Photosensitive type biomimetic drug delivery system and preparation method and application thereof

Abstract

This invention relates to nanodelivery, and particularly to a photosensitive biomimetic drug delivery system, its preparation method, and its applications. The photosensitive biomimetic drug delivery system, RSCP NPs, mainly consists of a core and a shell: the shell is composed of macrophage membrane RCP co-modified with a fat-targeting peptide P3 (CKGGRAKDC) and a photosensitive supramolecular complex CHD (composed of DSPE-2-(4-hydroxyphenylazo)-benzoic acid-β-cyclodextrin); the core is a pH-responsive SEP NP formed by the self-assembly of a triblock amphiphilic polymer. RSCP NPs possess high drug loading capacity and can also regulate drug release in stages in response to multiple signals (inflammatory microenvironment, red light, and pH), actively targeting the WAT inflammatory region, thereby effectively reducing the toxic side effects caused by off-target effects and improving the low-intensity inflammatory microenvironment of obese tissues.

Description

Technical Field

[0001] This invention relates to nanosystems, and particularly to a photosensitive biomimetic drug delivery system, its preparation method, and its applications. Background Technology

[0002] The World Health Organization has listed obesity as a disease, a multifactorial metabolic disorder characterized by excessive fat accumulation due to an imbalance between energy intake and expenditure. Global projections of high BMI (overweight + obesity) levels indicate that by 2035, nearly 3.3 billion adults will be at risk of being overweight and obese, a significant increase from 2.2 billion in 2020. The proportion of adults with high BMI will rise sharply from 42% in 2020 to over 54% in 2035. Equally concerning is the BMI situation among young people aged 5 to 19. The proportion of those with high BMI in this age group is projected to increase dramatically from 22% (430 million people) in 2020 to over 39% (770 million people) in 2035. Therefore, obesity has become a pressing global public health challenge.

[0003] Currently, treatment options include medication for individuals who have not achieved weight loss through lifestyle interventions. If lifestyle interventions do not result in at least a 5% weight loss within the first 3-6 months, obese or overweight individuals with complications may use medication, such as orlistat. Many weight-loss medications may have side effects, such as nausea, diarrhea, insomnia, or other health problems. Some medications may suppress appetite and affect nutrient intake. Drug treatment often leads to weight rebound after discontinuation, and there is a lack of effective long-term maintenance management methods. Furthermore, not all patients are suitable for medication; patients with comorbidities such as heart disease or diabetes need to choose carefully.

[0004] In the clinical treatment of most obesity complications, medications primarily act downstream of the inflammatory process (e.g., insulin or statins) rather than addressing the underlying cause. On the other hand, anti-inflammatory drugs such as glucocorticoids (dexamethasone) and aspirin have been clinically tested in obese and / or diabetic patients for over a decade. However, in the long term, high-dose and prolonged use of these drugs can lead to serious side effects, including inducing neurological disorders and gastrointestinal irritation, and often results in rapid relapse of obesity upon discontinuation.

[0005] Therefore, there is an urgent need to develop novel anti-obesity therapeutic targets and intervention strategies that directly target white adipose tissue, regulate lipid metabolism, alleviate "post-inflammatory" effects, and minimize off-target effects. Summary of the Invention

[0006] Purpose of the invention

[0007] This invention constructs a red-light-sensitive biomimetic nanoparticle drug delivery system targeting the inflammatory region of the white adipocyte (WAT). It promotes browning of white adipocytes while remodeling M1-like macrophage polarization, elucidates the causal relationship between obesity-induced macrophage polarization and WAT remodeling, and determines whether weight-loss therapies can additionally alleviate obesity-driven inflammatory phenotypes. Furthermore, by clarifying the synergistic long-acting targeting mechanism of the red-light-sensitive biomimetic nanoparticle drug delivery system's long circulation and the stability of the targeting molecules, this invention provides theoretical and practical basis for the research and application of novel drug delivery systems for metabolic diseases.

[0008] Technical solution

[0009] A photosensitive biomimetic drug delivery system is characterized by mainly consisting of two parts: a core and a shell. The shell is a photosensitive biomimetic material RCP, and the core is SEP NPs. The two are self-assembled to form a photosensitive biomimetic drug delivery system RSCP NPs.

[0010] The photosensitive biomimetic material RCP is composed of macrophage membrane RCP co-modified with a fat-targeting peptide P3 and a photosensitive supramolecular complex CHD; wherein the amino acid sequence of the fat-targeting peptide P3 is CKGGRAKDC; further, the fat-targeting peptide P3 is a DSPE-modified P3 polypeptide, namely the DSPE-P3 polypeptide. The photosensitive supramolecular complex CHD is prepared by the following steps: after reacting a mixture of carboxymethyl-β-cyclodextrin, carbodiimide and N-hydroxysuccinimide, 4-hydroxybenzoazo-benzoic acid and DSPE-PEG are added and reacted.

[0011] The macrophage membrane is an M1-like macrophage membrane formed after induction.

[0012] The SEP NPs are obtained by ultrasonic extrusion of the triblock amphiphilic polymer SEP.

[0013] The SEP is prepared by the following steps: sodium alginate, carbodiimide and N-hydroxysuccinimide are reacted, and then L-phenylalanine ethyl ester hydrochloride and PEG are added to form a triblock amphiphilic polymer SEP.

[0014] The aforementioned photosensitive biomimetic drug delivery system is characterized in that the photosensitive biomimetic drug delivery system encapsulates a weight-loss drug, such as astaxanthin and / or rosiglitazone.

[0015] The application of the aforementioned photosensitive biomimetic drug delivery system in the preparation of weight-loss drugs.

