A nanogel containing periplaneta americana extract and a preparation method and application thereof

Abstract

The application discloses a nanogel containing periplaneta americana extract and a preparation method and application thereof, and belongs to the technical field of biological medicines. The nanogel preparation is prepared by taking the periplaneta americana extract as an active ingredient and collocating carbomer 940, caprylocaproyl glycerides, polyoxyethylene (40) hydrogenated castor oil, 1,2-propanediol and other auxiliary materials; the nanogel has an average particle size of less than 200 nm, an absolute value of Zeta potential of more than 30 mV, and good stability under normal temperature, high and low temperature and centrifugal conditions. Animal experiments prove that the nanogel can significantly improve the alopecia symptom of a perhexiline-induced alopecia mouse, increase the number and depth of hair follicles, reduce the expression of inflammatory factors such as IFN-gamma, IL-15 and CXCL-9 in skin tissues, inhibit the CD8+T cell infiltration in the spleen and liver of the mouse, and promote the recovery of the hair follicle immune homeostasis. + NKG2D + T cell activation, restore hair follicle immune homeostasis, and promote hair growth, and has no obvious toxic and side effects. The preparation process of the periplaneta americana extract nanogel is simple, the nanogel has stable properties and high safety, can be used for preparing medicines for preventing and / or treating alopecia diseases such as alopecia areata and chemotherapy-induced alopecia, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nanogel containing extracts of American cockroaches, its preparation method, and its application in the preparation of drugs for the prevention and / or treatment of alopecia areata or chemotherapy-induced hair loss. Background Technology

[0002] Alopecia areata is a common inflammatory non-scarring hair loss. Pathologically, it is characterized by T-lymphocyte infiltration around the hair follicles, leading to disruption of the immune immunity of the hair follicles during the growth phase and damage to the hair follicle epithelial cells, thus causing abnormal hair growth. The main clinical manifestation is sudden onset of round or oval patches of hair loss with well-defined borders, smooth skin surface, no inflammation, scaling, or scarring, and usually no subjective symptoms. Alopecia areata can occur at any age, but is more common in young and middle-aged adults. The prevalence in my country is approximately 0.27%, with a lifetime prevalence of about 2%. Although the skin appears normal after hair loss and there are no subjective symptoms, it severely damages the patient's appearance, causing significant psychological burden, increasing the risk of mental illness, and leading to many complications such as rheumatoid arthritis, ulcerative colitis, systemic lupus erythematosus, and pernicious anemia, seriously affecting the patient's physical and mental health.

[0003] The pathogenesis of alopecia areata is extremely complex and influenced by multiple factors, such as genetics, immune abnormalities, psychological stress, infection, and endocrine factors. Among these, loss of hair follicle immune function is a significant cause. Hair follicles are the organs of hair growth. As tissues with immune-immune characteristics, hair follicles normally actively suppress immune surveillance and inflammatory responses through various mechanisms to protect themselves from immune-mediated damage. However, under the influence of susceptible factors, the immune-immune function of hair follicles collapses. Studies have shown that this collapse is accompanied by abnormal upregulation of MHC class I and II molecules in hair follicle epithelial cells, allowing self-antigens to be presented to immune cells, thereby triggering the activation and infiltration of autoreactive T cells. In this process, CD8... + NKG2D + Cytotoxic T cells have been identified as the core pathogenic population mediating hair follicle damage. In alopecia areata lesions, infiltrating CD8 cells... + T cells highly express the NKG2D receptor, forming characteristic CD8+ receptors. + NKG2D + T cell subsets. CD8 + NKG2D + T cells exhibit the phenotypic characteristics of effector memory T cells and can highly express IFN-γ, which can further disrupt the immune-immune state of hair follicles. Furthermore, IFN-γ can significantly enhance the secretion of IL-15 by hair follicle epithelial cells, and IL-15 can stimulate CD8... + NKG2D+ T cells produce more IFN-γ, which exacerbates the occurrence of alopecia areata.

[0004] Hair follicle immune exemption collapse and CD8 + NKG2D + T-cell-mediated autoimmune attack constitutes a core component of the pathogenesis of alopecia areata. Loss of immune immunity in hair follicles allows for the exposure of autoantigens, and CD8... + NKG2D + T cells, as the main effector cells, can promote the secretion of related factors, alter the immune environment around hair follicles, and trigger alopecia areata. Therefore, blocking CD8... + NKG2D + Improving the function of T cells, reducing the secretion level of related factors, and restoring the homeostasis of the hair follicle immune microenvironment are among the most promising intervention strategies in the treatment of alopecia areata.

