperature (LCST) adjusted to approximately 40-50℃, a drug dispersive index (DS) loading of approximately 65.82 μg / mg, and a significant temperature-responsive release behavior within the wound-related temperature range of 37-42℃.

[0079] Specifically, such as Figure 5 As shown in Figures a and b, the thermosensitive phase transition behavior of microspheres with different components was analyzed by rheological temperature scanning and differential scanning calorimetry (DSC). The results showed that the phase transition temperature of pure poly(N-isopropylacrylamide) microspheres was approximately 30 °C. However, with the introduction of methacrylamide gelatin, the phase transition temperature of PNIPAM / GelMA composite microspheres gradually increased. The lowest critical dissolution temperatures (LCST) of PG10 (poly(N-isopropylacrylamide) to methacrylamide gelatin mass ratio of 1:0), PG21 (poly(N-isopropylacrylamide) to methacrylamide gelatin mass ratio of 2:1), and PG11 (poly(N-isopropylacrylamide) to methacrylamide gelatin mass ratio of 1:1) microspheres were 30.4 °C, 41.8 °C, and 47.0 °C, respectively. This indicates that the temperature response range of the microspheres can be controlled by adjusting the ratio of poly(N-isopropylacrylamide) to methacrylamide gelatin.

[0080] Furthermore, the compression performance test results showed that as the proportion of methacrylamide gelatin increased, the compression stress-strain curve of the microspheres gradually became steeper, and the compression modulus increased from 10.50 kPa (PG10 / GH) to 21.9 kPa (PG12 / GH), indicating that the introduction of methacrylamide gelatin enhanced the compressive strength and structural stability of the microspheres. Figure 5(c, d). PG10 / GH, PG21 / GH, PG11 / GH, and PG12 / GH represent hydrogels prepared by mixing microspheres obtained by poly(N-isopropylacrylamide) and methacrylamide gelatin at mass ratios of 1:0, 2:1, 1:1, and 1:2, respectively, with GelMA-Hep microspheres in a GelMA / COS composite hydrogel precursor solution.

[0081] like Figure 5 As shown in Figure e, the loading of diclofenac sodium (DS) on PNIPAM / GelMA microspheres with different ratios were 53.43, 62.41, 65.82 and 63.84 μg / mg (corresponding to PG10Ms, PG21Ms, PG11Ms and PG12Ms).

[0082] Further analysis was conducted on the DS release behavior of PG11MGs. Figure 5 The results showed that PG11MGs had suitable temperature-responsive release characteristics in the wound inflammation-related temperature range of 37-42 ℃. PG11MGs represents a hydrogel prepared by mixing microspheres of poly(N-isopropylacrylamide) and methacrylamide gelatin in a 1:1 mass ratio in a GelMA / COS composite hydrogel precursor solution.

[0083] Therefore, the first microsphere with a mass ratio of poly(N-isopropylacrylamide) to methacrylamide gelatin of 1:1 was selected as the temperature-responsive anti-inflammatory drug delivery unit.

[0084] Example 5 This embodiment provides the preparation process of the second microsphere loaded with vascular endothelial growth factor, see [link to documentation]. Figure 6 .

[0085] GelMA microspheres were prepared using droplet microfluidic technology, and GelMA-Hep microspheres were obtained by heparin functionalization modification. The microspheres were then further loaded with vascular endothelial growth factor (VEGF) to prepare a second microsphere.

[0086] Specifically, GelMA precursor liquid is used as the dispersed phase, and mineral oil containing Span80 is used as the continuous phase. Uniform GelMA droplets are formed through a droplet microfluidic chip, and then photocrosslinking is performed under 405nm blue light to form a stable three-dimensional network structure of GelMA, thus obtaining GelMA microspheres.

[0087] After washing the obtained microspheres, they were added to the heparin coupling reaction system. Through the EDC / HOBt-mediated amide coupling reaction, the carboxyl groups in the heparin molecules were covalently bonded to the active amino groups in the GelMA network, thereby obtaining GelMA-Hep microspheres.

[0088] The heparin carboxyl group was activated using 0.02 mmol of EDC and 0.02 mmol of HOBt, and then 5 mg of heparin sodium was added for coupling reaction.

