A wet eczema targeted drug-loaded nano-liposome ointment and a preparation method thereof

Abstract

The application relates to a wet eczema targeted drug-loaded nanoliposome ointment and a preparation method thereof, and belongs to the technical field of external-use medicinal preparations; the preparation method comprises the following steps: (1) preparing Phl-CaCO3 nanoparticles by using a gas diffusion reaction method; (2) preparing a liposome with an L-selectin target head by using a membrane fusion method; and (3) drug loading of the liposome. The integrated drug delivery system has multiple functions of inflammation targeting enrichment, calcium carbonate stable drug loading, triple synergy of drugs and calcium ions, long-acting sustained release and skin barrier repair, can comprehensively improve the skin lesion state, long-acting maintain the treatment effect, and effectively reduce the recurrence risk of eczema; the transdermal penetration ability is excellent, the drug retention time at the skin lesion is longer, and the drug effect is durable; the ointment has the advantages of targeted precision, mild low stimulation and multi-effect synergy, greatly improves the comprehensive treatment effect of eczema, relieves clinical symptoms such as itching and swelling, and improves the use compliance of external-use medicinal drugs of patients.

Description

Technical Field

[0001] This invention relates to a targeted drug-loaded nanoliposome ointment for eczema and its preparation method, belonging to the field of topical drug formulation technology. Background Technology

[0002] Eczema is a common inflammatory skin disease in clinical practice. Treatment typically involves topical anti-inflammatory ointments, conventional liposome preparations, or single-active-ingredient formulations. Existing conventional preparations, with their advantages of simple formulations, mature manufacturing processes, and convenient mass production, have become the mainstream medications for eczema treatment. However, in actual clinical application, these traditional preparations still have many inherent technical defects, greatly limiting their therapeutic effects and easily causing recurrent flare-ups, thus exhibiting significant limitations in clinical application.

[0003] First, existing formulations generally lack lesion-targeting capabilities. The drugs lack selective targeting for skin inflammation, resulting in indiscriminate distribution between inflamed and normal skin after application. This fails to effectively increase the local drug concentration at the eczema lesion site, significantly reducing treatment effectiveness and easily causing irritation to surrounding normal skin, leading to poor safety and treatment precision. Second, traditional topical formulations have weak transdermal delivery capabilities, exhibiting low skin penetration efficiency and short drug retention time at the lesion site. Furthermore, commonly used hydrophobic active pharmaceutical ingredients such as phloretin have poor stability, easily undergoing oxidation, degradation, and inactivation, resulting in significantly reduced drug bioavailability, short duration of efficacy, and difficulty in achieving long-term treatment.

[0004] Meanwhile, most clinically used eczema treatments currently employ a single-effect modality, with limited functionality. They primarily focus on anti-inflammatory effects and fail to address the multiple treatment needs required in the pathological process of eczema, such as antioxidant effects, regulation of skin calcium ion homeostasis, and repair of damaged skin barriers. The lack of synergistic treatment across multiple pathways prevents the fundamental intervention of the eczema pathogenesis mechanism, which is one of the core reasons why eczema inflammation is recurrent and difficult to cure.

[0005] In addition, existing technologies lack structurally stable composite drug carrier systems, making it difficult to simultaneously load and stably deliver multiple active therapeutic components, and thus impossible to construct a multi-effect synergistic drug delivery system. Furthermore, current drug delivery technologies have not formed an integrated drug delivery strategy that combines targeted enrichment, dual-drug synergy, calcium ion regulation, and long-acting sustained release, failing to intervene in the pathological development of eczema from multiple pathways and dimensions. This results in limited overall therapeutic efficacy and fails to meet the current clinical needs for highly effective, long-lasting, safe, and curative treatment of eczema. Based on these numerous shortcomings of existing technologies, there is an urgent need to develop a drug delivery formulation specifically for eczema that possesses targeting, high stability, multi-effect synergy, and long-lasting effects. Summary of the Invention

[0006] This invention provides a targeted drug-loaded nanoliposome ointment for eczema and its preparation method to solve the technical problems existing in the prior art as described above.

