Preparation of cationic nano-micelle and application of cationic nano-micelle in lipid droplet degradation and targeted obesity therapy by combining photothermal therapy

Abstract

The invention provides a construction method of a fatty acid modified chitosan oligosaccharide derivative, a preparation method of cationic nano-micelles and application of the cationic nano-micelles in lipid droplet degradation and targeted obesity therapy by combining photothermal therapy. By covalently modifying fatty acid on a chitosan oligosaccharide skeleton, the cationic micelle capable of entrapping small molecule drugs and targeting adipose tissues is obtained. The compound is combined with medicines such as a photo-thermal agent or a browning agent for use, can be used for targeted therapy of obesity after being loaded on a microneedle, and can effectively reduce lipid accumulation of adipose tissues and regulate whole body metabolism.

Description

Technical Field

[0001] The invention belongs to the field of pharmacy, and relates to a method for preparing fatty acid-modified chitosan oligosaccharide and self-assembled nanoparticles thereof, and an anti-obesity application of nanoparticles co-loaded with a photothermal agent for targeting fat cells. Background Art

[0002] Obesity is an increasingly serious public health problem that can easily lead to complications such as cardiovascular disease, type 2 diabetes, and fatty liver. Currently, the main treatments for obesity either act on the central nervous system to suppress appetite (e.g., phentermine) or act on the gastrointestinal tract to inhibit fat absorption (e.g., orlistat), but most of them are not only limited in terms of use and applicable population, but may also increase the risk of other diseases. Therefore, it is urgent to develop new anti-obesity drug delivery systems to make obesity treatment safer and more effective.

[0003] Lipid droplets are the main storage site for neutral lipids (including triglycerides and cholesterol esters) in cells. Their abnormal accumulation is the root cause of obesity and metabolic syndrome. Therefore, promoting the degradation of lipid droplets may be a potential treatment for related diseases. Studies have shown that cationic nanoparticles can be enriched in adipose tissue containing a large amount of negatively charged glycosaminoglycans through charge interactions, and inhibit fat accumulation by regulating nutrient sensing signaling pathways. In addition, cationic materials have been shown to degrade organelles by increasing autophagy levels. Lipid droplets in adipocytes are also expected to be degraded through the autophagy-lysosome pathway to reduce lipid accumulation.

[0004] Chitosan oligosaccharide is obtained by deacetylation and depolymerization of chitin. It has good water solubility and biocompatibility, and is the only positively charged polysaccharide in nature. After chitosan oligosaccharide is modified with stearic acid, it can be made hydrophilic and lipophilic. The modified chitosan oligosaccharide can self-assemble to form nano-micelles, thereby increasing its positive charge density and further enhancing its fat targeting performance and lipid degradation efficacy. And when used in combination with photothermal agents or browning agents, it can promote the browning of fat cells, regulate systemic metabolism, and fight obesity.

[0005] Based on the above background, the applicant designed a chitosan oligosaccharide nanoparticle for carrying a photothermal agent for adipose tissue targeted drug delivery and anti-obesity treatment. Summary of the invention

[0006] One object of the present invention is to provide a chitosan oligosaccharide derivative modified by a long-chain fatty acid, which has a representative structural formula as follows Fig.15 .

[0007] in:

[0008] x is the percentage of sugar monocyclic rings substituted by fatty acids in chitosan oligosaccharide stearic acid micelles, ranging from 1 to 10%,

[0009] y is the percentage of unsubstituted sugar monocyclic rings in chitosan oligosaccharide stearic acid micelles, ranging from 75% to 90%

[0010] z is the percentage of undeacetylated sugar monocyclic rings in chitosan oligosaccharide stearic acid micelles, ranging from 1 to 15%

[0011] The second object of the present invention is to provide a method for synthesizing a fatty acid-modified chitosan oligosaccharide derivative, which is achieved by the following methods:

[0012] (4) dissolving chitosan oligosaccharide in water, and adding carbodiimide hydrochloride thereto for activation for 10 minutes to obtain solution A;

[0013] (5) Weighing a long-chain fatty acid and dissolving it in an organic solvent as solution B;

[0014] (6) Slowly add solution B dropwise to solution A, mix well, react at 90° C. for 24 hours, dialyze, remove impurities, and freeze-dry to obtain a fatty acid-modified chitosan oligosaccharide derivative.