[0089] Subsequently, 100 mg of GelMA-Hep microspheres were added to 3 mL of VEGF solution with a concentration of 250 ng / mL and incubated at 4 °C for 12 h. After incubation, the microspheres were collected by centrifugation to obtain the second microspheres loaded with vascular endothelial growth factor, which were denoted as VEGF@GelMA-Hep microspheres.

[0090] To enhance VEGF loading capacity and achieve long-term stable release, this embodiment modulates the interaction between GelMA microspheres and VEGF through heparin functionalization. Heparin molecules contain abundant sulfate groups, which can enhance the binding capacity of VEGF in the microsphere network through electrostatic interactions and specific affinity for VEGF, thereby reducing its rapid diffusion and release.

[0091] Experimental results show that... Figure 7 In the middle a, compared with unmodified heparin GelMA microspheres (denoted as GMs), GelMA-Hep microspheres (denoted as GHMs) can improve VEGF loading efficiency from 83.0% to 93.4%.

[0092] Next, hydrogels prepared by mixing unmodified heparinized GelMA microspheres in a gelatin-chitosan oligosaccharide composite hydrogel precursor solution (denoted as GMGs) were compared with hydrogels prepared by mixing GelMA-Hep microspheres in a gelatin-chitosan oligosaccharide composite hydrogel precursor solution (denoted as GHMGs). Figure 7 As can be seen from b, heparin modification enhances the binding between VEGF and the microsphere network, making its release process slower and more stable.

[0093] Figure 7 As can be seen from Figure c, in the 21-day in vitro release experiment, the cumulative release of VEGF@GelMA-Hep microspheres reached 16.43 ng / mL, with a cumulative release rate of 44.4%, which was lower than that of unmodified GelMA microspheres (17.20 ng / mL and 52.1%). This indicates that heparin functionalization can effectively reduce the VEGF diffusion rate, achieve continuous VEGF delivery, and help maintain long-term angiogenesis during wound repair.

[0094] Example 6 This embodiment provides the construction process of a spatially partitioned bilayer smart hydrogel.

[0095] The first microspheres of poly(N-isopropylacrylamide) and methacrylamide gelatin in Example 4 were mixed at a mass ratio of 1:1, and the VEGF@GelMA-Hep microspheres in Example 5 were mixed at a mass ratio of 1:1 and uniformly dispersed in the GelMA / COS composite hydrogel precursor solution. The total concentration of the two types of functional microspheres in the GelMA / COS composite hydrogel precursor solution was 60 mg / mL, thus obtaining the functional therapeutic layer precursor solution.

[0096] The GelMA / COS composite hydrogel precursor solution contained a GelMA concentration of 10% (w / v), a COS concentration of 10 mg / mL, and a LAP concentration of 0.3%.

[0097] Subsequently, using the PPG-NaCl ion-conducting hydrogel from Example 2 as the bottom temperature sensing layer, the precursor solution for the functional therapeutic layer was deposited on the surface of the PPG-NaCl ion-conducting hydrogel via 3D printing. Photocrosslinking was then performed using 405nm blue light, ultimately yielding a spatially partitioned bilayer smart hydrogel consisting of a lower temperature sensing layer and an upper drug delivery functional therapeutic layer. The 3D printing speed was 5 mm / s, the line spacing was 800 μm, and the single-layer printing thickness was 300 μm.

[0098] This embodiment integrates temperature sensing and drug delivery functions into different hydrogel layers through spatial partitioning design. This ensures that the PPG-NaCl ion-conducting network and the functional drug microspheres are independent, avoiding mutual interference between conductive components, drug carriers, and bioactive factors. The upper functional therapeutic layer is formed into a porous network with a regular pore structure through 3D printing, improving the uniformity of the functional microsphere distribution and increasing drug diffusion channels, which is beneficial for promoting the release of DS and VEGF.

[0099] The DS / VEGF@MGs / PPG-NaCl and GHMGs were compared. DS / VEGF@MGs / PPG-NaCl is the bilayer hydrogel prepared in Example 6, in which the functional therapeutic layer is printed on the temperature sensing layer by 3D printing. GHMGs refers to the hydrogel prepared by mixing GelMA-Hep microspheres in a gelatin-chitosan composite hydrogel precursor solution.