[0007] The technical solution provided by this invention is as follows: One objective of this invention is to provide a method for preparing a targeted drug-loaded nanoliposome ointment for eczema, comprising the following steps: (1) Phl-CaCO3 nanoparticles were prepared by gas diffusion reaction: calcium chloride CaCl2, phloretin Phl and dopamine hydrochloride DA were dispersed in a container containing anhydrous ethanol, ultrasonically treated with water bath, the container was placed in a desiccator containing ammonium bicarbonate, and then placed in a vacuum drying oven for gas diffusion reaction. After centrifugation, the precipitate was collected and freeze-dried to obtain Phl-CaCO3 nanoparticles. (2) L-selectin target liposomes were prepared by membrane fusion method: cell membranes containing L-selectin were extracted from leukocytes, resuspended and mixed with blank liposome prepreg solution, and then rotary evaporated to form L-selectin target liposome film; (3) Drug loading on liposomes: The Phl-CaCO3 nanoparticles obtained in step (1) are dispersed in water to obtain a Phl-CaCO3 nanoparticle dispersion. The Phl-CaCO3 nanoparticle dispersion is mixed with FAD aqueous solution and then transferred to a container carrying the L-selectin target liposome film obtained in step (2) for loading. Then it is transferred to a centrifuge tube, glycerol is added and the mixture is subjected to ice bath sonication to obtain the eczema-targeted drug-loaded nanoliposome ointment.

[0008] The advantages of the above-mentioned technical solution are that calcium chloride, as an exogenous calcium source, can react in situ with carbon dioxide generated from the decomposition of ammonium bicarbonate in the system to generate a homogeneous and stable calcium carbonate nanomatrix. This calcium carbonate matrix possesses good biocompatibility and a porous structure, enabling efficient loading of the hydrophobic drug phlorizin, achieving slow and controllable drug release. During delivery, it effectively protects the chemical structure and biological activity of phlorizin from degradation, while significantly improving the dispersibility and colloidal stability of the nanoparticle system, providing a structurally stable and high-performance inorganic carrier framework for subsequent liposome co-delivery systems. After the calcium carbonate microspheres are endocytosed by cells, they can gradually degrade in the acidic intracellular environment and continuously release calcium ions. The released calcium ions can produce multi-target synergistic effects with the co-delivered flavin adenine dinucleotide (FAD) and phlorizin, exerting a synergistic therapeutic effect on eczema through pathways such as regulating cellular calcium homeostasis, enhancing antioxidant stress capacity, inhibiting the release of inflammatory factors, and promoting skin barrier repair, significantly improving overall anti-inflammatory and tissue repair efficacy.

[0009] Dopamine hydrochloride (DA) can promote the nucleation and morphology regulation of calcium carbonate nanoparticles through the action of phenolic hydroxyl groups. At the same time, it can enhance the loading rate of phloretin on the calcium carbonate matrix and improve the stability of the nanoparticle system by utilizing the adhesive effect of dopamine.

[0010] Phloretin (Phl) possesses anti-inflammatory, antioxidant, skin barrier repair, and inflammatory factor inhibition properties, directly alleviating symptoms such as redness, itching, and scaling in eczema. It is a core active ingredient in eczema treatment, with long-lasting sustained release achieved through calcium carbonate nanoparticle loading. Phloretin exhibits strong antioxidant properties, with an antioxidant concentration of 10-30 ppm for lipids. It can scavenge free radicals in the skin, which are harmful substances produced by oxidation reactions and are associated with degenerative diseases. Topical application can prevent sugary components from entering epidermal cells, thereby inhibiting excessive sebum secretion and treating acne caused by excessive secretion. Studies have shown that under the influence of phloretin, the release of NO from macrophages significantly decreases under the stimulation of lipopolysaccharide and interferon-γ (IFN-γ); the phagocytic rate of macrophages also significantly decreases under the influence of phloretin. Therefore, phloretin has potential anti-inflammatory effects.

[0011] Flavin adenine dinucleotide (FAD), as a key coenzyme in cellular energy metabolism, can promote skin cell repair, enhance barrier function, reduce inflammation, and accelerate wound healing. It works synergistically with phloretin to improve the skin microenvironment of eczema and help restore normal skin physiological function.

