Double-targeted nanoparticle, preparation method therefor, and use thereof

Abstract

The present invention relates to the technical field of pharmaceutical formulations, and provides a dual-targeted nanoparticle, a preparation method therefor, and a use thereof. The dual-targeted particle comprises a browning inducer, i.e., Chiglitazar sodium, a photothermal reagent, i.e., indocyanine green, an adipocyte homing peptide, a cell penetrating peptide, a DSPE-PEG polymer, and a lipid matrix. The browning inducer and the photothermal reagent are encapsulated into a nanoparticle having excellent biocompatibility, and the surface of said nanoparticle is coated with the functionalized adipose homing peptide and the cell penetrating peptide to construct the dual-targeted nanoparticle. The combination of the dual-targeted nanoparticle and photothermal-drug therapy can effectively promote "browning" of white adipose, reduce weight and enhance glycolipid metabolism, and lay a foundation for developing safe and effective drugs against obesity and related metabolic disorders.

Description

A dual-targeting nanoparticle and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a dual-targeting nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Obesity is a global epidemic, with more than 1.9 billion overweight adults worldwide, of which more than 600 million are obese. Obesity is the source of many diseases, including cardiovascular disease, type 2 diabetes, hyperlipidemia, hypertension, sleep apnea syndrome, and other diseases, which pose a threat to human health and seriously affect the quality of life and increase the risk of death. Although weight loss can be achieved through effective exercise, energy intake restriction, and dietary changes, long-term effectiveness is suboptimal. In terms of drug treatment, only orlistat has been approved by the State Drug Administration of China for the treatment of obesity. However, due to gastrointestinal discomfort, fatty oily stools, and fatty diarrhea, its clinical application is limited. Some weight loss drugs on the market abroad also have systemic side effects that affect clinical application. Studies have shown that metabolic surgery for weight loss has significant effects on obesity-related hypertension, type 2 diabetes, and fatty liver disease. However, metabolic surgery still has problems such as difficulty in choosing the surgical procedure, 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. It can be divided into white adipose tissue (WAT) and brown adipose tissue (BAT) based on function and morphology. BAT contains a large number of small, multi-locular lipid droplets and a large number of mitochondria, with a large potential for heat production. Studies have shown that WAT can be directly converted into BAT after cold stimulation or beta3-adrenergic and Peroxisome proliferator-activated receptors (PPAR) agonist treatment. BAT plays an important role in regulating glucose and lipid metabolism, insulin sensitivity, and adipose tissue homeostasis. Therefore, promoting WAT "browning" is a new approach to weight loss and improving glucose and lipid metabolism. SUMMARY

[0004] Therefore, the present application aims to provide a dual-targeting nanoparticle and a preparation method and application thereof. The dual-targeting nanoparticle provided by the present application can effectively intervene in the expression of UCP1 and PPAR-gamma in adipocytes, promote white fat "browning", and effectively improve obesity and metabolic disorders.

[0005] In order to achieve the above object, the present application provides the following technical solutions:

[0006] A double-targeting nanoparticle comprises a browning inducer, a photothermal agent, 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 sodium sitagliptin CGZ, and the photothermal agent is indocyanine green ICG.

[0008] Preferably, 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.

[0009] Preferably, 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.

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

[0011] S1, mixing the adipocyte homing peptide and the DSPE-PEG5kD, oscillating the reaction at room temperature for 24 hours to prepare the adipocyte homing peptide-DSPE-PEG (DSPE-PEG5kDa-Pep).

[0012] S2, dissolving the lipid matrix in a chloroform solution, adding the stearyl cell penetrating peptide to obtain a lipid solution; mixing the lipid solution with an equal volume of diisopropyl ether, adding an equal volume of 10 mM HEPES buffer solution to the lipid solution to obtain a first mixture;

[0013] S3, dissolving one of the two combinations, combination one comprising DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Mal, and combination two comprising DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Pep, 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, adding the second mixture, the photothermal agent, and the browning inducer to the upper solution of the first mixture, stirring, and then homogenizing for 8 minutes using an ultrasonic cell disruptor, and then using an ultrasonic cleaner to treat the remaining solution for 5 minutes to obtain a liposome nanoparticle suspension;

[0015] S5, stirring and dialysis the NPs suspension in HEPES buffer with pH 7.4 and concentration of 10 mM for 2 h, to obtain the dual-targeting nanoparticles (LNPs).