[0100] Figure 8Experimental results show that the printed bilayer hydrogel exhibits superior drug delivery performance, with a cumulative VEGF release of 17.51 ​​ng / mL, higher than the unprinted system (16.43 ng / mL). Furthermore, within the temperature range of 35-42℃, the DS release increases with increasing temperature, maintaining a trend consistent with the resistance response of the PPG-NaCl sensing layer. This indicates that the bilayer system can achieve a synergistic response between temperature sensing signals and drug release behavior, providing a technological basis for dynamic wound monitoring and intelligent treatment.

[0101] Application Example 1 This application example utilizes the spatially partitioned bilayer smart hydrogel constructed in Example 6 to establish a temperature detection circuit.

[0102] A first electrode and a second electrode are placed at both ends of a PPG-NaCl ion-conductive hydrogel, and the first and second electrodes are connected to a resistance detection module to form a temperature detection circuit. By detecting the resistance change of the hydrogel under different temperature conditions, the temperature signal is converted into an electrical signal.

[0103] Using the initial resistance value R0 of the hydrogel at 35℃ as a reference value, the resistance value measured at the current temperature is denoted as R. The relative resistance change is calculated as follows: By establishing a correlation between temperature and relative resistance changes, quantitative monitoring of wound temperature changes is achieved. Real-time acquisition and transmission of hydrogel resistance signals are realized through connection to an external signal acquisition device using flexible conductive electrodes. To reduce the impact of moisture changes in the external environment on the resistance detection results, the hydrogel surface is encapsulated.

[0104] See Figure 9 Specifically, after applying a spatially partitioned, dual-layer smart hydrogel to a wound, changes in local wound temperature were characterized by real-time monitoring of resistance values. The hydrogel was applied to the wound on the back of diabetic mice, and conductive wires were led out from both sides of the hydrogel and connected to a Truebox 01RC device. Resistance values ​​were monitored and recorded in real-time via a mobile phone. To simulate a localized increase in wound temperature, an electric heater was placed above the wound, and changes in resistance within the hydrogel were continuously tracked.

[0105] Application Example 2 This application example utilizes the spatially partitioned bilayer smart hydrogel constructed in Example 6 for diabetic wounds.

[0106] A full-thickness skin defect with a diameter of 10 mm was created on the back of diabetic mice. The wound was constructed under anesthesia, and the prepared spatially partitioned double-layer smart hydrogel was applied to the wound area for treatment.

[0107] The experimental design included a control group, a PPG-NaCl group, a DS / VEGF@MGs group, and a DS / VEGF@MGs / PPG-NaCl group. The control group received no treatment. The PPG-NaCl group consisted of a hydrogel obtained in Example 2 with a NaCl concentration of 1 wt%, i.e., only a temperature-sensing layer. The DS / VEGF@MGs group consisted of a monolayer gel loaded with DS and VEGF, i.e., only a functional therapeutic layer. The DS / VEGF@MGs / PPG-NaCl group was the spatially partitioned bilayer smart hydrogel of Example 6, a complete bilayer hydrogel.

[0108] During treatment, the PPG-NaCl temperature sensing layer within the double-layer smart hydrogel was used to collect real-time information on local temperature changes in the wound, and temperature response signals were recorded at different time points. Images of the wound were acquired on days 0, 3, 7, 10, and 14 of treatment. Image analysis was used to calculate changes in wound area and closure rate to evaluate the promoting effect of different treatment systems on the healing process of diabetic wounds. (See below) Figure 10 .

[0109] Compared with the control group, all hydrogel treatment groups showed a faster wound contraction trend. On day 12 of treatment, the wound closure rate of all hydrogel treatment groups reached over 90%, with the DS / VEGF@MGs / PPG-NaCl group showing near-complete wound closure and the best wound repair effect; the wound closure rate of the DS / VEGF@MGs group reached 95.6%, which was higher than that of the PPG-NaCl group (92.4%) and the control group (81.5%).

