Icariin micro-needle and micro-needle patch for preventing and treating testis injury caused by DEHP (diethylhexyl phthalate) and preparation method of micro-needle patch

Abstract

The invention relates to the technical field of biomedical materials, and particularly discloses an icariin microneedle and microneedle patch for preventing and treating testis injury caused by DEHP and a preparation method of the icariin microneedle and microneedle patch. The microneedle comprises an active drug icariin, and polyvinylpyrrolidone K30 and chondroitin sulfate which are loaded with the icariin. The preparation method of the microneedle is characterized by comprising the following steps: S1, dissolving icariin in dimethyl sulfoxide, and then adding water for dilution to obtain an icariin solution; s2, polyvinylpyrrolidone K30 and chondroitin sulfate are mixed, then the icariin solution is added, and a microneedle matrix solution is obtained; and S3, preparing the microneedle by taking the microneedle matrix solution as a supporting layer. The icariin microneedle is safe, non-toxic and reliable, testis dysfunction induced by DEHP is effectively treated, and a targeted, effective and safe delivery method is provided for icariin to testis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, in particular to an icariin microneedle and a microneedle patch for preventing and treating testicular damage caused by DEHP, and a preparation method thereof. Background Art

[0002] Infertility is a growing concern, and environmental endocrine disruptors (EEDs) have attracted widespread attention as a contributing factor to male infertility due to their ubiquitous presence and potential health hazards. Di(2-ethylhexyl) phthalate (DEHP) is an endocrine disrupting chemical widely used in the manufacture of polyvinyl chloride (PVC) and several consumer products and has now become a ubiquitous environmental contaminant. DEHP has been associated with testicular dysfunction, including impaired spermatogenesis and increased oxidative stress, leading to reduced sperm quality and concentration. In addition, exposure to DEHP disrupts the testosterone / androgen receptor pathway, and this disruption may lead to impaired sperm motility and morphology, ultimately affecting reproductive health. In addition, DEHP has been shown to induce blood-testis barrier (BTB) dysfunction in mouse testicular tissue, and this dysfunction has serious implications for reproductive health because the BTB plays a crucial role in maintaining an appropriate environment for spermatogenesis and protecting developing sperm cells from harmful substances and immune responses. Disruption of BTB integrity can lead to germ cell loss and impaired spermatogenesis, resulting in male infertility.

[0003] DEHP can enter the human body through skin contact, oral ingestion, and inhalation. After absorption, DEHP is metabolized to the active metabolite mono(2-ethylhexyl) phthalate (MEHP), which has been found in a variety of human tissues, including the liver, blood, urine, and reproductive organs. The widespread distribution of MEHP in the body can disrupt endocrine function, especially affecting the male reproductive system. It can impair spermatogenesis, increase oxidative stress, lead to potential testicular damage, and decrease sperm quality and concentration. Despite the known harmful effects of phthalates on reproductive health, effective strategies are urgently needed to prevent the reproductive damage they cause.

[0004] Icariin (ICA), a natural isoprenyl flavonol glycoside derived from Epimedium, has recently been shown to have the potential to treat male infertility by targeting multiple pathways that improve spermatogenesis, reduce oxidative stress, and prevent inflammation. ICA has been found to modulate the PI3K / AKT, ERα / Nrf2, and Keap1 / Nrf2 / HO-1 signaling pathways, which are critical for preventing ROS production and maintaining sperm quality, and therefore, ICA is considered a promising candidate for alleviating DEHP-induced testicular dysfunction. Nevertheless, the therapeutic effect of ICA is limited by its low bioavailability and rapid metabolism when administered orally. Therefore, innovative delivery systems are needed to enhance the therapeutic potential of ICA. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an icariin microneedle patch for preventing and treating testicular damage caused by DEHP and a preparation method thereof, so as to at least provide a safe, non-toxic, reliable and effective microneedle for treating DEHP-induced testicular dysfunction.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] One of the purposes of the present invention is to provide an icariin microneedle for preventing and treating testicular damage caused by DEHP, wherein the arrowhead layer material of the microneedle comprises the active drug icariin, and polyvinyl pyrrolidone K30 and chondroitin sulfate loaded with the icariin.

[0008] Furthermore, the preparation method of the icariin microneedle comprises the following steps:

[0009] S1: dissolving icariin in dimethyl sulfoxide, and then diluting with water to obtain an icariin solution;

[0010] S2: mixing polyvinyl pyrrolidone K30 and chondroitin sulfate, and then adding the icariin solution to obtain a microneedle matrix solution;

[0011] S3: preparing microneedles using the microneedle matrix solution as the arrow layer.

