System for treating infantile hemangioma and microneedle and application thereof

Abstract

A system for treating infantile hemangioma includes a first active substance delivery portion and a second active substance delivery portion. The first active substance delivery part delivers a first active substance to the superficial surface of the skin; the second active material delivery unit delivers a second active material to the deep part of the skin. The first active substance contains a beta receptor blocker, and the second active substance contains an antibiotic. Through verification, the beta receptor blocker and the antibiotic are delivered subcutaneously, and the effect of synergistically enhancing anti-angiogenesis and hardening promotion on infantile hemangioma is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to a biomaterial, in particular to a system for treating infantile hemangioma, to a microneedle made thereof, and use in the manufacture of a medicament or medical device. BACKGROUND

[0002] Infantile hemangioma (IH) is the most common benign vascular tumor of infancy, characterized by a predictable natural history with early proliferation followed by spontaneous involution. However, a substantial proportion of IH fails to completely resolve and often leads to residual sequelae, such as telangiectasia, fibrofatty deposition, and skin atrophy. It has been reported that up to 54.9% of patients develop clinically significant sequelae requiring further intervention, which underscores the need for early effective treatment to mitigate long-term aesthetic and functional impairment.

[0003] Current first-line therapies include laser treatment for superficial lesions and oral propranolol for proliferating IH. However, their efficacy remains limited for thick or deep hemangiomas. In particular, oral propranolol is associated with systemic side effects, potential rebound growth upon discontinuation, and uncertain long-term outcomes, which highlights the unmet clinical need for more effective and localized treatment strategies.

[0004] Topical timolol maleate (TM), a non-selective beta-blocker, has emerged as a treatment option for superficial IH due to its ability to enhance vasoconstriction, inhibit VEGF-induced angiogenesis, and promote apoptosis. However, its therapeutic effectiveness is limited by poor skin penetration, rendering it insufficient for thicker IH. Therefore, there is an urgent need for a delivery platform that can enhance TM penetration.

[0005] Similarly, bleomycin (BLM), a sclerosing and antitumor antibiotic, has been used for intralesional injection to treat refractory IH and propranolol-resistant cases. Although BLM effectively induces vascular damage through DNA inhibition and fibrosis, its use is limited by the risk of pulmonary fibrosis from systemic exposure and the risk of local complications such as ulceration at high concentration administration. Therefore, there is an urgent need for a delivery system that can localize BLM activity while minimizing systemic exposure. SUMMARY

[0006] It is an object of the present application to provide a system for treating infantile hemangioma, enhancing anti-angiogenic and pro-sclerotic effects.

[0007] It is another object of the present application to provide a microneedle that delivers beta-blockers and antibiotics subcutaneously, synergistically enhancing anti-angiogenic and pro-sclerotic effects.

[0008] Still another object of the present application is to provide a medical device for facilitating the use of photothermal-browning therapy to promote subcutaneous fat browning, thereby achieving a synergistic anti-obesity effect.

[0009] Still another object of the present application is to provide a drug for synergistically enhancing the anti-angiogenic and pro-sclerotic effects of beta-blockers and antibiotics as active ingredients.

[0010] A fifth object of the present application is to provide a system for treating infantile hemangioma in the preparation of a medical device or a drug.

[0011] It has been verified that the delivery of beta-blockers and antibiotics to the subcutaneous layer has a synergistically enhanced anti-angiogenic and pro-sclerotic effect on infantile hemangioma.

[0012] Beta-blockers such as, but not limited to, timolol, metoprolol, and propranolol, and pharmaceutically acceptable salts thereof.

[0013] Antibiotics have sclerotic and anti-tumor effects, such as BLM, pingyangmycin, doxorubicin, epirubicin, and daunorubicin, etc.

[0014] A system for treating infantile hemangioma, beta-blockers are applied to the superficial layer of the skin, and antibiotics are applied to the deep layer of the skin (greater than 400 μm, especially greater than 500 μm, such as 550 μm ~ 650 μm), and gradually penetrate into the deep layer of the tumor through drug sustained release, thereby achieving a synergistically enhanced anti-angiogenic and pro-sclerotic effect on infantile hemangioma (thickened type).

[0015] Another system for treating infantile hemangioma, characterized in that it comprises:

[0016] A first active substance delivery part for delivering a first active substance to the superficial layer of the skin; and

[0017] A second active substance delivery part for delivering a second active substance to the deep layer of the skin.

