Photocrosslinked microneedle containing Cu@ZIF-8 particles and preparation method and application thereof

Abstract

The application discloses a photocrosslinking microneedle containing Cu@ZIF-8 particles and a preparation method and application thereof, wherein the microneedle is prepared from PEGDA / CMCS as a base material, the Cu@ZIF-8 particles are loaded in the base material, and a plurality of conical needle arrays are prepared through a mold forming and ultraviolet photocrosslinking process. The application combines the clinical requirements of a dressing to prepare the photocrosslinking microneedle loaded with the Cu@ZIF-8 particles; a series of microneedles with different proportions are prepared, and the microneedle product with good mechanical properties, broad-spectrum antibacterial properties, good blood vessel promoting effect and anti-inflammatory effect is preferred; and the obtained microneedle product is particularly suitable for the treatment of skin wounds and can also be used for bone repair and drug delivery of other organs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a light cross-linked microneedle containing Cu@ZIF-8 particles and a preparation method and application thereof. BACKGROUND

[0002] In recent years, wound healing has attracted widespread attention due to its severe challenges and serious economic burden. However, at present, we have very limited treatment measures for difficult-to-heal wounds, and need to continue to explore effective treatment methods. The wound healing process is usually divided into three stages: inflammation, proliferation and tissue remodeling. An excellent treatment method can not only improve the inflammation of the wound, but also accelerate the vascular regeneration, cell proliferation and tissue remodeling.

[0003] In order to solve the problem of difficult-to-heal wounds, people have developed various treatment strategies, such as hydrogel films, electrospun dressings and other various wound patches. Among them, microneedles (MN) are a very promising medical technology, which can be used for painless transdermal drug delivery through minimally invasive methods, and are widely used in the fields of vaccination, cancer treatment, skin treatment, cosmetics, etc. In particular, MNs containing active ingredients have been used in wound healing research. Compared with traditional dressings, MN patches can significantly increase the effect of promoting wound healing by increasing the contact area between them and the wound. However, for some MNs made of non-biodegradable materials such as stainless steel, the needle body of the MNs is very easy to form a small needle hole at the wound site. These holes are likely to cause secondary physical damage, and even cause serious microbial infection on the wound. These limitations limit the development of MNs in the field of promoting wound healing. Therefore, there is a great need for hydrogel MNs with improved minimally invasive therapeutic effect of drug delivery and excellent biocompatibility. At present, the application potential of microneedles needs to be further explored.

[0004] Polyethylene glycol diacrylate (PEGDA) hydrogel is a hydrophilic polymer network with good biocompatibility, which is widely used in the field of tissue engineering. In addition, carboxymethyl chitosan (CMCS) is a modified derivative of chitosan, which has good water solubility, stability and strong antibacterial performance, and is widely used in the field of biomedicine, and has broad application prospects. The combination of PEGDA / CMCS makes the MN have significant biocompatibility, and can make the loaded active ingredients be released to the wound stably.

[0005] Previous studies have shown that copper plays an effective role in antibacterial performance and proangiogenic effect in the wound healing process, and zinc ions are also believed to contribute to the antibacterial and wound healing effects. In recent years, copper and zinc-based zeolite-imidazolate frameworks (Cu@ZIF-8) have shown good antibacterial effects on wound models and bone repair models. These nanoparticles produce more reactive oxygen than ZIF-8, which contributes to their stronger antibacterial performance. The rough surface of Cu@ZIF-8 nanoparticles can increase the contact area between MOFs and bacteria, thereby achieving better antibacterial activity. In addition, zinc ions and copper ions play an important role in the vascularization process, which can stimulate vascular endothelial cells to form blood vessels and inhibit inflammatory reactions. However, as of now, the research on loading Cu@ZIF-8 particles into PEGDA / CMCS microneedles to endow the microneedles with strong performance is still in its infancy, and no research and development products have been marketed and applied. SUMMARY

[0006] To solve the above problems, the present application provides a light-crosslinked microneedle containing Cu@ZIF-8 particles and a preparation method and application thereof, and the specific technical scheme is as follows:

[0007] A light-crosslinked microneedle containing Cu@ZIF-8 particles, the microneedle is prepared by loading Cu@ZIF-8 particles in PEGDA / CMCS as a substrate, and then forming a plurality of conical needle arrays through a mold forming and ultraviolet light cross-linking process.

