Leptin modified targeted hair papilla cell liposome
By using leptin to modify the liposomes of the nipple cells in Aga treatment, the precision delivery of the drug and the synergy suppression of the premature faults of the hair follicles are achieved, and the problem of poor efficacy and high side reaction of the existing treatment method provides a one. High -efficiency and safe treatment plan.
Patent Information
- Application Number
- CN202410905280.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-08
- Publication Date
- 2025-01-28
AI Technical Summary
The existing AGA treatment methods have problems with poor efficacy and major side effects. Although drugs such as finasteride and minoxidil are effective, long-term use will cause sexual dysfunction and other side effects. The cost and concurrent surgical treatment is high. risk of symptoms.
Use leptin to modify the liposomes of the nipples cells, prepare nanoprotoxic nanometer at oxidation through the gum beam, and use the thin film hydration method to synthesize the lipid body. , Reduce DHT production and remove ROS, and coordinate premature faults of hair follicles.
The precise targeted delivery of drugs has been achieved, the treatment effect is enhanced, the side reaction is reduced, and a stable, low -toxic, and controllable nanometer medication system is provided, which promotes the transformation of the hair follicles from the rest period to the growth period.
Smart Images
Figure CN119367295A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of liposomes targeted to hair papilla cells, and specifically relates to leptin-modified liposomes targeted to hair papilla cells. Background Art
[0002] At present, the clinical treatments for AGA mainly include surgery and drug therapy. Hair transplantation is an effective technology for the treatment of permanent hair loss. It mainly uses surgery to transplant the patient's own hair that is not affected by androgens to the hair loss site to achieve the purpose of treatment. However, hair transplantation is expensive and may be accompanied by complications such as scars, folliculitis, local paresthesia, and skin necrosis in the donor and recipient areas. At present, the FDA-approved drugs for the treatment of AGA include finasteride and minoxidil, and drug treatment is still the first choice for the treatment of AGA hair loss. Finasteride is a 5α-reductase inhibitor that blocks the conversion of testosterone to its active form DHT by inhibiting 5α-reductase, thereby reducing the production of DHT. In a clinical study in which 1 mg / d of finasteride (FNS) was given to patients diagnosed with AGA for the first time as a treatment, 50% of the patients were well improved within one year. Although finasteride has no hepatotoxicity and renal toxicity, 5.1% of the patients stopped treatment due to erectile dysfunction, decreased libido, ejaculation disorders and other sexual dysfunction-related adverse reactions due to long-term medication. Minoxidil relaxes vascular smooth muscle by opening potassium channels that are sensitive to adenosine triphosphate, achieving a strong vasodilation effect. Minoxidil also has certain limitations in its use, such as limited dosing methods, poor patient compliance, and treatment accompanied by side effects such as scalp itching and contact dermatitis. Its therapeutic effect will disappear once the drug is stopped. In summary, the treatment of AGA still has the problem of poor efficacy and large side effects. Summary of the invention
[0003] The object of the present invention is to provide a leptin-modified liposome targeted to dermal papilla cells to solve the technical problems mentioned in the above background technology.
[0004] To achieve the above object, the present invention provides the following technical solution: a leptin-modified liposome targeting hair papilla cells, the synthesis of which comprises the following steps:
[0005] Step 1:
[0006] Take cerium acetate, oleylamine and xylene, stir and dissolve them into a mixed solution at room temperature, heat the mixed solution until it is clear, then add deionized water, heat the mixed solution again until it is clear, let it stand and cool, add ethanol for washing, centrifuge and discard the supernatant to obtain cerium oxide nanoparticles;
[0007] Step 2:
[0008] Take cholesterol, lecithin, and DSPE-PEG-NHS, dissolve finasteride and the cerium oxide nanoparticles prepared in step 1 in chloroform, add the dissolved mixture to the mixture of cholesterol, lecithin, and DSPE-PEG-NHS, and remove the organic reagent by rotary evaporation in a water bath to obtain a lipid film;
[0009] Step 3:
[0010] The lipid film synthesized in step 2 was added to PBS for hydration and ultrasonicated for 10-20 min to obtain liposomes without Leptin;
[0011] Step 4:
[0012] The liposomes synthesized in step 3 were diluted with pure water to 1 mg / ml, Leptin was added, and the mixture was incubated at 35-40°C for 1-2 hours and at 2-6°C overnight to obtain leptin-modified liposomes targeting dermal papilla cells.
[0013] Preferably, in step 1, the mass ratio of cerium acetate, oleylamine and xylene is 1-5:6:30.
[0014] Preferably, in step 1, the conditions for heating the mixed solution twice to clarify are both heating in an argon atmosphere at a temperature of 80-100° C. in a water bath.