[0018] The system is developed as a sustained-release drug for treating infantile hemangioma (thickened type). For example, beta-blockers are released in the superficial layer of the skin, and antibiotics are released in the deep layer of the skin.

[0019] Microneedle (MN) technology provides a promising alternative for transdermal drug delivery, which can bypass the stratum corneum and directly deposit therapeutic agents into the dermis in a minimally invasive manner. However, traditional microneedles often exhibit insufficient skin adhesion and premature shedding, resulting in poor drug delivery effect. To overcome these limitations, new microneedles with sufficient adhesion are needed to achieve sustained, depth-specific dual drug delivery.

[0020] The delivery vehicle of the present invention, in the form of microneedles, specifically constructed as a tower with more than two layers, presents a "barb" structure that prevents easy removal after penetration of the skin.

[0021] The overall shape of each tower layer of the microneedle is a square pyramid, with the edges of the square pyramid having a ridge structure. The microneedles are arranged in an array to form a microneedle patch, which is used as a medical device for treating thickened infantile hemangiomas.

[0022] Each MN patch contains an 8 x 8 array.

[0023] The beta-blocker and antibiotic are loaded into the microneedle in tower layers that penetrate the skin in a shallow to deep manner. For example, the antibiotic is loaded into the distal tower layer of the microneedle, which acts on the deep tissue of the skin, and the beta-blocker is loaded into the proximal tower layer of the microneedle, which acts on the superficial tissue of the skin. The distal end of the microneedle is understood to be the end of the microneedle that is farthest from the base and that is the first to contact the skin when the microneedle is inserted into the skin. The proximal end of the microneedle is understood to be the end of the microneedle that is closest to the base and that is the last to contact the skin when the microneedle is inserted into the skin.

[0024] The TM and BLM are incorporated into different hydrogel layers: BLM in the tip for deep tissue penetration, and TM in the base hydrogel layer for sustained superficial release

[0025] The microneedle material is, for example, PLGA, PLA, poly-gamma-glutamic acid (γ-PGA), hyaluronic acid, chitosan, silicon-based materials (e.g., PDMS), and metal materials.

[0026] A γ-PGA-based microneedle that loads BLM in the tip for deep tumor penetration and TM in the hydrogel base for vasoconstriction and inhibition of tumor growth. The released BLM and TM can simultaneously target different pathological levels of thickened IH, synergistically enhancing anti-angiogenic and pro-sclerotic effects. At the same time, the barb structure ensures long-term skin retention, overcoming the limitations of traditional microneedles that quickly fall off. Therefore, both in vitro and in vivo experiments have shown that this system significantly inhibits hemangioma cell proliferation, lumen formation, and tumor growth, providing a promising, minimally invasive, and highly transformative treatment strategy for the treatment of thickened IH.

[0027] This spatial separation allows for hierarchical-specific drug therapy, targeting the deep proliferative and superficial vascular components of thickened IH.

[0028] The system of the present invention for treating thickened infantile hemangiomas provides a safe, effective, and parent-friendly treatment option, particularly in situations where access to professional medical services is limited.

[0029] Compared to traditional treatments, the system of the present invention for treating (thickened) infantile hemangiomas, such as the TM-BLM@MN microneedles, offers several transformative advantages: (i) The spatially separated loading strategy allows BLM and TM to exert therapeutic effects at the deep and superficial layers respectively, achieving depth-specific drug delivery and producing synergistic anti-tumor and anti-angiogenic effects, suitable for enhancing the treatment of thickened IH. (ii) The barbed microstructure effectively anchors the microneedle patch to the skin, overcoming the limitation of rapid detachment and ensuring prolonged drug release and continuous therapeutic agent exposure at the lesion site. (iii) The minimally invasive nature of the MN and its potential for home administration may significantly improve treatment adherence in pediatric patients and reduce the clinical burden on families living in remote areas. Attached Figure Description

[0030] Figure 1 A schematic diagram illustrating the use of TM-BLM@MN to treat thickened IH;

[0031] Figure 2 Figure 1 shows the preparation and characterization results of TM-BLM@MN; where A is a schematic diagram of MN preparation, B is a local SEM image of MN at a scale of 200μm, C is a local SEM image of MN at a scale of 100μm, D is a local SEM image of MN at another angle at a scale of 100μm, E is a field of view of MN degradation at 85% humidity, F is a morphological image of skin with tumor tissue after MN insertion, and G is a field of view of the depth of MN insertion into tissue in vivo.