[0012] L-selectin on liposomes expresses a large amount of selectin ligands at the site of eczema. After grafting L-selectin targets, liposomes can be given active targeting ability at the site of inflammation, which can accurately accumulate in the inflammatory area of ​​eczema, increase the local drug concentration of the lesion, reduce the potential side effects of systemic drug distribution, and improve the treatment effect.

[0013] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, in step (1), the ratio of CaCl2, Phl, DA and anhydrous ethanol is 15 mg: 1 mg: 0.2~1 mg: 5 mL.

[0014] Furthermore, in step (1), the drying temperature is 35~45℃, the drying time is 20~28h, the centrifugation speed is 7000~9000rpm, and the centrifugation time is 10~20min. Further, in step (2), the specific steps for extracting cell membranes with L-selectin from leukocytes are as follows: extract mouse leukocytes, wash them with leukocyte separation solution, centrifuge to collect the precipitate, lyse them with hypotonic lysis buffer and treat them with an ice bath, then perform ultrasonic disruption under ice bath conditions, centrifuge to collect the supernatant, and centrifuge again to obtain cell membranes with L-selectin. Further, in step (2), the blank liposome prepreg solution is obtained by mixing an ethanol solution of soybean phospholipids and an ethanol solution of cholesterol at a volume ratio of 2 to 4:1. Furthermore, the concentrations of the ethanol solutions of soybean lecithin and cholesterol are both 8-12 mg / mL.

[0015] Further, in step (3), the FAD aqueous solution is prepared by dissolving FAD in water at a dosage ratio of 3~7 mg: 1 mL.

[0016] The second objective of this invention is to provide a targeted drug-loaded nanoliposome ointment for eczema, which is prepared using the method described above.

[0017] The technical solution provided by this invention has the following advantages compared with the prior art: 1. This invention uses a gas diffusion method to prepare calcium carbonate nanoparticles loaded with phlorizin. Relying on the spatial support and isolation protection of the inorganic matrix of calcium carbonate, hydrophobic phlorizin can be effectively encapsulated, inhibiting the oxidative degradation of active ingredients and significantly improving drug stability. At the same time, the calcium carbonate nanoparticles can be uniformly dispersed in the aqueous cavity of liposomes, improving the overall structural stability of the composite liposomes and avoiding the problems of easy rupture and premature drug leakage of ordinary liposomes.

[0018] 2. The composite liposomes of the present invention are coated with macrophage membranes. Relying on the L-selectin on the cell membrane surface to specifically recognize the site of inflammatory skin lesions, the drug delivery system can actively target and enrich the drug on the eczema lesion, which can significantly increase the local drug concentration at the lesion. It can also reduce the non-specific distribution of drugs in healthy skin tissue, reduce the irritation of drugs to normal skin, and improve the safety of topical preparations.

[0019] 3. After being taken up by the lesion cells, the calcium carbonate carrier of the present invention can be gradually degraded in the intracellular microenvironment and continuously release calcium ions. Calcium ions can regulate the calcium homeostasis of skin cells, inhibit over-activated inflammatory signaling pathways, and promote the proliferation and migration of keratinocytes and rebuild the tight junctions of the skin. This effect, together with the antioxidant and anti-inflammatory activity of phloretin and the mitochondrial protection and skin barrier repair effects of FAD, forms a triple synergistic therapeutic effect, breaking through the limitation of traditional preparations that only have single anti-inflammatory effects.

[0020] 4. The integrated drug delivery system of this invention integrates multiple functions such as inflammation-targeted enrichment, stable drug loading in calcium carbonate, triple synergy between drug and calcium ions, long-term sustained release, and skin barrier repair. It can simultaneously intervene in the pathogenesis of eczema from multiple dimensions such as inhibiting inflammatory response, blocking oxidative stress, maintaining skin calcium homeostasis, and repairing damaged skin barrier, comprehensively improve the skin lesion state, maintain the treatment effect for a long time, and effectively reduce the risk of eczema recurrence.