[0012] Furthermore, in step S1, 45-55 mg of the icariin is dissolved in 1 mL of the dimethyl sulfoxide.

[0013] Further, in step S1, the concentration of the icariin solution is 0.5-10 mg / mL;

[0014] Preferably, the concentration of the icariin solution is 4-6 mg / mL.

[0015] Furthermore, in step S2, the weight ratio of polyvinyl pyrrolidone K30 to chondroitin sulfate is 1:1.

[0016] Furthermore, in step S2, the concentration of polyvinyl pyrrolidone K30 in the microneedle matrix solution is 10% w / v.

[0017] Furthermore, in step S3, the specific method for preparing the microneedle is: injecting the microneedle matrix solution into a microneedle mold, and sequentially performing a first vacuuming, a first drying, applying a backing solution, a second vacuuming, a second drying, and demolding to obtain the microneedle.

[0018] Further, the drying temperature of the first drying is 25-42°C;

[0019] Preferably, the drying temperature of the first drying is 37°C;

[0020] And / or, the first drying time is 3-48 hours; preferably, the first drying time is 3-4 hours.

[0021] Furthermore, the first vacuuming time is 5-60 minutes; preferably, the first vacuuming time is 30 minutes;

[0022] And / or, the second vacuuming time is 5-60 min; preferably, the second vacuuming time is 30 min;

[0023] Furthermore, the backing solution is one of vinylpyrrolidone K30, chondroitin sulfate, hyaluronic acid, silk fibroin, polyvinyl alcohol, polylactic acid, maltose, trehalose and albumin.

[0024] Preferably, the backing solution is 10% polyvinyl pyrrolidone K30.

[0025] A second object of the present invention is to provide a microneedle patch prepared from the microneedles, wherein the microneedle patch comprises a substrate and the microneedles.

[0026] The beneficial effects of the present invention are:

[0027] 1. The microneedles of the present invention can safely and effectively treat DEHP-induced testicular dysfunction. The ratio of PVP-K30 to CS 1:1 shows strong mechanical properties in ICA-MNs, ensuring reliable transdermal drug delivery.

[0028] 2. In vitro evaluation showed that ICA-MNs significantly alleviated DEHP-induced oxidative stress and apoptosis in GC-1 cells by regulating the Keap1 / Nrf2 / HO-1 signaling pathway and reducing the expression of pro-apoptotic proteins. In vivo experiments in rat models confirmed that ICA-MNs treatment not only alleviated DEHP-induced testicular damage, but also was superior to traditional oral administration of ICA. Proteomic analysis showed that ICA MNs treatment restored the testicular proteome to normal levels, further confirming this effect.

[0029] 3. Comprehensive safety evaluation showed no adverse systemic effects, highlighting the biocompatibility and non-toxicity of ICA-MNs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 1a is the method for preparing drug-loaded microneedles by two-step method in Example 1, 1b is the result of mechanical testing of microneedles in Example 1, 1c-e are stereomicroscope views of a microneedle patch composed of 20×20 arranged microneedles, and 1f is a photo of the MN patch applied to intact skin 1 minute later:

[0031] Figure 2 4a is a flow chart of the experiment for evaluating the quality of semen in Example 2, 4b is a comparison chart of the morphology of the testis and epididymis of rats after exposure to DEHP, 4c is a comparison chart of the inflammation index of the testis and epididymis of rats after exposure to DEHP, 4d-e are semen quality (sperm count and motility) of rats after exposure to DEHP, 4f is a chart of the sperm staining results in Example 2, and 4g-h are H&E staining results of the testicular tissue sections of rats in Example 2;

[0032] Figure 3 The results of protein identification and pathway analysis in Example 2, wherein 6a is the false discovery rate, 6b is the principal component analysis diagram, 6c is the two-sample Student's t test result, 6d is the cluster analysis result, 6e is the heat map analysis result, and 6f is the subgroup analysis result. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited to the following.

[0034] Example 1

[0035] Prepare ICA-MNs as follows:

[0036] A 50 mg / mL stock solution was prepared by dissolving ICA (Chemconst, China) in DMSO and subsequently diluted to a concentration of 5 mg / mL in water. To determine the optimal ratio of PVP-K30 to chondroitin sulfate, combinations of PVP-K33 (Zhonglian, China) and CS (Guangyuan Shengwu, China) were created at ratios of 1:4, 2:1, 1:1, and 4:1 and integrated into the ICA solution. After vigorous mixing, the solution was left to stand overnight at low temperature to form a microneedle matrix solution. The support layer of the microneedles consisted of a 10% PVP-K30 solution (w / v).