[0005] The American cockroach (Periplaneta americana), belonging to the genus Periplaneta in the family Blattidae, is one of the oldest and most successfully reproducing insect groups in the world. It has a salty and cold nature, and enters the liver, spleen, and kidney meridians. It possesses the effects of promoting blood circulation, removing blood stasis, detoxifying, reducing malnutrition in children, and promoting diuresis and reducing swelling. In folk medicine and traditional medicine, the American cockroach is mainly used to treat injuries from falls and blows, carbuncles and boils, infantile malnutrition, and sore throat. Modern clinically used pharmaceutical products made from American cockroaches include Kangfuxin Liquid, Xinmailong Injection, and Ganlong Capsules. The American cockroach is rich in various chemical components such as proteins, amino acids, peptides, glycoproteins, fatty acids and esters, alkaloids, steroids, and isocoumarins. Studies have shown that it contains secondary metabolites such as dopamine derivatives, cyclic peptides, isoflavones, and phenolic compounds, as well as more than 16 amino acids, including 7 essential amino acids for humans. These abundant active substances constitute the material basis for its broad pharmacological effects. Modern pharmacological studies have shown that American cockroach extract possesses multifaceted biological activities, including antitumor, anti-inflammatory, antibacterial, antiviral, hepatoprotective, antioxidant, immune-enhancing, tissue-repair-promoting, microcirculation-improving, and anti-organ fibrosis effects. In terms of tissue repair, it can promote collagen production and deposition in wounds, accelerating mucosal healing; in terms of antitumor activity, it exerts its effects through mechanisms such as inhibiting tumor cell proliferation, inducing cell cycle arrest and apoptosis, and reversing drug resistance. Therefore, American cockroaches have significant development value, and their remarkable anti-inflammatory activity suggests a potential role in the treatment of alopecia areata.

[0006] Currently, treating alopecia areata remains a major clinical challenge. There are no specific drugs that can cure the condition or prevent recurrence; existing treatments primarily aim to control disease progression and promote hair regrowth. Traditional treatment regimens mainly include topical or systemic application of corticosteroids, topical minoxidil, contact immunotherapy (such as diphenylcyclopropenone), and photochemotherapy. For severe alopecia areata, traditional therapies often have low response rates or significant side effects; for example, long-term use of hormones can lead to skin atrophy, telangiectasia, acne, and metabolic disorders caused by systemic absorption. Immunosuppressants such as cyclosporine and methotrexate have been attempted for refractory patients, but their potential risks, such as nephrotoxicity, hepatotoxicity, and bone marrow suppression, limit their widespread clinical application.

[0007] In recent years, targeted drugs targeting key pathways in the pathogenesis of alopecia areata have made groundbreaking progress. Based on the core role of JAK-STAT pathway activation mediated by cytokines such as IFN-γ and IL-15 in the pathogenesis of alopecia areata, JAK inhibitors became the first approved targeted drugs for the treatment of severe alopecia areata. Currently, oral JAK inhibitors such as baricitinib and ritlecitinib have been approved in the United States, China, and other countries for the treatment of severe alopecia areata. Clinical studies have shown that they can significantly promote hair regeneration and improve patients' quality of life. However, these targeted small molecule drugs still face some challenges: First, they require long-term daily oral administration, and the disease is prone to relapse after discontinuation, suggesting the need for maintenance therapy; second, although the overall safety of JAK inhibitors is manageable, they are still accompanied by adverse reactions such as infection risks (e.g., upper respiratory tract infection, herpes zoster) and elevated blood lipids; in addition, these drugs are relatively expensive, and accessibility and economic burden remain prominent issues for patients requiring long-term treatment. Therefore, in-depth analysis of the complex immune regulatory network of alopecia areata, especially the pathological cells—CD8... + NKG2D + Discovering new T-cell therapies, identifying new therapeutic targets, and developing safer, more effective, convenient, and economical treatments remain the focus and challenge of current research on alopecia areata.

[0008] Furthermore, currently available commercially available formulations using American cockroach extract as the active ingredient are mainly oral or topical liquid preparations—such as Kangfuxin Liquid (KFX Liquid). However, the stratum corneum of the skin, as an important physical barrier of the body, has a natural barrier effect against hydrophilic macromolecules (such as peptides and polysaccharides). The active ingredients in American cockroach extract that exert anti-inflammatory and tissue-repairing activities are mostly hydrophilic macromolecules. When applied topically using ordinary solution formulations, the active ingredients have difficulty penetrating the stratum corneum barrier and cannot effectively reach the hair follicle target, resulting in low local bioavailability and slow onset of action, thus limiting its clinical application in the treatment of hair loss. In addition, ordinary liquid preparations have a short residence time on the skin surface, requiring frequent administration, leading to poor patient compliance.

