Biological nano composite hydrogel as well as preparation method and application thereof

Abstract

The invention provides biological nano composite hydrogel as well as a preparation method and application thereof, and the preparation method of the biological nano composite hydrogel comprises the following steps: dissolving VVPS and beta-CD in a NaOH aqueous solution, heating to 60-63 DEG C, and stirring for more than 30 minutes until the VVPS and the beta-CD are completely dissolved; slowly adding PEGDGE into the solution, continuously stirring until the cross-linking agent is dissolved, raising the temperature to 65 DEG C or above, and reacting for 4 hours or above to form hydrogel; and performing swelling treatment on the hydrogel, drying, soaking in an ethanol water solution containing capsaicin for more than 48 hours, filtering, and performing freeze drying. The biological nano composite hydrogel prepared by the preparation method disclosed by the invention has good application in delivery of capsaicin and alleviation of type II diabetes mellitus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of hydrogel preparation, in particular, to a biological nanocomposite hydrogel and a preparation method and application thereof. BACKGROUND

[0002] In recent years, the incidence of type 2 diabetes mellitus (T2DM) continues to rise globally, and has become one of the important chronic diseases affecting public health and national economy. More and more studies have shown that improving lipid metabolism, regulating inflammatory response and enhancing antioxidant defense are important strategies to alleviate T2DM. However, the low bioavailability of traditional drugs in vivo and the difficulty in achieving sustained release greatly limit their clinical application. Therefore, researchers have recently begun to explore the use of biomaterials to construct new drug delivery strategies, aiming to enhance drug stability, improve drug solubility and achieve targeted controlled release.

[0003] Capsaicin (CAP) as a naturally occurring active ingredient in chili peppers has attracted much attention due to its multiple biological effects in regulating energy metabolism, anti-inflammatory and antioxidant. Studies have shown that capsaicin can improve insulin sensitivity by activating specific signaling pathways, thereby regulating blood glucose levels. At the same time, the antioxidant and anti-inflammatory effects of CAP also help to alleviate chronic inflammation associated with T2DM. However, due to the hydrophobicity and irritancy of CAP, its bioavailability in vivo is relatively low, which limits its clinical translation and application.

[0004] In recent years, several reports have explored the use of various raw materials to construct composite nanomaterials, aiming to achieve efficient drug delivery and multifunctional therapy. Biocomposite nanohydrogels prepared by physical or chemical crosslinking methods have been proven to have significant advantages in drug release, biosensing and tissue engineering, etc. These nanocomposite hydrogels not only can encapsulate hydrophobic drugs in the molecular cavity of β-cyclodextrin, but also can form a three-dimensional network structure using natural polysaccharides, enhancing the overall biocompatibility and biodegradability of the system. In addition, the composite hydrogel system can achieve sustained drug release and targeting effect in vivo, further reducing the frequency of medication and side effects. However, the bioavailability of hydrogel CAP in the prior art is still not ideal.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The first object of the present application is to provide a biological nanocomposite hydrogel and a preparation method thereof, which is used for efficiently delivering capsaicin (CAP) and has a good relieving effect on T2DM. The results show that CAP is successfully encapsulated in the hydrophobic cavity of the hydrogel nanocomposite system HGNCs to form core-shell nanoparticles (average particle size 145.73±22.81 nm), and the encapsulation rate and drug loading capacity are 63.27% and 13.22%, respectively. In vivo pharmacodynamic studies show that the hydrogel nanocomposite system HGNCs / CAP system carrying capsaicin prolongs the peak time of CAP to 60 minutes through the slow-release effect, and significantly improves the bioavailability of CAP to 11.79%, which is three times that of free CAP. It can be seen that the bioavailability of CAP is significantly improved.

[0007] The second object of the present application is to provide the application of the above-mentioned biological nanocomposite hydrogel, which has a good application in the delivery of capsaicin and the preparation of the relief of type II diabetes.

[0008] In order to achieve the above-mentioned object of the present application, the following technical solutions are adopted:

[0009] The present application provides a preparation method of a biological nanocomposite hydrogel, comprising the following steps:

[0010] Dissolve VVPS and β-CD in NaOH aqueous solution, heat and stir at 60-63℃ for more than 30min until completely dissolved;

[0011] Slowly add PEGDGE to the above-mentioned solution and continue to stir until the crosslinking agent is dissolved, then increase the temperature to more than 65℃, and react for more than 4h to form a hydrogel;

[0012] Swelling treatment and drying of the hydrogel, soaking in an aqueous solution containing capsaicin for more than 48h, filtering and freeze-drying.

