A dual-targeting nanoparticle, its preparation method and application

Abstract

The present invention provides a dual-targeted nanoparticle and its preparation method and application, belonging to the technical field of pharmaceutical preparations. The dual-targeted nanoparticle described in the present invention comprises a browning inducer, ciglitazone sodium, a photothermal reagent, indocyanine green, an adipocyte homing peptide, a cell-penetrating peptide, a DSPE-PEG polymer, and a lipid matrix. By encapsulating the browning inducer and the photothermal reagent in nanoparticles with excellent biocompatibility and coating functionalized adipose homing peptides and cell-penetrating peptides on the surface, the present invention constructs a dual-targeted nanoparticle. Combining photothermal-drug therapy, it can effectively promote the "browning" of white fat, improve body weight and glycolipid metabolism, and lay a foundation for the development of safe and effective drugs for anti-obesity and related metabolic disorders.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and particularly relates to a dual-targeted nanoparticle and its preparation method and application. Background Art

[0002] Obesity is prevalent globally. Obesity is the root cause of numerous diseases, leading to various diseases including cardiovascular diseases, type 2 diabetes, hyperlipidemia, hypertension, sleep apnea syndrome, etc., which pose a threat to human health, seriously affect the quality of life and increase the risk of death. Although weight loss can be achieved through effective exercise, restricted energy intake, dietary structure modification, etc., the long-term effectiveness is poor. In terms of drug treatment, orlistat is a drug with obesity treatment indications, but its clinical application is limited due to adverse reactions such as gastrointestinal discomfort, fatty oily stools, and steatorrhea. Some weight loss drugs also have systemic side effects that affect clinical application. Existing studies have confirmed that metabolic surgery for weight loss has significant effects on obesity-related hypertension, type 2 diabetes, and non-alcoholic fatty liver disease. However, metabolic surgery still has problems such as difficulty in choosing the surgical method, insufficient patient acceptance, and the risk of postoperative complications.

[0003] In recent years, it has been gradually recognized that adipose tissue is an active metabolic organ that plays a key role in regulating whole-body energy homeostasis. According to function and morphology, it can be divided into white adipose tissue (WAT) and brown adipose tissue (BAT). BAT contains a large number of small, multilocular lipid droplets and a large number of mitochondria, with great thermogenic potential. Studies have confirmed that after cold stimulation or treatment with β3-adrenergic and peroxisome proliferator-activated receptors (PPAR) agonists, WAT can be directly converted into BAT, and BAT plays an important role in regulating glucose and lipid metabolism, insulin sensitivity, and adipose tissue homeostasis. Therefore, promoting the "browning" of WAT has become a new idea for weight loss and improving glucose and lipid metabolism. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a dual-targeted nanoparticle and its preparation method and application. The dual-targeted nanoparticle provided by the present invention can effectively intervene in the expression of UCP1 and PPAR-γ in adipocytes, promote the "browning" of white fat, and effectively improve obesity and metabolic disorders.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A dual-targeted nanoparticle, comprising a browning inducer, a photothermal reagent, an adipocyte homing peptide, a cell-penetrating peptide, a fluorescent label, a DSPE-PEG polymer, and a lipid matrix.

[0007] Preferably, the browning inducer is ciglitazone CGZ; the photothermal reagent is indocyanine green ICG.

[0008] Preferably, the amino acid sequence of the adipocyte homing peptide is as shown in SEQ ID NO.1, and the amino acid sequence of the cell-penetrating peptide is as shown in SEQ ID NO.2.

[0009] Preferably, the browning inducer accounts for 50 mol% of the total lipid, the photothermal reagent accounts for 20 mol% of the total lipid, the adipocyte homing peptide accounts for 4 mol% of the total lipid, the cell-penetrating peptide accounts for 20 mol% of the total lipid, and the DSPE-PEG polymer accounts for 5 mol% of the total lipid.

[0010] The present invention also provides a preparation method of the dual-targeted nanoparticle described in the above technical solution, comprising the following steps:

[0011] S1. Mix the adipocyte homing peptide and DSPE-PEG5kD, and react by shaking at room temperature for 24 h to prepare adipocyte homing peptide-DSPE-PEG (DSPE-PEG5kDa-Pep).