[0009] Nanogels, as novel topical formulations, possess advantages such as small particle size, strong transdermal permeability, long local retention time, and high bioavailability, enabling targeted delivery of active ingredients. Currently, there are no methods to prepare American cockroach extract into nanogels for treating alopecia areata or inhibiting CD8. + NKG2D + Reports on T-cell activation and restoration of hair follicle immune immunity have been published. Based on this, the present invention provides a *Periplaneta americana* extract nanogel, overcoming the shortcomings of existing technologies and offering a new drug option for the treatment of alopecia areata. Summary of the Invention

[0010] To address the problems of existing alopecia areata treatments, such as significant side effects, high recurrence rates, and poor transdermal absorption, as well as the lack of nano-targeted delivery of American cockroach extract formulations, this invention aims to provide an American cockroach extract nanogel and its preparation method. This nanogel exhibits good stability, strong transdermal permeability, and high safety, and can effectively inhibit CD8. + NKG2D + T cell activation reduces the expression of inflammatory factors, restores the immune immunity homeostasis of hair follicles, and significantly promotes hair growth in alopecia areata.

[0011] To achieve the above objectives, the present invention provides the following technical solution: On one hand, the present invention provides a nanogel containing American cockroach extract, comprising the following components: American cockroach extract, gelling agent, oil phase, surfactant, co-surfactant, and water.

[0012] Preferably, by weight, the American cockroach extract is 0.05-0.2 parts, the gelling agent is 0.025 parts, the oil phase is 0.1047-0.1136 parts, the surfactant is 0.4712-0.5111 parts, the co-surfactant is 0.4712-0.5111 parts, and the water is 3.7278-3.7893 parts.

[0013] Preferably, the mass fraction of the American cockroach extract in the nanogel containing the American cockroach extract is 1% to 4%.

[0014] Preferably, the gelling agent is carbomer 940; the oil phase is glyceryl caprylate; the surfactant is polyoxyethylene (40) hydrogenated castor oil; and the co-surfactant is 1,2-propanediol.

[0015] Preferably, the average particle size of the nanogel is <200 nm and the absolute value of the Zeta potential is >30 mV.

[0016] On the other hand, the present invention provides a method for preparing the above-mentioned nanogel containing American cockroach extract, comprising the following steps: (1) Weigh the oil phase, surfactant, co-surfactant and American cockroach extract, mix them, heat and stir, sonicate and continue stirring until homogeneous; (2) Add purified water dropwise to the system obtained in step (1), and stir under heating until the system becomes transparent to obtain nanoemulsion; (3) Weigh the gelling agent and dissolve it in the remaining purified water, let it swell overnight, and obtain the gel matrix; (4) Add the nanoemulsion obtained in step (2) to the gel matrix obtained in step (3) and stir to mix well to obtain the nanogel.

[0017] Preferably, the American cockroach extract is prepared by the following steps: pulverizing dried American cockroach bodies, extracting them by heating and reflux with an aqueous ethanol solution of 70%~95% by volume, filtering, concentrating the filtrate under reduced pressure, and drying to obtain the extract.

[0018] Preferably, in step (1), the ultrasonic power is 200~300 W and the ultrasonic time is 20 minutes; in steps (2) and (4), the stirring speed is 300~500 rpm; and in steps (1) and (2), the heating temperature is 60℃.

[0019] In another aspect, the present invention provides the use of the above-mentioned nanogel containing American cockroach extract in the preparation of medicaments for the prevention and / or treatment of alopecia areata or chemotherapy-induced hair loss.

[0020] Preferably, the nanogel containing American cockroach extract inhibits CD8. + NKG2D + T-cell function or improvement of hair follicle immune exemption collapse can prevent and / or treat alopecia areata or chemotherapy-induced hair loss.

[0021] Compared with the prior art, the beneficial effects of the present invention are: Significant advantages of the formulation: This invention uses a nanogel formulation with an average particle size of <200 nm and an absolute Zeta potential of >30 mV. It exhibits excellent colloidal stability, maintaining uniform properties under normal temperature, high and low temperature, and centrifugation conditions, without stratification or precipitation. The synergistic delivery of multiple substances in the formulation results in strong transdermal absorption, enabling targeted delivery of American cockroach extract to the hair follicle, achieving high local drug concentration and significantly improved bioavailability.

[0022] Outstanding therapeutic effects: The nanogel of this invention can significantly improve hair loss symptoms in mice with alopecia areata, increase the number and depth of hair follicles, and promote hair growth more effectively than the positive control drug minoxidil; it can directly target the core pathogenesis of alopecia areata and inhibit CD8. + NKG2D + T cell activation reduces the expression of key inflammatory factors such as IFN-γ and IL-15, effectively restoring the immune exemption homeostasis of hair follicles and treating alopecia areata from the root cause.

[0023] High safety: This invention uses natural American cockroach extract as the active ingredient, without the toxic side effects of drugs such as glucocorticoids and JAK inhibitors. Animal experiments have shown that it does not damage the main organs of mice, such as the heart, liver, spleen, lungs and kidneys, and has high safety for long-term use.

