Photosensitive bionic drug delivery system and preparation method and application thereof

Abstract

The invention relates to nano delivery, in particular to a photosensitive bionic drug delivery system as well as a preparation method and application thereof. The photosensitive bionic drug delivery system RSCP NPs is mainly composed of a core part and a shell part, wherein the shell is composed of a macrophage membrane RCP co-modified by fat targeting peptide P3 (CKGGRAKDC and a photosensitive supramolecular compound CHD (the compound is composed of DSPE-2-(4-hydroxyphenylazo)-benzoic acid-beta-cyclodextrin); an inner core is pH response type SEP NPs formed by self-assembly of a triblock amphiphilic polymer. The RSCP NPs has higher drug loading capacity, and can respond to multiple signals (inflammatory microenvironment, red light and pH) to regulate the capacity of gradual release of drugs and active targeting of WAT inflammatory areas, so that toxic and side effects caused by off-target effect of the drugs can be effectively reduced, and the effect of improving low-intensity inflammatory microenvironment of obese tissues is achieved.

Description

Technical Field

[0001] The present invention relates to a nanosystem, and in particular to a photosensitive bionic drug delivery system and a preparation method and application thereof Background Art

[0002] The World Health Organization has included "obesity" in the list of diseases. As a metabolic disorder caused by multiple factors, it is manifested as excessive fat accumulation caused by an imbalance in energy intake and expenditure. Global high BMI (overweight + obesity) level forecasts show that by 2035, nearly 3.3 billion adults will be at risk of overweight and obesity, a significant increase from 2.2 billion in 2020. The proportion of adults with high BMI will rise sharply from 42% in 2020 to more than 54% in 2035. Also worthy of attention is the BMI status of young people aged 5 to 19. It is expected that the proportion of high BMI in this age group will increase significantly from 22% (or 430 million people) in 2020 to more than 39% (or 770 million people) in 2035. Therefore, the problem of obesity has become a public health problem that needs to be solved urgently worldwide.

[0003] Individuals who have not successfully lost weight through lifestyle interventions can choose drug therapy. If lifestyle interventions do not achieve at least 5% weight loss within the first 3-6 months, obese, overweight individuals with complications can use drug therapy, such as orlistat. Many weight loss drugs may have adverse reactions, such as nausea, diarrhea, insomnia or other health problems, and some drugs may suppress appetite and affect nutritional intake. Drug therapy usually causes weight rebound after discontinuation, and there is a lack of effective long-term maintenance management methods. In addition, not all patients are suitable for taking medications, and patients with comorbidities such as heart disease and diabetes need to choose carefully.

[0004] Most of the drugs used in the clinical treatment of obesity complications mainly act downstream of the inflammatory process (such as insulin or statins, etc.) rather than addressing the root cause. On the other hand, anti-inflammatory drugs such as glucocorticoids (dexamethasone) and aspirin have been tested clinically in patients with obesity and / or diabetes for more than 10 years. However, in the long run, high-dose and long-term drug use is accompanied by serious side effects, including inducing neurological diseases and gastrointestinal irritation, and often triggers a rapid recurrence of obesity after drug withdrawal.

[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-inflammation,” and minimize off-target effects. Summary of the invention

[0006] Purpose of the Invention

[0007] The present invention constructs a red light-sensitive bionic nano-drug delivery system targeting the WAT inflammatory area, promotes the browning of white adipocytes while remodeling the polarization of M1-like macrophages, reveals the causal relationship between obesity-induced macrophage polarization and WAT remodeling, and determines whether weight loss therapy can additionally reduce the obesity-driven inflammatory phenotype. On the other hand, the present invention provides a theoretical and practical basis for the research and development and application of new drug delivery systems for metabolic diseases by clarifying the synergistic long-term targeting mechanism of the long circulation of the red light-sensitive bionic nano-drug delivery system and the stability of the targeting molecule.

[0008] Technical Solution

[0009] A photosensitivity bionic drug delivery system, characterized in that it is mainly composed of two parts: a core-shell: the shell is a photosensitivity bionic material RCP, and the core is SEP NPs, and the two are self-assembled to form a photosensitivity bionic drug delivery system RSCP NPs.

[0010] The photosensitive bionic material RCP is made of macrophage membrane RCP co-modified with fat-targeting peptide P3 and photosensitive supramolecular complex CHD; wherein the amino acid sequence of fat-targeting peptide P3 is CKGGRAKDC; further, fat-targeting peptide P3 is a DSPE-modified P3 polypeptide, i.e., DSPE-P3 polypeptide. The photosensitive supramolecular complex CHD is prepared by the following steps: carboxymethyl-β-cyclodextrin, carbodiimide and N-hydroxysuccinimide are mixed and reacted, and then 4-hydroxyphenylazo-benzoic acid and DSPE-PEG are added to react;

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

[0012] The SEP NPs are triblock amphiphilic polymer SEP obtained by ultrasound and extrusion.

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

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

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

[0016] Specifically:

[0017] Construction and characterization of photosensitizable 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) in turn and react at 25°C for 30 minutes. Then, add 4-hydroxyphenylazo-benzoic acid (4'-Hydroxyazobenzene-2-carboxylic acid, HABA) and DSPE-PEG, and react for 24 hours in a dark environment. After the reaction is completed, place the product in a dialysis bag with a molecular weight cutoff of 1000MW and dialyze it with deionized water to remove the unreacted reagents in the product.