[0016] Specifically:

[0017] Construction and characterization of the photosensitive supramolecular complex CHD:

[0018] Weigh an appropriate amount of carboxymethyl-β-cyclodextrin (CD) and dissolve it in deionized water. Then, add carbodiimide (EDC) and N-hydroxysuccinimide (NHS) sequentially, and react at 25°C for 30 min. Next, add 4'-hydroxyxyazobenzene-2-carboxylic acid (HABA) and DSPE-PEG, and react in the dark for 24 h. After the reaction is complete, place the product in a dialysis bag with a molecular weight cutoff of 1000 MW and dialyze it with deionized water to remove unreacted reagents.

[0019] Preparation process and characterization of photosensitive biomimetic material RCP:

[0020] RAW 264.7 cells were induced to polarize into M1-like macrophages by LPS (0.5 μg / mL) and IFN-γ (0.1 μg / mL). The M1-like macrophage membranes were then isolated and extracted for later use. Subsequently, RAW biofilms were obtained after ultrasonic extrusion. 5-10 mg of RAW biofilm was weighed, dissolved in PBS, and then 20 μL of CHD (20 mg / mL) and 10 μL of DSPE-P3 peptide (10 mg / mL) were added to the RAW biofilm solution. The mixture was vortexed and allowed to stand for 24 h to obtain the CHD and P3 peptide-modified photosensitive biomimetic material RCP. Then, through… 1 The main components of RCP biofilm were analyzed and characterized using techniques such as 1H NMR and FTIR.

[0021] Preparation process and characterization of triblock amphiphilic polymer SEP:

[0022] Weigh 50-80 mg of sodium alginate (SA) and dissolve it in 30-50 ml of deionized water, stirring thoroughly until dissolved. Next, add 10-20 mg of EDC and NHS and stir at 25°C for 30 min to activate the carboxyl groups. After the reaction is complete, add 40-60 mg of L-phenylalanine ethyl ester (LPE) and 20-30 mg of PEG sequentially, and continue the reaction at 25°C in the dark for 48 h. Then, place the product in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it with deionized water to remove unreacted reagents. Change the deionized water every 6 h for 3 consecutive days. The product obtained after dialysis is then freeze-dried under vacuum; the resulting white flocculent substance is the SEP polymer. 1 Physicochemical properties were characterized using techniques such as H NMR and FTIR.

[0023] Weigh 5 mg of SEP and dissolve it in 5 mL of deionized water. Stir in the dark at room temperature until fully dissolved. Place the resulting solution in a contact ultrasonic disruptor and sonicate for 3 minutes (100 W). After sonication, use a 0.45...

[0024] The SEP solution was filtered by squeezing through a μm filter membrane and then left to stand overnight at 4°C.

[0025] Preparation of RSCP NPs:

[0026] Preparation and characterization of the biomimetic nanomedicine delivery system RSCP NPs: 5-10 mg of SEP NPs and 5-10 mg of RCP biomembrane were dissolved in 1×PBS. Next, the SEP NPs and RCP biomembrane were thoroughly mixed at a 1:1 mass ratio for 10 min. Subsequently, the mixture was subjected to contact sonication (100 W, 5 min), and the sonicated solution was filtered through a 0.45 μm microporous membrane. Finally, the mixture was allowed to stand at 37 °C for 12 h, and the resulting pale yellow particles were obtained by vacuum freeze-drying, which were identified as RSCP NPs.

[0027] This invention constructs a red-light-sensitive biomimetic nanoparticle drug delivery system targeting the WAT (wasting adipocyte) inflammatory region, promoting browning of white adipocytes while reshaping M1-like macrophage polarization. Based on a novel red-light-sensitive biomimetic nanoparticle drug delivery system targeting the WAT inflammatory region, this system possesses dual-targeting capabilities, enabling the simultaneous delivery of two or more drugs to the WAT inflammatory region, reducing off-target effects and improving the safety of clinical drug use. The morphology and material stability of this red-light-sensitive biomimetic nanoparticle drug delivery system were evaluated using atomic force microscopy (AFM), transmission electron microscopy (TEM), and zeta potential analysis. Additionally, a biomimetic RSC (reactive scaffold cell) biofilm coating was constructed using an extrusion method. Meanwhile, this invention will analyze and characterize the main components of RSC biomembranes using fluorescent probe methods, polypropylene gel electrophoresis, and cell flow cytometry. We will also conduct in-depth research on the physicochemical properties and biological effects of RSCP NPs, including morphology, size, stability, drug loading rate, drug release kinetic parameters, targeting of adipose tissue, and safety. Furthermore, we will study the in vivo and in vitro efficacy of RSCP NPs. The results show that RSCP NPs not only have good safety and no obvious toxic side effects, but also have strong targeting ability and can precisely regulate the stepwise release of drugs (such as astaxanthin and rosiglitazone), thereby alleviating low-density lipoprotein inflammatory syndrome (WAT) and promoting browning of white adipocytes, thus achieving the purpose of weight loss.

[0028] Working principle of the invention

[0029] The photosensitive biomimetic drug delivery system RSCP NPs constructed in this invention mainly consists of two parts: a core and a shell. The shell is composed of macrophage membrane RCP co-modified with lipid-targeting peptide P3 (CKGGRAKDC) and a photosensitive supramolecular complex CHD; the core is SEP NPs (formed from the triblock amphiphilic polymer SEP). The two self-assemble to form the photosensitive biomimetic drug delivery system RSEPNPs. It has a high drug loading capacity and can also respond to multiple signals (inflammatory microenvironment, red light, and pH) to regulate the stepwise release of drugs and actively target the WAT inflammatory region. This effectively reduces the toxic side effects caused by off-target effects of drugs and achieves the effect of improving the low-intensity inflammatory microenvironment of obese tissues.