[0016] Preferably, the concentration of the adipocyte homing peptide solution and the DSPE-PEG polymer solution in S1 is 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 application also provides the dual-targeting nanoparticles as described in the above technical solutions, and the application is used for preparing 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 application provides a dual-targeting nanoparticle, a preparation method and an application thereof. The dual-targeting nanoparticle comprises a browning inducer, a photothermal reagent, an adipocyte homing peptide, a cell penetrating peptide, a DSPE-PEG polymer and a lipid matrix. The browning inducer and the photothermal reagent are encapsulated in a nanoparticle with excellent biocompatibility, and the surface is wrapped with a functionalized fat homing peptide and a cell penetrating peptide, to construct a dual-targeting nanoparticle. In combination with photothermal-drug therapy, the dual-targeting nanoparticle can effectively promote the "browning" of white adipose tissue, improve body weight and glycolipid metabolism, and lay a foundation for developing a safe and effective drug for resisting obesity and related metabolic disorders. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0023] Figure 3 is an oil red O staining imaging diagram of a cell experiment;

[0024] Figure 4 is a qPCR characterization statistical diagram of a cell experiment;

[0025] Figure 5 is a thermal imaging diagram of an animal experiment;

[0026] Figure 6 is a temperature change statistical diagram of an animal experiment;

[0027] Figure 7 is a body weight change diagram of an animal experiment;

[0028] Figure 8 is a fat and liver tissue section diagram of an animal experiment. DETAILED DESCRIPTION

[0029] The application provides a double-targeting nanoparticle, comprising a browning inducer, a photothermal agent, an adipocyte homing peptide, a cell penetrating peptide, a fluorescent label, a DSPE-PEG polymer and a lipid matrix.

[0030] In the application, the browning inducer is preferably chiglitazar CGZ; chiglitazar (CGZ) is the first PPAR full agonist, which can induce the expression of multiple downstream target genes, including genes related to insulin sensitivity, lipid metabolism / transport, heat production and energy conversion, and can not only reduce blood glucose, but also regulate blood lipids. In the application, chiglitazar CGZ is used as a browning promoter, which, together with the photothermal agent, can effectively promote the “browning” of white adipose tissue in the photothermal-drug therapy.

[0031] In the application, the photothermal agent is preferably indocyanine green ICG. Indocyanine green can be used as a photothermal agent to convert light energy into heat energy and produce singlet oxygen, which has been proved to be safe in clinical practice. Indocyanine green has near-infrared fluorescence imaging and good photothermal conversion ability under 808 nm laser irradiation, and has been accepted by FDA and widely used in clinical practice.

[0032] In the application, 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. In the application, the adipocyte homing peptide and the cell penetrating peptide can accurately identify adipocytes and promote the enrichment of the browning inducer and the photothermal agent 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 application, the DSPE-PEG polymer is preferably one or more of DSPE-PEG5kDa-Mal, DSPE-PEG5kDa-Pep, 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 and 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 and DSPE-PEG5kDa-Pep is preferably 1:1.25; the lipid matrix is preferably egg yolk phosphatidylcholine and cholesterol; the molar ratio of the egg yolk phosphatidylcholine and cholesterol is preferably 4:1.

[0036] In the present application, the browning inducer accounts for 50 mol% of the total lipid, the photothermal agent 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.

[0037] The present application also provides a preparation method of the double-targeting nanoparticle 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, oscillate and react at 30°C for 24h to obtain a first mixture;

[0039] S2, dissolve the lipid matrix in chloroform solution, add stearoylated cell penetrating peptide to obtain a lipid solution; mix the lipid solution with an equal volume of diisopropyl ether, add an equal volume of 10mM HEPES buffer solution to the lipid solution to obtain a second mixture;

[0040] S3, add the first mixture, the photothermal agent, and the browning inducer to the upper solution of the second mixture, stir, homogenize using an ultrasonic cell disruptor for 8min, stir to evaporate the organic solvent, and then use ultrasonic cleaning agent to treat the remaining solution for 5min to obtain a nanoparticle suspension;

[0041] S4, stirring and dialysis of the NPs suspension in HEPES buffer with pH 7.4 and concentration 10 mM for 2 h, to obtain the dual-targeting nanoparticles (LNPs).

[0042] The adipocyte homing peptide and the DSPE-PEG polymer are dissolved in distilled water respectively 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 the first mixture is obtained by oscillation reaction at 30 DEG C for 24 h.