[0019] Preparation and characterization of photosensitive bionic 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), and the M1-like macrophage membrane was separated and extracted for later use. Subsequently, the RAW biofilm was obtained after ultrasonic extrusion treatment. 5-10 mg of RAW biofilm was weighed, dissolved with PBS, and 20 μL CHD (20 mg / mL) and 10 μL DSPE-P3 peptide (10 mg / mL) were added to the RAW biofilm solution. The mixture was vortexed and allowed to stand for 24 hours to obtain the photosensitive biomimetic material RCP modified with CHD and P3 peptides. Then, by 1 H NMR and FTIR techniques were used to analyze and characterize the main components of RCP biofilm.

[0021] Preparation 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 to dissolve it. Secondly, add EDC and NHS10-20 mg and stir the reaction at 25°C for 30 minutes to activate the carboxyl group. After the reaction is completed, add 40-60 mg of L-phenylalanine ethyl ester hydrochloride (L-phenylalanine ethylester, LPE) and 20-30 mg of PEG in sequence, and continue to react at 25°C in the dark for 48 hours. Then, place the product in a dialysis bag with a molecular weight cutoff of 3500Da and dialyze it with deionized water to remove unreacted reagents in the product. Replace the deionized water every 6 hours and dialyze continuously for 3 days. The product obtained after dialysis is vacuum freeze-dried, and the white flocculent obtained is the SEP polymer, and it is used 1 The physicochemical properties were characterized by H NMR and FTIR techniques.

[0023] Weigh 5 mg of SEP and dissolve it in 5 mL of deionized water. Stir under light-shielded condition at room temperature to make it fully dissolved. Place the obtained solution in a contact ultrasonic crusher and ultrasonicate for 3 min (100 W). After the ultrasonication is finished, use 0.45

[0024] The SEP solution was squeezed and filtered through a μm filter membrane and allowed to stand at 4°C overnight.

[0025] Preparation of RSCP NPs:

[0026] Preparation and characterization of biomimetic nano drug delivery system RSCP NPs: 5-10 mg SEP NPs and 5-10 mg RCP biofilm were dissolved in 1×PBS respectively; secondly, SEP NPs and RCP biofilm were fully mixed for 10 min at a mass ratio of 1:1; then, the mixed solution was subjected to contact ultrasound (100 W, 5 min), and the ultrasound solution was filtered with a 0.45 μm microporous filter membrane. Finally, the light yellow particles obtained by standing at 37 ° C for 12 h and vacuum freeze drying were RSCP NPs.

[0027] The present invention constructs a red light-sensitive bionic nano drug delivery system targeting WAT inflammation area, which promotes browning of white adipocytes while reshaping M1-like macrophage polarization. The present invention is based on a novel red light-sensitive bionic nano drug delivery system targeting WAT inflammation area, which has a dual-targeting function and can deliver more than two drugs to WAT inflammation area at the same time, reducing the off-target effect of drugs and improving the safety of clinical medication. The red light-sensitive bionic nano drug delivery system evaluates the morphology and material stability of RSCP NPs by atomic force microscopy (atomic Force Microscope, AFM), transmission electron microscopy (transmission electronmicroscope, TEM) and Zeta potential. In addition, a bionic RSC biofilm coating is constructed by extrusion. At the same time, the present invention will analyze and characterize the main components of the RSC biofilm by fluorescent probe method, polypropylene gel electrophoresis method, and cell flow cytometry. We also conduct in-depth exploration of the physicochemical properties and biological effects of RSCP NPs, such as morphology, size, stability, drug loading rate, drug release kinetic parameters, targeting adipose tissue, and safety, and further 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 and side effects, but also have strong targeting ability and can accurately regulate the step-by-step release of drugs (such as astaxanthin and rosiglitazone), thereby alleviating WAT low-density inflammation and promoting the browning of white fat cells, thereby achieving the purpose of weight loss.

[0028] Working principle of the invention

[0029] The photosensitive bionic drug delivery system RSCP NPs constructed by the present invention is mainly composed of two parts: the shell is composed of macrophage membrane RCP co-modified with fat targeting peptide P3 (CKGGRAKDC) and photosensitive supramolecular complex CHD; the core is SEP NPs (formed by triblock amphiphilic polymer SEP), and the two self-assemble to form the photosensitive bionic 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 step-by-step release of drugs, and actively target the inflammatory area of ​​WAT, so as to effectively reduce the toxic side effects caused by the off-target effect of drugs, and achieve the effect of improving the low-intensity inflammatory microenvironment of obese tissue.

[0030] Beneficial Effects

[0031] In recent years, low-grade chronic inflammation and metabolic disorders caused by the expansion of white adipose tissue (WAT) in obese patients have attracted great attention from researchers. The latest studies have shown that in the WAT of obese patients, macrophages are easily recruited and aggregated, polarized into M1 macrophages, and produce a large number of proinflammatory cytokines, thereby forming low-grade chronic inflammation. Therefore, reducing the infiltration of M1 macrophages and inhibiting their proinflammatory programs may help reduce the pathological remodeling of WAT, alleviate the occurrence of obesity, and correct lipid metabolism disorders.