[0021] 5. The composite nanoliposomes of this invention are prepared as a topical ointment with excellent transdermal penetration ability, longer drug retention time at the skin lesion, and long-lasting efficacy; it also has the advantages of precise targeting, mild and low irritation, and synergistic effects, which greatly improves the overall treatment effect of eczema, relieves clinical symptoms such as itching and redness, and improves patients' compliance with the use of topical medications. Attached Figure Description

[0022] Figure 1 This is a comparison diagram showing the inhibitory effect of the present invention on mouse eczema; Figure 2 TEM images of Phl-CaCO3 nanoparticles and liposomes prepared in Example 1 of this invention; wherein, a) individual Phl-CaCO3 nanoparticles; b) free Phl-CaCO3 nanoparticles without liposomes; c) Phl-CaCO3 nanoparticles encapsulated inside a single liposome vesicle; d) composite nanoliposomes with a large number of Phl-CaCO3 nanoparticles. Figure 3 Zeta potential characterization of Phl-CaCO3 nanoparticles and liposomes loaded with Phl-CaCO3 nanoparticles prepared in Example 1 of the present invention; wherein, a) Phl-CaCO3 nanoparticles; b) L-selectin target liposomes; c) L-selectin target liposomes loaded with Phl-CaCO3 nanoparticles. Figure 4 The particle size distribution of Phl-CaCO3 nanoparticles and liposomes prepared in Example 1 of the present invention is shown in the figure; wherein, a) the particle size distribution of Phl-CaCO3 nanoparticles; b) L-selectin target liposomes loaded with Phl-CaCO3 nanoparticles. Detailed Implementation

[0023] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0024] Example 1 A method for preparing a targeted drug-loaded nanoliposome ointment for eczema includes the following steps: (1) Phl-CaCO3 nanoparticles were prepared by gas diffusion reaction: 150 mg CaCl2, 10 mg Phl and 5 mg DA were dispersed in a bottle containing 50 mL of anhydrous ethanol. After ultrasonic treatment in a water bath (100 W, 40 kHz) for 20 min, the bottle mouth was covered with tin foil. After punching holes in the tin foil, the bottle was placed in a beaker containing 5 g of ammonium bicarbonate. After drying in a vacuum drying oven at 40 °C for 24 h, the precipitate was collected by centrifugation (8000 rpm, 15 min). After pre-cooling at -40 °C for 1 h, the precipitate was freeze-dried at -80 °C to obtain Phl-CaCO3 nanoparticles. (2) Preparation of L-selectin target liposomes by membrane fusion method: Blood samples (5 ml each) were collected from the orbital cavity of 10 Balb / c mice. The blood was processed with 40 ml (4 ml corresponds to 500 μL of whole blood) of leukocyte separation buffer to obtain approximately 12.5 million leukocytes. The separated leukocyte pellet was then resuspended three times with 2 ml of washing buffer (Tianjin Haoyang Biological Products Technology Co., Ltd., Mouse Peripheral Blood Leukocyte Separation Kit). Each time, the pellet was centrifuged at 300 × g for 10 min, the supernatant was discarded, and the pellet was collected. Lysis was then performed with 1.25 ml of hypotonic lysis buffer (containing 1 mM MSF in 10 mM Tris-HCl and 1 mM EDTA, pH 7.4). The pellet was then placed in an ice bath for 30 min, followed by probe-based ultrasonic fragmentation under ice bath conditions (power 100 W, operating mode: on for 2 s, off for 1 s, total duration 3 minutes). After centrifugation for 10 min), the supernatant was obtained by two centrifugations: the first centrifugation was to remove the cell contents (centrifuge at 3000×g for 10 min at 4℃, retain the supernatant and discard the precipitate), and the second centrifugation was to enrich the cell membrane (the supernatant was transferred to an ultracentrifuge tube and centrifuged at 14000×g for 30 min at 4℃), and the supernatant was discarded to obtain the cell membrane precipitate. An ethanol solution of soybean lecithin and an ethanol solution of cholesterol, both with a concentration of 10 mg / mL, were mixed at a volume ratio of 3:1 to obtain a blank liposome pretreatment solution. The cell membrane with L-selectin obtained above was resuspended in 2 mL of pre-cooled PBS and mixed with 10 mL of blank liposome prepreg solution. Then, it was rotary evaporated (40℃ water bath, 150 r / min) for 35 min until the solvent evaporated to form an L-selectin target liposome film. (3) Liposome-encapsulated drugs: 5 mg FAD was dissolved in 1 mL of ultrapure water to prepare an aqueous solution of FAD. 0.5g of Phl-CaCO3 nanoparticles obtained in step (1) were dispersed in 9mL of ultrapure water to obtain a Phl-CaCO3 nanoparticle dispersion. Then, 1mL of FAD aqueous solution was added to it and the dispersion was subjected to ice bath sonication (100W, probe-type sonication with 2s on and 2s off) until the dispersion was complete. The liposomes were transferred to a flask containing the L-selectin target liposome film obtained in step (2) for encapsulation, and then transferred to a centrifuge tube. 40 mL of glycerin was added and the mixture was subjected to ice bath sonication (100 W, 40 K Hz) for 15 min to obtain the eczema-targeted drug-loaded nanoliposome ointment.