[0012] S2. Dissolve the lipid matrix in a chloroform solution, add the stearylated cell-penetrating peptide to obtain a lipid solution; mix the lipid solution with diisopropyl ether in equal volume, and add a HEPES buffer solution with a concentration of 10 mM and an equal volume to the lipid solution to obtain a first mixture.

[0013] S3. The DSPE-PEG polymer combination one comprises DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Mal, and the combination two comprises DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Pep. Select one of the two combinations and dissolve it in distilled water to prepare a DSPE-PEG polymer solution or a DSPE-PEG polymer solution containing the adipocyte homing peptide to obtain a second mixture.

[0014] S4. Add the second mixture, the photothermal reagent, and the browning inducer to the upper layer solution of the first mixture, stir and then homogenize with an ultrasonic cell disruptor for 8 min, stir and evaporate the organic solvent, and then ultrasonically treat the remaining solution with an ultrasonic cleaner for 5 min to obtain a liposome nanoparticle suspension.

[0015] S5. Stir and dialyze the NPs suspension in HEPES buffer with a pH of 7.4 and a concentration of 10 mM for 2 h to obtain dual-targeted nanoparticles (LNPs).

[0016] Preferably, the concentrations of the adipocyte homing peptide solution and the DSPE-PEG polymer solution in S1 are both 10 mM, and the ratio in S1 is 1:1.25.

[0017] Preferably, the concentration of the lipid matrix in the lipid solution in S2 is 20 μM.

[0018] The present invention also provides an application of the dual-targeted nanoparticles described in the above technical solution, and the application is for the preparation of a drug for improving obesity and / or glycolipid metabolism.

[0019] Preferably, the drug is a drug for photothermal-drug combination therapy.

[0020] Beneficial technical effects: The present invention provides a dual-targeted nanoparticle and its preparation method and application. The dual-targeted nanoparticle described in the present invention includes a browning inducer, a photothermal reagent, an adipocyte homing peptide, a cell-penetrating peptide, a DSPE-PEG polymer, and a lipid matrix. By encapsulating the browning inducer and the photothermal reagent in nanoparticles with excellent biocompatibility and coating functionalized fat-homing peptides and cell-penetrating peptides on the surface, the present invention constructs dual-targeted nanoparticles. Combining photothermal-drug therapy, it can effectively promote the "browning" of white fat, improve body weight and glycolipid metabolism, and lay a foundation for the development of safe and effective drugs for anti-obesity and related metabolic disorders. Description of the Drawings

[0021] Figure 1 In (A) is the particle size diagram of ICG LNPs; (B) is the particle size diagram of CGZ LNPs; (C) is the particle size diagram of ICG+CGZ LNPs;

[0022] Figure 2 is the release curve of CGZ and ICG in ICG+CGZ LNPs;

[0023] Figure 3 is the imaging diagram of Oil Red O staining in cell experiments;

[0024] Figure 4 is the statistical chart of qPCR characterization in cell experiments;

[0025] Figure 5 is the thermal imaging diagram in animal experiments;

[0026] Figure 6 is the statistical chart of temperature change in animal experiments;

[0027] Figure 7It is the weight change graph of animal experiments;

[0028] Figure 8 It is the histological section graph of adipose and liver tissues of animal experiments. Detailed implementation manners

[0029] The present invention provides a dual-targeted nanoparticle, which comprises a browning inducer, a photothermal reagent, an adipocyte homing peptide, a cell-penetrating peptide, a fluorescent label, a DSPE-PEG polymer, and a lipid matrix.

[0030] In the present invention, the browning inducer is preferably Chiglitazar CGZ; Chiglitazar (CGZ) is the first PPAR full agonist, which can induce the expression of a variety of downstream target genes, including genes related to insulin sensitivity, lipid metabolism / transport, thermogenesis, and energy conversion. It can not only reduce blood sugar but also regulate blood lipids. In the present invention, Chiglitazar CGZ is used as a browning promoter, together with a photothermal reagent, in combination with photothermal-drug therapy, which can effectively promote the "browning" of white fat.

[0031] In the present invention, the photothermal reagent is preferably Indocyanine Green ICG. Indocyanine Green can be used as a photothermal agent to convert light energy into heat energy and generate singlet oxygen. It has been clinically proven to be safe. It has near-infrared fluorescence imaging and good photothermal conversion ability under 808nm laser irradiation, and has been accepted by the FDA and widely used in clinical practice.