[0030] Beneficial effects

[0031] In recent years, the low-intensity chronic inflammation and metabolic disorders caused by the expansion of white adipose tissue (WAT) in obese patients have attracted significant attention from researchers. Recent studies have shown that macrophages are readily recruited and aggregated in the WAT of obese patients, polarizing into M1-type macrophages and producing large amounts of pro-inflammatory cytokines, thus leading to low-intensity chronic inflammation. Therefore, reducing the infiltration of M1-type macrophages and inhibiting their pro-inflammatory processes may help reduce pathological remodeling of WAT, alleviating obesity while correcting lipid metabolism disorders.

[0032] To this end, this invention presents a novel red-light-sensitive biomimetic nanoparticle drug delivery system targeting the inflamed area of ​​white adipocytes (WAT). This system possesses dual-targeting capabilities, enabling the simultaneous delivery of two or more drugs to the WAT inflamed area, thereby reducing off-target effects and improving the safety of clinical medication. Furthermore, leveraging the photosensitivity and pH sensitivity of this drug delivery system, the stepwise release of drugs can be precisely controlled, thereby alleviating low-density inflammation in WAT and promoting browning of white adipocytes.

[0033] This intervention strategy is our original and highly innovative design. Currently, existing drug delivery systems for metabolic diseases primarily focus on inhibiting lipid absorption or promoting lipid breakdown, with few studies exploring direct targeting to alleviate metabolic inflammation for the treatment of obesity and its complications. These shortcomings severely restrict the clinical translation of drug therapies. Therefore, focusing on the role of low-intensity inflammation in promoting obesity, we selected a previously unexplored area—the cross-disciplinary mechanism between macrophages, low-intensity inflammation, and lipid metabolism disorders—to investigate. This approach organically combines hot topics from interdisciplinary research, including biomimetic nanomedicine delivery systems, the WAT immune microenvironment, and lipid energy metabolism, proposing a macrophage-targeted precision treatment strategy for obesity. This provides a solid theoretical foundation for the development and application of novel drug delivery systems for metabolic diseases.

[0034] (1) Novel Therapeutic Targets for Obesity: In recent years, the expansion of white adipocytes (WATs) in obese patients, leading to low-grade chronic inflammation and metabolic disorders, has attracted considerable attention from researchers. Recent studies have found that macrophages in the WATs of obese patients are easily recruited, aggregated, and polarized into M1-like macrophages, producing a large number of pro-inflammatory cytokines, thus leading to low-grade chronic inflammation. Therefore, reducing the infiltration of M1-like macrophages and inhibiting their pro-inflammatory programs may reduce pathological remodeling of WATs, alleviating obesity while correcting lipid metabolism disorders. Furthermore, in previous co-culture experiments, we have demonstrated that Asta can reprogram M1-like macrophages into M2-like macrophages and effectively correct lipid metabolism disorders. Simultaneously, Rosi (rosiglitazone) can promote browning of white adipocytes, effectively alleviating the polarization of M1-like macrophages. All of these studies suggest that macrophages are potential therapeutic targets for obesity.

[0035] (2) Novel Biomimetic Nanoparticle Drug Delivery System: This invention prepares a photosensitive supramolecular complex CHD and modifies it together with P3 peptide onto the macrophage membrane surface to form a photosensitive biomimetic material RCP that can directly target the WAT inflammatory region. This biomimetic material has not been reported by any team before and has certain innovation. Subsequently, pH-sensitive SEPNPs are encapsulated with RCP to construct a red light-sensitive biomimetic nanoparticle drug delivery system RSCP NPs that targets the WAT inflammatory region. This invention utilizes the dual targeting of RSCP NPs to accurately deliver A&R to the WAT inflammatory region, reducing off-target effects and improving the safety of clinical drug use; at the same time, the multi-signal response of RSCP NPs (red light signal and microenvironment with different pH values) enables the stepwise release of drugs, thereby alleviating low-density inflammation in WAT and promoting browning of white adipocytes. This intervention strategy is our original construction and has strong innovation.

[0036] (3) Novel Concept for Treating Obesity and Insulin Resistance: In terms of academic approach, current drug delivery systems for metabolic diseases still focus more on inhibiting lipid absorption or promoting lipid breakdown, with few studies exploring direct targeting to alleviate "metabolic inflammation" for treating obesity and its complications. In fact, these shortcomings severely restrict the clinical translation of drug therapies. The applicant focuses on the role of low-density lipoprotein (LDL) inflammation in promoting obesity, selecting a previously unexplored area—the cross-disciplinary mechanism between macrophages and LDL inflammation and lipid metabolism disorders—to conduct research. This approach organically grasps the hot topics of interdisciplinary research, including the organic connection between biomimetic nanomedicine delivery systems, the WAT immune microenvironment, and lipid energy metabolism. In summary, this invention expands the pathological function of macrophages in the treatment of obesity and insulin resistance, proposes a precise treatment strategy for obesity and insulin resistance targeting macrophages, and provides a solid theoretical foundation for the research and application of novel drug delivery systems for metabolic diseases. Attached Figure Description

[0037] Figure 1 Synthesis, physicochemical characterization, and in vitro drug release kinetics of CHD. (A) CHD 1 (A) H NMR spectrum; (B) FTIR spectrum of CHD; (C) UV-Vis absorption spectrum of CHD; (D) Encapsulation efficiency and drug loading of CHD.

[0038] Figure 2 Preparation and physicochemical characterization of the photosensitive biomimetic material RCP. (A) P3 peptide 1 (A) 1H NMR spectrum; (B) DSPE-P3 peptide 1 H NMR image; (C) Chip of FITC-CHD with M1-like macrophage membrane; (C) Chip of FITC-P3 peptide with M1-like macrophage membrane; N=3.

[0039] Figure 3 Preparation and physicochemical characterization of SEP NPs. (A) SEP polymers 1 (A) H NMR spectrum; (B) FTIR spectrum of SEP polymer; (C) TEM spectrum of SEP NPs; (D) Hemolytic assay of SEP NPs.