[0043] In the present application, the oscillation reaction is preferably oscillation reaction at 30 DEG C for 24 h, and the concentration of the adipocyte homing peptide solution and the DSPE-PEG polymer solution is preferably 10 mM, and the proportion is preferably 1:1.25 in volume ratio.

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

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

[0046] The first mixture, a photothermal reagent, and a browning inducer are added to the upper solution of the second mixture, and the mixture is homogenized by using an ultrasonic cell disruptor for 8 min after stirring, the organic solvent is evaporated by stirring, and the remaining solution is treated by ultrasonic cleaning for 5 min by using an ultrasonic cleaning machine to obtain a nanoparticle suspension.

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

[0048] The NPs suspension is stirred and dialyzed in HEPES buffer with pH 7.4 and concentration 10 mM for 2 h to obtain the dual-targeting nanoparticles (LNPs).

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

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

[0051] In the present application, the drug is preferably a drug for photothermal-drug combination therapy. The double-targeting nanoparticles obtained by the present application, in combination with photothermal-drug therapy, can effectively promote the "browning" of white adipose tissue, improve body weight and glucose-lipid metabolism, and lay a foundation for developing safe and effective drugs against obesity and related metabolic disorders.

[0052] In order to better understand the present application, the content of the present application is further illustrated below in combination with examples, but the content of the present application is not limited only to the following examples. The materials, reagents and the like used in the examples and test examples of the present application can be obtained from commercial channels unless otherwise specified; the methods used in the examples and test examples of the present application are conventional methods unless otherwise specified.

[0053] Reagents:

[0054] CGZ was purchased from Shenzhen Microchip Biotechnology Co., Ltd. ICG was purchased from Shanghai Lixiayihua Chemical Industry Development Co., Ltd. DSPE-PEG was purchased from Guangzhou Pingsuo Biological Technology Co., Ltd. The two polypeptides were purchased from Nanjing Kingsrui Biological Technology Co., Ltd. Cell culture related reagents were purchased from Gilboc, USA. Dexamethasone was purchased from Shanghai Yisheng Biological Technology Co., Ltd. Bovine insulin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. 3-isobutyl-1-methyl xanthine was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.

[0055] Example 1

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

[0057] (2) In a mixture of egg yolk phosphatidylcholine and cholesterol at a molar ratio of 4:1, 2 mL of chloroform solution was added, and then 20 mol% of stearoylated cell penetrating peptide based on the total lipid was added to obtain a lipid solution. The lipid solution was mixed with an equal volume of diisopropyl ether, and then an equal volume of 10 mM HEPES buffer was added to obtain a second mixture;

[0058] (3) to the upper layer solution of the second mixture, add the first mixture (DSPE-PEG polymer accounts for 5 mol% of total lipid), CGZ (accounts for 50 mol% of total lipid), after stirring, homogenize using an ultrasonic cell disruptor for 8 min, after stirring to evaporate the organic solvent, use ultrasonic cleaner to ultrasonic treat the remaining solution for 5 min, to obtain a nanoparticle suspension;

[0059] (4) dialyze the NPs suspension in a HEPES buffer with a pH of 7.4 and a concentration of 10 mM for 2 h, to obtain the dual-targeting 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 ratio of 1:1 by volume, and oscillate the reaction at 30°C for 24 h to obtain the first mixture; confirm the combination 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 at a molar ratio of 4:1, and then add stearoylated cell penetrating peptide accounting for 4 mol% of total lipid to obtain a lipid solution; mix the lipid solution with an equal volume of diisopropyl ether, and then add an equal volume of a HEPES buffer with a concentration of 10 mM to the lipid solution to obtain the second mixture;

[0063] (3) to the upper layer solution of the second mixture, add the first mixture (DSPE-PEG polymer accounts for 5 mol% of total lipid), ICG (accounts for 20 mol% of total lipid), after stirring, homogenize using an ultrasonic cell disruptor for 8 min, after stirring to evaporate the organic solvent, use ultrasonic cleaner to ultrasonic treat the remaining solution for 5 min, to obtain a nanoparticle suspension;

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

[0065] Example 3

[0066] (1) The adipocyte homing peptide was dissolved in distilled water to prepare a 10 mM adipocyte homing peptide solution. DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Mal were dissolved in distilled water at a ratio of 1:1.25 to prepare a 10 mM DSPE-PEG polymer solution. The adipocyte homing peptide solution and the DSPE-PEG polymer solution were mixed at a ratio of 1:1 by volume, and the mixture was shaken at 30°C for 24 h to obtain a first mixture. The combination of the adipocyte homing peptide and the DSPE-PEG polymer was confirmed by MALDI-TOF-MS;