[0013] Preferably, as a further implementable scheme, the mass ratio of VVPS to β-CD is 4:(0-5), more preferably, the mass ratio of VVPS to β-CD is 4:(3-4), and most preferably, the mass ratio of VVPS to β-CD is 1:1. The best ratio of the two is optimized through subsequent experiments.

[0014] The present application shows good performance by the synergistic effect of β-cyclodextrin (β-CD) and Volvariella volvacea soluble polysaccharide (VVPS). As a cyclic oligosaccharide molecule containing seven glucose units, β-CD is connected by α-1, 4 glycosidic bond. Due to its unique molecular structure, it has the characteristics of internal hydrophobicity and external hydrophilicity, which can form inclusion compounds with different hydrophobic molecules, thereby significantly improving the water solubility and stability of drugs. In addition, the biological safety of β-CD and its derivatives has been fully recognized by toxicological evaluation. Based on the above characteristics, β-CD is not only practical but also harmless in drug delivery systems. In addition, VVPS is highly valued due to its structure and significant biological activity. These polysaccharides not only have antioxidant, immunomodulatory and lipid metabolism improving functions, but also can play a prebiotic role by regulating intestinal flora, thereby improving metabolic diseases. Molecular structure and functional analysis show that the rich β-glucan structure in VVPS is the key to its antioxidant and immunomodulatory functions. However, VVPS and β-cyclodextrin each have their own shortcomings. VVPS has low viscosity and poor mechanical properties. On the other hand, the water solubility of β-cyclodextrin is limited, resulting in the precipitation of solid β-cyclodextrin compounds in aqueous environments. In addition, it cannot be hydrolyzed by human saliva and pancreatic amylase. Therefore, encapsulating drugs with β-cyclodextrin inner core and using VVPS shell to endow the delivery system with hydrophilicity form a core-shell structure with hydrophilicity and good mechanical properties, effectively making up for their respective shortcomings. This core-shell structure swells less in the acidic environment of the stomach, protecting the drug from gastric acid damage, while fully swelling in the alkaline environment of the intestine, thereby accelerating drug release. This ensures better bioavailability of the drug in the human body.

[0015] The present application first proposes to construct a biological nanocomposite hydrogel based on β-CD and VVPS. By constructing a core-shell structure to load CAP, its efficient delivery is realized, in order to improve its in vivo bioavailability. This system is expected to play an important therapeutic role in regulating glycolipid metabolism, reducing oxidative stress and inhibiting inflammatory response through various synergistic effects, thereby exhibiting unique advantages in improving T2DM. This invention is expected to create a new method for integrating natural ingredients and traditional materials science to treat diabetes in the future, and provide reference and guidance for other diseases based on natural ingredient drugs.

[0016] Preferably, as a further implementable solution, the concentration of the aqueous NaOH solution is 1-2 mol / L.

[0017] Preferably, as a further implementable solution, the time for slowly adding PEGDGE into the above solution while continuing to stir is 15 min or more.

[0018] Preferably, as a further implementable solution, the swelling treatment method comprises immersing the hydrogel in deionized water, and changing the water every 6-8 h until all unreacted substances are completely removed.

[0019] Preferably, as a further implementable scheme, the aqueous ethanol solution containing capsaicin is an aqueous ethanol solution containing 1-2 mg / ml capsaicin, and the mass ratio of ethanol to water is 1:1.

[0020] The application also provides a biological nanocomposite hydrogel prepared by the above preparation method, which has high bioavailability after loading capsaicin.

[0021] In summary, the above biological nanocomposite hydrogel has good application in the delivery of capsaicin and the relief of type II diabetes. BRIEF DESCRIPTION OF DRAWINGS

[0022] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included to provide a description of the preferred embodiments and are not intended to limit the scope of the application. Moreover, like reference numerals designate like parts throughout the several views.