[0032] To this end, the present invention prepares a new type of red light-sensitive bionic nano drug delivery system targeting the WAT inflammation area. The system has a dual-targeting function and can deliver more than two drugs to the WAT inflammation area at the same time, thereby reducing the off-target effect of the drug and improving the safety of clinical medication. In addition, with the help of the physicochemical characteristics such as photosensitivity and pH sensitivity of the drug delivery system, the step-by-step release of the drug can be accurately controlled, thereby alleviating the low-density inflammation of WAT and promoting the browning of white adipocytes.

[0033] This intervention strategy is our original construction and is highly innovative. At present, the existing drug delivery systems for metabolic diseases mainly focus on the study of the effects of hindering lipid absorption or promoting lipid decomposition, while few studies have explored directly targeting the relief of "metabolic inflammation" to treat obesity and its complications. It is precisely because of these defects that the clinical transformation of drug therapy is severely restricted. Therefore, with the main line of promoting the occurrence and development of obesity by low-intensity inflammation, we chose a field that is still blank-the cross-dialogue mechanism between macrophages and low-intensity inflammation and lipid metabolism disorders for research, organically combining the hot spots of interdisciplinary research, including bionic nanodrug delivery systems, WAT immune microenvironment and lipid energy metabolism, and proposed a precise treatment strategy for obesity targeting macrophages, which provides a solid theoretical foundation for the research and development and application of new drug delivery systems for metabolic diseases.

[0034] (1) New therapeutic targets for obesity: In recent years, the expansion of WAT in obese patients has led to low-grade chronic inflammation and metabolic disorders, which has attracted great attention from researchers. The latest studies have found that macrophages in the WAT of obese patients are easily recruited, aggregated and polarized into M1-like macrophages, producing a large number of proinflammatory cytokines, thereby forming low-grade chronic inflammation. Therefore, reducing the infiltration of M1-like macrophages and inhibiting the proinflammatory program of M1-like macrophages may reduce the pathological remodeling of WAT, alleviate the occurrence of obesity and correct lipid metabolism disorders. In addition, in previous co-culture experiments, we have confirmed that Asta can reprogram M1-like macrophages into M2-like macrophages and can effectively correct lipid metabolism disorders. At the same time, Rosi (rosiglitazone) can promote the browning of white adipocytes and effectively alleviate the polarization of M1-like macrophages. The above studies all suggest that macrophages are potential targets for the treatment of obesity.

[0035] (2) New bionic nano drug delivery system: The present invention prepares a photosensitive supramolecular complex CHD, and modifies CHD and P3 peptide together on the surface of macrophage membrane to form a photosensitive bionic material RCP that can directly target the WAT inflammation area. This bionic material has not been reported by any team before and has certain innovation. Subsequently, pH-sensitive SEPNPs were wrapped with RCP to construct a red light-sensitive bionic nano drug delivery system RSCP NPs targeting the WAT inflammation area. The present invention uses the dual targeting of RSCP NPs to accurately deliver A&R to the WAT inflammation area, reduce the off-target effect of the drug, and improve the safety of clinical medication; at the same time, the multi-signal response of RSCP NPs (red light signal and microenvironment of different pH) is used to achieve the gradual release of the drug, thereby alleviating WAT low-density inflammation and promoting the browning of white adipocytes. This intervention strategy is our original construction and has strong innovation.

[0036] (3) New concept for treating obesity and insulin resistance: In terms of academic thinking, the existing drug delivery systems for metabolic diseases are still more focused on the effects of hindering lipid absorption or promoting lipid decomposition, but few studies have explored directly targeting the alleviation of "metabolic inflammation" for the treatment of obesity and complications. In fact, it is precisely because of these defects that the clinical transformation of drug therapy is seriously restricted. The applicant took low-density inflammation as the main line to promote the occurrence and development of obesity, and selected a field that is still blank - the cross-dialogue mechanism between macrophages and low-density inflammation and lipid metabolism disorders for research, organically grasping the hot spots of interdisciplinary research, including bionic nano-drug delivery systems, WAT immune microenvironment, and lipid energy metabolism. In summary, the present invention expands the pathological function of macrophages in the treatment of obesity and insulin resistance, and proposes a precise treatment strategy for obesity and insulin resistance targeting macrophages, which provides a solid theoretical basis for the development and application of new drug delivery systems for metabolic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0039] Figure 3 .Preparation and physicochemical characterization of SEP NPs. (A) SEP polymer 1 (B) SEP polymer FTIR image; (C) SEP NPs TEM image; (D) SEP NPs hemolytic experiment.

[0040] Figure 4 .Preparation and physicochemical characterization of RSCP NPs, a photosensitive biomimetic drug delivery system. (A) TEM and particle size distribution of RCM NPs; (B) TEM and particle size distribution of RSCP NPs; (C) Coomassie blue image of RSCP NPs; (D) Hemolytic test of RSCP NPs; N = 3.

[0041] Figure 5 .Study on the biological effects of RSCP NPs. (A) Uptake of RSCP NPs by primary adipocytes at different time points; (B) Uptake of NPs of different components by primary adipocytes; (C) Uptake mechanism of RSCP NPs by primary adipocytes; (D) Distribution of different NPs in mouse AT.

[0042] Figure 6 .In vitro and in vivo safety evaluation of RSCP NPs. (A) ALT level in serum; (B) AST level in serum; (C) BUN level in serum; (D) CRE level in serum.