[0040] Figure 4 Preparation and physicochemical characterization of the photosensitive biomimetic drug delivery system RSCP NPs. (A) TEM and particle size distribution of RSCP NPs; (B) TEM and particle size distribution of RSCP NPs; (C) Coomassie Brilliant Blue plot of RSCP NPs; (D) Hemolytic assay of RSCP NPs; N=3.

[0041] Figure 5 Biological effects of RSCP NPs. (A) Uptake of RSCP NPs by primary adipocytes at different time points; (B) Uptake of different NPs by primary adipocytes; (C) Mechanism of RSCP NP uptake by primary adipocytes; (D) Distribution of different NPs in mouse AT.

[0042] Figure 6 Safety assessment of RSCP NPs in vitro and in vivo. (A) Serum ALT level; (B) Serum AST level; (C) Serum BUN level; (D) Serum CRE level.

[0043] Figure 7A&R RSCP NPs remodel M1-like macrophage polarization and induce browning of white adipocytes in vitro. (A) Schematic diagram of in vitro efficacy evaluation; (B) Effect of red light signal on macrophage remodeling; (C) Efficacy evaluation of Asta RSCP NPs on macrophage remodeling; (D) Flow cytometry analysis of the proportion of M2-like macrophages.

[0044] Figure 8 A&R RSCP NPs remodel M1-like macrophage polarization and induce browning in white adipocytes in vitro. (A) Effect of M1 macrophages on lipid accumulation in primary adipocytes; (B) Effect of Asta RSCP NPs on macrophage inflammatory factor secretion.

[0045] Figure 9 Expression levels of Ucp1 and Cox7a1 in mouse primary adipocytes after treatment with Rosi RSCP NPs.

[0046] Figure 10 .ob / ob mouse CTL group and A&R RSCP NPs' GTT, ITT.

[0047] Figure 11 Changes in body weight and diet in HFD rats.

[0048] Figure 12 HE staining results of various tissues from .ob / ob mice.

[0049] Figure 13 Schematic diagram of the invention. Detailed Implementation

[0050] Example 1

[0051] 1. Research Plan

[0052] 1.1 Construction and biological effects of a red light-sensitive biomimetic nanomedicine delivery system

[0053] (1) Construction and physicochemical characterization of the photosensitive supramolecular complex CHD: such as Figure 13As shown, an appropriate amount of carboxymethyl-β-cyclodextrin (CD) was weighed and dissolved in deionized water, and carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added sequentially, and the reaction was carried out at 25°C for 30 min. Then, 4'-Hydroxyazobenzene-2-carboxylic acid (HABA) and DSPE-PEG were added, and the reaction was carried out in the dark for 24 h. After the reaction was completed, the product was placed in a dialysis bag with a molecular weight cutoff of 1000 MW and dialyzed with deionized water to remove unreacted reagents. Finally, the dialyzed product was freeze-dried under vacuum, and the resulting yellow flocculent substance was CHD, which was then analyzed by nuclear magnetic resonance spectroscopy. 1 The main components of the CHD biofilm were analyzed and characterized using techniques such as 1H NMR and Fourier Transform Infrared Spectrometry (FTIR). Furthermore, since astaxanthin absorbs blue light and emits green fluorescence, astaxanthin (Asta) was used as the encapsulation medium. We further investigated the drug loading capacity of the photosensitive supramolecular complex for CHD. Asta solution (5 mg / ml) was added to the CHD solution, mixed, and allowed to stand at room temperature for 24 h. The encapsulation efficiency and drug loading rate of CHD were calculated based on the concentration of free astaxanthin (Asta) in the supernatant.

[0054] Encapsulation rate

[0055] Drug loading rate Where M0 is the total mass of Asta; M1 is the free mass in the supernatant of Asta; and Q0 is the unloaded nanomass of CHD NPs.

[0056] result:

[0057] The photosensitive polymer CHD was prepared by esterification reaction, and then... 1 Physicochemical characterization by H NMR and FTIR revealed that DSPE and HAPA were successfully grafted onto CD. Figure 1 A and B). According to UV-Vis absorption spectroscopy analysis, the n-π* transition band of trans-CHD extends into the red region and is stronger than that of cis-CHD. Figure 1C). Therefore, trans-CHD can be excited by red light and converted to the cis form. To evaluate the drug loading capacity of CHD, we loaded Asta into the hydrophobic cavity of CD, forming a supramolecular valve with grafted HABA to close the pore (Asta & CHD). The encapsulation efficiency of Asta, as determined by high performance liquid chromatography, was 90.7%, and the optimal drug loading rate was achieved at an Asta:CHD ratio of 5:50. Figure 1 D). In summary, CHD can efficiently package Asta.

[0058] (2) Preparation and Characterization of Photosensitive Biomimetic Material RCP: RAW 264.7 cells were induced to polarize into M1-like macrophages by lipopolysaccharide (LPS) (0.5 μg / mL) and IFN-γ (0.1 μg / mL). The M1-like macrophage membranes were isolated and extracted for later use. Subsequently, RAW biomembranes were obtained by ultrasonic extrusion. In addition, 20 μL of CHD (20 mg / mL) and 10 μL of DSPE-P3 peptide (the amino acid sequence of P3 peptide is CKGGRAKDC, 10 mg / mL) were added to the RAW biomembrane solution, vortexed to mix, and allowed to stand for 24 h to obtain the CHD and P3 peptide-modified photosensitive biomimetic material RCP. Then, through... 1 The main components of RCP biofilm were analyzed and characterized using techniques such as 1H NMR and FTIR.

