CN116098855A

CN116098855A - Photo-crosslinking microneedle containing Cu@ZIF-8 particles, and preparation method and application thereof

Info

Publication number: CN116098855A
Application number: CN202211651069.1A
Authority: CN
Inventors: 郭亮; 蔡林; 向婕妤; 朱宇凡; 谢远龙; 陈丹洋; 周凌; 郭佳
Original assignee: Zhongnan Hospital of Wuhan University
Current assignee: Zhongnan Hospital of Wuhan University
Priority date: 2022-12-21
Filing date: 2022-12-21
Publication date: 2023-05-12

Abstract

A photo-crosslinking microneedle containing Cu@ZIF-8 particles, a preparation method and application thereof, wherein the microneedle uses PEGDA/CMCS as a base material, cu@ZIF-8 particles are loaded in the microneedle, and a plurality of conical needle arrays are prepared through a die forming and ultraviolet crosslinking process. According to the invention, the photo-crosslinking microneedle loaded with Cu@ZIF-8 particles is prepared by combining the clinical requirements of the dressing; preparing a series of microneedles with different proportions, preferably preparing a microneedle product with good mechanical properties and excellent broad-spectrum antibacterial property, vascular promoting effect and anti-inflammatory effect; the obtained microneedle product is especially suitable for treating skin wound surface, and can also be used for bone repair and other organ administration.

Description

Photo-crosslinking microneedle containing Cu@ZIF-8 particles, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a photo-crosslinking microneedle containing Cu@ZIF-8 particles, and a preparation method and application thereof.

Background

In recent years, wound healing has attracted considerable attention due to its serious challenges and severe economic burden. However, the current treatment measures for the wound surface difficult to heal are very limited, and effective treatment methods need to be continuously explored. The wound healing process is generally divided into three phases: inflammation, proliferation, and tissue remodeling. The excellent treatment means can not only improve the inflammation of the wound, but also accelerate the regeneration of blood vessels, the proliferation of cells and the remodeling of tissues.

In order to solve the problem of difficult healing of wound surfaces, various treatment strategies such as hydrogel films, electrospun dressings and other various wound patches have been developed. Among them, the Microneedle (MN) is a promising medical technology, can be administered without dysmenorrhea by a minimally invasive method, and is 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 studies. MNs patches can significantly increase the effect of promoting wound healing by increasing their contact area with the wound, as compared to conventional dressings. However, for some MNs made of non-biodegradable materials, such as stainless steel, the needle body of MNs easily forms tiny pinholes at the wound site. These holes are likely to cause secondary physical damage and even severe microbial infection on the wound. These limitations limit the development of MNs in the field of promoting wound healing. Therefore, hydrogel MNs having improved minimally invasive therapeutic effects of drug delivery while having excellent biocompatibility are highly desired. Currently, the potential for microneedle applications is still under deep development.

Polyethylene glycol diacrylate (PEGDA) hydrogel is a hydrophilic polymer network with good biocompatibility, and is widely applied to the field of tissue engineering. In addition, carboxymethyl chitosan (CMCS) is a modified derivative of chitosan, has good water solubility, stability and strong antibacterial property, and has wide application in the biomedical field and wide application prospect. The PEGDA/CMCS combination provides MN with significant biocompatibility and allows for consistent release of the loaded active ingredient to the wound.

Studies have demonstrated that copper exerts potent antibacterial and pro-angiogenic effects during wound healing, and zinc ions are also believed to contribute to antibacterial and wound healing effects. In recent years, the copper and zinc-based zeolite-imidazolium framework (Cu@ZIF-8) has good antibacterial effect on wound surface models and bone repair models. These nanoparticles produce more active oxygen than ZIF-8, which contributes to their more powerful antimicrobial properties. The rough surface of the Cu@ZIF-8 nano particles can increase the contact area between MOFs and bacteria, so that better antibacterial activity is obtained. In addition, zinc ions and copper ions play an important role in the vascularization process, and can stimulate vascular endothelial cells to form blood vessels and inhibit inflammatory reactions. However, until now, cu@ZIF-8 particles are loaded in a PEGDA/CMCS microneedle, and related researches for endowing the microneedle with strong performance are still in a starting stage, and no research and development product has been developed and popularized in the market.

Disclosure of Invention

In order to solve the problems, the invention provides a photo-crosslinking microneedle containing Cu@ZIF-8 particles, and a preparation method and application thereof, and the specific technical scheme is as follows:

a photo-crosslinking microneedle containing Cu@ZIF-8 particles takes PEGDA/CMCS as a base material, and is internally provided with the Cu@ZIF-8 particles, and a plurality of conical needle arrays are prepared by a die forming and ultraviolet crosslinking process.