[0037] The fabrication of drug-loaded microneedles involves two steps: First, 1 mL of microneedle matrix solution was injected into the PDMS mold, vacuumed for 30 minutes, and then dried in an incubator at 37°C for 3-4 hours. Once the microneedle tip was dry, 1 mL of 10% polyvinylpyrrolidone K30 was applied as a backing solution, vacuumed again for 30 minutes, and then air-dried at room temperature for 24 hours. Finally, the microneedles were carefully removed from the mold using tweezers to obtain ICA-loaded microneedles.

[0038] The microneedles were subjected to mechanical testing using a texture analyzer:

[0039] These microneedles exhibited an enhanced ability to withstand forces without displacement, indicating their robustness and suitability for transdermal drug delivery applications ( Figure 1 b). In addition, according to existing reports, MN needs to apply a minimum force of 0.058N per needle to penetrate human skin. The results show that a 1:1 ratio of PVP K30 to CS can withstand a force of >0.6N per needle ( Figure 1 b), indicating that the 1:1 ratio of PVP K30 / CS ensured the mechanical integrity of the microneedles during application. Therefore, in subsequent experiments, we chose to use MNs prepared with this specific concentration ratio to ensure their effectiveness and reliability in transdermal drug delivery applications. Figure 1 c shows a stereomicroscope view of a microneedle patch consisting of 20 × 20 microneedles. Each MN is cylindrical, a common geometric arrangement of microneedles designed to penetrate the stratum corneum of the skin while minimizing pain or damage to the underlying tissue. The MNs are arranged sequentially on the substrate ( Figure 1 ce). This sharp cone structure enables ICA MNs to be inserted quickly, non-invasively, and precisely into the testicular tunica albuginea. The height of the ICA MN is 700 μm and the base diameter is 300 μm ( Figure 1 e). To evaluate the safety of MN penetration, the MN patch was applied to intact skin for 1 min. Subsequently, the skin was observed to recover its original shape within approximately 10 min ( Figure 1 f), indicating that the MN patch is minimally invasive and has good biocompatibility.

[0040] The results showed that the microneedles fabricated with equal ratio of PVP-K30 to CS (1:1) exhibited excellent mechanical properties.

[0041] Example 2

[0042] Animal experiments were conducted to compare the effects of oral and transdermal ICA administration. The experimental methods are as follows:

[0043] Thirty 8-week-old Sprague-Dawley (SD) rats from Dossy, Chengdu, China, were acclimated for one week and then randomly divided into five groups: control group (Ctrl), DEHP, ICA, blank MNs, and ICA MNs, with six rats in each group. The treatment period was 30 days.

[0044] The control group (Ctrl) rats were given corn oil; the ICA group was given DEHP (500 mg / kg / d) and ICA (50 mg / kg / d) by intragastric gavage; the blank MNs group was treated with DEHP (500 mg / kg / d) and given blank MNs; and the ICA MNs group was treated with DEHP (500 mg / kg / d) and ICA MNs. Before each administration, ICA and DEHP solutions were freshly prepared using corn oil.

[0045] This animal study was approved by the Ethics Committee of Chengdu University of Traditional Chinese Medicine Hospital (approval number 2023DL-108).

[0046] The microneedles used in the test were all microneedle ICA-MNs manufactured in Example 1 with an equal ratio of PVP-K30 and CS (1:1).

[0047] The test methods and test results are as follows:

[0048] ① Semen quality evaluation: The left epididymis of rats was placed in 37℃ saline, minced and incubated to release sperm. Then 10μl of the sample was analyzed using a computer-assisted semen analysis system from Sujia, Beijing, China. The results showed that compared with the control group, DEHP exposure in rats resulted in changes in testicular and epididymal morphology ( Figure 2 b) Testicular and epididymitis index ( Figure 2 c) and semen quality (sperm count and motility) ( Figure 2 d, e) were significantly reduced. No significant improvement was observed after blank microneedle treatment ( Figure 2be). In contrast, oral ICA and ICA-MNs treatment significantly improved these parameters compared with DEHP treatment. Moreover, ICA MNs treatment was superior to oral ICA, indicating that microneedle delivery of ICA to the testis could be effective in preventing and treating DEHP-induced testicular damage. All these data suggest that microneedle technology may provide a targeted and effective method for the delivery of ICA to the testis, potentially circumventing the barriers faced by oral administration, such as metabolism and low bioavailability.