[0015] The third object of the present invention is to provide a targeted drug delivery system of chitosan oligosaccharide cationic nanoparticles and photothermal agents and its application in regulating adipocytes and anti-obesity treatment. The drug delivery system is prepared by the following method:

[0016] (1) dissolving a fatty acid-modified chitosan oligosaccharide derivative in water, and stirring or ultrasonicating the resulting chitosan oligosaccharide derivative to self-assemble into nano-micelles;

[0017] (2) The photothermal agent is dissolved in water and then co-loaded with the nanomicelles in a microneedle or a hydrogel matrix material to obtain a targeted drug delivery system of chitosan oligosaccharide cationic nanoparticles and the photothermal agent.

[0018] One of the preferred embodiments of the present invention is to select indocyanine green as the photothermal agent and use microneedles as the drug delivery method. The results show that the targeted drug delivery system has a significant effect of inhibiting lipid accumulation and inducing fat browning.

[0019] The benefits of the present invention are: achieving targeted delivery and local effects of drugs through microneedles or gel carriers, reducing systemic toxic side effects; being able to accurately regulate local fat browning, reduce lipid droplet accumulation, and treat obesity and improve metabolic syndrome in a gentle and effective manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : H NMR spectra of chitosan oligosaccharide derivatives modified with stearic acid;

[0021] Figure 2 :Transmission electron microscopy image of chitosan oligosaccharide micelles modified with stearic acid;

[0022] Figure 3 :Cytocompatibility of stearic acid modified chitosan oligosaccharide micelles in 3T3-L1 cell line;

[0023] Figure 4 :Effect of chitosan oligosaccharide-stearic acid micelles on inhibiting lipid accumulation in 3T3-L1 adipocyte model;

[0024] Figure 5 :Effects of chitosan oligosaccharide-stearic acid micelles on the levels of autophagy marker proteins in 3T3-L1 adipocytes;

[0025] Figure 6 :Investigation on the lipid droplet degradation effect after the combination of chitosan oligosaccharide-stearic acid micelles and autophagy inhibitors;

[0026] Figure 7 :Investigation on the lipid droplet degradation effect after chitosan oligosaccharide-stearic acid micelles combined with photothermal therapy;

[0027] Figure 8 :Effects of chitosan oligosaccharide-stearic acid micelles combined with photothermal therapy on browning and autophagy marker proteins;

[0028] Fig. 9 : Appearance photo of composite microneedles co-loaded with COA micelles and ICG;

[0029] Fig.10 : SEM images of composite microneedles;

[0030] Fig.11 : Body shape changes of obese mice after composite microneedle administration;

[0031] Fig.12 : Changes in adipose tissue morphology of obese mice after composite microneedle administration;

[0032] Fig.13 : H&E staining of adipose tissue of obese mice after composite microneedle administration;

[0033] Fig.14 : In vitro release profile of tripterygium wilfordii oligosaccharide-stearic acid micelles;

[0034] Fig.15 : A chitosan oligosaccharide derivative modified with a long-chain fatty acid, which has a representative general structural formula. DETAILED DESCRIPTION

[0035] The present invention is further described with reference to the accompanying drawings and embodiments.