[0032] Figure 3 The figures show the in vitro validation results of TM-BLM@MN on the activity and migration of HemSCs; where A is the statistical graph of HemSCs activity after 3 days of treatment with various concentrations of TM, B is the statistical graph of HemSCs activity after 3 days of treatment with various concentrations of BLM, C is the statistical graph of HemSCs activity after treatment with various types of MN, D is the double staining image of HemSCs live / dead cells, E is the scratch test image of HemSCs with various MN treatments or no treatment, F is the statistical graph of the quantitative gap closure rate of the scratch test, G is the field of view of the lumen formation test, and H is the quantitative statistical graph of the branching points and tube length of HemSCs in each experimental group.

[0033] Figure 4 The figure shows the in vivo therapeutic effect of TM-BLM@MN in inhibiting the growth of IH tumors; where A is a schematic diagram of the establishment of the IH animal model and its treatment, B is a field of view of the tumor tissue in each experimental group, C is a schematic diagram of the measurement of tumor diameter, and D is a statistical graph of the quantitative results of tumor diameter.

[0034] Figure 5The in vivo validation results of TM-BLM@MN in reducing human angiogenesis are shown in the figure; where A is the anti-human CD31 staining map of each experimental group, and B is the quantitative statistical map of anti-human CD31 staining (significance compared with the TM-BLM@MN group).

[0035] Figure 6 The in vivo validation results of TM-BLM@MN's effect on reducing angiogenesis are shown in the figure; where A is the anti-mouse CD31 staining map of each experimental group, and B is the quantitative statistical map of anti-mouse CD31 staining (significance compared with the TM-BLM@MN group). Detailed Implementation

[0036] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0037] The following embodiments of the present invention provide a barbed microneedle system based on poly-γ-glutamic acid (γ-PGA). BLM is loaded onto the needle tip for deep tumor penetration, and TM is loaded onto a hydrogel substrate for vasoconstriction and inhibition of tumor growth (Figure 1). The released BLM and TM can simultaneously target different pathological layers of thickened intrahepatic lesions (IH), synergistically enhancing anti-angiogenic and anti-sclerotic effects. Simultaneously, the barbed structure ensures prolonged skin retention, overcoming the limitation of rapid detachment in traditional microneedles.

[0038] The main materials used in the following embodiments of the present invention are sourced from the following sources:

[0039] γ-PGA (molecular weight = 1,000–15,000) was purchased from CITES Biotechnology Co., Ltd. (China). Polydimethylsiloxane (PDMS) microneedle molds were provided by Taizhou Microchip Technology Co., Ltd. (China). Endothelial cell culture medium (ECM) supplemented with endothelial growth factor was purchased from ScienceCell (Carlsbad, USA; catalog number #1001). Fetal bovine serum (FBS) was purchased from Gibco (UK). The CD133 microbead kit for cell sorting was purchased from Miltenyi Biotec (Germany).

[0040] All other reagents and chemicals were of analytical grade.

[0041] The specific experimental methods used in the following embodiments of the present invention are described below:

[0042] 1) Preparation and characterization of TM-BLM@MN

[0043] γ-PGA hydrogel substrates were prepared by dissolving 350 mg γ-PGA in 600 μL of double-distilled water. For drug-loaded hydrogels, the double-distilled water was replaced with an aqueous solution containing 3 mg / mL TM or 400 μg / mL BLM. The preparation method for dual-loaded TM-BLM@MN was as follows: BLM-containing hydrogels were first loaded into a PDMS mold and then centrifuged to ensure complete filling of the microneedle tips. Subsequently, TM-loaded hydrogels were stacked to form a backing matrix. The molds were dried in a 40°C oven for 24 hours before demolding. Control MNs, including TM@MN (containing timolol only in the substrate), BLM@MN (containing bleomycin only in the needle tips), and blank PGA@MN (no drug), were all prepared using a similar method.

[0044] Morphological examination of MN was performed using a Nikon Eclipse E100 optical microscope. To assess in vitro biodegradability, MN loaded with indocyanine green was placed at 25°C and 85% humidity and monitored for 60 minutes using a Leica DMi8 fluorescence microscope.