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

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

[0045] Fig. 9 .Ucp1 and Cox7a1 expression levels after Rosi RSCP NPs treatment in mouse primary adipocytes.

[0046] Fig.10 .GTT and ITT of ob / ob mouse CTL group and A&R RSCP NPs.

[0047] Fig.11 .Changes in body weight and diet of HFD mice.

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

[0049] Fig.13 .Schematic diagram of the structure of the present invention. DETAILED DESCRIPTION

[0050] Example 1

[0051] 1. Research plan

[0052] 1.1 Construction and biological effects of red light-sensitive bionic nano-drug delivery system

[0053] (1) Construction and physicochemical characterization of the photosensitized supramolecular complex CHD: Fig.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 in sequence, and the reaction was carried out at 25°C for 30 minutes. Then, 4-hydroxyphenylazo-benzoic acid (4'-Hydroxyazobenzene-2-carboxylic acid, HABA) and DSPE-PEG were added, and the reaction was shielded from light for 24 hours. After the reaction was completed, the product was placed in a dialysis bag with a molecular weight cutoff of 1000MW and dialyzed with deionized water to remove the unreacted reagents in the product. Finally, the dialyzed product was vacuum freeze-dried, and the yellow flocs obtained were CHD, and the nuclear magnetic resonance spectrometer (nuclear Magnetic Resonance, 1 H NMR) and Fourier Transform Infrared Spectrometer (FTIR) were used to analyze and characterize the main components of CHD biofilm. In addition, since astaxanthin can absorb blue light and emit green fluorescence, astaxanthin Asta was used as the encapsulation material. We will further explore the evaluation of the drug loading capacity of the photosensitive supramolecular complex CHD. Asta solution (5 mg / ml) was added to the CHD solution, mixed and allowed to stand at room temperature for 24 hours, and the encapsulation rate and drug loading rate of CHD were calculated by the concentration of free astaxanthin Asta in the supernatant.

[0054] Encapsulation efficiency

[0055] Drug loading rate Among them, M0 is the total mass of Asta; M1 is the free mass in the supernatant of Asta; Q0 is the empty nanomass of CHD NPs.

[0056] result:

[0057] The photosensitive polymer CHD is prepared by esterification reaction. 1 H NMR and FTIR characterization revealed that DSPE and HAPA were successfully grafted onto CD ( Figure 1 A and B). According to the UV-visible absorption spectrum analysis, the n-π* transition band of trans-CHD extends to 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 cis form. To evaluate the drug loading capacity of CHD, we loaded Asta into the hydrophobic cavity of CD to form a supramolecular valve with grafted HABA to close the pore (Asta&CHD). The Asta encapsulation efficiency measured by HPLC was 90.7%, and the optimal drug loading rate was achieved at the ratio of Asta to CHD of 5:50 ( Figure 1 D). In summary, CHD can efficiently package Asta.

[0058] (2) Preparation and characterization of photosensitive bionic material RCP: RAW 264.7 cells were polarized into M1-like macrophages by lipopolysaccharide (LPS) (0.5 μg / mL) and IFN-γ (0.1 μg / mL), and the M1-like macrophage membrane was separated and extracted for later use. Subsequently, the RAW biofilm was obtained after ultrasonic extrusion treatment. In addition, 20 μL CHD (20 mg / mL) and 10 μL DSPE-P3 peptide (the amino acid sequence of P3 peptide is CKGGRAKDC, 10 mg / mL) were added to the RAW biofilm solution, vortexed and allowed to stand for 24 hours to obtain the photosensitive bionic material RCP modified with CHD and P3 peptides. Then, by 1 H NMR and FTIR techniques were used to analyze and characterize the main components of RCP biofilm.

[0059] result:

[0060] In order to prepare the photosensitive biomimetic material RCP, we first modified the P3 peptide with DSEP and 1 H NMR and FTIR confirmed the formation of DSPE-P3 peptide polymer ( Figure 2 A and B). Secondly, by the principle of like dissolves like, with the help of DSPE lipid chains, CHD and P3 peptides were respectively embedded into the membrane of M1-like macrophages to generate photosensitive biomimetic material RCP. According to the results of fluorescence experiments, 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) Characterization of physicochemical properties and evaluation of drug loading capacity of SEP NPs:

[0062] Preparation process and characterization of triblock amphiphilic polymer SEP: Weigh an appropriate amount of sodium alginate (SA) and dissolve it in deionized water, stirring it thoroughly to dissolve. Secondly, add EDC and NHS and stir the reaction at 25°C for 30 minutes to activate the carboxyl group. After the reaction is completed, add L-phenylalanine ethyl ester hydrochloride (L-phenylalanineethylester, LPE) and PEG in sequence, and continue to react at 25°C in the dark for 48 hours. Then, the product is placed in a dialysis bag with a molecular weight cutoff of 3500Da and dialyzed with deionized water to remove unreacted reagents in the product. The deionized water is replaced every 6 hours, and the dialysis is continued for 3 days. The product obtained after dialysis is vacuum freeze-dried, and the white flocculent obtained is the SEP polymer, and it is used 1 The physicochemical properties were characterized by H NMR and FTIR techniques.