The invention further provides a preparation method of the photo-crosslinking microneedle containing Cu@ZIF-8 particles, which comprises the following steps of:

(1) Synthesizing Cu@ZIF-8 particles by a hydrothermal (solvent) method;

(2) Preparing a PDMS microneedle mould by adopting a photoresist technology;

(3) Preparing a PEGDA/CMCS mixed solution according to a certain proportion, adding Cu@ZIF-8 particles with different proportions into the mixed solution, uniformly mixing, and finally adding a photoinitiator 1173;

(4) Pouring the mixed solution into a microneedle mould, and centrifugally removing bubbles;

(5) And (3) placing the mould under an ultraviolet light source, and crosslinking to obtain the microneedle.

Further, the morphology parameters of the PDMS microneedle mold were: the needle height is 400-1500 μm, the diameter of the base is 150-500 μm, the diameter of the needle tip is 5-50 μm, the center-to-center spacing is 500-2000 μm, and the number of arrays is more than 5×5.

Further, the molecular weight of PEGDA is 600; the mass fraction of the CMCS solution was 2%;

the mixing proportion of PEGDA/CMCS is gradually increased from 0% to 60% by volume percent of CMCS solution;

adding Cu@ZIF-8 particles with different proportions into the solution, and uniformly mixing, wherein the concentration is gradually increased from 0mg/mL to 2.5mg/mL;

the photoinitiator 1173 is added in an amount of 0.02 to 0.1% by weight of the total mass of the solution.

Preferably, the PEGDA/CMCS blend volume ratio is 8:2.

Preferably, the Cu@ZIF-8 particles are added to the solution at a concentration of 1.5mg/mL.

Further, the parameters of centrifugal bubble removal are: the rotation speed is 3000-10000rpm, the time is 5-15min, and the process is repeated three times.

Further, the wavelength of the ultraviolet light source is 200-400nm, the power is 50-500W, and the crosslinking time is 3-10min.

The invention further provides an application of the photo-crosslinking microneedle containing Cu@ZIF-8 particles in a skin wound healing treatment product or other organ administration medical appliances.

The invention has the beneficial effects that:

(1) Preparing a photo-crosslinking microneedle loaded with Cu@ZIF-8 particles by combining the clinical requirements of the dressing;

(2) Preparing a series of microneedles with different proportions, preferably preparing a microneedle product with good mechanical properties and excellent broad-spectrum antibacterial property, vascular promoting effect and anti-inflammatory effect;

(3) The obtained microneedle product is especially suitable for treating skin wound surface, and can also be used for bone repair and other organ administration.

Drawings

FIG. 1 is an electron microscope image of Cu@ZIF-8 particles at different magnifications.

FIG. 2 is a graph of the morphology of microneedles prepared with varying mixing volume ratios of PEGDA/CMCS.

Fig. 3 is a topography of the microneedles (PEGDA/CMCS blend ratio=8:2) at different magnifications.

FIG. 4 is a graph showing the results of in vitro biocompatibility evaluation of the microneedles MN loaded with different Cu@ZIF-8 particle concentrations.

FIG. 5 is a graph showing the results of broad-spectrum antimicrobial evaluation of microneedles MN loaded with different Cu@ZIF-8 particle concentrations.

Fig. 6 is a graph showing the results of evaluation of in vitro proangiogenic activity of microneedles (PCCZ-mn=1.5 mg/mL).

Fig. 7 is a graph showing the results of evaluation of treatment of skin wounds in vivo with microneedles (PCCZ-mn=1.5 mg/mL).

Fig. 8 is a graph showing the evaluation results of inflammatory response-related markers for treating skin wounds in vivo with microneedles (PCCZ-mn=1.5 mg/mL).

Detailed Description

The invention is further described with reference to the accompanying drawings and specific embodiments:

the invention provides a photo-crosslinking microneedle containing Cu@ZIF-8 particles, which is a conical microneedle array prepared by taking PEGDA/CMCS as a substrate of the microneedle, loading Cu@ZIF-8 particles in the microneedle, and performing processes such as mold forming, ultraviolet crosslinking and the like. The microneedle can be inserted into tissues, and released Cu@ZIF-8 particles play roles of resisting bacteria, promoting angiogenesis and inhibiting inflammation; the microneedle also has good biocompatibility and biodegradability, and plays roles of promoting collagen deposition and wound healing aiming at wound surfaces.