[0024] The preparation process is simple: the preparation method of the present invention is simple to operate, has mild conditions, good repeatability, does not require complex equipment, is suitable for industrial production, has low cost, and has strong drug accessibility.

[0025] Wide range of applications: The nanogel of this invention can not only be used for the treatment of alopecia areata, but can also be extended to the prevention and treatment of other hair loss diseases, including chemotherapy-induced alopecia, and has broad clinical application prospects. Attached Figure Description

[0026] Figure 1 Characterization diagram of American cockroach extract nanogel; where A is 1% American cockroach extract nanogel, B is 2% American cockroach extract nanogel, and C is 4% American cockroach extract nanogel. Figure 2 Particle size and zeta potential of nanogels containing different concentrations of American cockroach extract; where A is the particle size distribution and B is the zeta potential. Figure 3 The graph shows the changes in particle size and zeta potential of the American cockroach extract nanogel under different conditions; where A and B represent the changes in particle size and zeta potential of 1% American cockroach extract nanogel under different placement conditions, respectively; C and D represent the changes in particle size and zeta potential of 2% American cockroach extract nanogel under different placement conditions, respectively; and E and F represent the changes in particle size and zeta potential of 4% American cockroach extract nanogel under different placement conditions, respectively. Figure 4 Effect of American cockroach extract nanogel on hair growth in mice with alopecia areata (patchy alopecia areata) Figure 5 The effect of American cockroach extract nanogel on the skin color score on the back of mice with alopecia areata; Figure 6 The effect of American cockroach extract nanogel on the quality of new hair growth in mice with alopecia areata; where A represents the length of new hair growth in each group of mice on day 31; B represents the length of new hair growth in each group of mice on day 32; and C represents the weight of new hair growth in each group of mice on day 32. Figure 7The effect of American cockroach extract nanogel on the number and structure of hair follicles in mice with alopecia areata; where A is the result of HE staining of the back skin, magnification 5×; B is the result of HE staining of the back skin, magnification 10×; C is the result of HE staining of the back skin, hair follicle count; D is the result of HE staining of the back skin, hair follicle depth. Figure 8 The effect of American cockroach extract nanogel on the expression of inflammatory factors mRNA in the skin of mice with alopecia areata; where A, E, and F represent the results of mRNA expression analysis of IFN-γ, IL-12, IL-15, CXCL-9, and CXCL-10 in the dorsal skin of mice, respectively. Figure 9 American cockroach extract nanogel on CD8 in spleen of mice with alopecia areata + NKG2D + The influence of T cell subsets is shown in the graph; where A is a scatter plot of flow cytometry data of spleen lymphocytes in each group of mice; B is a graph of CD8+ in the spleen of each group of mice. + NKG2D + A quantitative statistical bar chart of the proportion of T cells to lymphocytes; Figure 10 Figure showing the effect of American cockroach extract nanogel on organ indices in mice with alopecia areata. Figure 11 In vivo fluorescence imaging of Nile Red-labeled Kangfuxin Liquid and American cockroach extract nanogel on the back of alopecia areata mice (0, 2, 4, 6, 12 h after application). Detailed Implementation

[0027] To address the problems of existing alopecia areata treatments, such as significant side effects, high recurrence rates, and poor transdermal absorption, as well as the lack of nano-targeted delivery of American cockroach extract formulations, this invention provides an American cockroach extract nanogel and its preparation method. This nanogel exhibits good stability, strong transdermal permeability, and high safety, and can effectively inhibit CD8. + NKG2D + T cell activation reduces the expression of inflammatory factors, restores the immune immunity homeostasis of hair follicles, and significantly promotes hair growth in alopecia areata.

[0028] The present invention will be further described below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications, equivalent substitutions, or improvements made based on the principles of the present invention should be included within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments were performed under conventional conditions. Unless otherwise specified, the materials and reagents used are commercially available. All animal experiments were approved by the institution's animal ethics committee and complied with laboratory animal ethics guidelines.

[0029] Example Example 1: Preparation of American cockroach extract Dried American cockroach bodies were pulverized and passed through a 40-mesh sieve. 500 g of the powder was weighed and added to 4 L of a 75% (v / v) ethanol aqueous solution. The mixture was heated under reflux and extracted twice, 2 hours each time. The extracts were combined. The extract was filtered through a 200-mesh filter cloth and concentrated under reduced pressure at 60°C to a thick paste with a relative density of 1.10–1.20. The paste was then freeze-dried to obtain the American cockroach extract.

[0030] It should be noted that the preparation method of American cockroach extract given in this embodiment is only an example. Those skilled in the art can use other conventional extraction methods to prepare American cockroach extract. As long as American cockroach extract containing effective ingredients can be obtained, the technical effects of this invention can be achieved.