[0063] Weigh 5 mg of SEP and dissolve it in 5 mL of deionized water. Stir it under light-shielded conditions at room temperature to fully dissolve it. The obtained solution was placed in a contact ultrasonic crusher and ultrasonically crushed for 3 minutes (100w). After the ultrasonication, the SEP solution was squeezed and filtered using a 0.45 μm filter membrane and allowed to stand overnight at 4°C. Then, the shape, size and distribution of SEP NPs were observed by TEM, and the surface charge, particle size and polydispersity index (PdI) of SEP NPs were detected by laser dynamic light scattering (DLS). In order to further evaluate the drug loading capacity of SEP, a SEP solution with a concentration of 5 mg / mL was prepared for use. Then, the optimal drug encapsulation formula was determined based on the different mass ratios of the drug to the carrier. The specific experimental steps are as follows: Weigh rosiglitazone Rosi (5-20 mg) and add it to 1 mL of SEP solution. Stir it under light-shielded conditions at room temperature for 30 minutes. Subsequently, the above samples were ultrasonically treated for 10 minutes under 100W. After the ultrasonication, it was allowed to stand at 4°C for 24 hours. The sample was then centrifuged at 24000 rpm / min, 4°C for 1 h, and the supernatant was taken for later use. The bottom precipitate was Rosi SEP NPs. At the same time, the encapsulation efficiency and drug loading rate of SEPNPs 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 by esterification. 1 The structure of SEP was analyzed by HNMR and FTIR. The results showed that SEP is composed of three monomers: SA, LEP and PEG. Figure 3A and B). Since SEP is an amphiphilic polymer, it can self-assemble into a spherical structure after ultrasonic treatment in the reaction medium. Figure 3 As shown in C, TEM and DLS showed that SEPNPs were spherical, uniform in size, and evenly dispersed. According to the hemolysis experiment analysis, the hemolysis rate of SEPNPs at a concentration of 250 μg / mL was only 1.43%, which showed that SEP NPs had good biocompatibility ( Figure 3 D).

[0066] (4) Preparation and characterization of biomimetic nano drug delivery system RSCP NPs: Take appropriate amounts of SEP NPs and RCP and dissolve them in 1×PBS respectively; secondly, mix SEP NPs and RCP thoroughly for 10 minutes according to a mass ratio of 1:1; then, the mixed solution is subjected to contact ultrasound (100W, 5min), and the sonicated solution is filtered with a 0.45μm microporous filter membrane. Finally, the mixture is allowed to stand at 37°C for 12h, and the light yellow particles obtained by vacuum freeze drying are RSCP NPs. To further verify the physicochemical properties of RSCP NPs, the present invention will verify whether the RCP biofilm is coated on the surface of SEP NPs by proteomics, Malvern particle size analyzer, transmission electron microscopy, polyacrylamide gel electrophoresis and flow cytometry. At the same time, the stability and hemolysis of RSCP NPs are detected. In addition, we will further explore the in vitro drug release mechanism of A&R RSCP NPs with multi-level response (photosensitivity and pH sensitivity).

[0067] The photosensitive bionic drug delivery system RSCPNPs is mainly composed of two parts: the inner core SEPNPs (drug-carrying core) and the outer shell of the macrophage membrane (targeting WAT, red light sensitive) co-modified with P3 peptide and CHD. Through TEM, it can be observed that RSCPNPs have a clear core-shell structure, with a uniform outer membrane layer visible on the outer layer, the thickness of the membrane layer is consistent with that of RCPNPs, and the inner part is a dark sphere with a diameter similar to that of SEPNPs, and the overall surface is smooth, the size is uniform and it is dispersed separately ( Figure 4 A and B). As shown in Figure B, the average particle size of RSCPNPs was 170.5 nm. In addition, according to the experimental results of Coomassie Brilliant Blue and Western blot, RCP biofilm was successfully coated on the surface of SEP NPs to form RSCP NPs ( Figure 4 C). The results of the hemolytic assay showed that when the concentration of RSCP NPs was as high as 250 μg / mL, the hemolytic rate was only 0.24% ( Figure 4 D) The above studies all show that RSCPNPs have good biocompatibility.

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

[0069] (1) Isolation and differentiation induction of primary adipocytes: 10 male C57BL / 6J mice aged 3-4 weeks were selected, euthanized, and then immersed in 75% ethanol solution for 3-5 minutes; then, the mice were taken out on absorbent paper and the alcohol was slightly absorbed for later use; then, WAT from the inguinal area of ​​the mice was obtained, cut into pieces and transferred to HEPES-collagenase solution for digestion. Finally, the digestion was terminated, filtered, washed with PBS, and centrifuged to obtain cells, which are adipocytes. In addition, the obtained adipocytes were inoculated into a cell culture dish, and inducers I to III were added in sequence for induction to obtain white adipocytes.

[0070] (2) Investigation of the uptake capacity and mechanism of RSCP NPs by adipocytes: In order to investigate the release of RSCP NPs in adipocytes, RSCP NPs were first labeled with Cy5 fluorescent probe. Secondly, adipocytes were inoculated into confocal cell culture dishes for culture. After the cells adhered to the wall, culture medium containing Cy5-RSCP NPs (dose of Cy5-RSCP NPs: 40 μg / mL) was added and cultured for 0, 2, 4, and 8 h, respectively. Finally, the uptake of RSCP NPs by adipocytes was observed by confocal laser scanning microscopy (CLSM). In addition, in order to further verify the molecular mechanism of RSCP NPs entering adipocytes, the present invention uses different inhibitors (Filipin (1 μg / mL) to block caveolin-mediated endocytosis; Chlorpromazine (CPZ, 10 μg / mL) to block clathrin-mediated endocytosis; 5-(N-ethyl-N-isopropyl) Amiloride (EIPA, 10 μg / mL) to block macropinocytosis) to pretreat adipocytes, and then add Cy5-RSCP NPs for co-incubation for 24 hours. Then, the fluorescence signal in adipocytes is observed through RSCPNPs to reveal the molecular mechanism of RSCP NPs entering adipocytes.