[0032] In the present invention, the amino acid sequence of the adipocyte homing peptide is preferably as shown in SEQ ID NO.1, and the amino acid sequence of the cell-penetrating peptide is preferably as shown in SEQ ID NO.2. Through the adipocyte homing peptide and the cell-penetrating peptide, the present invention can accurately identify adipocytes and enrich the browning inducer and the photothermal reagent in adipocytes. The specific amino acid sequences of SEQ ID NO.1 to SEQ ID NO.2 are as follows:

[0033] SEQ ID NO.1: CNFGHVGGC;

[0034] SEQ ID NO.2: STR-RRRRRRRR.

[0035] In the present invention, the DSPE-PEG polymer is preferably one or more of DSPE-PEG5kDa-Mal, DSPE-PEG5kDa-Pep, and DSPE-PEG2kDa-Mal, more preferably a combination of DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Mal, or a combination of DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Pep. When the DSPE-PEG polymer is a combination of DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Mal, the molar ratio of DSPE-PEG2kDa-Mal to DSPE-PEG5kDa-Mal is preferably 1:1.25; when the DSPE-PEG polymer is a combination of DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Pep, the molar ratio of DSPE-PEG2kDa-Mal to DSPE-PEG5kDa-Pep is preferably 1:1.25; the lipid matrix is preferably egg yolk phosphatidylcholine and cholesterol; the molar ratio of egg yolk phosphatidylcholine to cholesterol is preferably 4:1.

[0036] In the present invention, the browning inducer accounts for 50 mol% of the total lipids, the photothermal reagent accounts for 20 mol% of the total lipids, the adipocyte homing peptide accounts for 4 mol% of the total lipids, the cell-penetrating peptide accounts for 20 mol% of the total lipids, and the DSPE-PEG polymer accounts for 5 mol% of the total lipids.

[0037] The present invention also provides a method for preparing the dual-targeted nanoparticles described in the above technical solution, comprising the following steps:

[0038] S1. Dissolve the adipocyte homing peptide and the DSPE-PEG polymer in distilled water respectively to prepare an adipocyte homing peptide solution and a DSPE-PEG polymer solution. Mix the adipocyte homing peptide solution and the DSPE-PEG polymer solution in proportion and react with shaking at 30°C for 24 h to obtain a first mixture.

[0039] S2. Dissolve the lipid matrix in a chloroform solution, add the stearylated cell-penetrating peptide to obtain a lipid solution; mix the lipid solution with diisopropyl ether in equal volume, and add a 10 mM HEPES buffer solution with the same volume as the lipid solution to obtain a second mixture.

[0040] S3. Add the first mixture, the photothermal reagent, and the browning inducer to the upper layer solution of the second mixture, stir and then homogenize with an ultrasonic cell disruptor for 8 min. After stirring and evaporating the organic solvent, ultrasonically treat the remaining solution with an ultrasonic cleaner for 5 min to obtain a nanoparticle suspension.

[0041] S4. Stir and dialyze the NPs suspension in HEPES buffer with a pH of 7.4 and a concentration of 10 mM for 2 h to obtain dual-targeted nanoparticles (LNPs).

[0042] In the present invention, the adipocyte-homing peptide and the DSPE-PEG polymer are respectively dissolved in distilled water to prepare an adipocyte-homing peptide solution and a DSPE-PEG polymer solution. The adipocyte-homing peptide solution and the DSPE-PEG polymer solution are mixed in proportion and reacted with shaking at 30 °C for 24 h to obtain a first mixture.

[0043] In the present invention, the shaking reaction is preferably a shaking reaction at 30 °C for 24 h; the concentrations of both the adipocyte-homing peptide solution and the DSPE-PEG polymer solution are preferably 10 mM, and the proportion is preferably 1:1.25 by volume ratio.

[0044] In the present invention, the lipid matrix is dissolved in a chloroform solution, and the stearylated cell-penetrating peptide is added to obtain a lipid solution; the lipid solution is mixed with diisopropyl ether in an equal volume, a HEPES buffer with a concentration of 10 mM and an equal volume to the lipid solution is added, and a fluorescent label is added to obtain a second mixture.