[0110] Wound tissue samples were collected at predetermined treatment time points for histological and immunohistochemical analysis. H&E staining was used to observe wound tissue structure recovery and re-epithelialization; Masson staining was used to analyze collagen deposition and tissue remodeling; CD31 and α-SMA immunostaining were used to detect angiogenesis and vascular maturation; and changes in wound inflammation levels were analyzed using TNF-α and IL-10-related indicators to evaluate the anti-inflammatory regulatory capacity and tissue repair effect of the hydrogel system. By combining dynamic wound temperature monitoring signals with tissue repair results, the application effect of the constructed bilayer intelligent hydrogel in real-time wound monitoring and synergistic treatment regulation was verified.

[0111] 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 changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A spatially partitioned bilayer intelligent hydrogel, characterized in that, It includes a temperature sensing layer as the bottom layer, and a functional therapeutic layer disposed on the upper surface of the temperature sensing layer; The functional therapeutic layer contains a first microsphere loaded with anti-inflammatory drugs and a second microsphere loaded with vascular endothelial growth factor.

2. The spatially partitioned bilayer smart hydrogel according to claim 1, characterized in that, The temperature sensing layer is an ion-conductive hydrogel, and the functional therapeutic layer is a gelatin-chitosan oligosaccharide composite hydrogel.

3. The spatially partitioned bilayer intelligent hydrogel according to claim 1, characterized in that, The first microsphere is obtained by crosslinking gelatin methacrylate with poly(N-isopropylacrylamide), and the minimum critical dissolution temperature of the first microsphere is 40-50℃.

4. The spatially partitioned bilayer smart hydrogel according to claim 1, characterized in that, The second microsphere was obtained by grafting heparin onto a gelatin methacrylate network via an EDC / HOBt-mediated coupling reaction; the loading efficiency of the second microsphere for vascular endothelial growth factor was higher than 83%.

5. A method for preparing the spatially partitioned bilayer smart hydrogel as described in claim 1, characterized in that, include: Step (1): Dissolve acrylamide, photoinitiator, crosslinking agent, gelatin, polyvinyl alcohol and NaCl in deionized water, mix evenly and then photocrosslink to form the first chemical crosslinking network, and then form the physical crosslinking network through borax treatment and freeze-thaw cycle to obtain the temperature sensing layer. Step (2): Methacrylamide gelatin, N-isopropylacrylamide, crosslinking agent and photoinitiator are mixed in proportion and microdroplets are prepared by droplet microfluidics. The first microspheres are obtained by photocrosslinking. The first microspheres are freeze-dried and incubated in an anti-inflammatory drug solution to obtain the first microsphere loaded with anti-inflammatory drugs. Step (3): Heparin was grafted onto the gelatin methacrylic acid network using an EDC / HOBt-mediated coupling reaction to obtain the second microspheres. The second microspheres were then freeze-dried and incubated in a vascular endothelial growth factor solution to obtain the second microspheres loaded with vascular endothelial growth factor. Step (4): The first microsphere loaded with anti-inflammatory drugs and the second microsphere loaded with vascular endothelial growth factor are mixed and dispersed in the gelatin-chitosan composite hydrogel precursor solution to obtain the functional therapeutic layer precursor solution. Step (5): The functional therapeutic layer precursor liquid is deposited on the upper surface of the temperature sensing layer by 3D printing and then photocrosslinked.

6. The method for preparing the spatially partitioned bilayer smart hydrogel according to claim 5, characterized in that, In step (4), the first microsphere loaded with anti-inflammatory drugs and the second microsphere loaded with vascular endothelial growth factor are mixed at a mass ratio of 1:3 to 3:

1.

7. The method for preparing the spatially partitioned bilayer smart hydrogel according to claim 5, characterized in that, In step (4), the total concentration of the first microsphere loaded with anti-inflammatory drugs and the second microsphere loaded with vascular endothelial growth factor in the gelatin-chitosan composite hydrogel precursor solution is 10-100 mg / mL.

8. The application of a spatially partitioned bilayer intelligent hydrogel as described in claim 1 or a spatially partitioned bilayer intelligent hydrogel prepared by the preparation method described in claim 5 in the preparation of chronic wound repair materials.

9. The application according to claim 8, characterized in that, The chronic wound is a diabetic wound.

10. The application according to claim 8, characterized in that, When applying, the functional therapeutic layer of the spatially partitioned double-layer smart hydrogel is attached to the wound surface, with the temperature sensing layer facing the air side.

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