[0059] result:

[0060] To prepare the photosensitive biomimetic material RCP, we first modified the P3 peptide with DSEP, and then... 1 The formation of the DSPE-P3 peptide polymer was verified by 1H NMR and FTIR. Figure 2 (A and B). Secondly, using the principle of "like dissolves like," and with the aid of DSPE lipid chains, CHD and P3 peptides were respectively embedded into the membrane of M1-like macrophages to generate the photosensitive biomimetic material RCP. Fluorescence experiments showed that the P3 peptide (P3 was labeled with FITC) and CHD were successfully embedded into the membrane of M1-like macrophages. Figure 2 (C and D).

[0061] (3) Physicochemical characterization and drug loading capacity assessment of SEP NPs:

[0062] Preparation and Characterization of the Triblock Amphiphilic Polymer SEP: An appropriate amount of sodium alginate (SA) was dissolved in deionized water and stirred thoroughly until dissolved. Next, EDC and NHS were added and the mixture was stirred at 25°C for 30 min to activate the carboxyl groups. After the reaction was complete, L-phenylalanine ethyl ester hydrochloride (LPE) and PEG were added sequentially, and the reaction was continued at 25°C under light for 48 h. Then, the product was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water to remove unreacted reagents. The deionized water was changed every 6 h, and dialyzed continuously for 3 days. The product obtained after dialysis was then freeze-dried under vacuum. The resulting white flocculent substance was the SEP polymer. 1 Physicochemical properties were characterized using techniques such as H NMR and FTIR.

[0063] 5 mg of SEP was dissolved in 5 mL of deionized water and stirred under light at room temperature until fully dissolved. The resulting solution was placed in a contact ultrasonic disruptor and ultrasonicated for 3 min (100 W). After ultrasonication, the SEP solution was filtered through a 0.45 μm filter membrane and allowed to stand overnight at 4 °C. The shape, size, and distribution of SEP NPs were then observed using TEM, and the surface charge, particle size, and polydispersity index (PdI) of the SEP NPs were detected using dynamic light scattering (DLS). To further evaluate the drug loading capacity of SEP, a 5 mg / mL SEP solution was prepared for later use. The optimal drug loading formulation was then determined based on different drug-to-carrier mass ratios. The specific experimental steps were as follows: Rosiglitazone (5-20 mg) was added to 1 mL of SEP solution and stirred under light at room temperature for 30 min. Subsequently, the sample was ultrasonicated at 100 W for 10 min. After ultrasonication, the sample was allowed to stand at 4 °C for 24 h. The sample was then centrifuged at 24,000 rpm / min, 4°C, for 1 hour. The supernatant was collected for later use, and the precipitate at the bottom was the Rosi SEP NPs. The encapsulation efficiency and drug loading rate of the SEP NPs were calculated based on the concentration of free Rosi in the supernatant.

[0064] result:

[0065] In this study, we prepared a triblock amphiphilic polymer, SEP, using esterification. We then... 1 HNMR and FTIR analyses revealed that SEP is composed of polymers of three monomers: SA, LEP, and PEG. Figure 3(A and B). Because SEP is an amphiphilic polymer, it can self-assemble into a spherical structure after ultrasonic treatment in the reaction medium. For example... Figure 3 As shown in Figure C, TEM and DLS revealed that SEPNPs were spherical, uniform in size, and evenly dispersed. Hemolytic activity analysis showed that the hemolysis rate at a SEPNPs concentration of 250 μg / mL was only 1.43%, indicating that SEPNPs possess good biocompatibility. Figure 3 D).

[0066] (4) Preparation and Characterization of the Biomimetic Nanoparticle Drug Delivery System RSCP NPs: Appropriate amounts of SEP NPs and RCP were dissolved in 1×PBS. Next, SEP NPs and RCP were thoroughly mixed at a 1:1 mass ratio for 10 min. Subsequently, the mixture was subjected to contact sonication (100W, 5 min), and the sonicated solution was filtered through a 0.45 μm microporous membrane. Finally, the mixture was allowed to stand at 37℃ for 12 h, and the resulting pale yellow particles obtained by vacuum freeze-drying were identified as RSCP NPs. To further verify the physicochemical properties of RSCP NPs, this invention will use proteomics, Malvern particle size analyzer, transmission electron microscopy, polypropylene gel electrophoresis, and flow cytometry to verify whether the RCP biofilm is coated on the surface of SEP NPs. Simultaneously, the stability and hemolytic activity of RSCP NPs will be tested. Furthermore, we will further investigate the multi-level response (photosensitivity and pH sensitivity) in vitro drug release mechanism of A&R RSCP NPs.

[0067] The photosensitive biomimetic drug delivery system RSCPNPs mainly consists of two parts: a core of SEPNPs (drug-carrying core) and a macrophage membrane shell co-modified with P3 peptide and CHD (targeting WAT, red light sensitive type). TEM observations reveal that RSCPNPs exhibit a distinct core-shell structure, with a uniform outer membrane layer of similar thickness to that of RSCPNPs. The interior contains dark spheres with a diameter similar to that of SEPNPs, and the overall surface is smooth, uniform in size, and individually dispersed. Figure 4 Figures A and B show the RSCP NPs. As shown in Figure B, the average particle size of the RSCP NPs is 170.5 nm. Furthermore, based on Coomassie Brilliant Blue and Western blot results, RCP biofilms were successfully coated onto the surface of SEP NPs to form RSCP NPs. Figure 4 C). Hemolytic assay results showed that at RSCP NPs concentrations as high as 250 μg / mL, the hemolysis rate was only 0.24% ( Figure 4 D). All of the above studies indicate that RSCPNPs have good biocompatibility.

[0068] 1.2 Cellular uptake mechanism and in vitro / in vivo targeting of RSCP NPs

[0069] (1) Isolation and induction of primary adipocytes: Ten 3-4 week old male C57BL / 6J mice were selected. After euthanasia, the mice were immersed in 75% ethanol solution for 3-5 minutes. Then, the mice were removed and placed on absorbent paper to slightly dry them. WAT (waxygenated adipocytes) from the groin area of ​​the mice was then obtained, chopped, and transferred to HEPES-collagenase solution for digestion. Finally, digestion was terminated, the cells were filtered, washed with PBS, and centrifuged to obtain adipocytes. The obtained adipocytes were then seeded into cell culture dishes and induced sequentially with inducers I through III to obtain white adipocytes.