[0045] For in vivo insertion analysis, MN patches were applied to the hairless dorsal skin of anesthetized NOD-SCID mice. Tissue sections were collected and stained with hematoxylin and eosin (H&E) to assess penetration depth and local tissue response.

[0046] 2) Isolation and culture of hemangioma stem cells (HemSCs)

[0047] Specimens used were obtained from the biobank in accordance with institutional guidelines and ethical approval. All patients signed written informed consent forms before the procedure. This study was approved by the Ethics Committee of the Ninth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine (Ethics Approval No. SH9H-2023-T304-2). Tissue samples were minced and digested with collagenase and DNase at 37°C for 2 hours. The resulting cell suspension was magnetically sorted using CD133 microbeads to separate HemSCs. Cells were cultured in ECM supplemented with 10% FBS at 37°C and 5% CO2.

[0048] 3) Preparation of RES@hmPDA-Dpep / MN

[0049] First, 10 mg or 5 mg of each nanoparticle of RES was uniformly dispersed in 600 μL of deionized water. 350 mg of γ-PGA was completely dissolved in the mixed solution to prepare a precursor hydrogel. Then, 200 μL of the mixture was added to a microneedle mold and centrifuged at 3750 rpm to obtain the needle tip. The mold was then placed in a 40°C drying oven overnight. After demolding, a patch was obtained and stored in a low-temperature environment with a desiccant.

[0050] Similarly, the control group, consisting only of γ-PGA microneedles, was prepared using the same procedure.

[0051] 4) CCK-8 method

[0052] HemSCs were seeded at a density of 2000 cells per well in 96-well plates and starved with serum for 12 hours. For drug toxicity assessment, cells were treated for 3 days with different concentrations of TM (0, 100, 150, 200, 250, 300 μg / mL) or BLM (0, 10, 20, 30, 40, 50 μg / mL). Cell viability was assessed using the CCK-8 assay according to the manufacturer's protocol. Absorbance was measured at 450 nm using a SpectraMAX iD3 microplate reader.

[0053] To verify the cytotoxicity of PGA@MN, TM@MN, BLM@MN, and TM-BLM@MN, MN was first immersed in 5 mL of complete cell culture medium for 1 hour to obtain MN extract. 2000 HemSCs were seeded in one well of a 96-well plate and cultured together with a solution containing 90 μL of complete cell culture medium and 10 μL of the corresponding extract. HemSCs cultured only in complete cell culture medium served as a control group.

[0054] 5) Double staining of live / dead cells

[0055] HemSCs were 4×10 5 Cells were seeded at a density in 6-well plates and then divided into seven groups: control, PGA@MN, TM, BLM, TM@MN, BLM@MN, and TM-BLM@MN, each interacting with its corresponding extract. After a 3-day cycle, Calcein-AM / PI assay reagent (300 µL) was added to each well to distinguish between live and dead cells according to the manufacturer's instructions. After incubation at 37°C for 15 minutes, fluorescence images were acquired using a Leica DMi8 microscope.

[0056] 6) In vitro cell migration analysis

[0057] Cell migration was assessed using a wound healing scratch assay. HemSCs were seeded in 6-well plates and serum starved for 12 hours. Linear scratches were carefully made in each confluent monolayer using a sterile pipette tip. Cells were then treated with 1 mL of complete culture medium combined with 1 mL of extract from PGA@MN, TM@MN, BLM@MN, or TM-BLM@MN. Control cells received only 2 mL of complete culture medium. After 12 hours of incubation, images of the scratched areas were captured at specified time points using a Nikon Eclipse TS100 microscope. The degree of migration was quantified by measuring the gap closure percentage using ImageJ software (National Institutes of Health).

[0058] 7) Lumen formation analysis

[0059] The anti-angiogenic activity of MN extract was evaluated using a Matrigel-based lumen formation assay. In short, 2.5 × 10⁻⁶ ppm was used. 4 One HemSC was resuspended in 100 µL of low-serum medium (1% FBS) containing PGA@MN, TM@MN, BLM@MN, TM-BLM@MN extracts, or a carrier control. The cell suspension was plated into Matrigel-coated 96-well plates and incubated at 37°C for 3 hours. Lumen formation was observed using a Nikon Eclipse TS100 microscope. The number of branching points and total tube length were quantified using ImageJ software and its Angiogenesis Analyzer plugin.