[0031] Example 2: Preparation of American cockroach extract nanogel 1. The formulation of the American cockroach extract nanogel of the present invention The present invention relates to a nanogel of American cockroach extract, which is composed of American cockroach extract, carbomer 940, caprylic / capric triglyceride, polyoxyethylene (40) hydrogenated castor oil, 1,2-propanediol and water; wherein American cockroach extract is the active ingredient, carbomer 940 is the gel matrix material, caprylic / capric triglyceride is the oil phase, polyoxyethylene (40) hydrogenated castor oil is the surfactant, 1,2-propanediol is the co-surfactant, and water is the solvent.

[0032] In a preferred embodiment of the present invention, the mass fraction of the American cockroach extract is 1%, 2%, or 4%, and the specific formulation is as follows: 1% American cockroach extract nanogel: American cockroach extract 0.0500 g, carbomer 0.0250 g, caprylic / capric triglyceride 0.1136 g, polyoxyethylene (40) hydrogenated castor oil 0.5111 g, 1,2-propanediol 0.5111 g, water 3.7893 g; 2% American cockroach extract nanogel: American cockroach extract 0.1000 g, carbomer 0.0250 g, caprylic / capric triglyceride 0.1108 g, polyoxyethylene (40) hydrogenated castor oil 0.4986 g, 1,2-propanediol 0.4986 g, water 3.7688 g; 4% American cockroach extract nanogel: American cockroach extract 0.2000 g, carbomer 0.0250 g, caprylic / capric triglyceride 0.1047 g, polyoxyethylene (40) hydrogenated castor oil 0.4712 g, 1,2-propanediol 0.4712 g, water 3.7278 g.

[0033] 2. Preparation method 1) Weigh the oil phase (caprylic / capric triglyceride), surfactant (polyoxyethylene (40) hydrogenated castor oil), co-surfactant (1,2-propanediol) and American cockroach extract into a beaker, heat and stir at 60°C until homogeneous, sonicate at 250 W for 20 min, transfer to a magnetic stirrer, and stir at 400 rpm for 15 min. 2) Add the prescribed amount of purified water dropwise to the above system and continue magnetic stirring at 60°C and 400 rpm until the system is completely transparent to obtain drug-loaded nanoemulsion; 3) Weigh the prescribed amount of Carbomer 940 and dissolve it in the remaining purified water. Let it stand at room temperature overnight to swell and obtain a transparent gel matrix; 4) Slowly add the drug-loaded nanoemulsion to the gel matrix and stir at 400 rpm to obtain the American cockroach extract nanogel.

[0034] Example 3: Quality evaluation of American cockroach extract nanogels 1. Experimental Methods: 1) Initial state assessment: The 1%, 2% and 4% concentrations of American cockroach extract nanogels prepared in Example 2 were taken and their appearance was observed under natural light. The particle size was measured by dynamic light scattering instrument and the zeta potential was measured by electrophoretic light scattering method.

[0035] 2) Stability test at room temperature: Three batches of American cockroach extract nanogels were stored at room temperature (25℃ ± 2℃). Samples were taken after one week to observe the appearance of the American cockroach extract nanogels, determine their particle size using a dynamic light scattering instrument, and measure their zeta potential using electrophoretic light scattering to evaluate their stability at room temperature.

[0036] 3) Low-temperature stability test: Three batches of American cockroach extract nanogels were stored at low temperature (-20℃). Samples were taken after 24 hours, and after thawing, the appearance of the American cockroach extract nanogels was observed. The particle size was determined by dynamic light scattering, and the zeta potential was determined by electrophoretic light scattering to evaluate its low-temperature stability.

[0037] 4) High-temperature stability test: Three batches of American cockroach extract nanogels were stored at high temperature (40℃). Samples were taken after 24 hours to observe the appearance of the American cockroach extract nanogels, determine their particle size using a dynamic light scattering instrument, and measure their zeta potential using electrophoretic light scattering to evaluate their high-temperature stability.

[0038] 5) Centrifugal stability test: Take appropriate amounts of three batches of American cockroach extract nanogels and place them in 1.5 mL centrifuge tubes. Centrifuge at 3000 rpm for 30 min. Immediately after centrifugation, observe whether the samples show stratification, precipitation, or flocculation. Evaluate the precipitation. If precipitation occurs, apply a small amount of mechanical force to mix it. Observe the appearance of the American cockroach extract nanogels, determine their particle size using a dynamic light scattering instrument, and determine the zeta potential using electrophoretic light scattering to evaluate their centrifugal stability.

[0039] 2. Experimental Results (1) Initial state assessment like Figure 1 As shown, the 1% American cockroach extract nanogel is white and transparent, the 2% American cockroach extract nanogel is slightly yellow and transparent, and the 4% American cockroach extract nanogel is yellow and transparent. All three have a fine texture, intact shape, moderate consistency, and are easy to apply. There are no visible particles, sediments, or stratification.