[0070] (2) Investigation of the uptake capacity and mechanism of RSCP NPs by adipocytes: To investigate the release of RSCP NPs in adipocytes, RSCP NPs were first labeled with a Cy5 fluorescent probe. Next, adipocytes were seeded into confocal cell culture dishes. After cell attachment, culture medium containing Cy5-RSCP NPs (Cy5-RSCP NPs dosage: 40 μg / mL) was added, and the cells were cultured for 0, 2, 4, and 8 h, respectively. Finally, the uptake of RSCP NPs by adipocytes was observed using confocal laser scanning microscopy (CLSM). In addition, to further verify the molecular mechanism of RSCP NPs entering adipocytes, this invention pretreated adipocytes with different inhibitors (filipin (1 μg / mL) to block caveolin-mediated endocytosis; chlorpromazine (CPZ, 10 μg / mL) to block clathrin-mediated endocytosis; and 5-(N-Ethyl-N-isopropyl)amiloride (EIPA, 10 μg / mL) to block macropinocytosis), and then co-incubated them with Cy5-RSCP NPs for 24 h. The fluorescence signal in adipocytes was then observed using RSCP NPs to reveal the molecular mechanism of RSCP NPs entering adipocytes.

[0071] (3) RSCP NPs-specific targeting of WAT: To determine whether the targeting performance of RSCP NPs also occurs in vivo, Cy5-RSCP NPs were injected into C57BL / 6J mice via the tail vein. The distribution of Cy5-RSCP NPs in vivo was observed using a small animal in vivo imaging system. After 24 hours, the mice were euthanized, and their hearts, livers, spleens, lungs, kidneys, subcutaneous fat, epididymal fat, visceral fat, and brown WAT were collected. The fluorescence intensity in each tissue was observed using a small animal in vivo imaging system to determine the enrichment capacity of Cy5-RSCP NPs in different WATs. Simultaneously, WAT tissue was collected, macrophages in the WAT were labeled, and the distribution of Cy5-RSCP NPs in adipocytes and macrophages was observed under CLSM.

[0072] result:

[0073] To investigate the specific uptake mechanism of RSCP NPs by primary adipocytes, we first labeled RSCP NPs with Cy5 (Cy5 RSCP NPs). Figure 5 As shown in Figure A, the red fluorescence signal within adipocytes is directly proportional to the incubation time, and the red fluorescence signal gradually increases with prolonged incubation time. Therefore, RSCP NPs are taken up by primary adipocytes in a time-dependent manner. After entering the cell, the nuclear SEP NPs of RSCP NPs are captured by lysosomes and release the drug in response to the low pH microenvironment within the lysosome. To further investigate the molecular mechanism of RSCP NPs uptake by primary adipocytes, the fluorescence intensity in cells treated with the inhibitor CPZ was significantly reduced compared to that in the control group. Figure 5 B) indicates that RSCP NPs mainly rely on clathrin-mediated endocytosis to enter the cell. Furthermore, compared to other single-component nanomaterials, RSCP NPs exhibit specific uptake by primary adipocytes (B). Figure 5 C). Therefore, we will further evaluate the ability of RSCP NPs to specifically target WAT in mice. Figure 5 As shown in Figure D, after administration via tail vein injection, the fluorescence intensity of the WAT region in mice in the SEP NPs group was higher than that in mice in the free Cy5 group, while the fluorescence signal in the RSCP NPs group was the strongest. This indicates that the dual-targeting drug delivery system RSCP NPs has a stronger ability to specifically recognize the WAT inflammatory region.

[0074] 1.3 In vitro and in vivo safety evaluation of RSCP NPs

[0075] (1) In vitro safety evaluation of RSCP NPs: To assess the safety of RSCP NPs, we selected primary mouse hepatocytes and primary adipocytes as model cells. Therefore, primary mouse hepatocytes and primary adipocytes were seeded into 96-well plates and allowed to adhere. Complete culture medium containing different concentrations of RSCP NPs (0, 50, 100, 200, 250, and 500 μg / mL) was added, and the cells were cultured for 48 h. Cell viability was then assessed using a CCK-8 assay kit to reveal the in vitro safety of RSCP NPs.

[0076] (2) In vivo safety evaluation of RSCP NPs: To assess the safety of RSCP NPs in vivo, ten 6-week-old male C57BL / 6J mice were randomly divided into two groups. Mice in the experimental group were injected with RSCP NPs via the tail vein, while mice in the control group were injected with PBS. The administration was repeated every 3 days for 21 days. Before each administration, mouse weight and dietary changes were recorded. After 21 days of administration, the mice were euthanized, and whole blood was collected and incubated overnight at 4°C. The blood was then centrifuged at 3000 rpm for 15 minutes at 4°C. Serum was collected, and the levels of aspartate transaminase (AST), alanine transaminase (ALT), creatinine (CRE), and blood urea nitrogen (BUN) in the serum of the two groups were measured using a commercially available kit to determine the differences in liver and kidney function. In addition, various tissues and organs of mice (heart, liver, spleen, lung, kidney, subcutaneous fat, epididymal fat, visceral fat and brown WAT) were collected for H&E staining to assess the morphological changes of major organs after drug administration.

[0077] result:

[0078] Furthermore, this study also examined the changes in serum levels of liver injury markers ALT and AST, as well as kidney injury markers BUN and CRE. The results showed no significant difference between the RSCP NPs experimental group and the PBS control group. Figure 6 The above results confirm that the RSCP NPs drug delivery system does not cause acute damage to the liver and kidneys of mice.