[0015] Preferably, in step 1, the amount of deionized water added is 1-5 ml, and the amount of ethanol added is 50 ml.
[0016] Preferably, in step 2, the mass ratio of cholesterol, lecithin and DSPE-PEG-NHS is 9-12:3:0.1.
[0017] Preferably, in step 2, the mass ratio of finasteride to cerium oxide nanoparticles is 1-3:1.
[0018] Preferably, in step 2, the water bath rotary evaporation time is 40-50 min
[0019] The beneficial effects of the present invention are:
[0020] (1) A leptin-modified liposome targeted to hair papilla cells. Cerium oxide nanoparticles are prepared by a reverse micelle method, and leptin-LP-Fi / CeO2 is synthesized by a thin film hydration method. The liposome is a spherical lipid bilayer nanostructure with an outer lipid layer and an aqueous core. The structure of the liposome allows it to load hydrophilic drugs in the core and hydrophobic drugs between the bilayers. The phospholipids in the liposome can rearrange intercellular lipids, provide space for drug accumulation, and enhance skin delivery. In addition, the lipophilicity of epithelial cells makes it an effective delivery system for penetrating hair follicles.
[0021] (2) Leptin can target DPC via LEPR. The liposomes use LEPR as a target and leptin as a prospective targeting tool. Leptin is linked to liposomes to target dermal papilla cells (DPC) to achieve the purpose of precise treatment of androgenic alopecia (AGA).
[0022] (3) By targeting specific proteins on the surface of hair papilla cells, the drug is anchored around the hair papilla cells, achieving precise targeted delivery of the drug to hair follicles. After the liposomes that are efficiently targeted to the hair follicles are internalized by the hair papilla cells, the finasteride and cerium oxide contained in the liposomes synergistically inhibit premature aging of hair follicles through two different mechanisms: reducing DHT production and clearing ROS, respectively. This promotes the release of signals by hair papilla cells to activate hair follicle stem cells, causing the hair follicles to transition from the resting phase to the growth phase ahead of time, thereby enhancing the therapeutic effect of androgenic alopecia and providing new ideas for the treatment of the disease and the development of formulations.
[0023] (4) Liposomes were used to effectively load finasteride and the antioxidant cerium oxide nanoparticles, with leptin as the target, to construct a stable, low-toxic, and size-controlled nano-drug delivery system.
[0024] (5) After percutaneous administration, the liposomes can actively target DPCs through the leptin polypeptide target head and accumulate in DPCs. After being taken up by DPCs, finasteride and cerium oxide can inhibit the premature aging of DPCs, regulate the activation of hair follicle stem cells, and promote the transformation of hair follicles from the resting phase to the growth phase. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 The co-localization immunofluorescence of the target LEPR and the target head Leptin on human dermal papilla cells (HDPC) in the example;
[0026] Figure 2 This is an experimental diagram of the co-localization of the target LEPR and the targeted liposome (L-LP-Fi / CeO2) in the mouse skin in the embodiment;
[0027] Figure 3 A is a graph showing the in vitro transdermal test of common liposomes and targeted liposomes in the examples. Figure 3 B is a 24h in vitro mouse skin retention experiment;
[0028] Figure 4 This is a diagram showing the in vitro AGA modeling of C57 / BL6 mice using testosterone in the examples. DETAILED DESCRIPTION
[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0030] Embodiment 1:
[0031] Synthesis of cerium oxide nanoparticles: Weigh cerium acetate, oleylamine and xylene in a ratio of 1:6:30 in a penicillin bottle, stir to dissolve at room temperature, heat in a water bath at 80°C under an argon atmosphere until the liquid is clear, quickly add 4 ml of deionized water, heat in a water bath at 80°C under an argon atmosphere until the liquid is clear again, let stand to cool, add 50 ml of ethanol to wash, centrifuge and discard the supernatant to obtain cerium oxide nanoparticles.
[0032] Synthesis of targeted liposomes: Weigh the corresponding proportions of cholesterol, lecithin, and DSPE-PEG-NHS (9:3:0.1, w / w), add a certain amount of finasteride and nano-cerium oxide (1:1, w / w) and dissolve in 5 ml of chloroform, and remove the organic reagent by rotary evaporation in a water bath for 45 minutes to obtain a lipid film.
[0033] Add 5 ml PBS for hydration and sonicate for 10 min to obtain liposomes without Leptin;
[0034] The obtained liposomes were diluted with pure water to 1 mg / ml, Leptin was added, and the mixture was incubated at 35°C for 1 hour and at 2°C overnight to obtain leptin-modified liposomes targeting dermal papilla cells.