[0060] 8) Establishment of IH xenograft model and in vivo treatment

[0061] All experimental protocols were approved according to the *Laboratory Animal Care and Use Guidelines* and by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine (Approval No.: SH9H-2024-A1007-1), ensuring adherence to ethical standards. Thirty-five male NOD-SID mice (8 weeks old) were anesthetized, shaved, and had their dorsal skin disinfected. Two × 10^6 HemSCs were suspended in a mixture of 75 µL serum-free culture medium and 75 µL Matrigel matrix and subcutaneously injected into each mouse. After complete Matrigel gelation, the mice were divided into seven groups: control group, PGA@MN group, TM group, BLM group, TM@MN group, BLM@MN group, and TM-BLM@MN group. MN patches were applied once and fixed to the tumor site. Simultaneously, the TM group and BLM group received topical TM and BLM injections, respectively, as treatment to mimic current clinical treatment for IH. Ten days later, all mice were euthanized, and the tumors were carefully removed for further analysis.

[0062] 9) In vivo cytotoxicity analysis

[0063] To assess systemic toxicity, major organs (liver, heart, and kidney) were collected from mice in the TM-BLM@MN group after sacrifice. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E to identify histopathological changes.

[0064] 10) In vivo tumor regression and angiogenesis analysis

[0065] The excised tumor tissue was photographed and its diameter measured. The tissue was fixed in 4% PFA, paraffin-embedded, and sectioned for histological examination. H&E staining and Masson's trichrome staining were performed to assess tumor morphology and collagen deposition.

[0066] Immunohistochemical staining for CD31 (targeting human and mouse CD31) was performed to assess microvessel density and angiogenesis within the tumor.

[0067] 11) Statistical Analysis

[0068] All experimental results were replicated in at least three independent trials. Data are presented as mean ± standard deviation. Unpaired two-tailed Student's t-tests were used for comparisons between two groups. One-way ANOVA with appropriate post-hoc tests was used for comparisons among multiple groups. ImageJ was used for quantitative analysis of wound closure, lumen formation, and immunohistochemical signals. All statistical analyses were performed using GraphPad Prism (version 9.0). Significance levels are expressed as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, 'ns' indicates no significance.

[0069] Example 1: Preparation and Characterization of TM-BLM@MN

[0070] To overcome the limitations of traditional microneedle systems, particularly their tendency to detach prematurely and insufficient drug delivery efficiency, this embodiment designs and fabricates a barbed microneedle system for simultaneous dual drug delivery. The microneedles are composed of the biodegradable and biocompatible polymer γ-PGA. TM and BLM are integrated into separate hydrogel layers: BLM is located within the needle tip for deep tissue penetration, while TM is located within the base hydrogel layer for sustained superficial release (Figure 2A). This spatial separation allows for layer-specific drug therapy, targeting both the deep proliferative and superficial vascular portions of thickened IH.

[0071] Scanning electron microscopy (SEM) confirmed the successful fabrication of the barb microstructures, which facilitated enhanced fixation in tissue (Figs. 2B, 2C, and 2D). Each MN patch contained an 8 × 8 array with the needle body in a square pyramidal shape.

[0072] Under physiologically relevant conditions (85% humidity, 25°C), the drug release capacity and biodegradability of MN were evaluated using indocyanine green as a fluorescent tracer. Significant degradation of the microneedle tip was observed within 45 minutes (Fig. 2E), indicating rapid drug release after insertion. Mechanical testing confirmed that MN possessed sufficient robustness to penetrate mouse skin without breaking (Fig. 2F). Histological analysis using H&E-stained tissue sections showed an insertion depth of approximately 300 μm (Fig. 2G), sufficient to bypass the epidermal barrier and deliver the drug to the dermis and subcutaneous layer to enhance IH treatment.

[0073] In summary, this embodiment successfully developed a novel barbed microneedle system, TM-BLM@MN, for spatiotemporally controlled co-delivery of TM and BLM. The unique barbed design enhances skin adhesion, prolongs retention time, and enables deep, specific drug delivery. These characteristics collectively address key limitations of existing local and invasive treatments for thickened IH. This system represents a promising minimally invasive strategy that can improve drug efficacy and patient compliance, supporting its potential for clinical translation in the management of progressive IH.