[0008] The present application further provides a preparation method of a light-crosslinked microneedle containing Cu@ZIF-8 particles, comprising the following steps:

[0009] (1) synthesizing Cu@ZIF-8 particles by hydrothermal method;

[0010] (2) preparing a PDMS microneedle mold by using photoresist technology;

[0011] (3) preparing a PEGDA / CMCS mixed solution according to a certain proportion, adding Cu@ZIF-8 particles in different proportions to the mixed solution and uniformly mixing, and finally adding a photoinitiator 1173;

[0012] (4) pouring the mixed solution into the microneedle mold and centrifuging to remove bubbles;

[0013] (5) placing the mold under an ultraviolet light source to cross-link to obtain the microneedle.

[0014] Further, the morphological parameters of the PDMS microneedle mold are as follows: needle height 400-1500 μm, base diameter 150-500 μm, needle tip diameter 5-50 μm, center distance 500-2000 μm, and array number greater than 5x5.

[0015] Furthermore, the molecular weight of PEGDA is 600; the mass fraction of the CMCS solution is 2%;

[0016] The mixing ratio of PEGDA / CMCS, expressed as a volume percentage of the CMCS solution, gradually increases from 0% to 60%.

[0017] Different proportions of Cu@ZIF-8 particles were added to the above solution and mixed well, with the concentration gradually increased from 0 mg / mL to 2.5 mg / mL;

[0018] The amount of photoinitiator 1173 added accounts for 0.02-0.1% of the total mass of the solution.

[0019] Preferably, the volume ratio of PEGDA / CMCS is 8:2.

[0020] Preferably, the concentration of Cu@ZIF-8 particles added to the solution is 1.5 mg / mL.

[0021] Furthermore, the parameters for centrifugation and degassing are: rotation speed 3000-10000 rpm, time 5-15 min, repeated three times.

[0022] Furthermore, the ultraviolet light source has a wavelength of 200-400nm, a power of 50-500W, and a crosslinking time of 3-10min.

[0023] The present invention further provides the application of photocrosslinked microneedles containing Cu@ZIF-8 particles in skin wound healing treatment products or other organ delivery medical devices.

[0024] Beneficial effects of this invention:

[0025] (1) Based on the clinical needs of dressings, photocrosslinked microneedles loaded with Cu@ZIF-8 particles were prepared;

[0026] (2) Prepare a series of microneedles with different ratios, and select the microneedle products with good mechanical properties and excellent broad-spectrum antibacterial, angiotensin-promoting and anti-inflammatory effects;

[0027] (3) The obtained microneedle products are particularly suitable for the treatment of skin wounds and can also be used for bone repair and drug delivery to other organs. Attached Figure Description

[0028] Figure 1 Electron micrographs of Cu@ZIF-8 particles at different magnifications.

[0029] Figure 2 Microneedle morphology images prepared for different mixing volume ratios of PEGDA / CMCS.

[0030] Figure 3The images show the morphology of the microneedles (PEGDA / CMCS mixing ratio = 8:2) at different magnifications.

[0031] Figure 4 The figure shows the results of the in vitro biocompatibility evaluation of microneedles MN loaded with different concentrations of Cu@ZIF-8 particles.

[0032] Figure 5 The figure shows the results of evaluating the broad-spectrum antibacterial activity of microneedles MN loaded with different concentrations of Cu@ZIF-8 particles.

[0033] Figure 6 The figure shows the results of the in vitro evaluation of the angiogenesis-promoting effect of microneedles (PCCZ-MN = 1.5 mg / mL).

[0034] Figure 7 This is a graph showing the evaluation results of in vivo treatment of skin wounds using microneedles (PCCZ-MN = 1.5 mg / mL).