[0023] Figure 1 FTIR spectra of hydrogel nanocomposites (HGNCs) with different β-cyclodextrin (β-CD) contents (A) and HGNCs / capsaicin (CAP) complexes (B) in the range of 4000-500 cm -1 ;

[0024] Figure 2 TEM and AFM characterization of HGNCs / CAP composite carriers; (A) TEM image of HGNCs / CAP in the form of spherical nanoparticles (SNPs) (scale bar 1 μm); (B) TEM image of HGNCs / CAP in the form of SNPs (scale bar 100 nm); (C) particle size distribution of HGNCs / CAP; (D) AFM two-dimensional image of HGNCs / CAP (white patches); (E) AFM three-dimensional image of HGNCs / CAP (white patches);

[0025] Figure 3 In vitro inclusion and dynamic release of CAP by HGNCs; (A) encapsulation efficiency (EE%) and drug loading (LC%) of HGNCs (VVPS / β-CD) samples; different lowercase letters represent significant differences in LC, and different uppercase letters represent significant differences in EE (P < 0.05); (B) in vitro release kinetics curve of CAP from HGNCs in PBS;

[0026] Figure 4Bioavailability and distribution of free CAP and HGNCs / CAP; (A) Serum pharmacokinetic curve of CAP after intragastrical administration of different treatments; (B) CAP content in different parts of the gastrointestinal tract 24 hours after treatment with different formulations, the color block indicates the increase of CAP content (%) from white to dark green; * indicates P < 0.05, ** indicates P < 0.01, and NS indicates no significant difference;

[0027] Figure 5 Dynamic changes of body weight and blood glucose of rats after intragastrical administration of HGNCs / CAP for 6 weeks; (A) Changes in body weight of rats in each group during the intervention period; (B) Fasting blood glucose (FBG) of rats in each group; different lowercase letters indicate significant differences between groups (P < 0.05); (C) Oral glucose tolerance test (OGTT) curve and area under the curve (AUC) of rats in each group; the color block indicates the difference in AUC from dark green to light green; *** indicates P < 0.001, **** indicates P < 0.0001;

[0028] Figure 6 Insulin levels and insulin resistance of rats after intragastrical administration of HGNCs / CAP for 6 weeks; (A) Serum insulin levels of rats in each group; (B) HOMA-IR index of rats in each group; (C) Insulin tolerance test (ITT) curve and AUC of rats in each group, the color block indicates the difference in AUC from dark green to light green; *** indicates P < 0.001, **** indicates P < 0.0001;

[0029] Figure 7 Histopathological section images of kidneys and pancreases of different groups of diabetic rats, (A) Kidney (H&E staining, scale bar 100 μm); (B) Pancreas (H&E staining, scale bar 100 μm);

[0030] Figure 8 Immunofluorescence images of pancreases of different groups of diabetic rats; red is islet beta cells, green is alpha cells, and blue is cell nuclei; images under different light sources represent different immunohistochemical results, scale bar = 100 μm;

[0031] Figure 9 Preparation process of the biological nanocomposite hydrogel of the application and subsequent application flowchart. DETAILED DESCRIPTION

[0032] The embodiments of the application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the application and should not be regarded as limiting the scope of the application. If no specific conditions are specified in the examples, they are carried out under conventional conditions or according to the manufacturer's recommendations. If no manufacturer of the reagents or instruments is specified, they are all conventional products that can be purchased on the market.

[0033] The Volvariella volvacea soluble polysaccharide (VVPS) (purity > 90%, protein 0.8%, sugar content 98.2%, ->4)-Glcp-(1-> is the main backbone of VVPS polysaccharide chain) and capsaicin (CAP) (purity > 95%) in the following examples were purchased from Lanzhou Rice Biotechnology Co., Ltd. The β-cyclodextrin (β-CD) (> 97%) and polyethylene glycol diglycidyl ether (PEGDGE) were purchased from Macklin Reagent Network.

[0034] Example 1

[0035] Four bio-nano composite hydrogels with different raw material mass were prepared by dissolving 4 g of VVPS and four different masses of 0, 3, 4 or 5 g of β-CD in 10 ml of 1.5 mol / L NaOH aqueous solution, heated to 60°C, and magnetically stirred for 30 min until completely dissolved. Next, 4 g (8 mmol) of PEGDGE was slowly added to the solution at 60°C, and stirring was continued for 15 min to ensure the dissolution of the crosslinking agent. Then, the temperature was raised to 65°C, and the reaction was carried out for 4 h to form the hydrogel. The hydrogel was immersed in deionized water, and the water was changed every 6 h until all unreacted substances were completely removed. Finally, the swollen hydrogel was freeze-dried and stored in a desiccator for later use. To load CAP, the freeze-dried hydrogel (1 g) was weighed and soaked in 20 ml of an aqueous ethanol solution containing 1.5 mg / ml of capsaicin (ethanol: water = 1: 1). After soaking for 48 h, the excess solution was removed by filtration. Finally, the freeze-dried hydrogel was stored in a desiccator for later use.