[0036] Example 1: Synthesis and characterization of stearic acid modified chitosan oligosaccharide derivatives

[0037] (1) Weigh 1g of chitosan oligosaccharide (relative molecular weight 2.5kDa) (hereinafter referred to as COS) and place it in a 250mL round-bottom flask, add 50mL of deionized water and stir to dissolve for use. According to the molar ratio of stearic acid to COS (in units) of 0.2:1, weigh stearic acid and dissolve it in 50mL of hot anhydrous ethanol, stir and dissolve. After the stearic acid is completely dissolved, slowly add it to the COS solution under stirring at 90℃ oil bath and mix well. Then, according to the molar ratio of carbodiimide hydrochloride to COS (in units) of 2:1, add carbodiimide hydrochloride to the above mixture, and stir and react at 90℃ for 12h. After the reaction is completed, add the reaction mixture into a dialysis bag (MWCO 1000Da) and place it in 5000mL of 10% ethanol for 2 days at room temperature, change the dialysate twice a day, and freeze-dry. Next, the freeze-dried product was dispersed with anhydrous ethanol, vortexed for 2 minutes, and then centrifuged at 4000 rpm for 5 minutes at room temperature, and the supernatant was discarded. This was repeated 3 times to fully remove the unreacted stearic acid. The precipitate was kept in a fume hood overnight to evaporate the ethanol, and 50 mL of deionized water was added to re-dissolve the precipitate. The lyophilized product was freeze-dried again to obtain a brown fluffy powder, which was stearic acid-modified chitosan oligosaccharide (i.e., COS-SA, hereinafter referred to as COA). A portion of the product was weighed and analyzed by nuclear magnetic resonance spectroscopy ( 1 H-NMR) confirmed the structure (see Figure 1 ), the characteristic peak of methylene on stearic acid appeared at δ = 1.2 ppm, indicating that stearic acid was successfully modified on COS, and the calculated modification degree was about 1.08%.

[0038] (2) Pyrene fluorescence probe method for measuring critical micelle concentration

[0039] Weigh 10.1 mg of pyrene and dissolve it in THF. Dilute it to a concentration of 5 × 10 -6 M pyrene tetrahydrofuran solution. Take 0.8 mL of pyrene solution and place it in multiple 10 mL centrifuge tubes. Volatilize the tetrahydrofuran in a 37°C water bath. Then take the prepared COA and dilute it to 9.8×10 -4 ~1mg / mL of a series of aqueous solutions, 8mL of each was added to the centrifuge tube containing pyrene, 200W water bath ultrasound for 30min to promote dissolution, and shaken at 37℃ in the dark overnight. Take 3mL of solution from each tube, and measure the fluorescence intensity of pyrene with a fluorescence spectrophotometer, set the excitation wavelength to 335nm, scan the emission wavelength range to 350~450nm, and the slit widths of emission and excitation are both 5nm. The fluorescence intensity I1 is measured at 372nm, and the fluorescence intensity I3 is measured at 392nm. LogC (mg / mL) is the horizontal axis, I1 / I3 is the vertical axis, and the curve fitting is performed according to the Boltzmann formula using Origin8.0 software, and the CMC of the system is calculated to be 7.47μg / mL.

[0040] Example 2: Preparation and characterization of chitosan oligosaccharide stearic acid micelles

[0041] (1) Preparation of chitosan oligosaccharide stearic acid micelles: 10.0 mg of COA dry powder was weighed and dissolved in 10 mL of deionized water. After high-speed stirring at room temperature for 3 h, the COA micelle solution was obtained by probe ultrasonication at a power of 200 W for 8 min (5 s on, 5 s off).

[0042] (2) Particle size and potential: The particle size, polydispersity index (PDI) and potential were characterized by dynamic light scattering (DLS). The results are shown in Table 1.

[0043] Table 1: Particle size, PDI and potential of COA

[0044] sample Particle size (nm) PDI Potential (mV) COA 99.1±0.54 0.262±0.021 33.6±1.90

[0045] (3) Transmission electron microscopy (TEM): TEM images of COA micelles are shown in Figure 2 , COA micelles were spherical and exhibited a complete nanostructure.