[0049] ② Sperm morphology examination: 10 μL of epididymal fluid sample was spread on a slide, fixed with 95% alcohol, rehydrated, and stained with two dyes built in Nanjing, China. After rinsing and drying, the sample was examined under a microscope. The sperm staining results showed that exposure of rats to DEHP resulted in abnormal sperm morphology ( Figure 2 f). Notably, no significant improvements were observed in the blank microneedle intervention group. In contrast, oral and microneedle-delivered ICA interventions significantly improved sperm morphology parameters compared with treatment with DEHP alone. Importantly, the repair effect of ICA treatment was significantly better than that of oral ICA administration, highlighting the enhanced efficacy of local ICA delivery directly to the testis using microneedle technology. H&E staining results showed that rats exposed to DEHP exhibited significant irregularities in seminiferous tubule arrangement and spermatogonia separation compared with the normal control group ( Figure 2 g). No significant difference was observed between the blank MNs intervention group and the DEHP group ( Figure 2 g). In contrast, ICA intervention significantly alleviated these pathological phenomena ( Figure 2 g). It is noteworthy that the repair effect of ICA MNs was significantly better than that of oral ICA ( Figure 2 g).

[0050] ③ Histology: Testicular tissue was fixed in testicular fixative solution for one week, and other tissue samples, including heart, liver, spleen, lung, and kidney, were fixed in 4% paraformaldehyde for the same period of time. After fixation, the tissue was embedded in paraffin and cut into 5-micron-thick sections for histological examination. The paraffin-embedded tissue sections were then dewaxed to remove the paraffin, which is a necessary step before staining. Staining with hematoxylin for 5 minutes stained the nuclei. After staining, the sections were rinsed with tap water to remove any unbound stains. To enhance contrast, the sections were briefly differentiated in a 1% hydrochloric acid alcohol solution and then rinsed again with tap water. The sections were then counterstained with a 0.6% ammonia solution to further determine the cell structure and rinsed again under tap water. After hematoxylin staining, the sections were stained with eosin for 1-3 minutes to stain the cytoplasm and other acidic components of the tissue. The eosin-stained sections were then dehydrated in a series of alcohol solutions to remove water and cleared with xylene to make the tissue transparent. Finally, processed sections were examined under a light microscope to assess tissue morphology and any potential pathological changes.

[0051] ④ Apoptosis detection: Prepare rat testicular tissue sections for TUNEL detection to display DNA fragments indicating apoptosis. The sections were stained with diaminobenzidine (DAB) and hematoxylin ( Figure 2 h), and then examined under an optical microscope. The results showed that after DEHP treatment, a large number of apoptotic cells (yellow cells) appeared in the testis, and after ICA-MNs intervention, apoptotic cells decreased ( Figure 2 h). These findings suggest that transdermal administration of ICA into the testis via MNs produces a higher therapeutic effect than oral administration, thereby achieving the optimal therapeutic effect.

[0052] ⑤Proteomic analysis: Testicular tissue proteins were extracted, quantified, and analyzed by mass spectrometry. These data were processed using Spectronaut software and further analyzed on the Majorbio Cloud platform for protein identification and pathway analysis.

[0053] A total of 10,816 proteins were identified in the testis, with false discovery rates (FDRs) of less than 1% for both protein and peptide identifications ( Figure 3 a). The results of principal component analysis (PCA) showed that the protein profiles were significantly different between the DEHP and ICA MNs groups, indicating that the proteome of testis exposed to DEHP was altered after ICA-MNs treatment ( Figure 3b). Using two-sample Student's t test, a total of 3050 DEPs with statistical significance (p < 0.05, fold change (FC) > 1.2 or < 0.83) were detected between the Ctrl and DEHP groups, of which 1349 DEPs were up-regulated and 1701 DEPs were down-regulated; a total of 2682 DEPs were detected between the ICA MNs and DEHP groups, of which 1540 DEPs were up-regulated and 1142 DEPs were down-regulated; a total of 602 DEPs were detected between the ICA MNs and Ctrl groups, of which 1540 DEPs were up-regulated and 1142 DEPs were down-regulated ( Figure 3 c). Cluster analysis showed that after ICA-MNs treatment, proteins in the ICA group clustered on the same branch as those in the Ctrl group ( Figure 3 d). Heatmap and subgroup analysis showed that the expression level of DEP was consistent in biological replicates, and there was a significant difference between the DEHP-exposed group and the ICA microneedle-treated group, which is consistent with Figure 3 The PCA results in b are consistent ( Figure 3 e, f). These results indicate that the changes in protein expression induced by DEHP exposure were somewhat affected after intervention with ICA-MNs in the testis.