The PEGDA/CMCS is selected as the base material of the microneedle, cu@ZIF-8 particles are loaded in the microneedle, the performance defects of single dressing effect and low drug delivery efficiency are effectively overcome, and the clinical requirements of the wound dressing are better met. The PEGDA/CMCS is a biocompatible and biodegradable polymer material, so that the micro-needle has good mechanical properties, and has no obvious stimulation reaction (such as swelling, erythema and the like) to the skin due to being minimally invasive. The Cu@ZIF-8 particles loaded in the microneedles have good angiogenesis promoting, inflammation inhibiting and broad-spectrum antibacterial effects. Therefore, cu@ZIF-8 particles are loaded in the PEGDA/CMCS-based microneedle, and the multifunctional microneedle dressing with more comprehensive performance can be prepared.

The method comprises the following steps:

a preparation method of a photo-crosslinking microneedle containing Cu@ZIF-8 particles comprises the following steps:

(1) Synthesizing Cu@ZIF-8 particles by a hydrothermal (solvent) method;

the synthesis of Cu@ZIF-8 particles by a hydrothermal method or a solvothermal method is a disclosed technique and is not described in detail herein; the electron microscope pictures of the obtained Cu@ZIF-8 particles under different magnifications are shown in figure 1.

(2) Preparing a PDMS microneedle mould by adopting a photoresist technology;

as shown in fig. 2, 3, pre-experiments were performed with CMCS solutions at volume ratios of 0%, 20%, 40%, and 60%, respectively (i.e., PEGDA/CMCS mixed volume ratios of 10:0, 8:2, 6:4, and 4:6, respectively); wherein, the mixing volume ratio of PEGDA/CMCS is 6:4 and 4: the tip portions of group 6 were not completely demolded. When the mixing ratio of PEGDA/CMCS is 8:2, the obtained morphology patterns of the micro needle under different magnifications show that the tip demoulding and forming of the micro needle are good; thus, PEGDA/CMCS mixing volume ratio of 8:2 is the optimum ratio, and subsequent experiments and examples will use PEGDA/CMCS mixing volume ratio = 8:2 to make a microneedle loaded with cu@zif-8 particles.

the parameters of centrifugal bubble removal are as follows: the rotation speed is 3000-10000rpm, the time is 5-15min, and the process is repeated three times.

The wavelength of the ultraviolet light source is 200-400nm, the power is 50-500W, and the crosslinking time is 3-10min.

According to the preparation method of the microneedle, the aspects of in-vitro biocompatibility, antibacterial property, in-vitro angiogenesis promotion effect, in-vivo skin wound treatment and the like of the microneedle loaded with Cu@ZIF-8 particles with different concentrations are described by combining with specific tests.

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

The cytotoxicity of the microneedles was detected using the CCK-8 method. The microneedles were immersed in PBS for 24h (37 ℃); l929 fibroblasts were seeded in 96-well plates and incubated for 24 hours before testing to allow adhesion to the plates. Next, 100. Mu.L of the filtrate loaded with microneedles of different Cu@ZIF-8 particle concentrations (0, 0.5, 1.5, 2.5mg/mL, respectively) was used in place of the original medium. CCK-8 assays were performed on

days

1, 2, and 3, respectively, after incubation.

The graph shows that the OD value at 450nm detected by CCK-8 shows that when the concentration of the loaded Cu@ZIF-8 particles exceeds 1.5mg/mL, the proportion of living cells is obviously reduced, and the cell growth is obviously inhibited; the corresponding normalized cell viability (fig. 4) also shows the same results.

As shown in FIG. 5, the broad-spectrum antibacterial property of the microneedles was evaluated when the concentration of Cu@ZIF-8 particles was 0, 0.5, 1.5 and 2.5mg/mL, respectively.

The antimicrobial ability of the microneedles was evaluated using staphylococcus aureus and escherichia coli. Microneedles loaded with different Cu@ZIF-8 particle concentrations (0, 0.5, 1.5, 2.5mg/mL, respectively) were immersed in PBS solution, mixed with 100. Mu.L of bacterial solution (1.5 OD), and incubated at 37℃for 24h. mu.L of the bacterial suspension was dropped into a petri dish containing a solid medium, and cultured in an incubator at 37℃for 24 hours to form colonies. Colonies were photographed with a camera, their numbers were estimated with ImageJ, and their mortality was finally calculated.

The graph shows that the colony count of the Cu@ZIF-8 loaded group (PCCZ-MN) is obviously smaller than that of the PC-MN group and the control group, and the colony count of the PC-MN group is smaller than that of the control group. It can be seen that the bacteriostatic activity is proportional to the concentration of Cu@ZIF-8 nanoparticles, which indicates that higher concentration of Cu@ZIF-8 can kill bacteria more effectively, while the bacteriostatic activity of PC-MN is lower. In combination with the results of the biocompatibility experiments, cu@ZIF-8 particles with the concentration of 1.5mg/mL are suitable for preparing the microneedles with acceptable cell biocompatibility and good antibacterial effect.

Therefore, the Cu@ZIF-8 particles can be loaded to remarkably improve the antibacterial activity of the microneedles, and PCCZ-MN has outstanding antibacterial action potential.