[0045] In the present invention, the concentration of the lipid matrix in the lipid solution is preferably 20 μM.

[0046] In the present invention, the first mixture, a photothermal reagent, and a browning inducer are added to the upper layer solution of the second mixture, stirred, and homogenized with a ultrasonic cell disruptor for 8 min. After stirring and evaporating the organic solvent, the remaining solution is ultrasonically treated with a ultrasonic cleaner for 5 min to obtain a nanoparticle suspension.

[0047] In the present invention, the power of the ultrasonic cell disruptor is preferably 9 W; the homogenization is preferably 8 min of homogenization; the stirring and evaporation is preferably a rotary evaporator at 25 °C and 100 MPa; the ultrasonic treatment is preferably 5 min of ultrasonic treatment; the ultrasonic cleaning machine is preferably Xinzhi SB-5200DTD with a power of 100 W.

[0048] In the present invention, the NPs suspension is stirred and dialyzed in HEPES buffer with a pH of 7.4 and a concentration of 10 mM for 2 h to obtain dual-targeted nanoparticles (LNPs).

[0049] In the present invention, the dialysis is preferably a dialysis bag with a molecular weight cut-off of 3500, brand Thermo Fisher, and dialysis at room temperature.

[0050] The present invention also provides an application of the dual-targeted nanoparticles described in the above technical solution, and the application is for the preparation of a drug for improving obesity and / or glycolipid metabolism.

[0051] In the present invention, the drug is preferably a drug for combined photothermal-drug therapy. The dual-targeted nanoparticles obtained in the present invention, in combination with photothermal-drug therapy, can effectively promote the "browning" of white fat, improve body weight and glycolipid metabolism, and lay a foundation for the development of safe and effective drugs against obesity and related metabolic disorders.

[0052] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments. However, the content of the present invention is not limited to the following embodiments. The materials, reagents, etc. used in the examples and test examples of the present invention can be obtained from commercial channels without special instructions; the methods used in the examples and test examples of the present invention are conventional methods without special instructions.

[0053] Reagents:

[0054] CGZ was purchased from Shenzhen MicroPort Biotherapeutics Co., Ltd. ICG was purchased from Tokyo Chemical Industry Co., Ltd. DSPE-PEG was purchased from Guangzhou Pengshuo Biotechnology Co., Ltd. The two polypeptides were purchased from Nanjing Genscript Biotechnology Co., Ltd. The reagents related to cell culture were all purchased from Gibco, USA. Dexamethasone was purchased from Shanghai Yeasen Biotechnology Co., Ltd. Bovine insulin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. 3-Isobutyl-1-methylxanthine was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0055] Example 1

[0056] (1) Dissolve the adipocyte homing peptide in distilled water to prepare an adipocyte homing peptide solution with a concentration of 10 mM; dissolve DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Mal in distilled water at a ratio of 1:1.25 to prepare a DSPE-PEG polymer solution with a concentration of 10 mM. Mix the adipocyte homing peptide solution and the DSPE-PEG polymer solution at a volume ratio of 1:1.25, and react with shaking at 30 °C for 24 h to obtain a first mixture; the binding of the adipocyte homing peptide and the DSPE-PEG polymer was confirmed by MALDI-TOF-MS;

[0057] (2) Add 2 mL of chloroform solution to a mixture of egg yolk phosphatidylcholine and cholesterol with a molar ratio of 4:1, and then add stearoylated cell-penetrating peptide accounting for 20 mol% of the total lipids to obtain a lipid solution; mix the lipid solution with diisopropyl ether in equal volume, and then add a HEPES buffer solution with a concentration of 10 mM in an equal volume to the lipid solution to obtain a second mixture;

[0058] (3) Add the first mixture (DSPE-PEG polymer accounts for 5 mol% of the total lipids) and CGZ (accounts for 50 mol% of the total lipids) to the upper layer solution of the second mixture. After stirring, homogenize it with an ultrasonic cell disruptor for 8 min. After stirring and evaporating the organic solvent, ultrasonically treat the remaining solution with an ultrasonic cleaner for 5 min to obtain a nanoparticle suspension;

[0059] (4) Stir and dialyze the NPs suspension in a HEPES buffer solution with a pH of 7.4 and a concentration of 10 mM for 2 h to obtain the dual-targeted liposome nanoparticles CGZ LNPs.