[0025] Comparative Example 1 Only the blank liposome preform solution from Example 1 was used.

[0026] Comparative Example 2 Referring to Example 1, the only difference from Example 1 is that Phl is not added when preparing Phl-CaCO3 nanoparticles in step (1).

[0027] Comparative Example 3 Referring to Example 1, the difference from Example 1 is that FAD is not added when encapsulating the drug in liposomes in step (3).

[0028] Step (3) is as follows: 0.5 g of Phl-CaCO3 nanoparticles obtained in step (1) were dispersed in 9 mL of ultrapure water to obtain a Phl-CaCO3 nanoparticle dispersion. The liposomes were transferred to a flask containing the L-selectin target liposome film obtained in step (2) for encapsulation, and then transferred to a centrifuge tube. 40 mL of glycerin was added and the mixture was subjected to ice bath sonication (100 W, 40 K Hz) for 15 min to obtain the eczema-targeted drug-loaded nanoliposome ointment.

[0029] Comparative Example 4 Referring to Example 1, the difference from Example 1 is that in step (2), cell membranes with L-selectin are not prepared, and only blank liposome prepreg is obtained.

[0030] A method for preparing a nanoliposome ointment specifically includes the following steps: (1) Phl-CaCO3 nanoparticles were prepared by gas diffusion reaction: 150 mg CaCl2, 10 mg Phl and 5 mg DA were dispersed in a bottle containing 50 mL of anhydrous ethanol. After ultrasonic treatment in a water bath (100 W, 40 kHz) for 20 min, the bottle mouth was covered with tin foil. After punching holes in the tin foil, the bottle was placed in a beaker containing 5 g of ammonium bicarbonate. After drying in a vacuum drying oven at 40 °C for 24 h, the precipitate was collected by centrifugation (8000 rpm, 15 min). After pre-cooling at -40 °C for 1 h, the precipitate was freeze-dried at -80 °C to obtain Phl-CaCO3 nanoparticles. (2) Mix the ethanol solutions of soybean lecithin and cholesterol, both with a concentration of 10 mg / mL, at a volume ratio of 3:1 to obtain a blank liposome pretreatment solution. (3) Liposome-encapsulated drugs: 5 mg FAD was dissolved in 1 mL of ultrapure water to prepare an aqueous solution of FAD. 0.5g of Phl-CaCO3 nanoparticles obtained in step (1) were dispersed in 9mL of ultrapure water to obtain a Phl-CaCO3 nanoparticle dispersion. Then, 1mL of FAD aqueous solution was added to it and the dispersion was subjected to ice bath sonication (100W, probe-type sonication with 2s on and 2s off) until the dispersion was complete. The liposomes were transferred to a flask containing the L-selectin target liposome film obtained in step (2) for encapsulation, and then transferred to a centrifuge tube. 40 mL of glycerin was added and the mixture was subjected to ice bath sonication (100 W, 40 K Hz) for 15 min to obtain the eczema-targeted drug-loaded nanoliposome ointment.

[0031] Experiment on the inhibitory effect on mouse eczema: Eight-week-old female bal / BC mice, weighing 18-22g.

[0032] Husbandry conditions: After arriving at the laboratory, males and females were separated, with 5 mice per cage. Mice were put into service after 3 days and managed by designated personnel. The laboratory had ample lighting, good ventilation and air conditioning, with the room temperature controlled at 20-25℃ and humidity at 55-65%.