[0035] Figure 8 The graph shows the evaluation results of inflammatory markers related to in vivo treatment of skin wounds with microneedles (PCCZ-MN = 1.5 mg / mL). Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0037] This invention provides a photocrosslinked microneedle containing Cu@ZIF-8 particles. Using PEGDA / CMCS as the substrate, Cu@ZIF-8 particles are loaded into the microneedles, and a conical microneedle array is fabricated through molding, ultraviolet light crosslinking, and other processes. The microneedles can be inserted into tissues, and the released Cu@ZIF-8 particles exert antibacterial, angiogenesis-promoting, and anti-inflammatory effects. The microneedles also exhibit good biocompatibility and biodegradability, and promote collagen deposition and wound healing in wound environments.

[0038] Using PEGDA / CMCS as the substrate for microneedles and loading Cu@ZIF-8 particles into them effectively overcomes the performance defects of dressings, such as limited function and low drug delivery efficiency, thus better meeting the clinical needs of wound dressings. Both PEGDA and CMCS are biocompatible and biodegradable polymers, giving the microneedles good mechanical properties. Furthermore, due to their minimally invasive nature, they cause no significant skin irritation (such as swelling or erythema). The Cu@ZIF-8 particles loaded into the microneedles exhibit good angiogenesis-promoting, anti-inflammatory, and broad-spectrum antibacterial effects. Therefore, loading Cu@ZIF-8 particles into PEGDA / CMCS-based microneedles can produce more comprehensive and multifunctional microneedle dressings.

[0039] Specifically as follows:

[0040] A method for preparing photocrosslinked microneedles containing Cu@ZIF-8 particles includes the following steps:

[0041] (1) Cu@ZIF-8 particles were synthesized by hydrothermal method;

[0042] It should be noted that the synthesis of Cu@ZIF-8 particles using hydrothermal or solvothermal methods is a publicly available technique and will not be described in detail here; the electron microscope images of the obtained Cu@ZIF-8 particles at different magnifications are shown below. Figure 1 As shown.

[0043] (2) PDMS microneedle molds were prepared using photoresist technology;

[0044] (3) Prepare a PEGDA / CMCS mixed solution according to a certain ratio, add different proportions of Cu@ZIF-8 particles to the mixed solution and mix well, and finally add photoinitiator 1173.

[0045] like Figure 2 , 3 As shown, preliminary experiments were conducted with CMCS solution volume ratios of 0%, 20%, 40%, and 60% (i.e., PEGDA / CMCS mixing volume ratios of 10:0, 8:2, 6:4, and 4:6, respectively). Among these, the needle tips in the PEGDA / CMCS mixing volume ratios of 6:4 and 4:6 could not be completely demolded. When the PEGDA / CMCS mixing ratio was 8:2, the morphology images of the obtained microneedles at different magnifications showed that the microneedle tips were well demolded. Therefore, a PEGDA / CMCS mixing volume ratio of 8:2 is the optimal ratio, and subsequent experiments and all embodiments will use a PEGDA / CMCS mixing volume ratio of 8:2 to fabricate microneedles loaded with Cu@ZIF-8 particles.

[0046] (4) Pour the mixed solution into the microneedle mold and centrifuge to remove air bubbles;

[0047] The parameters for centrifugation to remove air bubbles are: speed 3000-10000 rpm, time 5-15 min, repeated three times.

[0048] (5) Place the mold under an ultraviolet light source to crosslink and obtain microneedles.

[0049] The ultraviolet light source has a wavelength of 200-400nm, a power of 50-500W, and a crosslinking time of 3-10min.

[0050] Based on the above-mentioned microneedle preparation method, and combined with specific experiments, the in vitro biocompatibility, antibacterial properties, in vitro angiogenesis-promoting effects, and in vivo treatment of skin wounds of microneedles loaded with different concentrations of Cu@ZIF-8 particles are explained.