[0067] (2) A chloroform solution was added to a mixture of egg yolk phosphatidylcholine and cholesterol at a molar ratio of 4:1, and 5 mol% of the total lipids of stearoylated cell-penetrating peptide was added to obtain a lipid solution. The lipid solution was mixed with an equal volume of diisopropyl ether, and an equal volume of a 10 mM HEPES buffer was added to obtain a second mixture;

[0068] (3) The first mixture (5 mol% of DSPE-PEG polymer of the total lipids), ICG (20 mol% of the total lipids), and CGZ (50 mol% of the total lipids) were added to the upper solution of the second mixture, which was stirred and homogenized using an ultrasonic cell disruptor for 8 min. After the organic solvent was evaporated by stirring, the remaining solution was treated with ultrasonic cleaning agent for 5 min to obtain a nanoparticle suspension;

[0069] (4) The NPs suspension was dialyzed in a 10 mM HEPES buffer at pH 7.4 for 2 h to obtain the dual-targeting liposome nanoparticle ICG+CGZ LNPs.

[0070] Test Example 1: Performance test of dual-targeting nanoparticles

[0071] The size and zeta potential of the dual-targeting nanoparticles obtained in Examples 1-3 were determined by dynamic light scattering (Zeta sizer), and the test results are shown in FIG. 1. As shown in FIG. 1, the average hydrodynamic diameter of the ICG+CGZ LNPs was 95.2 nm, and the polydispersity index (PdI) was 0.38, indicating that the ICG+CGZ LNPs were in a uniformly dispersed state. The surface potential of the ICG+CGZ LNPs was 5.2 mV. Since the stearoylated 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 for the subsequent internalization and enrichment in cells.

[0072] The drug content in the dual-targeting nanoparticles obtained in Examples 1-3 was quantified by UV-Vis spectrophotometer. To determine the drug encapsulation efficiency, the drug content before and after dialysis was determined, and the results showed that the encapsulation efficiency of ICG and CGZ in ICG+CGZ LNPs was 88.39% and 81.12%, respectively.

[0073] The above tests were also performed on ICG LNPs, which had a size of 117.4 nm (as shown in FIG. 1) 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 LNPs, which had a size of 156.5 nm (as shown in FIG. 1) and a surface potential of 27.3 mV. The encapsulation efficiency of CGZ was 91.21%.

[0075] To analyze the drug release curve, an aliquot of each NPs suspension in dialysis was extracted at a predetermined time point for absorbance analysis, and the results of absorbance analysis are shown in FIG. 2. As can be seen from FIG. 2, CGZ LNPs can release 51.62% of the drug in the solution within 120 h.

[0076] Test Example 2

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

[0078] The mice were divided into 12 groups: (1) Blank group, blank control group; (2) NIR group, only 808 nm laser irradiation (0.33 W cm -2 , 5 min); (3) ICG-L group, only ICG LNPs incubation (5 μg mL -1 ); (4) ICG-M group, only ICG LNPs incubation (10 μg mL -1 ); (5) ICG+NIR-L group, ICG LNPs incubation (5 μg mL -1 ) followed by 808 nm laser irradiation (0.33 W cm -2 , 5 min); (6) ICG+NIR-M group, ICG LNPs incubation (10 μg mL -1After that, it was irradiated with an 808nm laser (0.33W cm⁻¹). -2 (7) CGZ-L group, incubated with only CGZ LNPs (15 μmol L) for 5 min); -1 (8) CGZ-M group, incubated only with CGZ LNPs (30 μmol L); -1 (9) ICG+CGZ-L group, incubated with ICG+CGZ LNPs (ICG, 5 μg mL); -1 (10) ICG+CGZ+NIR-L group, incubated with ICG+CGZ LNPs (ICG, 5 μg mL); -1 CGZ, 15 μmol L -1 After that, it was irradiated with an 808nm laser (0.33W cm⁻¹). -2 (11) ICG+CGZ-M group, incubated with ICG+CGZ LNPs (ICG, 10 μg mL-1; CGZ, 30 μmol L-1); (12) ICG+CGZ+NIR-M group, incubated with ICG+CGZ LNPs (ICG, 10 μg mL-1); -1 CGZ, 30 μmol L -1 After that, it was irradiated with an 808nm laser (0.33W cm⁻¹). -2 (5 min). Completely differentiated adipocytes were treated three times on days 10, 12, and 14 of 3T3L1 cell differentiation. On day 15, isoproterenol (10 μM HEPES solution) was added, and after 4 h of stimulation, the treated cells were harvested. Lipid droplets were observed using Oil Red O staining. The expression of UCP1 and PPARγ genes in adipocytes was detected by qPCR.