[0036] Example 2

[0037] A bio-nano composite hydrogel was prepared by dissolving 4 g of VVPS and 4 g of β-CD in 10 ml of 2 mol / L NaOH aqueous solution, heated to 63°C, and magnetically stirred for 35 min until completely dissolved. Next, 4 g (8 mmol) of PEGDGE was slowly added to the solution at 63°C, and stirring was continued for 15 min to ensure the dissolution of the crosslinking agent. Then, the temperature was raised to 66°C, and the reaction was carried out for 4.5 h to form the hydrogel. The hydrogel was immersed in deionized water, and the water was changed every 7 h until all unreacted substances were completely removed. Finally, the swollen hydrogel was freeze-dried and stored in a desiccator for later use. To load CAP, the freeze-dried hydrogel (1 g) was weighed and soaked in 20 ml of an aqueous ethanol solution containing 2 mg / ml of capsaicin (ethanol: water = 1: 1). After soaking for 48 h, the excess solution was removed by filtration. Finally, the freeze-dried hydrogel was stored in a desiccator for later use.

[0038] Example 3

[0039] Bio-nano composite hydrogels were prepared by dissolving 4 g of VVPS and 4 g of β-CD in 10 ml of 1 mol / L NaOH aqueous solution, heated to 62 °C, and magnetically stirred for 32 min until completely dissolved. Next, 4 g (8 mmol) of PEGDGE was slowly added to the solution at 62 °C, and stirring was continued for 15 min to ensure the dissolution of the crosslinking agent. Then, the temperature was raised to 67 °C, and the reaction was allowed to proceed for 4 h to form the hydrogel. The hydrogel was immersed in deionized water, and the water was changed every 8 h until all unreacted materials were completely removed. Finally, the swollen hydrogel was freeze-dried and stored in a desiccator for later use. To load CAP, the freeze-dried hydrogel (1 g) was weighed and immersed in 20 ml of an aqueous ethanol solution containing 1 mg / ml of capsaicin (ethanol:water = 1:1). After 48 h of immersion, the excess solution was removed by filtration. Finally, the freeze-dried hydrogel was stored in a desiccator for later use.

[0040] Comparative Example 1

[0041] Self-assembled nanotube loaded capsaicin in the document “Intestinal-targeted nanotubes-in-microgels composite carriers for capsaicin delivery and their effect for alleviation of Salmonella induced enteritis”.

[0042] Experimental Example 1

[0043] FT-IR spectroscopy was performed on the four bio-nano composite hydrogels prepared in Example 1:

[0044] The infrared spectrum of the sample was measured using a Nicolet 470 Fourier Transform Infrared Spectrometer (FTIR) (Thermo Fisher Scientific, USA). The sample was prepared by the press pellet method, and the analysis was performed using a potassium bromide (KBr) background. The scanning speed was 21 min / scan, and the spectral range was 4000 to 400 cm -1 with a resolution of 2 cm -1 . The baseline-corrected spectrum was processed using Omnic software.

[0045] Figure 1 The infrared spectra of the freeze-dried HGNCs containing different amounts of β-CD are shown. As the concentration of β-CD increased, specific positions in the spectrum (3410 cm -1 , 2920 cm -1 , 1630 cm -1 , 1150 cm -1 , and 1030 cm -1) also increased Figure 1 A) at 1150 cm -1 Figure 1 B) distinguished the two HGNC treatments from β-CD and VVPS, the latter of which did not show such a peak. Confirmation of this phenomenon indicated that HGNCs were the result of bio-macromolecular chemical crosslinking.

[0046] As shown in Figure 1 B, CAP had distinct characteristic peaks at 3380, 3190, 2770, 1660 and 1470 cm -1 The composition of CAP included aromatic rings, polar groups (hydroxyl, amide and carbonyl) and hydrophobic groups. The weakening of the absorption peak intensity at 1660 and 1470 cm -1 for the HGNCs (1 : 1) / CAP sample, possibly due to the partial entry of the N-H bond of the benzene ring into the β-CD cavity through hydrophobic interaction without destroying the structure of the cyclodextrin, indicating that CAP had been successfully loaded into HGNCs (1 : 1) / CAP.