[0051] like Figure 4 As shown, the in vitro biocompatibility of microneedles loaded with Cu@ZIF-8 particles at concentrations of 0, 0.5, 1.5, and 2.5 mg / mL was evaluated.

[0052] The cytotoxicity of microneedles was assessed using the CCK-8 assay. Microneedles were soaked in PBS for 24 h (37°C). Before the assay, L929 fibroblasts were seeded into 96-well plates and incubated for 24 h to allow them to adhere to the plates. Then, 100 μL of filtrate loaded with microneedles at different concentrations of Cu@ZIF-8 particles (0, 0.5, 1.5, and 2.5 mg / mL) was used to replace the original culture medium. The CCK-8 assay was performed on days 1, 2, and 3 after incubation.

[0053] As shown in the figure, the OD value at 450 nm, as detected by CCK-8, indicates that when the concentration of the loaded Cu@ZIF-8 particles exceeds 1.5 mg / mL, the proportion of viable cells decreases significantly, demonstrating a clear inhibitory effect on cell growth; the corresponding normalized cell viability (NCL) also shows a significant decrease. Figure 4 The same result was also shown.

[0054] like Figure 5 As shown, the broad-spectrum antibacterial activity of microneedles loaded with Cu@ZIF-8 particles at concentrations of 0, 0.5, 1.5, and 2.5 mg / mL was evaluated.

[0055] The antibacterial activity of microneedles was evaluated using Staphylococcus aureus and Escherichia coli. Microneedles loaded with different concentrations of Cu@ZIF-8 particles (0, 0.5, 1.5, and 2.5 mg / mL) were immersed in PBS solution and mixed with 100 μL of bacterial solution (1.5 OD value), and incubated at 37 °C for 24 h. 100 μL of the bacterial suspension was then added dropwise to a petri dish containing solid culture medium and incubated at 37 °C for 24 h to form colonies. The colonies were photographed, their number was estimated using ImageJ, and the lethality rate was calculated.

[0056] As shown in the figure, the number of colonies in the Cu@ZIF-8 loaded group (PCCZ-MN) was significantly lower than that in the PC-MN group and the control group, while the number of colonies in the PC-MN group was lower than that in the control group. This indicates that the antibacterial activity is directly proportional to the concentration of Cu@ZIF-8 nanoparticles, suggesting that higher concentrations of Cu@ZIF-8 can more effectively kill bacteria, while PC-MN exhibits lower antibacterial activity. Combined with the biocompatibility experiment results, a concentration of 1.5 mg / mL of Cu@ZIF-8 particles is suitable for preparing microneedles that simultaneously possess acceptable cell biocompatibility and good antibacterial effects.

[0057] Therefore, loading Cu@ZIF-8 particles can significantly improve the antibacterial activity of microneedles, and PCCZ-MN has outstanding antibacterial potential.

[0058] like Figure 6 As shown, the in vitro angiogenesis-promoting effect of microneedles loaded with Cu@ZIF-8 particles at a concentration of 1.5 mg / mL was evaluated.

[0059] In the HUVEC tube formation experiment, 1*10 5 HUVECs were seeded onto matrix gel membranes in 24-well plates and treated with 50% microneedle (PC-MN, PCCZ-MN) filtrate and 50% ECM; cells were incubated for 6 hours and imaged under a bright-field microscope. Tube length was quantified using angiography software.

[0060] As shown in the figure, PCCZ-MN is more efficient than PC-MN in promoting HUVEC tube formation, characterized by a more visible tubular structure and a longer tube length.

[0061] like Figure 7 As shown, this study evaluates the in vivo treatment of skin wounds using microneedles loaded with Cu@ZIF-8 particles at a concentration of 1.5 mg / mL.

[0062] To further investigate the practical value of PCCZ-MN in wound healing, a full-thickness skin defect rat model was established by creating a 1cm diameter circular wound on the back. Rats were randomly divided into three groups: a control group, a PC-MN group, and a PCCZ-MN group. During the wound healing process, changes in the wound bed were photographed and measured on days 0, 4, 8, and 16. The figures show that the wound in the PCCZ-MN group was almost completely healed after 16 days, while the wound healing rate in the PC-MN group was slower. The ability of Cu@ZIF-8 encapsulated microneedles to promote wound healing in vivo is due to their strong antibacterial properties and significant angiogenesis during degradation.