[0035] Embodiment 2:
[0036] Synthesis of cerium oxide nanoparticles: Weigh cerium acetate, oleylamine and xylene in a ratio of 1:3:15 in a penicillin bottle, stir to dissolve at room temperature, heat in a water bath at 90°C under an argon atmosphere until the liquid is clear, quickly add 2 ml of deionized water, heat in a water bath at 90°C under an argon atmosphere until the liquid is clear again, let stand to cool, add 50 ml of ethanol to wash, centrifuge and discard the supernatant to obtain cerium oxide nanoparticles.
[0037] Synthesis of targeted liposomes: Weigh the corresponding proportions of cholesterol, lecithin, and DSPE-PEG-NHS (10:3:0.1, w / w), add a certain amount of finasteride and nano-cerium oxide (2:1, w / w) and dissolve in 5 ml of chloroform, and remove the organic reagent by rotary evaporation in a water bath for 45 minutes to obtain a lipid film.
[0038] Add 5 ml PBS for hydration and sonicate for 15 min to obtain liposomes without Leptin;
[0039] The obtained liposomes were diluted with pure water to 1 mg / ml, Leptin was added, and the mixture was incubated at 37°C for 2 hours and at 3°C overnight to obtain leptin-modified liposomes targeting dermal papilla cells.
[0040] Embodiment 3:
[0041] Synthesis of cerium oxide nanoparticles: Weigh cerium acetate, oleylamine and xylene in a ratio of 1:2:10 into a penicillin bottle, stir to dissolve at room temperature, heat in a water bath at 95°C under an argon atmosphere until the liquid is clear, quickly add 3 ml of deionized water, heat in a water bath at 95°C under an argon atmosphere until the liquid is clear again, let stand to cool, add 50 ml of ethanol to wash, centrifuge and discard the supernatant to obtain cerium oxide nanoparticles.
[0042] Synthesis of targeted liposomes: Weigh the corresponding proportions of cholesterol, lecithin, and DSPE-PEG-NHS (11:3:0.1, w / w), add a certain amount of finasteride and nano-cerium oxide (3:1, w / w) and dissolve in 5 ml of chloroform, and remove the organic reagent by rotary evaporation in a water bath for 45 minutes to obtain a lipid film.
[0043] Add 5 ml PBS for hydration and sonicate for 20 min to obtain liposomes without Leptin;
[0044] The obtained liposomes were diluted with pure water to 1 mg / ml, Leptin was added, and the mixture was incubated at 38°C for 1 hour and at 5°C overnight to obtain leptin-modified liposomes targeting dermal papilla cells.
[0045] Embodiment 4:
[0046] Synthesis of cerium oxide nanoparticles: Weigh cerium acetate, oleylamine and xylene in a ratio of 2:3:15 in a penicillin bottle, stir and dissolve at room temperature, heat in a water bath at 100°C under an argon atmosphere until the liquid is clear, quickly add 5 ml of deionized water, heat in a water bath at 100°C under an argon atmosphere until the liquid is clear again, let stand and cool, add 50 ml of ethanol to wash, centrifuge and discard the supernatant to obtain cerium oxide nanoparticles.
[0047] Synthesis of targeted liposomes: Weigh the corresponding proportions of cholesterol, lecithin, and DSPE-PEG-NHS (12:3:0.1, w / w), add a certain amount of finasteride and nano-cerium oxide (1:1, w / w) and dissolve in 5 ml of chloroform, and remove the organic reagent by rotary evaporation in a water bath for 45 minutes to obtain a lipid film.
[0048] Add 5 ml PBS for hydration and sonicate for 15 min to obtain liposomes without Leptin;
[0049] The obtained liposomes were diluted with pure water to 1 mg / ml, Leptin was added, and the mixture was incubated at 40°C for 1 hour and at 6°C overnight to obtain leptin-modified liposomes targeting dermal papilla cells.
[0050] Embodiment 5:
[0051] Synthesis of cerium oxide nanoparticles: Weigh cerium acetate, oleylamine and xylene in a ratio of 5:6:30 in a penicillin bottle, stir and dissolve at room temperature, heat in a water bath at 100°C under an argon atmosphere until the liquid is clear, quickly add 5 ml of deionized water, heat in a water bath at 100°C under an argon atmosphere until the liquid is clear again, let stand and cool, add 50 ml of ethanol to wash, and discard the supernatant after centrifugation to obtain cerium oxide nanoparticles.
[0052] Synthesis of targeted liposomes: Weigh the corresponding proportions of cholesterol, lecithin, and DSPE-PEG-NHS (12:3:0.1, w / w), add a certain amount of finasteride and nano-cerium oxide (2:1, w / w) and dissolve in 5 ml of chloroform, and remove the organic reagent by rotary evaporation in a water bath for 45 minutes to obtain a lipid film.