[0047] Similar Figure 1 results were also obtained for FT-IR spectroscopy measurements of the 3-nanocomposite hydrogels.

[0048] Experimental Example 2

[0049] TEM and AFM analysis was performed on the bio-nanocomposite hydrogel prepared from 4g VVPS, 4g β-CD (mass ratio 1 : 1) of Example 1:

[0050] Transmission electron microscopy (TEM) measurement sample preparation: 10 μL of 0.5 mg / mL hydrogel dispersion was dropped on a carbon-coated copper grid and dried at room temperature for 24 hours. The samples were examined using a Hitachi-7650 transmission electron microscope (Hitachi, Japan) at 110 kV.

[0051] Atomic force microscopy (AFM) (Bruker Dimension Icon Nanoscope V, Germany) was used to study the two-dimensional (2D) and three-dimensional (3D) morphology of HGNCs. About 10 μL of diluted freeze-dried HGNCs aqueous solution (0.5 mg / mL) was dropped on a mica sheet and dried at 25 °C for 12 hours. AFM images of the dried sample were acquired in tapping mode at a scan rate of 0.5 Hz.

[0052] TEM and AFM were used to study the morphology and size distribution of HGNCs. In the TEM image, 361 spherical nanoparticles (SNPs) were observed, which were uniformly dispersed in the HGCNs Figure 2 ​A). The morphology of SNPs was the same as the previous study by Zou et al., i.e. CAP particles loaded into the core of the micelles had uniform spherical nanosize. CAP SNPs size ranged from 59.31 nm to 194.71 nm, with an average size of 145.73 ± 22.81 nm, and the polydispersity index (PDI) obtained by Gaussian fitting analysis was 21.61% (Fig. 2A). Figure 2 C). SNPs in HGNCs / CAP formed a three-layered core-shell structure, indicating that CAP was encapsulated in the β-CD sites within the WPS shell core Figure 2 B). AFM images confirmed that SNPs were uniformly dispersed in HGNCs as spherical nanoparticles with a spherical morphology Figure 2 D). The surface of SNPs was observed to be sharp and rough with no distinct features Figure 2 E). It can be inferred that SNPs exhibited good dispersibility due to their negatively charged surface, which originated from galacturonic acid. The electronegative surface led to electrostatic repulsion between SNPs. This further demonstrated that WPS is the outer shell.

[0053] Experimental Example 3

[0054] In vitro encapsulation and dynamic release of CAP by the four bio-nanocomposite hydrogels prepared in Example 1:

[0055] The ability of HGNCs to encapsulate CAP was evaluated by the swelling method as follows: each hydrogel sample (100 mg) was immersed in a 50 mL test tube containing 25 mL of deionized water and 25% (w / w) CAP ethanol solution. After the addition of CAP, a stable white turbid dispersion (emulsion-like) was formed. The test tube was then sealed and shaken at 150 rpm for 48 hours at 25°C. Next, the hydrogel was removed from the solution and rinsed with a water / ethanol (1:1) mixture to remove the surface-adsorbed CAP. Subsequently, the hydrogel was pulverized and sonicated in absolute ethanol. Magnetic stirring was performed for more than 48 hours to ensure complete extraction of the encapsulated CAP by pure ethanol. The extract was filtered and analyzed by UV spectroscopy at 295 nm. The concentration of the CAP sample was determined by a calibration curve. The concentration of CAP added was estimated by the highest β-CD content inside the hydrogel in a theoretical conversion of 1:1 ratio of β-CD to CAP. The encapsulation efficiency (EE) and the drug loading (LC) of each hydrogel for CAP were calculated using equations (1) and (2):

[0056] Encapsulation efficiency (EE %) = (total amount of capsaicin - free capsaicin content in supernatant) / total amount of capsaicin) x 100 (1)

[0057] Drug loading (LC %) = ( (total capsaicin amount - free capsaicin content in supernatant) / total hydrogel weight) x 100 (2)

[0058] The release kinetics of CAP from HGNCs were investigated using 0.05 M phosphate buffered saline (PBS) solution at pH 7.4. To improve the solubility of uncomplexed CAP in aqueous media, Tween-20 (0.5% w / v) was included in the release solution. Dry HGNCs / CAP samples (100 mg) were incubated in the release medium at 25 °C with continuous shaking at 150 rpm for 30 min. As the hydrogels swelled and transformed from a glassy to a rubbery state, 1 mL of fresh buffer was exchanged for 1 mL of release medium at specific time points. The cumulative release of CAP was quantified by measuring the UV absorbance at 295 nm. Control and blank groups were hydrogels without CAP and release buffer, respectively.