[0079] 1.4 A&R RSCP NPs in vitro efficacy evaluation

[0080] (1) Mechanism of Asta RSCP NPs in remodeling macrophage polarization: RAW 264.7 cells were induced to polarize into M1-like macrophages by LPS (0.5 μg / mL) and IFN-γ (0.1 μg / mL). First, to investigate whether red light signaling intervention affects macrophage remodeling, polarized M1-like macrophages were treated with red light signaling for 0.5 h and then incubated for 24 h. Cell samples were collected and the M1 / M2 macrophage ratio was detected by flow cytometry to determine whether red light affects macrophage remodeling. Second, the efficacy and mechanism of Asta RSCP NPs in remodeling macrophages were investigated. Free Asta and Asta RSCP NPs (with the same dose of Asta) were added to M1-like macrophages, and after red light signaling intervention, they were incubated for 24 h. Then, cell supernatants were collected, and the levels of IL-10, IL-1β, and IL-6 in the supernatants were detected using an ELISA kit. Cell samples were collected, stained, and the M1 / M2 macrophage ratio was determined by CLSM and flow cytometry. At the molecular level, total protein and RNA were extracted from the treated cell samples, and the expression levels of M2-like macrophage markers CD206, Arg1, and DECTIN-1 were detected using RT-qPCR and Western blot techniques.

[0081] The preparation method of Asta RSCP NPs is as follows: 50 mg / ml Asta is added to RSCP NPs, vortexed and mixed, and reacted at 4°C overnight;

[0082] result:

[0083] Astaxanthin (Asta) has anti-inflammatory properties. Here, astaxanthin is used to reprogram macrophages, reprogramming M1 macrophages into M2 macrophages.

[0084] As a photosensitive nanomedicine delivery system, RSCP NPs were first investigated to determine whether red light signaling intervention affected macrophage polarization. Figure 7 A). The study found that red light treatment alone did not affect the polarization of M1-like macrophages. Figure 7 B). Figure 7 The CD description aims to further explore the efficacy and mechanism of Asta RSCP NPs in remodeling macrophages. For example... Figure 7 As shown in C, free Asta can reprogram M1-like macrophages into M2-like macrophages, and compared with the untreated Asta RSCPNPs group, the Asta RSCPNPs significantly increased the M2 / M1 macrophage ratio after red light intervention. Figure 7 D).

[0085] (2) Investigating the correlation between macrophage polarization and lipid metabolism disorders in adipocytes: To reveal the effect of macrophage polarization on lipid metabolism in adipocytes, this invention co-cultured macrophages under different polarization states with primary adipocytes in a Trans-well chamber. First, M0-polarized macrophages were seeded in the upper layer of the chamber, and primary adipocytes were seeded in the bottom layer. Second, M0 macrophages were polarized to M1-like macrophages using LPS (0.5 μg / mL) and IFN-γ (0.1 μg / mL). After co-culturing for 48 h, primary adipocytes in the bottom layer were collected, and total protein and RNA were extracted. The expression levels of lipid metabolism genes such as Pparα, ATGL, HSL, and Perilipin1 were detected using RT-qPCR and Western blot techniques. In addition, Asta RSCP NPs were added to reprogram M1-like macrophages, and they were co-cultured with primary adipocytes. Changes in the expression levels of lipid metabolism genes were detected using molecular biology techniques to reveal whether macrophage reprogramming can improve lipid metabolism in adipocytes.

[0086] result:

[0087] like Figure 8 As shown in Figures AB, after red light signal intervention, Asta RSCP NPs significantly increased the M2 / M1 macrophage ratio while inhibiting the expression of pro-inflammatory cytokines. Furthermore, to investigate the effect of macrophage polarization on adipocyte lipid metabolism, macrophages under different polarization states were co-cultured with primary adipocytes in a Trans-well chamber.

[0088] (3) Study on the mechanism of Rosi RSCP NPs inducing browning in white adipocytes:

[0089] Rosiglitazone (Rosi), a thiazolidinedione insulin sensitizer, works by acting on the specific peroxisome proliferator-activated gamma receptor (PPARγ). It lowers blood glucose by increasing the sensitivity of skeletal muscle, liver, and adipose tissue to insulin, thereby enhancing cellular glucose utilization. It significantly reduces fasting blood glucose, insulin, and C-peptide levels, and also has a lowering effect on postprandial blood glucose and insulin. It can promote browning of white adipocytes and effectively alleviate the polarization of M1-like macrophages.

[0090] Free Rosi and Rosi RSCP NPs were added to induced differentiated leukoplakia cells and incubated for 48 h. After incubation, lipid droplets were labeled using a BODIPY probe, and lipid accumulation in adipocytes before and after treatment was observed using CLSM. At the molecular level, the ability of Rosi RSCP NPs to regulate browning in leukoplakia cells was revealed by detecting the gene expression of leukoplakia browning marker Ucp1 and cytochrome C oxidase polypeptide 7a1 (Cox7a1).

[0091] The preparation method of Rosi RSCP NPs is as follows: 120 mg / ml Rosi is added to RSCP NPs, sonicated for 5 minutes, with a 3-second sonication pause and a 2-second pause, and the reaction is allowed to proceed overnight.

[0092] result:

[0093] To further investigate whether Rosi RSCP NPs can promote browning in leukoplakia, we added free Rosi and Rosi RSCP NPs to primary leukoplakia cells and co-incubated them. Figure 9 As shown, the gene expression of white fat browning markers Ucp1 and Cox7a1 was significantly upregulated after treatment, indicating that single Rosi RSCP NPs can effectively promote white fat cell browning and alleviate lipid accumulation in fat cells.

[0094] 1.5 To investigate the efficacy and mechanism of action of A&R RSCP NPs in improving low-density inflammation and complications in obese mice of multiple models.