[0079] As shown in Figure 3, observation using an inverted microscope reveals that the lipid droplets in the Blank, NIR, and ICG+NIR groups are large and mostly aggregated together. The lipid droplet size in the CGZ group is significantly reduced, but the number is still relatively large and occasionally aggregated. In contrast, the lipid droplet size in the ICG+CGZ+NIR group is significantly reduced and relatively dispersed, with fewer small lipid droplets visible in the same magnification field of view.

[0080] As shown in FIG. 4, in the qPCR test, compared with the control group, the ICG LNPs co-incubated with cells and using light stimulation can make the thermogenic gene UCP1 have a rising trend, and increase with the increase of concentration, while the PPARy gene shows a downward trend. As for the drug itself, CGZ drug can make the thermogenic gene UCP1 show a rising trend, but the low concentration seems to rise more than the medium concentration. In addition, the results show that ICG+CGZ LNPs under NIR light can combine the light effect and the drug itself effect to make the UCP1 gene rise, showing a 1+1>2 effect. However, the PPARy gene shows a downward effect.

[0081] Test Example 3

[0082] To establish an obese mouse model, 6-week-old male C57 / 6J mice weighing about 22 g were selected, and 60% fat energy high-fat feed was used for feeding for more than 6 weeks, and the body weight was more than 120% of the body weight of the control group mice fed with ordinary feed, which was considered to be a successful modeling.

[0083] The obese mice were divided into seven groups, five in each group: (1) Blank group, blank control group; (2) NIR group, only using 808 nm laser for irradiation (0.33 W cm -2 , 5 min); (3) ICG group, only unilateral inguinal fat injection of ICG LNPs, the injection dose of ICG LNPs was 50 μL, and the concentration of ICG was 0.5 mg mL -1 ; (4) ICG+NIR group, first unilateral inguinal fat injection of ICG LNPs, the injection dose of ICG LNPs was 50 μL, and the concentration of ICG was 0.5 mg mL -1 , and then using 808 nm laser for irradiation (0.33 W cm -2 , 5 min); (5) CGZ group, only unilateral inguinal fat injection of CGZ LNPs, the injection dose of CGZ LNPs was 10 mg kg -1 ; (6) ICG+CGZ group, only unilateral inguinal fat injection of ICG+CGZ LNPs, containing ICG injection dose of 50 μL, and the concentration of ICG was 0.5 mg mL -1 , and the injection dose of CGZ was 10 mg kg -1 ; (7) ICG+CGZ+NIR group, first unilateral inguinal fat injection of ICG+CGZ LNPs, containing ICG injection dose of 50 μL, and the concentration of ICG was 0.5 mg mL -1 , containing CGZ injection dose of 10 mg kg -1 , and then using 808 nm laser for irradiation (0.33 W cm-2 The mice were treated for four days, with the first two days of daily administration and light stimulation, and the last two days of rest. The body weight of the mice was continuously observed and the change of the body temperature of the mice under light stimulation was recorded.

[0084] After completing the treatment of four cycles, the inguinal white adipose tissue (IgWAT), epididymal white adipose tissue (EpWAT), mouse scapular BAT, and mouse liver of the mice were fixed for hematoxylin-eosin (HE) section staining observation.

[0085] As shown in FIG. 5, the mouse surface temperature detection showed that the temperature of the injection site of the ICG LNPs and the ICG+CGZ LNPs under the action of NIR light was about 45°C. When the temperature of the fat was higher than 42°C and lower than 48°C, it was conducive to the browning of the fat. When the temperature was higher than 48°C, the intense heat stimulation would not only cause pain, but also would burn the skin for a long time. As shown in FIG. 6, the temperature of the mice could be maintained at about 46°C within four days, and only slightly increased on the fifth day when the next cycle of administration began, which indicated that the drug accumulation caused by one cycle of treatment would not cause the temperature to be too high under the photothermal stimulation. This was mainly due to the metabolism of the drug and the photolysis of ICG.