[0060] Example 2

[0061] (1) Dissolve the adipocyte homing peptide in distilled water to prepare an adipocyte homing peptide solution with a concentration of 10 mM; dissolve DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Mal in distilled water at a ratio of 1:1.25 to prepare a DSPE-PEG polymer solution with a concentration of 10 mM. Mix the adipocyte homing peptide solution and the DSPE-PEG polymer solution at a volume ratio of 1:1 and react with shaking at 30 °C for 24 h to obtain the first mixture; confirm the binding of the adipocyte homing peptide and the DSPE-PEG polymer by MALDI-TOF-MS;

[0062] (2) Add a chloroform solution to a mixture of egg yolk phosphatidylcholine and cholesterol with a molar ratio of 4:1, and then add stearoylated cell-penetrating peptide accounting for 4 mol% of the total lipids to obtain a lipid solution; mix the lipid solution with diisopropyl ether in equal volume, and then add a HEPES buffer solution with a concentration of 10 mM and an equal volume to the lipid solution to obtain the second mixture;

[0063] (3) Add the first mixture (DSPE-PEG polymer accounts for 5 mol% of the total lipids) and ICG (accounts for 20 mol% of the total lipids) to the upper layer solution of the second mixture. After stirring, homogenize it with an ultrasonic cell disruptor for 8 min. After stirring and evaporating the organic solvent, ultrasonically treat the remaining solution with an ultrasonic cleaner for 5 min to obtain a nanoparticle suspension;

[0064] (4) Stir and dialyze the NPs suspension in a HEPES buffer solution with a pH of 7.4 and a concentration of 10 mM for 2 h to obtain the dual-targeted liposome nanoparticles ICG LNPs.

[0065] Example 3

[0066] (1) Dissolve the adipocyte homing peptide in distilled water to prepare an adipocyte homing peptide solution with a concentration of 10 mM; dissolve DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Mal in distilled water at a ratio of 1:1.25 to prepare a DSPE-PEG polymer solution with a concentration of 10 mM. Mix the adipocyte homing peptide solution and the DSPE-PEG polymer solution at a volume ratio of 1:1, and react with shaking at 30 °C for 24 h to obtain a first mixture; confirm the binding of the adipocyte homing peptide and the DSPE-PEG polymer by MALDI-TOF-MS;

[0067] (2) Add a chloroform solution to a mixture of egg yolk phosphatidylcholine and cholesterol with a molar ratio of 4:1, and then add stearylated cell-penetrating peptide accounting for 5 mol% of the total lipids to obtain a lipid solution; mix the lipid solution with diisopropyl ether in an equal volume, and then add a HEPES buffer solution with a concentration of 10 mM and an equal volume to the lipid solution to obtain a second mixture;

[0068] (3) Add the first mixture (DSPE-PEG polymer accounting for 5 mol% of the total lipids), ICG (accounting for 20 mol% of the total lipids), and CGZ (accounting for 50 mol% of the total lipids) to the upper layer solution of the second mixture, stir and then homogenize with an ultrasonic cell disruptor for 8 min, stir and evaporate the organic solvent, and then ultrasonically treat the remaining solution with an ultrasonic cleaner for 5 min to obtain a nanoparticle suspension;

[0069] (4) Stir and dialyze the NPs suspension in a HEPES buffer solution with a pH of 7.4 and a concentration of 10 mM for 2 h to obtain the dual-targeted liposome nanoparticles ICG+CGZ LNPs.

[0070] Test Example 1 Performance Test of Dual-Targeted Nanoparticles

[0071] Determine the size and ζ potential of the dual-targeted nanoparticles obtained in Examples 1 to 3 by a dynamic light scattering instrument (Zetasizer), and the test results are as Figure 1 shown. It can be seen from Figure 1 that the average hydrodynamic diameter of ICG+CGZ LNPs is 95.2 nm, and the polydispersity index (PdI) is 0.38, indicating that ICG+CGZ LNPs are in a state of uniform dispersion. The surface potential of ICG+CGZ LNPs is 5.2 mV. Since stearylated R8 is positively charged, the positive potential on the surface of the nanoparticles indicates that R8 is modified on the surface of the nanoparticles, which is beneficial to the next internalization and enrichment in cells.