[0071] (3) RSCP NPs specifically target WAT: To determine whether the targeting performance of RSCP NPs also occurs in vivo. Therefore, we injected Cy5-RSCP NPs into C57BL / 6J mice through the tail vein and observed the distribution of Cy5-RSCP NPs in vivo using a small animal in vivo imaging system. After 24 hours, the mice were euthanized, and the heart, liver, spleen, lung, kidney, subcutaneous fat, epididymal fat, visceral fat, and brown WAT of the mice were collected. The fluorescence intensity in each tissue was observed in the small animal in vivo imaging system to determine the enrichment ability of Cy5-RSCP NPs in different WAT. At the same time, WAT tissues were collected, macrophages in WAT were labeled, and the distribution of Cy5-RSCP NPs in adipocytes and macrophages was observed under CLSM.

[0072] result:

[0073] In order to study 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 A, the red fluorescence signal in the adipocytes is proportional to the incubation time, and as the incubation time increases, the red fluorescence signal gradually increases. Therefore, RSCP NPs are taken up by primary adipocytes in a time-dependent manner. After RSCP NPs enter the cells, the inner core SEP NPs will be captured by the lysosomes and release the drugs in response to the low pH microenvironment inside the lysosomes. In order to further explore the molecular mechanism of RSCP NPs being taken up by primary adipocytes. Compared with the fluorescence intensity in the control group cells, the fluorescence intensity in the cells treated with the inhibitor CPZ was significantly reduced ( Figure 5 B), indicating that RSCP NPs mainly rely on clathrin-mediated endocytosis to enter the cell. In addition, compared with other single-component nanomaterials, RSCP NPs show specific uptake by primary adipocytes ( Figure 5 C) Therefore, we will further evaluate the ability of RSCP NPs to specifically target WAT in mice. Figure 5 As shown in D, after drug administration via tail vein injection, the fluorescence intensity of the WAT site of the SEP NPs group mice was higher than that of the free Cy5 group mice, while the fluorescence signal of the RSCP NPs group was the strongest, indicating that the dual-targeted drug delivery system RSCP NPs has a stronger ability to specifically recognize the WAT inflammatory area.

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

[0075] (1) In vitro safety evaluation of RSCP NPs: In order to evaluate the safety of RSCP NPs, we selected primary hepatocytes and primary adipocytes of mice as model cells. Therefore, primary hepatocytes and primary adipocytes of mice were inoculated into 96-well plates respectively and allowed to adhere to the wall. Complete culture medium containing different concentrations of RSCP NPs (0, 50, 100, 200, 250 and 500 μg / mL) was added and cultured for 48 h. The viability of each group of cells was then detected by the CCK-8 kit to reveal the in vitro safety of RSCP NPs.

[0076] (2) In vivo safety evaluation of RSCP NPs: In order to evaluate the safety of RSCP NPs in vivo, the present invention selected 10 6-week-old male C57BL / 6J mice and randomly divided them into two groups. The experimental group mice were injected with RSCP NPs through the tail vein, and the control group mice were injected with PBS. The administration was once every 3 days for 21 days. Before each administration, the weight and dietary changes of the mice were recorded. After 21 days of administration, the mice were euthanized, and the whole blood was taken and left to stand at 4°C overnight, and then centrifuged at 3000rpm and 4°C for 15min. The serum was collected and the content of aspartate transaminase (AST), alanine transaminase (ALT), creatinine (CRE) and blood urea nitrogen (BUN) in the serum of the two groups of mice was detected by commercial kits to determine whether the physiological functions of the liver and kidneys were normal. In addition, various tissues and organs (heart, liver, spleen, lung, kidney, subcutaneous fat, epididymal fat, visceral fat and brown WAT) of mice were collected for H&E staining to evaluate the morphological changes of major organs after administration.

[0077] result:

[0078] In addition, this study further detected the changes in serum levels of liver injury markers ALT and AST, as well as kidney injury markers BUN and CRE. The results showed that there was no significant difference between the RSCP NPs experimental group and the PBS control group ( Figure 6 AD). The above results confirmed that the RSCP NPs drug delivery system did not cause acute damage to the liver and kidneys of mice.

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

[0080] (1) Study on the mechanism of Asta RSCP NPs remodeling macrophage polarization: RAW 264.7 cells were polarized into M1-like macrophages by LPS (0.5 μg / mL) and IFN-γ (0.1 μg / mL). First, we explored whether red light signal intervention affects macrophage remodeling through IL-4. Then, polarized M1-like macrophages were intervened with red light signal for 0.5 h and then incubated for 24 h. Cell samples were collected and the ratio of M1 / M2 macrophages was detected by flow cytometry to determine whether red light affects macrophage remodeling. Secondly, we explored the efficacy and mechanism of Asta RSCP NPs remodeling macrophages. We added free Asta and Asta RSCP NPs (the Asta dose was the same) to M1-like macrophages, and continued to incubate for 24 h after red light signal intervention. Then, the cell supernatant was collected and the levels of IL-10, IL-1β and IL6 in the supernatant were detected by ELISA kits; the cell samples were collected and stained, and the M1 / M2 macrophage ratio was determined by CLSM and flow cytometry. At the molecular level, the total protein and RNA of the treated cell samples were extracted, and the expression levels of M2-like macrophage markers CD206, Arg1 and DECTIN-1 were detected by RT-qPCR and Western blot techniques.