[0059] The encapsulation capacity of HGNCs for CAP was investigated by UV-Vis spectrophotometry and the effect of the β-CD / VVPS weight ratio on the drug loading (LC) % and CAP encapsulation efficiency (EE) % was determined. All HGCNs exhibited a certain degree of EE % and LC % as CAP molecules entered the hydrogel pores and then bound to the already covalently embedded β-CD hydrophobic inner surface as the HGNCs swelled Figure 3 A). In combination with the TEM results, it can be inferred that CAP diffused and distributed in the β-CD cavities inside HGNCs, while the VVPS shell covered the molecules, which was beneficial for protecting CAP and supporting its slow release. HGNCs.VVPS / β-CD (4 / 3) exhibited the best encapsulation effect with an EE % of 63.27 and a LC % of 13.22, and its cumulative release of CAP was greater than that of HGNCs.VVPS / β-CD (4 / 4) and HGNCs.VVPS / β-CD (4 / 5) Figure 3 B). Furthermore, HGNCs.VVPS / β-CD (4 / 3) exhibited a high release rate of nearly 43% in the last 12 h, and the release rate reached nearly 65% when the release time was extended to 200 h. In contrast, when the VVPS / β-CD ratio increased to 4 / 5, only 18% of CAP was released in the first 12 h, and the release amount decreased to 32% after 200 h. At the same time, encapsulation with HGNCs resulted in a significant enhancement of the water solubility of the lipophilic CAP Figure 3 C). In this work, the model analysis and numerical values indicated that HGNCs with low β-CD content exhibited higher complexation efficiency and loading capacity, while HGNCs with high β-CD content exhibited a longer release rate.

[0060] Experimental Example 4

[0061] The corresponding animal experiment design was performed on the bio-nanocomposite hydrogel prepared from 4g VVPS, 4g β-CD (mass ratio 1:1) of Example 1:

[0062] Male ZDF rats (8 weeks old) and male Zucker Lean (ZL) rats were purchased from Charles River Laboratories, Inc. and were SPF grade. All animal experiments were approved by Chongqing Institute of Chinese Materia Medica and were performed in accordance with the Guide for the Care and Use of Laboratory Animals. ZL rats were fed with maintenance chow as the CK group, and ZDF rats were fed with Purina #5008 chow. After four weeks of feeding, ZDF rats with random blood glucose ≥11.1 mmol / L were considered to have T2DM. T2DM rats were divided into the DM group and the DM+HGNCs / CAP group. From the 12th week to the 18th week, the DM group and the CK group were intragastrically administered with normal saline (200 mg / kg body weight) once a day, and the DM+HGNCs / CAP group was intragastrically administered with HGNCs / CAP (200 mg / kg body weight) once a day. Daily food and water intake were recorded, and body weight and fasting blood glucose were measured every week.

[0063] Based on the relevant index determination methods of animal experiments, including in vivo pharmacokinetic curves, oral glucose tolerance test, and rat kidney single cell analysis, etc.

[0064] 1) Evaluation of CAP bioavailability and pharmacokinetics in rats in vivo

[0065] The use of micelles, nanoemulsions, and liposomes can enhance the water solubility and bioavailability of capsaicin and reduce the irritation to the gastrointestinal tract. From the curve trend, free CAP showed rapid absorption (rapid peak concentration) and rapid elimination (rapid concentration decline), while the curve of HGNCs (1:1) / CAP was smoother, with a delayed peak time, a longer time of maintaining a higher blood drug concentration Figure 4 A). Figure 4A shows that free CAP reached the maximum release CAP concentration of 293.6 ± 14.68 ng mL-1 at 15 minutes, while HGNCs (1:1) / CAP reached 391.7 ± 19.58 ng mL-1 at 60 minutes. This behavior is similar to previous studies, which concluded that gel carriers can fine-tune the drug release rate due to their 3D network structure and good biocompatibility. On the other hand, the area under the curve (AUC) of the two samples differs greatly, at 2528.05 ± 1016.35 ng·h mL-1 and 6794.8 ± 762.86 ng·h mL-1, respectively. The larger AUC of HGNCs (1:1) / CAP means that its bioavailability is increased to 11.79 ± 1.73 FAbs (%), which is about three times that of free CAP. After 24 hours of ingestion, CAP is mainly retained in the stomach; a small and medium portion is retained in the intestine, and the content in the colon is negligible Figure 4 B). HGNCs delay the release of CAP, reduce the destruction of gastric acid, and prolong the stability of CAP in the stomach. HGNCs improve the bioavailability of CAP through a sustained-release effect; prolong the maintenance time of blood CAP concentration, and combine with the reasonable distribution of gastrointestinal residues, ultimately achieving more efficient and safer CAP delivery.