[0040] like Figure 2 As shown, the average particle sizes of the 1%, 2%, and 4% nanogels were 185.3 nm, 189 nm, and 193.3 nm, respectively, all <200 nm, indicating that the American cockroach extract nanogels prepared in this invention have good particle size distribution; the Zeta potentials were -33.6 mV, -31.5 mV, and -31.5 mV, respectively, all with absolute values ​​>30 mV, indicating that the American cockroach extract nanogels prepared in this invention can effectively prevent the nanogels from agglomerating during storage and have excellent colloidal stability.

[0041] (2) Stability test like Figure 3 As shown, the 1%, 2%, and 4% *Periplaneta americana* extract nanogels exhibited uniform and transparent appearance after one week of storage at room temperature, and 24 hours of storage under two different conditions (-20℃ and 40℃), with no flocculation, precipitation, or phase separation. After centrifugation at 3500 r / min for 30 min, slight stratification occurred in the *Periplaneta americana* extract nanogels; however, this was easily remixed and the stratification disappeared after applying a small amount of mechanical force (gentle shaking). Particle size and Zeta potential results indicated that, at the same dilution factor, the particle size and Zeta potential of the *Periplaneta americana* extract nanogels remained within the effective range after one week of storage at room temperature, 24 hours of storage at low or high temperatures, and before and after high-speed centrifugation, without significant changes, demonstrating the stable and controllable quality of the *Periplaneta americana* extract nanogels.

[0042] Example 4: Pharmacodynamic evaluation of American cockroach extract nanogels 1. Experimental Materials SPF-grade female C57BL / 6J mice, 8-10 weeks old, weighing 18-22 g, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., Laboratory Animal Production License No.: SCXK(Su)2025-0012; Laboratory Animal Quality Certificate No.: B202511240385. They were housed in an SPF-grade animal facility at a temperature of 25±2℃ and a relative humidity of 55%, with a 12-hour light-dark cycle. The animals had free access to food and water.

[0043] Unless otherwise specified, all reagents used in the following examples are commercially available analytical grade or biochemical reagents. 2. Experimental Methods (1) Animal model establishment and grouping After one week of acclimatization feeding, the mice were plucked from their backs to establish a model and randomly divided into 6 groups of 10 mice each: control group, model group, minoxidil group, 1% nanogel group (L-PAEGel), 2% nanogel group (M-PAEGel), and 4% nanogel group (H-PAEGel).

[0044] (2) Modeling and drug administration On the 10th day after hair removal, except for the normal group, all other groups were injected intraperitoneally with cyclophosphamide 150 mg / kg to induce alopecia areata model. Starting from the 2nd day after hair removal, the normal group and the model group were coated with blank matrix, the minoxidil group was coated with 5% minoxidil, and the nanogel group was coated with the corresponding preparation, twice a day for 32 consecutive days.

[0045] (3) Observation indicators and detection methods 1) Hair growth observation: Observe the hair growth on the back of the mice every day, take pictures and record the data, and score the skin color. The scoring criteria are as follows: pink skin is 0 points; turning from pink to grayish-white is 1 point; turning from grayish-white to black is 2 points; black skin with a few hairs growing out is 3 points; and skin with no difference from the hair in the non-haired area is 4 points. 2) Hair quality test: On days 31 and 32, the length of the hair on the back of each mouse was measured using calipers. On day 32, all the new hair growing in the bald area on the back of the mouse was shaved off using a razor, and the weight of the new hair was measured using an analytical balance. 3) Histological examination: Mice were sacrificed, and the skin on the back of the mice was collected. A portion of the skin was fixed in 4% paraformaldehyde and stained with hematoxylin and eosin (HE). Histological changes in the skin were observed. The number of hair follicles in the back skin was observed under 5× field of view, and the depth of 6 hair follicles was randomly measured under 10× field of view, and changes in hair follicle structure were observed. 4) Detection of inflammatory factors: The skin on the back of another group of mice was flash-frozen in liquid nitrogen and stored at -80℃. The expression of multiple inflammatory factors such as IFN-γ, IL-12, IL-15, CXCL-9, and CXCL-10 was analyzed by real-time quantitative PCR (PT-qPCR). 5) Immune cell detection: After separating the mouse skin, the spleens of each group of mice were isolated. Three mice from each group were randomly selected for their spleens. Single-cell suspensions were extracted, and flow cytometry was used to detect CD8+ in the mouse spleens. + NKG2D + T cell ratio.