[0074] Example 2: In vitro therapeutic effects of TM-BLM@MN

[0075] The cytotoxicity and optimal concentrations of TM and BLM against HemSCs were determined using the CCK-8 assay. HemSCs were treated with a series of concentrations of TM or BLM for 72 hours. The results showed that the optimal concentration of TM was 150 μg / mL and that of BLM was 20 μg / mL (Figures 3A and 3B). These concentrations were selected for all subsequent in vitro experiments to simulate combined treatment.

[0076] Subsequently, the cytotoxicity of MN was investigated by co-culturing HemSCs with extracts of different MNs. Compared with the control group and the single-drug-loaded MN or blank PGA@MN groups, the TM-BLM@MN group showed significantly stronger cytotoxicity (Figure 3C). Live / dead cell double staining experiments further confirmed this result (Figure 3D), visually demonstrating a significant increase in cell death after treatment with TM-BLM@MN extract, highlighting the enhanced efficacy of the dual-drug combination.

[0077] The anti-migration effect of MN extract was evaluated using a wound healing scratch assay. Representative images captured at 0 and 12 hours post-scratching are shown in Figure 3E. Quantitative analysis showed that TM-BLM@MN treatment reduced gap closure rate by 2.24-fold compared to the control group. Furthermore, the dual-drug system exhibited superior inhibitory effects on cell migration compared to TM@MN or BLM@MN alone, reducing gap closure rate by 1.76-fold and 1.49-fold, respectively (Figure 3F). These findings suggest a potent synergistic effect between TM and BLM in inhibiting HemSCs migration.

[0078] Furthermore, the anti-angiogenic potential of the treatment was evaluated using a Matrigel lumen formation assay. HemSCs were incubated with various MN extracts or no material (control group) and then plated onto cured Matrigel. Representative images after 3 hours are shown in Figure 3G. TM-BLM@MN treatment profoundly disrupted vascular network formation, reducing the number of branch points by 2.50-fold compared to the control group. This inhibitory effect was significantly stronger than either single-drug MN group, reducing branch points by 1.82-fold and 1.94-fold, respectively, compared to the TM@MN and BLM@MN groups. Similarly, the total vessel length in the TM-BLM@MN group was reduced by 3.19-fold compared to the control group, and by 2.32-fold and 2.52-fold, respectively, compared to the TM@MN and BLM@MN groups (Figure 3H).

[0079] In summary, these in vitro results confirm that the dual-drug loaded TM-BLM@MN system is superior to the single-drug formulation. The enhanced cytotoxicity, migration inhibition, and lumen formation inhibition exhibited by TM-BLM@MN indicate a strong synergistic interaction between timolol and bleomycin.

[0080] Example 3: In vivo therapeutic effects of TM-BLM@MN

[0081] To evaluate the in vivo antitumor efficacy of the TM-BLM@MN system, 2×10⁻⁶ cells were injected subcutaneously. 6 A hematopoietic stem cell transplantation (IH) model was established by injecting HemSCs (suspended in a mixture of 75 µL serum-free medium and 75 µL Matrigel) into 35 NOD-SCID mice (8 weeks old, male). After tumor formation, the mice were randomly divided into seven treatment groups (n = 5 per group), as shown in Figure 4A. Tumors were removed 10 days after treatment for macroscopic and histological evaluation. Representative images of the removed tumors clearly show a significant reduction in tumor size in the TM-BLM@MN group (Figure 4B).

[0082] Tumor diameter was precisely measured using calipers (Figure 4C). Quantitative analysis confirmed that TM-BLM@MN treatment resulted in a 1.93-fold reduction in tumor growth compared to the control group (Figure 4D). Monotherapy (TM@MN or BLM@MN) also induced statistically significant tumor suppression compared to the control group, while the dual-drug TM-BLM@MN system was significantly superior, with tumor size reductions of 1.58-fold and 1.45-fold, respectively, compared to the TM@MN and BLM@MN groups. These results clearly demonstrate the superior efficacy of combination therapy over monotherapy. Crucially, to assess the biocompatibility and potential systemic toxicity of the novel delivery system, histological analysis of major organs (heart, liver, and kidney) was performed using H&E staining. No significant pathological abnormalities were observed in the TM-BLM@MN group, indicating excellent in vivo biocompatibility and good safety.