[0066] 2) Effect of HGNCs / CAP on blood glucose and insulin levels in diabetic rats

[0067] The body weight of diabetic rats (DM group and DM+HGNCs / CAP group) was almost the same, but significantly higher than that of the CK group Figure 5 A). Compared with the CK group, the fasting blood glucose (FBG) level of the DM group was four times that of the CK group, while the FBG value of the DM+HGNCs / CAP group was significantly lower than that of the DM group, which means that CAP can significantly reduce the blood glucose level of diabetic rats Figure 5 B). In the oral glucose tolerance test (OGTT), the blood glucose levels of all treatment groups showed a trend of first rising and then falling. However, two hours after the OGTT test, the blood glucose of rats receiving HGNCs / CAP decreased much faster than that of the DM group. At the same time, the AUC value of DM+HGNCs / CAP treated rats was much smaller than that of the DM group, indicating that CAP has the effect of improving glucose tolerance and reducing blood sugar in diabetic rats Figure 5 C). These results are consistent with previous studies, which showed that dietary CAP reduced insulin, fasting blood glucose, and leptin levels in rats and significantly reduced impaired glucose tolerance.

[0068] Insulin is a protein hormone secreted by pancreatic islet beta cells and is the only hypoglycemic hormone in the body. Experimental data show that not only the insulin content of rats in the DM+HGNCs / CAP group was significantly higher than that in the DM groupFigure 6 A), and the insulin HOMA-IR index was also significantly reduced Figure 6 B). Meanwhile, after fasting oral insulin, the blood glucose of the DM+HGNCs / CAP group was significantly reduced, and the insulin resistance was much lower than that of the DM group Figure 6 C). By directly binding to TRPV1, CAP not only stimulates pancreatic beta cells to secrete insulin, but also can activate adjacent neurons and endocrine cells to further regulate insulin sensitivity. In addition to directly regulating pancreatic function, CAP can also enhance insulin action and further reduce insulin resistance by regulating downstream signaling molecules such as AMPK and AKT. Experimental data show that CAP-loaded HGNCs intervention can significantly slow down the rate of blood glucose rise and enhance glucose tolerance by improving insulin sensitivity and insulin resistance.

[0069] 3) Histopathological observation of kidney and pancreas in different groups

[0070] From the pathological sections, it can be found that the kidney and pancreas tissue structure of diabetic rats has undergone great changes. The kidney section of the CK group shows that the normal glomerulus is surrounded by the Bowman's capsule, and there is no obvious structural change Figure 7 A). But in the DM group, diffuse mesangial matrix expansion, mesangial cell proliferation, and significant glomerular basement membrane thickening in diabetic nephropathy can be seen, which is consistent with previous descriptions of diabetic nephropathy. In the DM+HGNCs / CAP group, the hypertrophy and hyperplasia of glomerular and tubular structures were significantly improved, which means that the pathological damage of kidney tissue has been repaired and alleviated to some extent by CAP. At the same time, compared with CK, the decrease in the number of beta cells in the DM group also indicates that the local pancreatic microenvironment has undergone a similar damage process as previously reported Figure 7 B). After six weeks of HGNCs / CAP treatment, the structure of islets in the pancreas section of the DM+HGNCs / CAP group tended to recover to some extent, indicating that CAP alleviated the progression of pancreatic lesions. The experimental results show various pathological lesions caused by chronic hyperglycemia in diabetes at the histological level, further revealing the significant effect of CAP in improving T2DM kidney structural damage, alleviating inflammatory response, and regulating pancreatic function.