[0054] ⑥ In vitro safety assessment: Rats were treated with ICA MNs for 15 days, and body weight and biochemical parameters were monitored. Histological changes in organ tissues were collected, processed, and examined. After the 14-day treatment regimen, blood samples were collected to quantify alanine aminotransferase (ALT), aspartate aminotransferase, alkaline phosphatase (ALP), blood urea nitrogen (BUN), uric acid (UA), and creatinine (CR) levels, all of which were determined to be within the physiological parameter range (Figure S2). Histological examination using hematoxylin and eosin (H&E) staining showed no obvious pathological changes in major organs (including heart, liver, spleen, lung, and kidney) (Figure S3). Throughout the treatment period, the body weight of rats fluctuated within the expected range, with no obvious downward trajectory (Figure S4). These comprehensive findings collectively indicate that ICA MNs have no adverse systemic effects and have good biocompatibility, and are therefore considered a non-toxic and effective treatment modality to improve DEHP-induced testicular damage.

[0055] In summary, the present invention verifies that ICA-loaded CS and PVP K30-MN patches have good efficacy and safety for the treatment of DEHP-induced testicular dysfunction. The optimized 1:1 ratio of PVP K30 to CS in ICAMNs showed strong mechanical properties, ensuring reliable transdermal delivery. In vitro evaluation showed that ICAMNs significantly alleviated DEHP-induced oxidative stress and apoptosis in GC-1 cells. In vivo experiments in a rat model confirmed that ICAMNs treatment not only alleviated DEHP-induced testicular damage, but also outperformed traditional oral administration of ICA. These findings were further supported by proteomic analysis, indicating that ICA MNs treatment restored the testicular proteome to normal levels. In addition, a comprehensive safety assessment showed no adverse systemic effects, highlighting the biocompatibility and non-toxicity of ICA MNs. It is worth noting that microneedle patches are generally used for transdermal delivery of macromolecular drugs and are often used to treat skin diseases. Compared with direct oral administration of icariin, the microneedle patch loaded with icariin of the present invention has an effect of about 50% in treating testicular damage caused by DEHP, which not only overcomes the limitations of the application field of microneedle patches, but also proves the specificity of icariin in treating testicular damage caused by DEHP.

[0056] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. Icariin microneedle for preventing and treating testicular damage caused by DEHP, characterized in that: The raw materials of the arrowhead layer of the microneedle include active drug icariin, and polyvinyl pyrrolidone K30 and chondroitin sulfate loaded with the icariin.

2. The method for preparing the icariin microneedle according to claim 1, characterized in that: The following steps are involved: S1: dissolving icariin in dimethyl sulfoxide, and then diluting with water to obtain an icariin solution; S2: mixing polyvinyl pyrrolidone K30 and chondroitin sulfate, and then adding the icariin solution to obtain a microneedle matrix solution; S3: preparing microneedles using the microneedle matrix solution as the arrow layer.

3. The preparation method according to claim 2, characterized in that: In step S1, 45-55 mg of the icariin is dissolved in 1 mL of the dimethyl sulfoxide.

4. The preparation method according to claim 2, characterized in that: In step S1, the concentration of the icariin solution is 0.5-10 mg / mL.

5. The preparation method according to claim 2, characterized in that: In step S2, the weight ratio of polyvinyl pyrrolidone K30 to chondroitin sulfate is 1:

1.

6. The preparation method according to claim 2, characterized in that: In step S2, the concentration of polyvinyl pyrrolidone K30 in the microneedle matrix solution is 10% w / v.

7. The preparation method according to claim 2, characterized in that: In step S3, the specific method for preparing the microneedle is: injecting the microneedle matrix solution into a microneedle mold, and sequentially performing a first vacuuming, a first drying, applying a backing solution, a second vacuuming, a second drying, and demolding to obtain the microneedle.

8. The preparation method according to claim 7, characterized in that: The drying temperature of the first drying is 25-42°C; And / or, the first drying time is 3-48h; And / or, the vacuuming time of the first vacuuming is 5-60min; And / or, the second vacuuming time is 5-60 min.

9. The preparation method according to claim 8, characterized in that: The backing solution includes one of polyvinyl pyrrolidone K30, chondroitin sulfate, hyaluronic acid, silk fibroin, polyvinyl alcohol, polylactic acid, maltose, trehalose and albumin.

10. An icariin microneedle patch, characterized in that: The invention comprises a substrate and the microneedle according to claim 1 or a microneedle prepared by the preparation method according to any one of claims 2 to 8.

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