As shown in FIG. 6, evaluation of in vitro proangiogenic effects at a concentration of 1.5mg/mL of Cu@ZIF-8 particles loaded on the microneedles.

In the HUVEC tube formation experiments, 1×10 ⁵ HUVECs were seeded onto a 24-well plate matrigel membrane 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 angiographic tool software.

The illustrations show that PCCZ-MN is more efficient at promoting HUVEC tube formation than PC-MN, and is characterized by a more visible tubular structure and a higher tube length.

As shown in FIG. 7, treatment evaluation of skin wound in vivo at a concentration of 1.5mg/mL of Cu@ZIF-8 particles loaded on the microneedle.

In order to further study the practical value of PCCZ-MN in wound healing, a full-thickness skin defect rat model is established by creating a circular wound with the diameter of 1cm on the back. Rats were randomly divided into three groups, control, PC-MN and PCCZ-MN. During the wound healing process, photographs were taken on

days

0, 4, 8, 16, respectively, and changes in the wound bed were measured. The figures show that the PCCZ-MN group showed almost complete wound healing after 16 days, compared to the PC-MN group which showed slower wound healing. The ability of cu@zif-8 encapsulated microneedles to promote wound healing in vivo is due to their strong antimicrobial ability, as well as significant angiogenic effects during degradation.

As shown in FIG. 8, the evaluation of inflammatory response-related markers for treating skin wounds in vivo at a concentration of 1.5mg/mL Cu@ZIF-8 particles loaded on the microneedles.

CD206 has been demonstrated to be classified as a marker of the selectively activated M2 phenotype and as a hemoglobin scavenger receptor on macrophages. By performing immunohistochemical staining of CD206 on the wound tissue on day 8, the number of M2 macrophages is counted, and the wound repair condition can be reflected. The graph shows that each group of M2 macrophages is positive to different degrees; wherein the density of PCCZ-MN groups in the wound bed is highest in each group and the number of M2-like cells of CD206 in the PC-MN group is greater than that in the control group. This phenomenon indicates that the Cu@ZIF-8 nanoparticle has outstanding properties in reducing wound inflammation, and is beneficial to killing microorganisms and promoting regeneration of new epithelial tissues. Thus, the ability of cu@zif-8 encapsulated microneedles to promote wound healing in vivo is due to their strong antimicrobial ability, as well as significant angiogenic and anti-inflammatory effects during degradation.

Wherein the relevant abbreviations are as follows:

PC-MN: microneedles without cu@zif-8 particles loaded;

PCCN-MN: microneedle loaded with Cu@ZIF-8 particles;

PCCNMN-L: microneedle with Cu@ZIF-8 particle concentration of 0.5 mg/mL;

PCCNMN-M: microneedle with Cu@ZIF-8 particle concentration of 1.5 mg/mL;

PCCNMN-H: microneedle with Cu@ZIF-8 particle concentration of 2.5 mg/mL.

Claims

1. A photo-crosslinking microneedle containing Cu@ZIF-8 particles is characterized in that the microneedle uses PEGDA/CMCS as a base material, the Cu@ZIF-8 particles are loaded in the microneedle, and a plurality of conical needle arrays are prepared through a die forming and ultraviolet crosslinking process.

2. The method for preparing the photo-crosslinking microneedle containing Cu@ZIF-8 particles according to claim 1, which comprises the following steps:

(1) Synthesizing Cu@ZIF-8 particles by a hydrothermal (solvent) method;

(2) Preparing a PDMS microneedle mould by adopting a photoresist technology;

3. The method of claim 2, wherein the morphology parameters of the PDMS microneedle mould are: the needle height is 400-1500 μm, the diameter of the base is 150-500 μm, the diameter of the needle tip is 5-50 μm, the center-to-center spacing is 500-2000 μm, and the number of arrays is more than 5×5.

4. The method of claim 2, wherein in step (3), the PEGDA has a molecular weight of 600; the mass fraction of the CMCS solution was 2%;

5. The method of claim 4, wherein the PEGDA/CMCS blend volume ratio is 8:2.

6. The method of claim 4, wherein the concentration of Cu@ZIF-8 particles added to the solution is 1.5mg/mL.

7. The method according to claim 2, wherein the parameters of the centrifugal bubble removal are: the rotation speed is 3000-10000rpm, the time is 5-15min, and the process is repeated three times.

8. The method according to claim 2, wherein the ultraviolet light source has a wavelength of 200-400nm, a power of 50-500W and a crosslinking time of 3-10min.

9. Use of a photocrosslinked microneedle comprising cu@zif-8 particles according to claim 1 in a skin wound healing treatment product or other organ administration medical device.