[0086] As shown in FIG. 7, the body weight of the mice was recorded as an important reference data for weight loss during the treatment. Under the feeding of high-fat diet, the body weight of the mice in the blank control group continued to slowly increase, while the body weight of the mice treated with LNPs drug could be controlled or obviously decreased. Among them, the body weight of the mice in the CGZ and ICG+CGZ and ICG groups was slightly lighter than the initial body weight after completing the treatment of four cycles. The most obvious was the ICG+CGZ+NIR group, with a weight loss of 14.6%. Far more than the obvious weight loss effect of other administration groups, which indicated that the ICG+CGZ LNPs achieved the effect of “1+1>2” under the synergistic effect of photothermal-drug.

[0087] In 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 large energy storage organ. There are a lot of liver glycogen in the liver, after food intake, the sugar in the food will be concentrated in the liver in the form of glycogen storage, this part of energy will be released in time to regulate blood glucose concentration when blood glucose decreases, and when the patient is in an obese or overweight state, under the stimulation of hormones, etc., the sugar substances in the liver are more likely to be converted into fat and cholesterol and accumulated in the liver. As shown in FIG. 8, by comparing the small fat and liver HE staining pathological sections of each group, it can be seen that compared with the Blank group of mice, the fat size of the CGZ group of mice is reduced, and the size of the lipid droplets in the liver is reduced, and the aggregation degree of the lipid droplets is relatively low; the fat size of the ICG+CGZ+NIR group of mice is very obviously reduced, and the number of lipid droplets is sharply reduced, and the number of lipid droplets is less than one percent of the number of liver lipid droplets of the Blank group of mice. This shows that the synergistic effect of photothermal-drug treatment of the ICG+CGZ+NIR group can effectively reduce the fat size in the near and far ends, and can effectively treat fatty liver problems through hormone adjustment and other stimuli.

[0088] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A dual-targeting nanoparticle, characterized in that, The browning inducer, the photothermal agent, the adipocyte homing peptide, the cell penetrating peptide, the DSPE-PEG polymer, and the lipid matrix.

2. The dual-targeting nanoparticle of claim 1, wherein, The browning inducer is sodium sitagliptin CGZ, and the photothermal agent is indocyanine green ICG.

3. The dual-targeting nanoparticle of claim 2, wherein, 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.

4. The dual targeting nanoparticle according to any one of claims 1 to 3, wherein, The DSPE-PEG polymer is one or more of DSPE-PEG5kDa-Mal, DSPE-PEG5kDa-Pep, and DSPE-PEG2kDa-Mal, and the lipid matrix is egg yolk phosphatidylcholine and cholesterol.

5. The dual-targeting nanoparticle of claim 4, wherein, 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.

6. The dual-targeting nanoparticle of claim 5, wherein, The preparation method comprises the following steps: S1, mixing the adipocyte homing peptide and the DSPE-PEG5kD, oscillating the mixture at 30°C for 24 hours to obtain the adipocyte homing peptide-DSPE-PEG; S2, dissolving the lipid matrix in a chloroform solution, adding the stearyl cell penetrating peptide to obtain a lipid solution; mixing the lipid solution with an equal volume of diisopropyl ether, adding an equal volume of 10 mM HEPES buffer solution to the lipid solution to obtain a first mixture; S3, dissolving the DSPE-PEG polymer 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, wherein the DSPE-PEG polymer solution comprises DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Mal, or DSPE-PEG2kDa-Mal and DSPE-PEG5kDa-Pep; S4, adding the second mixture, the photothermal agent, and the browning inducer to the upper solution of the first mixture, stirring, homogenizing for 8 minutes using an ultrasonic cell disruptor, and then stirring to evaporate the organic solvent, and then ultrasonic cleaning the remaining solution for 5 minutes using an ultrasonic cleaner to obtain a nanoparticle suspension; S5, stirring and dialyzing the NPs suspension in a 10 mM HEPES buffer solution with a pH of 7.4 for 2 hours to obtain the dual-targeting nanoparticles.

7. The method for preparing dual-targeted nanoparticles according to claim 6, characterized in that, The concentration of the DSPE-PEG polymer solution in the combination one or the combination two in S1 is 10 mM, and the ratio in S1 is 1:1.

25.

8. The method for preparing dual-targeted nanoparticles according to claim 6, characterized in that, The concentration of the lipid matrix in the lipid solution in S2 is 20 μM.

9. Use of the dual targeting nanoparticle of any one of claims 4, wherein the nanoparticle is used for the treatment of cancer. The application is used for preparing a drug for improving obesity and / or glycolipid metabolism.

10. The use of the dual targeting nanoparticle according to claim 9, wherein, The drug is a drug for photothermal-drug combination therapy.

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