[0071] 4) Immunohistochemical observation of pancreas

[0072] In the CK group, alpha cells and beta cells are evenly distributed in the islets, showing a ring-like pattern of alpha cells surrounding beta cells Figure 8). However, in the DM group, this circular pattern was disrupted, accompanied by a decrease in β-cell number. Notably, after six weeks of HGNCs / CAP treatment, there was an increase in β-cell area and number compared to the DM group. In addition, the circular pattern of α-cells surrounding β-cells was restored to some extent. Pancreatic cells, including α and β cells, are regulated by a variety of receptor-mediated signaling pathways. From the perspective of β cells, various agonists and modulators directly affect insulin secretion through receptor-mediated signaling activation. The results of this experiment show that CAP can repair the damage to the pancreatic structure caused by diabetes to some extent and restore the reduced number of pancreatic β cells.

[0073] In summary, the present application developed a HGNCs complex delivery system based on β-CD and VVPS for efficient delivery of CAP, and systematically evaluated its remission effect on type II diabetes. The research results show that CAP is successfully loaded into HGNCs, and the problem of low water solubility is also effectively improved. The in vivo pharmacokinetic study shows that the HGNCs / CAP system delays the peak concentration of CAP in the blood to 60 minutes through the slow-release effect, and significantly increases the bioavailability to 11.79%, which is three times that of free CAP. The bioavailability of the self-assembled nanotube loaded with capsaicin in Comparative Example 1 is only 5.69% compared with the present application. In the diabetes rat animal experiment, HGNCs / CAP effectively reduces fasting blood glucose and insulin resistance, enhances oral glucose tolerance, and promotes insulin secretion. In addition, HGNCs / CAP significantly improves dyslipidemia (TG and LDL-C are reduced by 30%-35%), reduces BUN and UA levels, and repairs the thickening of the glomerular basement membrane and the reduction of the number of pancreatic β cells caused by diabetes. Single-cell transcriptome analysis further reveals that CAP inhibits the fibrosis process and relieves the inflammatory microenvironment by regulating the fatty acid metabolism, mitochondrial function and inflammation-related pathways of renal tubular cells (PT). In summary, the present application not only provides a new type of biocompatible carrier for efficient delivery of CAP, but also confirms its potential in treating T2DM through multi-dimensional mechanism analysis, laying an experimental foundation for precise intervention strategies for diabetes and its complications. The specific whole experiment process is shown in Figure 9

[0074] Although the present application has been illustrated and described with reference to specific embodiments, it is to be understood that numerous other changes and modifications can be made therein without departing from the spirit and scope of the present application. Accordingly, it is intended to embrace all such alterations and modifications as fall within the scope of the appended claims.​

Claims

1. A method for preparing a bio-nanocomposite hydrogel, characterized in that, Includes the following steps: Dissolve VVPS and β-CD in NaOH aqueous solution, heat to 60-63℃ and stir for more than 30 minutes until completely dissolved; PEGDGE was slowly added to the above solution and stirred until the crosslinking agent dissolved. Then the temperature was raised to above 65°C and the reaction was carried out for more than 4 hours to form a hydrogel. The hydrogel was swollen and dried, then soaked in an ethanol aqueous solution containing capsaicin for more than 48 hours, filtered, and freeze-dried.

2. The preparation method according to claim 1, characterized in that, The mass ratio of VVPS to β-CD is 4:(0-5).

3. The preparation method according to claim 1, characterized in that, The mass ratio of VVPS to β-CD is 4:(3-4).

4. The preparation method according to claim 1, characterized in that, The mass ratio of VVPS to β-CD is 1:

1.

5. The preparation method according to claim 1, characterized in that, The concentration of the NaOH aqueous solution is 1-2 mol / L.

6. The preparation method according to claim 1, characterized in that, Slowly add PEGDGE to the above solution and continue stirring for at least 15 minutes.

7. The preparation method according to claim 1, characterized in that, The swelling treatment method includes immersing the hydrogel in deionized water, changing the water every 6-8 hours, until all unreacted substances are completely removed.

8. The preparation method according to claim 1, characterized in that, The capsaicin-containing ethanol-water solution is an ethanol-water solution containing 1-2 mg / ml capsaicin, with a mass ratio of ethanol to water of 1:

1.

9. The bio-nanocomposite hydrogel prepared by the preparation method according to any one of claims 1-8.

10. The application of the bio-nanocomposite hydrogel prepared by the preparation method according to any one of claims 1-8 and the bio-nanocomposite hydrogel according to claim 9 in the delivery of capsaicin and the preparation of a drug for the relief of type II diabetes.

Images