[0046] Example 3: In vitro cytological study of COA micelles

[0047] (1) Cell compatibility test of COA micelles: according to 5×10 3 3T3-L1 cells were inoculated into 96-well culture plates at a density of cells / well. After the cells adhered to the wall, the culture medium was discarded and serum-free and antibiotic-free culture medium containing different concentrations of COA micelles was added. At the same time, a negative control group and a blank control group were set up, and 6 replicates were set up in each group. Among them, the negative control group was not inoculated with cells to correct the background, and the blank control group was blank cells that were not treated with drugs. After 24 hours of drug action, the culture medium was carefully aspirated, and 100 μL of blank culture medium containing 10% (v / v) CCK-8 reagent was added. After incubation in a 37°C cell culture incubator for 1 hour, the absorbance was measured at 450nm, and the relative cell viability was calculated according to the following formula.

[0048] Cell viability (%) = (OD x -OD b ) / (OD c -OD b )×100%

[0049] OD in the formula x is the absorbance of the experimental group, OD b is the absorbance of the negative control group, OD c is the absorbance of the blank control group. Figure 3, COA micelles showed no cytotoxicity below 500 μg / mL and had good cytocompatibility with 3T3-L1 cells at subsequent experimental concentrations.

[0050] (2) Effect of COA micelles on reducing intracellular lipid accumulation

[0051] 3T3-L1 cells were induced into adipocyte models, and adipocytes were incubated with culture medium containing different concentrations (125-500 μg / mL) of free COS or COA micelles for 48 hours. 3T3-L1 adipocytes without drug administration were used as controls. The results of Oil Red O staining are shown in Figure 4 Compared with the control group, free COS and COA micelles treated with concentrations above 250 μg / mL reduced the lipid content in 3T3-L1 adipocytes. The quantitative results showed statistical differences and concentration dependence. COA micelles at the same concentration had better efficacy in degrading lipid droplets. The WB image of the autophagy marker protein LC3 is shown in Figure 5 The results showed that COA micelle treatment could increase the expression of LC3-Ⅱ in cells and improve the level of autophagy in cells. The results of Oil Red O staining after incubation of COA micelles with autophagy inhibitors chloroquine (CQ, 10 μM) or bafilomycin A1 (Baf A1, 200 nM) are shown in Figure 6 The results showed that the lipid droplet degradation process caused by COA micelles was reversed after the addition of autophagy inhibitors, suggesting that the lipid droplet degradation caused by COA micelles may be mediated by autophagy.

[0052] (3) Effect of COA micelles combined with photothermal therapy (PTT) on inhibiting intracellular lipid accumulation

[0053] 3T3-L1 cells were induced into adipocyte models, and adipocytes were incubated with culture medium containing 250 μg / mL COA micelles and 20 μg / mL indocyanine green (ICG) for 48 hours. After administration, the cells were irradiated with an 808 nm near-infrared laser for photothermal therapy (1.45 w / cm 2 , 5min).

[0054] The results of Oil Red O staining of adipocytes after COA micelle and photothermal treatment are shown in Figure 7 The results showed that both treatments could effectively reduce the lipid content in adipocytes. Compared with the single treatment group, the combination group showed better therapeutic effect, and the difference was statistically significant. The expression of intracellular proteins was investigated by WB (see Figure 8), it can be seen that COA micelle incubation for 12h can greatly increase the expression of LC3-Ⅱ in cells, while photothermal therapy can also slightly increase the expression of LC3-Ⅱ, and both can induce an increase in intracellular autophagy levels. Compared with COA micelles, the efficacy of photothermal therapy is mainly reflected in the ability to more effectively increase the expression of intracellular UCP-1 and reduce lipid accumulation by inducing browning of adipocytes. When the two are used in combination, they show the best effect of increasing the level of autophagy and inducing browning.

[0055] Example 4: Preparation of microneedles co-loaded with COA micelles and ICG

[0056] Methacrylated hyaluronic acid (HA-MA) and photoinitiator (LAP) were dissolved in ultrapure water as matrix materials, then mixed with COA micelles and ICG at 30°C and added to the microneedle mold. After drying and photocrosslinking, 30% PVP ethanol solution was added as the backing layer, and after drying, microneedles co-loaded with COA micelles and ICG were prepared.

[0057] Appearance of microneedles (see Fig. 9 ) and SEM images (see Fig.10 ) showed that the needle tip was in the shape of a quadrangular pyramid with a height of about 1 mm, clear edges and corners, and a complete structure.