[0072] The drug content in the dual-targeted nanoparticles obtained in Examples 1 to 3 was quantified by an ultraviolet-visible spectrophotometer. To determine the drug encapsulation efficiency, the drug content before and after dialysis was measured. The results showed that the encapsulation efficiencies of ICG and CGZ in ICG+CGZ LNPs were 88.39% and 81.12%, respectively.

[0073] The above tests were also performed on ICG LNPs, with a size of 117.4 nm (as Figure 1 shown), and a surface potential of 19.9 mV. The encapsulation efficiency of ICG was 97.08%.

[0074] The above tests were also performed on CGZ LNP, with a size of 156.5 nm (as Figure 1 shown), and a surface potential of 27.3 mV. The encapsulation efficiency of CGZ was 91.21%.

[0075] To analyze the drug release curve, aliquots of each NPs suspension in dialysis were extracted at preset time points for absorbance analysis. The results of the absorbance analysis are as Figure 2 shown. It can be seen from Figure 2 that 51.62% of the drug can be released from CGZ LNPs in solution within 120 h.

[0076] Test Example 2

[0077] Mouse embryonic fibroblasts 3T3L1 were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS). 1 mL of the stock solution of 3-isobutyl-1-methylxanthine (IBMX, 50 mM DMSO solution) and 0.1 mL of the stock solution of dexamethasone (Dex, 1 mM DMSO solution) were added to the mixture, and 1 mg of lyophilized insulin was added and cultured and differentiated for 3 days at 37 °C in a 5% CO2 environment. Adipocytes were cultured in complete DMEM medium containing 10% FBS for 7 days.

[0078] Divided into 12 groups: (1) Blank group, blank control group; (2) NIR group, irradiated only with an 808 nm laser (0.33 W cm -2 , 5 min); (3) ICG-L group, incubated only with ICG LNPs (5 μg mL -1 ); (4) ICG-M group, incubated only with ICG LNPs (10 μg mL -1 ); (5) ICG+NIR-L group, incubated with ICG LNPs (5 μg mL -1 ) and then irradiated with an 808 nm laser (0.33 W cm -2, 5 min); (6) ICG + NIR - M group, incubated with ICG LNPs (10 μg mL -1 ), and then irradiated with an 808 nm laser (0.33 W cm -2 , 5 min); (7) CGZ - L group, only incubated with CGZ LNPs (15 μmol L -1 ); (8) CGZ - M group, only incubated with CGZ LNPs (30 μmol L -1 ); (9) ICG + CGZ - L group, incubated with ICG + CGZ LNPs (ICG, 5 μg mL -1 ; CGZ, 15 μmol L-1); (10) ICG + CGZ + NIR - L group, incubated with ICG + CGZ LNPs (ICG, 5 μg mL -1 ; CGZ, 15 μmol L -1 ), and then irradiated with an 808 nm laser (0.33 W cm -2 , 5 min); (11) ICG + CGZ - M group, incubated with ICG + CGZ LNPs (ICG, 10 μg mL-1; CGZ, 30 μmo l L-1); (12) ICG + CGZ + NIR - M group, incubated with ICG + CGZ LNPs (ICG, 10 μg mL -1 ; CGZ, 30 μmol L -1 ), and then irradiated with an 808 nm laser (0.33 W cm -2 , 5 min). On the 10th, 12th, and 14th days of 3T3L1 cell differentiation, fully differentiated adipocytes were treated three times. On the 15th day, isoproterenol (in a 10 μM HEPES solution) was added, and after 4 h of stimulation, the treated cells were harvested. Oil Red O kit staining was used to observe lipid droplets. qPCR was used to detect the expression of UCP1 and PPARγ genes in adipocytes.

[0079] As Figure 3 shown, it can be seen by observing with an inverted microscope that the lipid droplets in the cells of the Blank group, NIR group, and ICG + NIR group are large and mostly aggregated together. In the cells of the CGZ group, the size of the lipid droplets is significantly reduced, but the number is still relatively large and there are occasional aggregations; while in the cells of the ICG + CGZ + NIR group, the size of the lipid droplets is significantly reduced and relatively more dispersed, and fewer small lipid droplets can be seen in the same magnification field of view.