[0033] The laboratory is disinfected regularly according to standard procedures.

[0034] Thirty-two healthy mice were selected and randomly divided into eight groups of four mice each.

[0035] Negative control group: Healthy mice were photographed after shaving and were not treated with drugs. The model control group consisted of mice with eczema that were shaved and photographed without any drug treatment. The positive control group was given phlorizin (Meilun Biotechnology, MB5985-1) at a dose of 20 μg / cm³. 2 Once daily; Example 1 group: The ointment prepared in Example 1 was applied at a dosage of 20 μg / cm³. 2 Once daily; Comparative Example 1: The ointment prepared in Comparative Example 1 was applied at a dosage of 20 μg / cm³. 2 Once daily; Comparative Example 2: The ointment prepared in Comparative Example 2 was applied at a dosage of 20 μg / cm³. 2 Once daily; Comparative Example 3: The ointment prepared in Comparative Example 3 was applied at a dosage of 20 μg / cm³. 2 Once daily; Comparative Example 4: The ointment prepared in Comparative Example 4 was applied at a dosage of 20 μg / cm³. 2 Once daily.

[0036] Except for the negative control group, the backs of mice in all other groups were shaved uniformly, and the solution was applied at a concentration of 20 μg / cm². 2 The dosage was sensitized by topical application of 5% phthalic anhydride. After 7 days, the drug was administered to the skin lesions of mice at a dosage of 20 μg / cm³. 2 The mice were administered the drug once daily for 7 consecutive days. Twenty-four hours after the last administration, the visible changes in the skin lesions were observed with the naked eye. The results are as follows: Figure 1 As shown.

[0037] from Figure 1 As can be seen from the results, this invention successfully prepared a CaCO3 composite nanoliposome grafted with an L-selectin target and co-loaded with phloretin (Phl) and FAD dual active components, and its targeted therapeutic effect was verified using a mouse eczema model. The control group mice exhibited typical pathological eczema lesions such as obvious redness, swelling, erosion, desquamation, thickening, and crusting. The negative control group mice had smooth, intact skin without inflammatory damage. The positive control group showed mild relief of skin inflammation after intervention with free phloretin, confirming the reliability of the eczema animal model and the effectiveness of the overall experimental system. Example 1: The complete formulation of nanoliposomes showed the best therapeutic effect, with basic reduction of redness and swelling in mouse skin lesions, complete shedding of scales and scabs, and complete repair of the skin barrier. Comparative Example 1: The blank liposome group without a target showed no significant improvement in skin lesions, confirming that pure liposome carriers have no anti-inflammatory or repairing pharmacological activity. Comparative Example 2: The absence of phlorizin as an active drug significantly reduced the degree of inflammation relief, confirming that phlorizin is the core active ingredient for anti-inflammatory treatment of eczema. Comparative Example 3: The absence of FAD as an active ingredient resulted in oxidative damage to the skin and incomplete barrier repair, confirming that FAD can synergistically enhance and assist in repairing the skin barrier. Comparative Example 4: Removal of the L-selectin target decreased the drug accumulation capacity at lesions, and the therapeutic effect was significantly inferior to Example 1, demonstrating that cell membrane targeted modification can effectively increase drug accumulation at lesions and enhance the therapeutic efficacy of eczema.