[0058] Example 5: In vivo pharmacodynamic study of microneedles co-loaded with COA micelles and ICG

[0059] After HFD mice were obtained by feeding mice with a high-fat diet for 10 weeks, mice fed with a normal diet (CD) were used as negative controls, and the blank microneedle group was used as a blank control. The COA-loaded microneedle group, ICG-loaded microneedle group, and COA- and ICG-loaded microneedle group were treated respectively. The 808nm near-infrared laser was used for irradiation after each administration, and the temperature of the hyperthermia was controlled at 45°C for 5 minutes each time. The administration was carried out in a cycle of 3 days for a total of 4 weeks. The appearance of the mice was photographed after the experiment (see Fig.11 ) and the morphology of isolated epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) (see Fig.12 ), and H&E staining analysis of adipose tissue was performed (see Fig.13 The results showed that microneedle therapy with co-loaded COA micelles and ICG could significantly reduce the body weight and fat weight of mice, reduce the size of mouse adipocytes, and had a significant weight loss effect.

[0060] Example 6: Preparation and characterization of COA micelles loaded with tripterygium wilfordii (CLT)

[0061] (1) Preparation of COA / CLT micelles: Weigh 20.0 mg of COA dry powder and dissolve it in 20 mL of deionized water. Weigh 10 mg of CLT powder and dissolve it in 10 mL of anhydrous ethanol to prepare a 1 mg / mL stock solution. According to different mass ratios (COA:CLT = 50:1, 30:1, 20:1, 10:1), slowly add the CLT stock solution to the COA solution, stir at room temperature for 3 hours, and then ultrasonicate at a power of 200 W for 8 minutes (5s on, 5s off). Then, remove the organic solvent anhydrous ethanol by reduced pressure evaporation at 37°C to obtain the CLT-loaded micelle solution (COA / CLT), and characterize the particle size and potential.

[0062] (2) Determination of encapsulation efficiency and drug loading of drug-loaded micelles: Take 0.4 mL of COA / CLT and place it in the inner tube of a 3K ultrafiltration tube and centrifuge it at 5000 rpm / min for 1 hour. After centrifugation, add 1.2 mL of methanol to the inner tube and mix well. Then dilute the liquid 10 times with methanol, use water bath ultrasound for 5 minutes to fully break the emulsion, filter with a 0.22 μm filter membrane, and use the method described in 2.1 of this section to detect the content of drug CLT by HPLC C1. Take another 100 μL of freshly prepared COA / CLT micelles, break the emulsion with methanol, dilute and make the volume to 10 mL, filter with 0.22 μm, and measure the drug concentration by HPLC, which is the total drug concentration C0 in the micelles (including the drug encapsulated in the micelles and the drug free outside the micelles). Calculate the encapsulation efficiency and drug loading according to the following formula:

[0063] Encapsulation efficiency (EE%) = (C1 / C0) × 100%

[0064] Drug loading (DL%) = (drug loading / total micelle mass) × 100%

[0065] Table 2: Particle size, PDI and potential of COA at different dosing ratios

[0066]

[0067] COA / CLT 10 Compared with micelles with other dosing ratios, micelles have the worst transmittance, are slightly turbid, and have an encapsulation rate of only about 42%. There are more free drugs that are not encapsulated in micelles. With the increase of drug loading ratio and the decrease of CLT feed amount, the encapsulation rate and drug loading gradually increase. In addition, there is no significant difference in the potential of blank micelles and drug-loaded micelles, both of which are around 33-34mV, indicating that the encapsulation of CLT has no effect on the potential of the micelles themselves. Taking into account the above physicochemical properties and subsequent dosage, we finally preferred COA / CLT with an encapsulation rate of more than 95%. 50 Conduct follow-up research.