[0080] As Figure 4As shown, in the qPCR test, compared with the control group, co-incubating ICG LNPs with cells and stimulating with light irradiation showed an increasing trend in the thermogenic gene UCP1, which increased with the increase in concentration, while the PPARγ gene showed a decreasing trend. In terms of the drug itself, the CGZ drug could promote an increasing trend in the thermogenic gene UCP1, but the low concentration seemed to increase more than the medium concentration. In addition, the results showed that NIR irradiation of ICG+CGZ LNPs could combine the light irradiation effect and the drug itself effect, resulting in an increase in the UCP1 gene, showing an effect of 1+1>2. However, the PPARγ gene showed a decreasing effect in all cases.

[0081] Experimental Example 3

[0082] To establish an obese mouse model, male C57 / 6J mice at 6 weeks old and about 22 g were selected and fed with a high-fat diet providing 60% of energy from fat for more than 6 weeks. When their body weight exceeded 120% of that of the control group mice fed with normal diet, the model was considered successfully established.

[0083] The obese mice were divided into seven groups, with 5 mice in each group: (1) Blank group, blank control group; (2) NIR group, irradiated only with an 808 nm laser (0.33 W cm -2 , 5 min); (3) ICG group, injected with ICG LNPs only into the unilateral inguinal fat, with an injection dose of 50 μL of ICG LNPs, and the concentration of ICG was 0.5 mg mL -1 ; (4) ICG+NIR group, first injected with ICG LNPs into the unilateral inguinal fat, with an injection dose of 50 μL of ICG LNPs, and the concentration of ICG was 0.5 mg mL -1 , and then irradiated with an 808 nm laser (0.33 W cm -2 , 5 min); (5) CGZ group, injected with CGZ LNPs only into the unilateral inguinal fat, with an injection dose of 10 mg kg -1 ; (6) ICG+CGZ group, injected with ICG+CGZ LNPs only into the unilateral inguinal fat, with an injection dose of 50 μL of ICG, and the concentration of ICG was 0.5 mg mL -1 , and the injection dose of CGZ was 10 mgkg -1 ; (7) ICG+CGZ+NIR group, first injected with ICG+CGZ LNPs into the unilateral inguinal fat, with an injection dose of 50 μL of ICG, and the concentration of ICG was 0.5 mg mL -1 , and the injection dose of CGZ was 10 mg kg -1, and then irradiated with an 808 nm laser (0.33 W cm -2 , 5 min). The treatment cycle was four days, with drug administration and light stimulation once a day for the first two days and rest for the next two days. During the treatment process, the body weight of the mice was continuously monitored and the body temperature changes during light exposure were recorded.

[0084] After 4 cycles of treatment, the inguinal white adipose tissue (IgWAT), epididymal white adipose tissue (EpWAT), brown adipose tissue (BAT) of the mice, and the livers of the mice were fixed and stained with hematoxylin-eosin (HE) for histological observation.

[0085] As Figure 5 shown, during the treatment process, the body surface temperature detection of the mice showed that the temperatures at the injection sites of ICG LNPs and ICG+CGZ LNPs were the highest under NIR light irradiation, both around 45°C. When the fat temperature is higher than 42°C and lower than 48°C, it is beneficial for fat browning; while when the temperature is higher than 48°C, the overly strong heat stimulation will not only cause pain but also burn the skin at high temperature for a long time. As Figure 6 shown, the body temperature of the mice could be maintained around 46°C within 4 days, and only slightly increased when the next cycle of drug administration started on the 5th day, indicating that the drug accumulation brought by one cycle of treatment would not cause the temperature to be too high during photothermal stimulation. This is mainly due to drug metabolism and the photolysis of ICG.

[0086] As Figure 7 shown, during the treatment process, the body weights of the mice, which are important reference data for weight loss, were recorded. Under the high-fat diet, the body weights of the mice in the blank control group continued to increase slowly, while the body weights of the mice treated with LNPs drugs could be controlled or significantly decreased. Among them, the body weights of the mice in the CGZ, ICG+CGZ, and ICG groups were slightly lighter than the initial weights after 4 cycles of treatment. The most obvious weight loss was in the ICG+CGZ+NIR group, with a 14.6% weight loss, far exceeding the obvious weight loss effects of other drug administration groups, indicating that ICG+CGZ LNPs achieved the effect of "1+1>2" under the synergistic action of photothermal-drug.