[0095] (1) A&R RSCP NPs improve T2D in ob / ob mice: To investigate the efficacy of A&R RSCP NPs in alleviating metabolic inflammation and improving T2D in ob / ob mice, 7-week-old ob / ob mice were randomly divided into 7 groups (n=10 per group) and treated with PBS, Free Asta, Free Rosi, A&R, Asta RSCP NPs, Rosi RSCP NPs, and A&R RSCP NPs via tail vein injection, respectively. The tail vein administration was repeated every 3 days for 27 days. During each treatment period, changes in food intake and body weight were recorded. After the administration of the drug, the following experimental studies were conducted: Changes in blood glucose metabolism in mice after treatment were detected using the Glucose Tolerance Test (GTT) and the Insulin Tolerance Test (ITT); the overall metabolic level of mice after treatment was monitored using a small animal metabolic system; after euthanasia, serum samples were collected, and the levels of inflammatory cytokines in the serum of each group of mice were detected using an ELISA kit; at the tissue level, different types of adipocytes (WAT) from mice were collected and weighed, and H&E staining and immunofluorescence staining were performed on the WAT to statistically analyze adipocyte size and inflammatory cell infiltration, and the M1 / M2 macrophage ratio in the WAT was analyzed by flow cytometry; at molecular water levels, the expression levels of inflammatory cytokines in the WAT and the expression levels of the white adipose tissue browning marker genes Ucp1 and Cox7a1 were detected using RT-qPCR and Western blot techniques.

[0096] The preparation of A&R RSCP NPs is based on Asta RSCP NPs and Rosi RSCP NPs.

[0097] result:

[0098] like Figure 10 As shown in Figures AB, the glucose tolerance test results indicated that the A&R RSCP NPs administration group improved glucose tolerance in ob / ob mice. That is, treatment with A&R RSCP NPs significantly improved glycemic homeostasis in ob / ob mice. Furthermore, the ITT results confirmed that A&R RSCP NPs could improve insulin resistance in ob / ob mice.

[0099] (2) Mechanism study of A&R RSCP NPs in improving metabolic disorders in HFD obese mice: Sixty 8-week-old male C57BL / 6J mice raised in a standard environment were randomly divided into 5 groups. One group was fed a normal diet (ND), and the other 7 groups were fed a high-fat diet for 11 weeks, and weight changes were recorded. Then, the HFD mice were randomly divided into 7 groups of 10 mice each. They were treated with PBS, Free Asta, Free Rosi, A&R, Asta RSCP NPs, Rosi RSCP NPs, and A&R RSCP NPs via tail vein injection, respectively, once every 3 days for 27 days. After the treatment, the glucose tolerance and insulin resistance levels, overall metabolic level, serum inflammatory cytokine levels, adipocyte size, M2 / M1 macrophage ratio in WAT, expression levels of WAT inflammatory cytokines, and expression levels of white adipose tissue browning marker genes Ucp1 and Cox7a1 were detected.

[0100] result:

[0101] like Figure 11 As shown, HFD mice were randomly divided into 7 groups of 10 mice each. Each group was treated with PBS, Free Asta, Free Rosi, A&R, Asta RSCP NPs, Rosi RSCP NPs, and A&R RSCP NPs via tail vein injection, respectively. Monitoring the body weight of the HFD-fed mice showed that, compared to the HFD-CTL group, A&R RSCP NPs prevented weight gain in mice fed a high-fat diet. The body weight of mice in the Free Asta and Asta RSCP NPs groups was similar to that of the control mice.

[0102] (3) In vivo biocompatibility evaluation of A&R RSCP NPs: To assess the safety of A&R RSCP NPs, blood samples were collected for serum biochemical analysis. Major organs of mice in the treatment and control groups were collected, fixed with 4% paraformaldehyde, embedded, sectioned, and stained with H&E to assess the safety of A&R RSCP NPs.

[0103] result:

[0104] like Figure 12 As shown, we observed the physiological structures of the mouse heart, liver, spleen, lungs, and kidneys using H&E staining. The results indicated that the tissue structures of the two groups of mice were similar, and no tissue lesions were observed in either group. In conclusion, the A&R RSCP NPs drug nanodelivery system exhibits good biocompatibility and safety.

Claims

1. A photosensitive biomimetic drug delivery system, characterized in that, It consists of two parts: the outer shell is made of photosensitive biomimetic material RCP, and the inner core is SEP NPs. The two self-assemble to form a photosensitive biomimetic drug delivery system RSCP NPs. in: The photosensitive biomimetic material RCP is formed by co-modifying macrophage membranes with fat-targeting peptide P3 and photosensitive supramolecular complex CHD. The amino acid sequence of the fat-targeting peptide P3 is CKGGRAKDC. The photosensitive supramolecular complex CHD was prepared by the following steps: carboxymethyl-β-cyclodextrin, carbodiimide and N-hydroxysuccinimide were mixed and reacted, and then 4-hydroxybenzoazo-benzoic acid and DSPE-PEG were added to react to obtain CHD; The macrophage membrane is an M1-like macrophage membrane formed after induction; The SEP NPs are obtained by ultrasonic extrusion of the triblock amphiphilic polymer SEP. The triblock amphiphilic polymer SEP is prepared by the following steps: sodium alginate, carbodiimide and N-hydroxysuccinimide are reacted, and then L-phenylalanine ethyl ester hydrochloride and PEG are added to form the triblock amphiphilic polymer SEP.

2. The photosensitive biomimetic drug delivery system according to claim 1, characterized in that, The fat-targeting peptide P3 is a DSPE-modified P3 polypeptide, namely the DSPE-P3 polypeptide.

3. A pharmaceutical composition, characterized in that, The photosensitive biomimetic drug delivery system according to claim 1 or 2 encapsulates astaxanthin and / or rosiglitazone.

4. The use of the pharmaceutical composition according to claim 3 in the preparation of a weight-loss drug.

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