[0053] Add 5 ml PBS for hydration and sonicate for 20 min to obtain liposomes without Leptin;
[0054] The obtained liposomes were diluted with pure water to 1 mg / ml, Leptin was added, and the mixture was incubated at 35°C for 2 hours and at 5°C overnight to obtain leptin-modified liposomes targeting dermal papilla cells.
[0055] Examples 1-5 Liposome in vitro percutaneous permeation experiment (the results obtained in the five examples are similar, and the experimental results of Example 1 are shown)
[0056] See also Figure 1 , Figure 1 Co-localization immunofluorescence of target LEPR and target head Leptin on human dermal papilla cells (HDPC). Figure 1 It can be observed that the target head Leptin and the target point LEPR co-localize in the cytoplasm, that is, Leptin can target the dermal papilla cells as a target head.
[0057] See also Figure 2 , Figure 2 This is an experimental picture of the co-localization of the target LEPR and the targeted liposome (L-LP-Fi / CeO2) in mouse skin. Figure 2 It can be observed that the target and liposomes are co-localized in the hair follicles. The liposomes can anchor LEPR, allowing the drug to accurately enter the hair follicles and exert its efficacy at the target cell DPC.
[0058] See also Figure 3 , Figure 3A is a picture of the in vitro transdermal test of common liposomes and targeted liposomes. It can be observed that the liposomes with target heads can accurately enter the hair follicles. Figure 3 B is a 24h in vitro mouse skin retention experiment. HPLC detection shows that the retention amount of the liposomes (L-LP-Fi / CeO2) prepared in Example 1 in mouse skin is 150% and 15% higher than that of the free drug (Fi / CeO2) and the ordinary liposomes (LP-Fi / CeO2), respectively. It can be seen that the liposomes prepared in Example 1 can accurately deliver the drug to the hair follicles and increase the retention of the drug in the skin.
[0059] Animal experiments of Examples 1-5 (the results obtained in the five examples are similar, and the experimental results of Example 1 are shown)
[0060] See also Figure 4 In vitro, C57 / BL6 mice were treated with testosterone to establish AGA model (Model) and were divided into groups for drug administration. There were significant differences in hair growth rate and hair growth coverage between the targeted co-loaded liposome group (L-LP-Fi / CeO2) and the model group (Model) and the common liposome group (LP-Fi / CeO2). Figure 4 It can be seen that the hair growth coverage rate of the targeted co-loaded liposome group (L-LP-Fi / CeO2) is the best.
[0061] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A leptin-modified liposome targeting dermal papilla cells, characterized in that: The synthesis of the liposome comprises the following steps: Step 1: Take cerium acetate, oleylamine and xylene, stir and dissolve them into a mixed solution at room temperature, heat the mixed solution until it is clear, then add deionized water, heat the mixed solution again until it is clear, let it stand and cool, add ethanol for washing, centrifuge and discard the supernatant to obtain cerium oxide nanoparticles; Step 2: Take cholesterol, lecithin, and DSPE-PEG-NHS, dissolve finasteride and the cerium oxide nanoparticles prepared in step 1 in chloroform, add the dissolved mixture to the mixture of cholesterol, lecithin, and DSPE-PEG-NHS, and remove the organic reagent by rotary evaporation in a water bath to obtain a lipid film; Step 3: The lipid film synthesized in step 2 was added to PBS for hydration and ultrasonicated for 10-20 min to obtain liposomes without Leptin; Step 4: The liposomes synthesized in step 3 were diluted with pure water to 1 mg / ml, Leptin was added, and the mixture was incubated at 35-40°C for 1-2 hours and at 2-6°C overnight to obtain leptin-modified liposomes targeting dermal papilla cells.
2. The leptin-modified liposome targeted to dermal papilla cells according to claim 1, characterized in that: In step 1, the mass ratio of cerium acetate, oleylamine and xylene is 1-5:6:
30.
3. The leptin-modified liposome targeted to dermal papilla cells according to claim 2, characterized in that: In step 1, the conditions for heating the mixed solution twice to clarify are both heating in an argon atmosphere at a temperature of 80-100° C. in a water bath.
4. The leptin-modified liposome targeted to dermal papilla cells according to claim 3, characterized in that: In step 1, the amount of deionized water added is 1-5 ml, and the amount of ethanol added is 50 ml.
5. The leptin-modified liposome targeted to dermal papilla cells according to any one of claims 1 to 4, characterized in that: In step 2, the mass ratio of cholesterol, lecithin and DSPE-PEG-NHS is 9-12:3:0.
1.
6. The leptin-modified liposome targeted to dermal papilla cells according to claim 5, characterized in that: In step 2, the mass ratio of finasteride to cerium oxide nanoparticles is 1-3:
1.
7. The leptin-modified liposome targeted to dermal papilla cells according to claim 6, characterized in that: In step 2, the water bath rotary evaporation time is 40-50 min.