[0087] Among obese and overweight people, the most common complication is fatty liver, because the liver is not only the largest metabolic organ in the human body, but also a major energy storage organ. There is a large amount of glycogen in the liver. After ingesting food, the sugar in the food will be concentrated in the liver and stored in the form of glycogen. This part of the energy will be released in time when the blood sugar drops to regulate the blood sugar concentration. When the patient is obese or overweight, under the stimulation of hormones and the like, sugar substances are more likely to be converted into fat and cholesterol in the liver and accumulate in the liver. As Figure 8 shown, by comparing the HE-stained pathological sections of small fat and liver in each group, it can be seen that compared with the mice in the Blank group, the fat sizes of the mice in the CGZ group have all decreased, and at the same time, the sizes of the lipid droplets in the liver have become smaller and the degree of lipid droplet aggregation is relatively low; while the fat sizes of the mice in the ICG+CGZ+NIR group have all decreased significantly, the number of lipid droplets has decreased sharply, and the number of lipid droplets is less than one percent of the number of liver lipid droplets in the Blank group mice. This shows that the photothermal-drug therapy synergistic effect of the ICG+CGZ+NIR group can effectively reduce the fat size both proximally and distally, and can effectively treat fatty liver problems through stimulation such as hormone adjustment.

[0088] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A dual-targeting nanoparticle, characterized in that: It includes a browning inducer, a photothermal agent, an adipocyte homing peptide, a cell-penetrating peptide, a DSPE-PEG polymer, and a lipid matrix; the browning inducer is sitagliptin sodium CGZ; the photothermal agent is indocyanine green ICG; the amino acid sequence of the adipocyte homing peptide is shown in SEQ ID NO.1, and the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.2; the lipid matrix is ​​egg yolk phosphatidylcholine and cholesterol; The preparation method of the dual-targeted nanoparticles comprises the following steps: S1. Mix the adipocyte homing peptide and DSPE-PEG5kD, and shake the mixture at 30°C for 24 hours to prepare DSPE-PEG5kDa-Pep; S2, dissolving the lipid matrix in a chloroform solution, adding the stearoyl cell penetrating peptide to obtain a lipid solution; mixing the lipid solution with an equal volume of diisopropyl ether, adding an equal volume of a 10 mM HEPES buffer to the lipid solution to obtain a first mixture; S3, dissolving DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Pep in distilled water to prepare a DSPE-PEG polymer solution containing adipocyte homing peptide to obtain a second mixture; S4, adding the second mixture, the photothermal reagent, and the browning inducing agent to the upper layer solution of the first mixture, stirring and homogenizing using an ultrasonic cell disruptor for 8 minutes, stirring and evaporating the organic solvent, and then using an ultrasonic cleaning agent to ultrasonically treat the remaining solution for 5 minutes to obtain a nanoparticle suspension; S5. The NPs suspension was dialyzed in a HEPES buffer with a pH of 7.4 and a concentration of 10 mM for 2 h to obtain dual-targeted nanoparticles.

2. The dual-targeted nanoparticle according to claim 1, characterized in that: The browning inducer accounts for 50 mol% of the total lipids, the photothermal agent accounts for 20 mol% of the total lipids, the adipocyte homing peptide accounts for 4 mol% of the total lipids, the cell penetrating peptide accounts for 20 mol% of the total lipids, and the DSPE-PEG polymer accounts for 5 mol% of the total lipids.

3. The dual-targeted nanoparticle according to claim 2, characterized in that: The concentration of the DSPE-PEG polymer solution in S3 is 10 mM.

4. The dual-targeting nanoparticle according to claim 3, characterized in that: The concentration of the lipid matrix in the lipid solution in S2 is 20 μM.

5. A use of the dual-targeted nanoparticles according to any one of claims 1 to 4, characterized in that: The application is for preparing medicines for improving obesity, sugar and lipid metabolism, and non-alcoholic fatty liver.

6. The use of the dual-targeted nanoparticles according to claim 5, characterized in that: The drug is a drug used for photothermal-drug combined therapy.

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