[0068] (3) Study on the in vitro release behavior of COA / CLT micelles: Prepare several dialysis bags (MWCO=1000Da) with a length of about 5 cm in advance, tie one end with a ligature, add 1 mL of free CLT solution or COA / CLT solution into the dialysis bag, and quickly tie the other end with a ligature. Then place the dialysis bag in a 50 mL round-bottom flask, put it in a 37°C constant temperature shaker, set the speed to 100 rpm / min, and immediately add 30 mL of PBS solution containing 0.2% Tween 80 as the release medium into the flask, and start timing. Then, take 1 mL of dialysate from the flask for testing at 1h, 2h, 4h, 8h, 16h, 24h, 48h, 72h, 96h, and 120h, and add 1 mL of fresh release medium. Use high performance liquid chromatography to determine the content of CLT in the release medium, and calculate the cumulative release rate. The results are as follows: Fig.14 As shown in the figure, the free CLT solution has a cumulative release rate of about 50% in 1 hour, and more than 80% of the drugs are basically released in 12 hours. In contrast, the release behavior of the drug-loaded micelles is slower, with a cumulative release rate of nearly 50% at 48 hours, and a cumulative release rate of about 80% at the last time point of 120 hours. COA micelles can delay the release of the drugs contained therein, and have certain sustained-release characteristics.

Claims

1. A cationic nanomicelle, characterized in that: Cationic nanomicelles formed by self-assembly of amphiphilic chitosan oligosaccharide derivatives.

2. The cationic nano micelle according to claim 1, characterized in that By covalently linking the hydrophobic groups to the amino groups on the chitosan oligosaccharide skeleton, the chitosan oligosaccharide has positive charge and self-assembly properties and can target adipose tissue and adipocytes. The particle size is 50 to 200 nm and the zeta potential is 20 to 40 mV.

3. The hydrophobic group as claimed in claim 2 is selected from long-chain fatty acids such as palmitic acid, stearic acid, arachidic acid, etc. The molecular weight of the chitosan oligosaccharide material is 500-3200 Da, and the modification degree of the fatty acid-modified chitosan oligosaccharide is 1-50%.

4. The chitosan oligosaccharide derivative according to claim 2 is prepared by the following method: (1) Dissolve chitosan oligosaccharide in water, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide thereto, and activate for 10 minutes to obtain solution A; (2) Weighing a long-chain fatty acid and dissolving it in an organic solvent as solution B; (3) Slowly add solution B to solution A, mix well, react at 90° C. for 24 hours, dialyze, remove impurities, and freeze-dry to obtain a fatty acid-modified chitosan oligosaccharide derivative.

5. A combination of cationic nanoparticles and photothermal therapy for targeted regulation of adipose tissue and lipid droplet degradation, characterized in that: It comprises an amphiphilic chitosan oligosaccharide derivative carrier and a photothermal agent.

6. The photothermal agent as claimed in claim 5 is selected from at least one or more of indocyanine green (ICG), heptamethine cyanine (IR780), etc.

7. The drug delivery system according to claim 1 is prepared according to the following method: (1) dissolving a fatty acid-modified chitosan oligosaccharide derivative in water, and stirring or ultrasonicating the chitosan oligosaccharide derivative to form nanomicelles by self-assembly; (2) The hydrophobic small molecule drug is encapsulated in the nanomicelles and then co-loaded in the microneedles or hydrogel matrix materials as needed to obtain a targeted drug delivery system of chitosan oligosaccharide cationic nanoparticles and photothermal agents.

8. The micelle as described in claim 7 can encapsulate one of the photothermal agents, browning agents or anti-inflammatory drugs, etc., to achieve therapeutic effects such as local hyperthermia targeting fat cells, inducing fat browning, and local anti-inflammatory.

9. The administration method of the drug delivery system according to claim 7, characterized in that Nanoparticles and photothermal agents can be delivered to the vicinity of subcutaneous fat tissue using thermosensitive gel or microneedles as carriers, and photothermal therapy can be achieved through 808nm wavelength near-infrared light irradiation.

10. Use of the drug delivery system according to claim 7, characterized in that Targeted regulation of adipose tissue browning and reduction of lipid accumulation, inhibiting obesity and related metabolic complications.

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