[0038] Figure 2 The images show TEM images of the Phl-CaCO3 nanoparticles and their encapsulated liposomes prepared in Example 1. It can be seen that the free Phl-CaCO3 nanoparticles... Figure 2 In particle a, the particles are regular and nearly spherical; in the monodisperse state, the particle size is small and controllable, and the individual particles are well dispersed. However, if... Figure 2 As shown in b, free calcium carbonate nanoparticles lacking the protection of a liposome shell are prone to agglomeration into micron-sized clumps due to their high surface energy. Agglomerated particles will significantly weaken the skin's transdermal ability and will also cause phloretin to be directly exposed and undergo oxidative degradation. The overall stability is poor, making it difficult to use directly in topical eczema preparations. Figure 2 c. Liposome vesicles with intact and undamaged phospholipid bilayer membranes can be clearly observed. Phl-CaCO3 nanoparticles are completely encapsulated in the aqueous core of the liposomes, which confirms that the process of the present invention can successfully complete the liposome encapsulation of nanoparticles. The phospholipid shell can not only isolate air and external water environment and inhibit the oxidative inactivation of phloretin in the core, but also avoid the problem of calcium carbonate nanoparticle aggregation. Figure 2d represents the drug-loaded composite liposome, the final product of this invention. The liposomes are uniformly spherical with a narrow particle size distribution, no obvious aggregation, and excellent dispersion performance. The vesicle structure is intact without rupture or leakage, proving that the entire preparation process is stable and controllable. The liposome shell not only improves the water solubility and skin compatibility of the system, but also enhances the transdermal absorption efficiency of drugs at the lesion site by relying on the permeation-promoting effect of phospholipids. It can also achieve sustained release of phloretin through the vesicle structure, prolonging the retention time of the drug on the eczema lesion, effectively overcoming the shortcomings of simple Phl-CaCO3 nanoparticles, such as easy aggregation, poor storage stability, and short duration of drug action.

[0039] Figure 3 Zeta potential characterization of Phl-CaCO3 nanoparticles and liposomes prepared in Example 1; from Figure 3 As can be seen from a, the potential of the free Phl-CaCO3 nanoparticles is generally negative, ranging from approximately -9 to -5 mV. The absolute value of the potential is low, indicating weak electrostatic repulsion between particles, which cannot effectively counteract the high surface energy. This corresponds to the phenomenon observed in the TEM results where the free nanoparticles easily aggregate, exhibiting poor dispersion stability and making them difficult to store stably on their own. Figure 3 As shown in b, the L-selectin target liposomes exhibit a strongly positively charged surface with a potential of approximately 40 mV. This high positive potential provides sufficient electrostatic repulsion, ensuring uniform dispersion of the blank liposomes. Simultaneously, the positively charged phospholipid vesicles can bind to the negatively charged Phl-CaCO3 nanoparticles through electrostatic adsorption, providing the driving force for nanoparticle encapsulation. Figure 3 As shown in Figure c, the potential of the L-selectin target liposome encapsulating Phl-CaCO3 nanoparticles dropped to approximately 14 mV, while still maintaining a stable positive charge. This indicates that the negatively charged Phl-CaCO3 core is completely encapsulated by a positively charged phospholipid bilayer membrane, and the nanoparticles are not exposed on the particle surface, directly proving the formation of a complete encapsulation structure of the liposomes. Compared to free nanoparticles, the composite liposomes have a higher absolute potential value, and the electrostatic repulsion between particles is significantly improved. This addresses the defect of easy aggregation of free Phl-CaCO3 at the charge level, improving the long-term storage stability of the formulation. At the same time, the positively charged liposomes can generate electrostatic adsorption with the negatively charged stratum corneum of eczema-damaged skin, prolonging the drug's residence time at the lesion site and improving the transdermal drug delivery effect.

[0040] Figure 4 The particle size distribution diagrams are shown for the Phl-CaCO3 nanoparticles and their encapsulated liposomes prepared in Example 1; Figure 4As can be seen, the free Phl-CaCO3 nanoparticles have an extremely wide particle size distribution with multiple distinct particle size peaks. The main peak is distributed in the micrometer range, accompanied by a large number of ultra-large aggregated particles with significant differences in particle size. This demonstrates that the free nanoparticles are prone to aggregation and have poor polydispersity when not encapsulated by liposomes. This is consistent with the conclusions of aggregation and poor stability reflected by TEM and Zeta potential. In this state, the particles are difficult to penetrate the stratum corneum of eczema skin and are not suitable for topical administration. Figure 4 b represents the composite drug-loaded liposomes after liposome encapsulation. The overall structure exhibits only a single, concentrated particle size peak, without obvious large-particle aggregation peaks. The particle size distribution range is significantly narrowed, and the degree of dispersion is greatly reduced. This indicates that the phospholipid shell of the liposomes can isolate nanoparticles through electrostatic encapsulation, effectively inhibiting the aggregation of Phl-CaCO3 particles and forming uniformly sized nanoscale vesicles. The composite liposomes have a concentrated and uniform particle size distribution. The appropriate nanoscale size can penetrate the damaged eczema epidermis with the help of phospholipids to increase the drug accumulation in the lesion site. It can also achieve long-term sustained release of phloretin through the vesicle structure. At the same time, the uniform particle size distribution ensures batch-to-batch stability of the formulation, which is conducive to the large-scale preparation of topical eczema creams.