[0046] 2. Experimental Results (1) Hair growth In the control group, the back fur of mice was dark and smooth, with no hair loss observed; in the model group, the back fur of mice was completely lost, and the newly grown fur was sparse and easily lost; in the minoxidil group, hair growth was obvious in the early stage, but significant hair loss occurred in the later stage; in each nanogel group, the back skin of mice turned black earlier, and hair growth was vigorous. By day 32, the 2% and 4% concentration groups had almost full hair growth, and the effect was significantly better than that of the minoxidil group. Figure 4 , Figure 5 ).

[0047] The above results indicate that different doses of the American cockroach extract nanogel of the present invention can improve the clinical symptoms of alopecia areata to a certain extent and promote hair growth in mice with alopecia areata, with better effects than the positive control drug minoxidil.

[0048] (2) Hair quality Compared with the model group, the hair length and weight of mice in the nanogel group were significantly increased (P<0.01 or P<0.001), with the 4% concentration group showing the best effect. Furthermore, the nanogel group was superior to the minoxidil group in all aspects. Figure 6 ).

[0049] The above results indicate that the American cockroach extract nanogel can improve the back hair growth and the quality of new back hair in mice with cyclophosphamide-induced alopecia areata, and its effect is better than that of minoxidil.

[0050] (3) Hair follicle repair HE staining results showed that the number of hair follicles in the model group was significantly reduced, with most follicles in the regression or resting phase, the hair bulb atrophied, and inflammatory cell infiltration visible around the hair follicles; the number and depth of hair follicles in the nanogel group were significantly increased (P<0.001), most hair follicles were in the growth phase, the hair bulb was full, the hair follicle structure was intact, and the repair effect was better than that in the minoxidil group. Figure 7 ).

[0051] The above results indicate that the American cockroach extract nanogel of the present invention can effectively promote hair follicle regeneration and repair in alopecia areata model mice.

[0052] (4) Inhibition of inflammatory factors The results showed that the expression of IFN-γ, IL-12, IL-15, CXCL-9, and CXCL-10 mRNA was significantly increased in the model group mice (P<0.001), while the expression of IFN-γ, IL-12, IL-15, CXCL-9, and CXCL-10 mRNA was significantly decreased in the skin of the nanogel group mice (P<0.01 or P<0.001). This indicates that the nanogel of the present invention can effectively inhibit the high expression of IFN-γ, IL-12, IL-15, CXCL-9, and CXCL-10 genes in the skin of mice with cyclophosphamide-induced alopecia areata. Figure 8 ).

[0053] (5) Regulation of immune cells The results showed that the spleen CD8 in the model group mice + NKG2D + The proportion of T cells was significantly higher in the nanogel group than in the control group (P<0.001), and the spleen CD8 count was higher in the nanogel group. + NKG2D + The proportion of T cells was significantly reduced (P < 0.01 or P < 0.001), indicating that the American cockroach extract nanogel of the present invention can effectively inhibit CD8. + NKG2D + T cell activation reduces the immune attack on hair follicles, restores the immune immunity homeostasis of hair follicles, thereby helping hair follicles restore or rebuild the immune immunity environment and promote hair regeneration. Figure 9 ).

[0054] Example 5: Safety Evaluation Experiment This invention uses natural American cockroach extract as the active ingredient, delivered topically via a nanogel, which should theoretically have good safety. To further verify this, a systematic safety evaluation was conducted on mice administered the drug continuously for 32 days in this embodiment.

[0055] Experimental method: After continuous administration for 32 days (same as in Example 4), mice were sacrificed, and the heart, liver, spleen, lung, kidney, and thymus were collected, weighed, and organ indices were calculated.

[0056] Experimental results: such as Figure 10As shown, there were no significant differences in the heart, liver, lung, kidney, and thymus indices of mice in each nanogel administration group compared with the normal group (P>0.05), and the spleen index was also not significantly different from the model group (P>0.05). No obvious tissue damage was observed in any organ, indicating that continuous external application of the nanogel of this invention for 32 days did not cause significant systemic toxicity, had no obvious toxic side effects, and demonstrated high safety. Furthermore, no abnormal behaviors (such as decreased appetite, lethargy, and reduced activity) were observed in mice during the administration period. The weight gain curves were basically consistent with those of the normal group, and no abnormal changes in color, size, or texture were observed in the anatomy of any organ (data not shown).

[0057] The above results indicate that the American cockroach extract nanogel of the present invention has no obvious toxic side effects at the administered dosage, has high safety, and has good prospects for clinical application.

[0058] Comparative Example 1: Follicle-targeted delivery of conventional liquid formulations versus the nanogel of this invention Existing American cockroach hair growth preparations are mostly ordinary liquid formulations with poor transdermal absorption. To verify whether the nanogel of this invention can achieve a hair follicle-targeted delivery effect superior to ordinary liquid formulations, a hair follicle-targeted delivery experiment was conducted.