[0083] In summary, in vivo results strongly demonstrate that the barbed dual-drug loaded TM-BLM@MN system achieves potent and synergistic inhibition of IH tumor growth. Compared with monotherapy and conventional administration routes, TM-BLM@MN treatment significantly enhances efficacy. TM-BLM@MN represents a transformable and highly effective strategy for treating thickened, proliferative infantile hemangiomas. Its combination of potency, minimal invasiveness, and excellent safety profile makes it a promising candidate for future clinical application, potentially improving the prognosis of patients with lesions resistant to conventional therapies.

[0084] Example 4: In vivo anti-angiogenic effect of TM-BLM@MN

[0085] To elucidate the mechanism by which TM-BLM@MN inhibits IH tumor growth, this embodiment examined its effect on tumor-associated angiogenesis on day 10 post-treatment.

[0086] CD31 (anti-human) immunohistochemical staining was performed to assess its specific effect on the human vascular network within the tumor. As shown in Figure 5A, TM-BLM@MN treatment resulted in a significant reduction in CD31-positive vessels.

[0087] Quantitative analysis showed that TM-BLM@MN inhibited angiogenesis by 4.46 times compared to the control group. Although both topical TM and intralesional BLM monotherapy produced significant anti-angiogenic effects, the dual-drug TM-BLM@MN system was significantly more effective, with an additional 4.08-fold and 2.54-fold reduction in vessel density compared to the TM@MN and BLM@MN groups, respectively (Figure 5B). These results highlight the superior anti-angiogenic efficacy resulting from the synergistic delivery of the two therapeutic agents.

[0088] The effects of TM-BLM@MN on the host-derived vascular system in the peritumoral region were further investigated using anti-mouse CD31 staining. Consistent with the findings within the tumor, TM-BLM@MN also effectively inhibited angiogenesis in mouse endothelial cells (Figure 6A), reducing angiogenesis by 2.34 times compared to the control group. Furthermore, its effect was superior to the two single-drug MN groups, achieving a 1.97-fold reduction relative to each single-drug group (Figure 6B).

[0089] In summary, the significantly enhanced inhibition of angiogenesis by the TM-BLM@MN group highlights the synergistic interaction between TM and BLM. The sustained release achieved by the MN system ensures continuous exposure of the tumor microenvironment to both drugs, leading to comprehensive vascular ablation. The significant reduction in CD31-positive structures in humans and mice (host-derived) indicates that TM-BLM@MN not only targets the tumor vascular system but also normalizes the blood supply to the surrounding matrix, effectively cutting off the tumor's nutrient and oxygen supply. Therefore, the TM-BLM@MN system primarily mediates potent inhibition of IH growth through a synergistic anti-angiogenic mechanism, demonstrating its clinical potential as an effective and minimally invasive therapeutic strategy for proliferative IH.

Claims

1. A system for treating infantile hemangiomas, characterized in that It comprises: a first active substance delivery part for delivering a first active substance to the superficial layer of the skin; and a second active substance delivery part for delivering a second active substance to the deep layer of the skin. The first active substance contains a beta-receptor blocker, and the second active substance contains an antibiotic. The beta-receptor blocker is selected from one or more of timolol, metoprolol and propranolol, and pharmaceutically acceptable salts thereof.

2. The system of claim 1, wherein The antibiotic has a sclerosing and anti-tumor effect.

3. The system of claim 1, wherein The antibiotic is bleomycin.

4. The system of claim 1, wherein 5. Use of the system of claim 1 in the preparation of a medical device or a drug. The beta-receptor blocker is applied to the superficial layer of the skin, and the antibiotic is applied to the deep layer of the skin.

6. A microneedle characterized by The beta-receptor blocker is selected from one or more of timolol, metoprolol and propranolol, and pharmaceutically acceptable salts thereof. The antibiotic has a sclerosing and anti-tumor effect. It comprises two or more tower layers, each tower layer has a quadrangular pyramid shape, and the radial edge has a prismatic structure.

7. The microneedle of claim 6, wherein, The beta-receptor blocker is loaded on the tower layer near the proximal end of the microneedle, and is applied to the superficial layer of the skin; the antibiotic is loaded on the tower layer near the distal end of the microneedle, and is applied to the deep layer of the skin.

8. The microneedle of claim 7, wherein, The microneedles are arranged in an array to form a microneedle patch.

9. The microneedle of claim 7, wherein, 10. A medical device comprising the microneedle of claim 6. ​

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