[0081] Wherein: Asta RSCP NPs were prepared as follows: 50 mg / ml Asta was added to RSCP NPs, vortexed and reacted at 4°C overnight;

[0082] result:

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

[0084] RSCP NPs are a photosensitive nano-drug delivery system. We first investigated whether red light signal intervention affects the polarization of macrophages ( Figure 7 A). The results showed that red light treatment alone did not affect the polarization of M1-like macrophages ( Figure 7 B). Figure 7 The CD description is to further explore the efficacy and mechanism of Asta RSCP NPs in remodeling macrophages. Figure 7 As shown in C, free Asta can reprogram M1-like macrophages into M2-like macrophages, and compared with the Asta RSCPNPs group without light treatment, Asta RSCP NPs can significantly increase the M2 / M1 macrophage ratio after red light signal intervention ( Figure 7 D).

[0085] (2) Exploring the correlation between macrophage polarization and lipid metabolism disorders in adipocytes: In order to reveal the effect of macrophage polarization on lipid metabolism in adipocytes. In the present invention, macrophages in different polarization states were co-cultured with primary adipocytes through Trans-well chambers. First, polarized M0 macrophages were inoculated into the upper layer of the chamber, and primary adipocytes were inoculated into the bottom layer. Secondly, M0 macrophages were polarized into M1-like macrophages using LPS (0.5 μg / mL) and IFN-γ (0.1 μg / mL). After continuing to co-culture for 48 hours, the bottom primary adipocytes were collected, total protein and RNA were extracted, and RT-qPCR and Western blot techniques were used to detect the expression levels of lipid metabolism genes such as Pparα, ATGL, HSL and Perilipin1. In addition, after adding Asta RSCP NPs to reprogram M1-like macrophages, they continued to be co-cultured with primary adipocytes, and the changes in the expression levels of lipid metabolism genes were detected by molecular biology techniques to reveal whether remodeling macrophage reprogramming can improve the lipid metabolism of adipocytes.

[0086] result:

[0087] like Figure 8 As shown in AB, after red light signal intervention, Asta RSCP NPs can significantly increase the ratio of M2 / M1 macrophages and inhibit the expression of pro-inflammatory cytokines. In addition, in order to explore the effect of macrophage polarization on adipocyte lipid metabolism, macrophages in different polarization states were co-cultured with primary adipocytes through Trans-well chambers.

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

[0089] Rosiglitazone is a thiazolidinedione insulin sensitizer, and its mechanism of action is related to the specific peroxisome proliferator-activated receptor γ (PPARγ). It can reduce blood sugar by increasing the sensitivity of skeletal muscle, liver, and adipose tissue to insulin and improving the utilization of glucose by cells. It can significantly reduce fasting blood sugar and insulin and C-peptide levels, and also has a lowering effect on postprandial blood sugar and insulin. It can promote the browning of white adipocytes and effectively alleviate the polarization of M1-like macrophages.

[0090] Free Rosi and Rosi RSCP NPs were added to the induced differentiated white adipocytes and incubated for 48 hours. After the incubation, the lipid droplets were labeled with the BODIPY probe, and the lipid accumulation in the adipocytes before and after treatment was observed by CLSM. At the molecular level, the gene expression of white fat browning markers Ucp1 and cytochrome C oxidase polypeptide 7a1 (Cox7a1) was detected to reveal the ability of Rosi RSCP NPs to regulate the browning of white adipocytes.

[0091] The preparation method of Rosi RSCP NPs is as follows: 120 mg / ml Rosi is added to RSCP NPs, sonicated for 5 minutes, supersonicated for 3 seconds and stopped for 2 seconds, and reacted overnight.

[0092] result:

[0093] To further explore whether Rosi RSCP NPs can promote the browning of white adipocytes, we added free Rosi and Rosi RSCP NPs to primary white adipocytes for co-incubation. Fig. 9 As shown, the gene expressions of white fat browning markers Ucp1 and Cox7a1 were significantly upregulated after treatment, indicating that a single Rosi RSCP NPs can effectively promote the browning of white adipocytes and alleviate lipid accumulation in adipocytes.

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

[0095] (1) A&R RSCP NPs improve T2D in ob / ob mice: In order to explore the efficacy of A&R RSCP NPs in alleviating "metabolic inflammation" and improving T2D in ob / ob mice. In the present invention, 7-week-old ob / ob mice were randomly divided into 7 groups (10 mice in each group) and treated with PBS, Free Asta, Free Rosi, A&R, Asta RSCP NPs, Rosi RSCP NPs and A&R RSCP NPs by tail vein injection, respectively, once every 3 days for 27 days. During each treatment, the food intake and weight changes of the mice were recorded. After the end of drug administration, the following experimental studies were carried out: Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) were used to detect changes in blood glucose metabolism in mice after treatment; the small animal life metabolic system was used to monitor the overall metabolic level of mice after treatment; the mice were euthanized, and their serum samples were collected, and the levels of inflammatory cytokines in the serum of each group of mice were detected using ELISA kits; at the tissue level, different WAT of the mice were collected and weighed, and the WAT was subjected to H&E staining and immunofluorescence staining to count the size of adipocytes and inflammatory cell infiltration, and the M1 / M2 macrophage ratio in WAT was analyzed by flow cytometry; at the molecular level, RT-qPCR and Western blot techniques were used to detect the expression levels of WAT inflammatory cytokines and the changes in the expression levels of white fat browning marker genes Ucp1 and Cox7a1.