[0059] Experimental Methods: After one week of acclimatization feeding, the hair on the backs of mice was shaved to avoid damaging the hair follicles. Mice were randomly divided into two groups: a Nile Red-labeled KFX solution group (KFX applied topically) and a Nile Red-labeled nanogel group (the nanogel of this invention applied topically), with five mice in each group. After 24 hours, the corresponding formulation was evenly applied to the back skin of each group, and gently massaged to promote penetration. The fluorescence signal intensity and retention time of the drug on the mouse backs were observed at different time points using an in vivo imaging system. After observation, the mouse back skin was harvested, embedded, sectioned, and the drug follicle enrichment intensity and retention time were observed.

[0060] Experimental results: such as Figure 11 As shown, the fluorescence signal of the ordinary liquid formulation group was significantly weakened 4 h after application, and the fluorescence signal at the administration site was significantly reduced at 12 h; the fluorescence signal of the nanogel of the present invention was weakened 2 h after application, but the fluorescence signal remained stable thereafter, and there was still a significant fluorescence signal in the administration area at 12 h.

[0061] The above results demonstrate that, compared to conventional solution formulations, the nanogel of this invention can stably and specifically deliver the active ingredients to the deep hair follicles and prolong their retention time, thereby improving local bioavailability, reducing the frequency of administration, and enhancing patient compliance. This delivery advantage is attributed to the permeation-enhancing and microcirculation-improving effects of 1,2-propanediol, the sebaceous gland pathway-targeting effect of caprylic / capric triglyceride, and the oil-water two-phase solubilizing and stabilizing effect of polyoxyethylene (40) hydrogenated castor oil—these three factors work together to achieve synergistic delivery, enabling the active ingredients to effectively penetrate the stratum corneum barrier and reach the hair follicle target. This is the important formulation basis for the superiority of this invention over existing topical formulations.

[0062] In summary, the preparation process of the American cockroach extract nanogel of this invention is simple, stable, safe, and has significant therapeutic effects. It can effectively achieve targeted delivery of active drugs to the hair follicle, effectively prevent or treat alopecia areata, and can also be extended to other hair loss diseases, including chemotherapy-induced alopecia. It has broad clinical application prospects and related hair loss diseases, and has outstanding inventiveness and practicality.

[0063] Any aspects of this invention not described in detail are well-known to those skilled in the art.

[0064] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A nanogel containing extracts of the American cockroach, characterized in that, It contains the following ingredients: American cockroach extract, gelling agent, oil phase, surfactant, co-surfactant, and water.

2. The nanogel containing American cockroach extract according to claim 1, characterized in that, By weight, the American cockroach extract is 0.05-0.2 parts, the gelling agent is 0.025 parts, the oil phase is 0.1047-0.1136 parts, the surfactant is 0.4712-0.5111 parts, the co-surfactant is 0.4712-0.5111 parts, and the water is 3.7278-3.7893 parts.

3. The nanogel containing American cockroach extract according to claim 1, characterized in that, The mass fraction of the American cockroach extract in the nanogel containing the American cockroach extract is 1% to 4%.

4. The nanogel containing American cockroach extract according to claim 1, characterized in that, The gelling agent is carbomer 940; the oil phase is caprylic / capric glyceride; the surfactant is polyoxyethylene (40) hydrogenated castor oil; and the co-surfactant is 1,2-propanediol.

5. The nanogel containing American cockroach extract according to claim 1, characterized in that, The nanogel containing American cockroach extract has an average particle size of <200 nm and an absolute value of zeta potential >30 mV.

6. The method for preparing nanogels containing American cockroach extract according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Weigh the oil phase, surfactant, co-surfactant and American cockroach extract, mix them, heat and stir, sonicate and continue stirring until homogeneous; (2) Add purified water dropwise to the system obtained in step (1), and stir under heating until the system becomes transparent to obtain nanoemulsion; (3) Weigh the gelling agent and dissolve it in the remaining purified water, let it swell overnight, and obtain the gel matrix; (4) Add the nanoemulsion obtained in step (2) to the gel matrix obtained in step (3) and stir to mix well to obtain the nanogel.

7. The preparation method according to claim 6, characterized in that, In step (1), the ultrasonic power is 200~300 W and the ultrasonic time is 20 minutes; in steps (2) and (4), the stirring speed is 300~500 rpm; in steps (1) and (2), the heating temperature is 60℃.

8. The use of the nanogel containing American cockroach extract according to any one of claims 1 to 5 in the preparation of a medicament for the prevention and / or treatment of alopecia areata or chemotherapy-induced hair loss.

9. The application according to claim 8, characterized in that, The nanogel containing American cockroach extract inhibits CD8. + NKG2D + T-cell function or improvement of hair follicle immune exemption collapse can prevent and / or treat alopecia areata or chemotherapy-induced hair loss.

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