[0041] In summary, the L-selectin targeted composite nanoliposomes of this invention have good biocompatibility and strong synergistic effects among their functional components. They can target eczema lesions, inhibit skin inflammation, and repair damaged epidermis. Compared with free raw materials and simplified formulations, they have significant therapeutic advantages, providing a novel nanomedicine delivery solution for targeted and precise topical treatment of eczema.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a targeted drug-loaded nanoliposome ointment for eczema, characterized in that, Includes the following steps: (1) Phl-CaCO3 nanoparticles were prepared by gas diffusion reaction: calcium chloride CaCl2, phloretin Phl and dopamine hydrochloride DA were dispersed in a container containing anhydrous ethanol, ultrasonically treated with water bath, the container was placed in a desiccator containing ammonium bicarbonate, and then placed in a vacuum drying oven for gas diffusion reaction. After centrifugation, the precipitate was collected and freeze-dried to obtain Phl-CaCO3 nanoparticles. (2) L-selectin target liposomes were prepared by membrane fusion method: cell membranes containing L-selectin were extracted from leukocytes, resuspended and mixed with blank liposome prepreg solution, and then rotary evaporated to form L-selectin target liposome film; (3) Drug loading on liposomes: The Phl-CaCO3 nanoparticles obtained in step (1) are dispersed in water to obtain a Phl-CaCO3 nanoparticle dispersion. The Phl-CaCO3 nanoparticle dispersion is mixed with a flavin adenine dinucleotide (FAD) aqueous solution and then transferred to a container carrying the L-selectin target liposome film obtained in step (2) for loading. Then it is transferred to a centrifuge tube, glycerol is added and the mixture is subjected to ice bath sonication to obtain the eczema-targeted drug-loaded nanoliposome ointment.

2. The method for preparing the eczema-targeted drug-loaded nanoliposome ointment according to claim 1, characterized in that, In step (1), the ratio of CaCl2, Phl, DA and anhydrous ethanol is 15mg:1mg:0.2~1mg:5mL.

3. The method for preparing the eczema-targeted drug-loaded nanoliposome ointment according to claim 1, characterized in that, In step (1), the drying temperature is 35~45℃ and the drying time is 20~28h; the centrifugation speed is 7000~9000rpm and the centrifugation time is 10~20min.

4. The method for preparing the eczema-targeted drug-loaded nanoliposome ointment according to claim 1, characterized in that, In step (2), the specific steps for extracting cell membranes with L-selectin from leukocytes are as follows: extract mouse leukocytes, wash them with leukocyte separation solution, centrifuge to collect the precipitate, lyse them with hypotonic lysis buffer and treat them with an ice bath, then perform ultrasonic disruption under ice bath conditions, centrifuge to collect the supernatant, and centrifuge again to obtain cell membranes with L-selectin.

5. The method for preparing the eczema-targeted drug-loaded nanoliposome ointment according to claim 1, characterized in that, In step (2), the blank liposome prepreg solution is obtained by mixing an ethanol solution of soybean phospholipids and an ethanol solution of cholesterol at a volume ratio of 2 to 4:

1.

6. The method for preparing the eczema-targeted drug-loaded nanoliposome ointment according to claim 5, characterized in that, The concentrations of the ethanol solutions of soybean phospholipids and cholesterol are both 8-12 mg / mL.

7. The method for preparing the eczema-targeted drug-loaded nanoliposome ointment according to claim 1, characterized in that, In step (3), the FAD aqueous solution is prepared by dissolving FAD in water at a ratio of 3~7 mg: 1 mL.

8. A targeted drug-loaded nanoliposome ointment for eczema, characterized in that, It is prepared using the preparation method of the eczema-targeted drug-loaded nanoliposome ointment as described in any one of claims 1 to 7.

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