[0063] like Figure 8As shown, this study evaluated the inflammatory markers associated with in vivo treatment of skin wounds when microneedles loaded with Cu@ZIF-8 particles at a concentration of 1.5 mg / mL.

[0064] Previous studies have demonstrated that CD206 is classified as a marker of the selectively activated M2 phenotype and acts as a hemoglobin scavenger receptor on macrophages. Immunohistochemical staining of wound tissue on day 8 with CD206 and counting the number of M2 macrophages can reflect wound healing progress. The figure shows that M2 macrophages were positive to varying degrees in all groups; the PCCZ-MN group had the highest density in the wound bed, and the number of CD206-containing M2-like cells in the PC-MN group was greater than that in the control group. This phenomenon indicates that Cu@ZIF-8 nanoparticles have outstanding performance in reducing wound inflammation, not only helping to kill microorganisms but also promoting the regeneration of new epithelial tissue. Therefore, the ability of Cu@ZIF-8 encapsulated microneedles to promote wound healing in vivo is due to their strong antibacterial ability and significant angiogenesis and anti-inflammatory effects during degradation.

[0065] The relevant abbreviations are as follows:

[0066] PC-MN: Microneedles without Cu@ZIF-8 particles loaded;

[0067] PCCN-MN: Microneedles loaded with Cu@ZIF-8 particles;

[0068] PCCNMN-L: Microneedles loaded with Cu@ZIF-8 particles at a concentration of 0.5 mg / mL;

[0069] PCCNMN-M: Microneedles loaded with Cu@ZIF-8 particles at a concentration of 1.5 mg / mL;

[0070] PCCNMN-H: Microneedles loaded with Cu@ZIF-8 particles at a concentration of 2.5 mg / mL.

Claims

1. A photocrosslinked microneedle containing Cu@ZIF-8 particles, characterized in that, The microneedles are made of PEGDA / CMCS as substrate, with Cu@ZIF-8 particles loaded inside, and are multiple conical needle arrays prepared by molding and ultraviolet crosslinking process. The preparation method of the photocrosslinked microneedles includes the following steps: (1) Cu@ZIF-8 particles were synthesized by hydrothermal method; (2) PDMS microneedle molds were fabricated using photoresist technology; (3) Prepare a PEGDA / CMCS mixed solution according to a certain ratio, add different proportions of Cu@ZIF-8 particles to the mixed solution and mix well, and finally add photoinitiator 1173; (4) Pour the mixed solution into the microneedle mold and centrifuge to remove air bubbles; (5) Place the mold under an ultraviolet light source to crosslink and obtain microneedles; The PEGDA / CMCS mixing volume ratio was 8:2, and the concentration of Cu@ZIF-8 particles added to the mixed solution was 1.5 mg / mL.

2. The method according to claim 1, characterized in that, The morphological parameters of the PDMS microneedle mold are as follows: needle height 400-1500µm, base diameter 150-500µm, needle tip diameter 5-50µm, center-to-center spacing 500-2000µm, and array number greater than 5×5.

3. The method according to claim 1, characterized in that, In step (3), the molecular weight of PEGDA is 600; the mass fraction of the CMCS solution is 2%. The amount of photoinitiator 1173 added accounts for 0.02-0.1% of the total mass of the solution.

4. The method according to claim 1, characterized in that, The parameters for centrifugation to remove air bubbles are: speed 3000-10000 rpm, time 5-15 min, repeated three times.

5. The method according to claim 1, characterized in that, The ultraviolet light source has a wavelength of 200-400nm, a power of 50-500W, and a crosslinking time of 3-10min.

6. The application of the photocrosslinked microneedles containing Cu@ZIF-8 particles as described in claim 1 in the preparation of skin wound healing treatment products or other organ delivery medical devices.

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