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

[0097] result:

[0098] like Fig.10 As shown in AB, the glucose tolerance results showed that the A&R RSCP NPs administration group improved the glucose tolerance of ob / ob mice. That is, A&R RSCP NPs administration treatment can significantly improve the blood glucose homeostasis of ob / ob mice. And the ITT results confirmed that A&R RSCP NPs can improve the insulin resistance of ob / ob mice.

[0099] (2) Study on the mechanism of A&R RSCP NPs improving metabolic disorders in HFD obese mice: 60 8-week-old C57BL / 6J male mice raised under standard conditions were randomly divided into 5 groups, one of which was fed with normal feeding (ND) and the other 7 groups were fed with high-fat diet for 11 weeks, and the weight changes were recorded. Then, the HFD mice were randomly divided into 7 groups, with 10 mice in each group. PBS, Free Asta, Free Rosi, A&R, Asta RSCP NPs, Rosi RSCP NPs and A&R RSCP NPs were respectively given by tail vein injection, once every 3 days for 27 days. After the treatment, the glucose tolerance and insulin resistance levels of the treated mice, the overall metabolic level of the mice, the level of inflammatory cytokines in the serum, the size of adipocytes, the ratio of M2 / M1 macrophages in WAT and the expression level of inflammatory cytokines in WAT, and the expression level of white fat browning marker genes Ucp1 and Cox7a1 were detected.

[0100] result:

[0101] like Fig.11 As shown, HFD mice were randomly divided into 7 groups, with 10 mice in each group. PBS, Free Asta, FreeRosi, A&R, Asta RSCP NPs, Rosi RSCP NPs, and A&R RSCP NPs were respectively given by tail vein injection. By monitoring the body weight of HFD-fed mice, the experimental results showed that A&R RSCP NPs could prevent the weight gain of mice fed with a high-fat diet compared with the HFD-CTL group. The body weights of mice in the Free Asta and Asta RSCP NPs groups were similar to those of control mice.

[0102] (3) In vivo biosafety evaluation of A&R RSCP NPs: In order to evaluate the safety of A&R RSCP NPs, blood was collected for serum biochemical testing. The main organs of mice in the treatment group and the control group were collected, fixed with 4% paraformaldehyde, then embedded, sectioned, and stained with H&E to evaluate the safety of A&R RSCP NPs.

[0103] result:

[0104] like Fig.12 As shown in the figure, we observed the physiological structure of the mouse heart, liver, spleen, lung, and kidney by H&E staining. The results showed that the tissue structures of the two groups of mice were similar, and no tissue lesions were observed in the mice. In summary, the A&R RSCP NPs drug nanodelivery system has good biocompatibility and safety.

Claims

1. A photosensitized bionic drug delivery system, characterized in that: It is mainly composed of two parts: the shell is the photosensitivity bionic material RCP, and the core is SEP NPs. The two are self-assembled to form the photosensitivity bionic drug delivery system RSCP NPs.

2. A photosensitive bionic drug delivery system according to claim 1, characterized in that: The photosensitive bionic material RCP is made of macrophage membrane RCP co-modified by fat targeting peptide P3 and photosensitive supramolecular complex CHD.

3. A photosensitive bionic drug delivery system according to claim 2, characterized in that: The photosensitive supramolecular complex CHD is prepared by the following steps: carboxymethyl-β-cyclodextrin, carbodiimide and N-hydroxysuccinimide are mixed and reacted, and then 4-hydroxyphenylazo-benzoic acid and DSPE-PEG are added to react to obtain CHD.

4. A photosensitive bionic drug delivery system according to claim 2, characterized in that: The amino acid sequence of the fat-targeting peptide P3 is CKGGRAKDC; and the macrophage membrane is an M1-like macrophage membrane formed after induction.

5. A photosensitive bionic drug delivery system according to claim 4, characterized in that: The fat targeting peptide P3 is a DSPE-modified P3 polypeptide, namely, a DSPE-P3 polypeptide.

6. The photosensitive bionic drug delivery system according to claim 1, characterized in that: The SEP NPs are triblock amphiphilic polymer SEP obtained by ultrasound and extrusion.

7. The light-sensitive bionic drug delivery system according to claim 6, characterized in that: The triblock amphiphilic polymer SEP is prepared according to the following steps: after sodium alginate, carbodiimide and N-hydroxysuccinimide react, L-phenylalanine ethyl ester hydrochloride and PEG are added to react to form the triblock amphiphilic polymer SEP.

8. A pharmaceutical composition, characterized in that The photosensitized bionic drug delivery system according to claims 1-7 encapsulates astaxanthin and / or rosiglitazone.

9. Use of the pharmaceutical composition according to claim 8 in the preparation of weight loss drugs.

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