Water-soluble self-assembled chitosan nanocomposite as well as preparation method and application thereof

Abstract

The invention discloses a water-soluble self-assembled chitosan nano-composite and a preparation method thereof.The nano-composite is an amphiphilic polymer NCH composite formed by quaternized chitosan (O-HTCC) and 5 beta-cholanic acid through covalent coupling, the particle size of nano-particles is 110 + / -8.48 nm, the solubility of Alda-1 can be effectively improved by wrapping the Alda-1, the encapsulation efficiency is 36.1%, and the nano-particles can be used for preparing nano-particles. IRI injury after DCD kidney transplantation is remarkably reduced, mitochondrial ALDH2 activity is improved, and serum creatinine is reduced to the lowest level 3 days after rat transplantation. The technology is suitable for improvement of mechanical perfusate of isolated organs, and has clinical transformation potential.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, in particular to a water-soluble self-assembled chitosan nanocomposite and a preparation method thereof. Background Art

[0002] Kidney transplantation is the best treatment for end-stage renal disease, but the shortage of donor organs has led to an increase in the use of organs donated after cardiac death. However, DCD organs are associated with more severe ischemia-reperfusion injury (IRI) and a higher incidence of delayed recovery of renal function (DGF). Therefore, improving the quality of DCD donor kidneys has become a bottleneck in the field of kidney transplantation. Donor organ preservation strategies have a significant impact on the restoration of donor kidney quality, with cold preservation (CS) and mechanical perfusion being the primary methods of donor organ preservation. Previous clinical studies have shown that cold mechanical perfusion (HMP) significantly reduces the incidence of DGF and significantly improves the one- and three-year survival rates of transplanted kidneys. Studies have shown that HMP can enhance aldehyde dehydrogenase 2 (ALDH2) activation, inhibit mitochondrial autophagy, reduce apoptosis of renal tubular epithelial cells, and improve the quality of DCD donor kidneys. However, ALDH2 expression in HMP-treated kidneys remains low compared with normal kidneys. Therefore, finding methods to further increase ALDH2 expression in the kidneys is crucial for improving donor kidney quality. ALD-1, a specific activator of ALDH2, can increase ALDH2 activity by 30% to 60%. Numerous studies have demonstrated that ALD-1 plays a role in alleviating oxidative stress, protecting mitochondrial function, and inhibiting apoptosis, and plays a significant protective role against ischemia-reperfusion injury in various organs, including the heart, liver, kidney, and intestine. However, ALD-1 is insoluble in water and only soluble in organic solvents such as dimethyl sulfoxide (DMSO), posing several challenges to its clinical application and translation.

[0003] Therefore, in order to solve the problem of poor water solubility of Alda-1 in the prior art, it is necessary to develop a method for preparing a water-soluble self-assembled chitosan nanocomposite. Summary of the Invention

[0004] The present invention aims to provide a method for preparing a water-soluble self-assembled chitosan nanocomposite, which solves the problem of poor water solubility of Alda-1 by forming an amphoteric polysaccharide carrier through quaternization and cholanic acid grafting.

[0005] To achieve the above-mentioned purpose, the present invention adopts the following technical solutions: In a first aspect of the present invention, a method for preparing a water-soluble self-assembled chitosan nanocomposite is provided. N-trimethyl chitosan is used as a raw material, the C-2 amino group is protected by benzaldehyde, and a quaternary ammonium salt reaction is carried out with ammonium chloride in isopropanol to obtain O-hydroxypropyl trimethyl ammonium chloride chitosan, namely O-HTCC; O-hydroxypropyl trimethyl ammonium chloride chitosan is subjected to a cholanic acid coupling reaction with 5β-cholanic acid under NHS / EDC catalysis, and the water-soluble self-assembled chitosan nanocomposite is obtained after purification, referred to as NCH.

[0006] Furthermore, the method uses N-trimethyl chitosan as a raw material, protects the C-2 amino group with benzaldehyde, and reacts with ammonium chloride in isopropanol to form a quaternary ammonium salt to obtain O-hydroxypropyl trimethyl ammonium chloride chitosan, which specifically includes: Dissolve N-trimethyl chitosan powder in acetic acid solution, add an equal volume of ethanol, stir and heat to 68-72°C, slowly add benzaldehyde dropwise, and after the reaction is completed, purify to obtain chitosan Schiff base; Ammonium chloride and the chitosan Schiff base are dissolved in isopropyl alcohol to carry out quaternary ammonium salt reaction to obtain O-hydroxypropyltrimethylammonium chloride chitosan.

[0007] Preferably, the N-trimethyl chitosan is selected to have a degree of deacetylation ≥85%, which facilitates its solubility in neutral and alkaline environments. The degree of quaternization in the quaternization reaction depends primarily on the number of reactions, reaction time, and the deacetylation level of CS. The degree of quaternization can be adjusted by adjusting the amount of N-trimethyl chitosan (TMC) to control its water solubility.

[0008] Furthermore, the temperature of the quaternary ammonium salt reaction is 68° C.-72° C., and the reaction time is 14-18 hours.

[0009] Furthermore, the mass ratio of the N-trimethyl chitosan powder to the ammonium chloride is 1:2-1:4.

[0010] Furthermore, the molar feed ratio of the 5β-cholanoic acid to O-HTCC is 1:2-1:5.

[0011] In a second aspect of the present invention, a water-soluble self-assembled chitosan nanocomposite prepared by the method is provided.

[0012] Furthermore, the average particle size of the nanoparticle micelles is 110±8.48 nm; the water solubility is 6.34±0.23 mg / ml, and the solubility in PBS is 5.12±0.36 mg / ml.

[0013] In a third aspect of the present invention, there is provided a use of a water-soluble self-assembled chitosan nanocomposite in preparing a nanocarrier for encapsulating Alda-1 drug.

[0014] Furthermore, the water-soluble self-assembled chitosan nanocomposite improves the solubility of the Alda-1 drug.

[0015] Furthermore, in the application, the nanocomplex and Alda-1 are ultrasonically treated in an ethanol / water mixed solvent to obtain a nanocarrier containing the Alda-1 drug.

[0016] In a fourth aspect of the present invention, nanoparticles containing Alda-1 are provided. The nanoparticles are obtained by ultrasonically treating the water-soluble self-assembled chitosan nanocomplex with Alda-1 in an ethanol / water mixed solvent.

[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: The present invention provides a method for preparing a water-soluble, self-assembled chitosan nanocomposite. The acetylamino group is used as a protecting group for the amino group at C-2. The C-6 group is first quaternized and then deacetylated to obtain O-quaternized chitosan (O-hydroxypropyl trimethyl ammonium chloride chitosan, O-HTCC) to improve the chitosan's water solubility. 5β-Cholecanoic acid is then covalently bonded to O-HTCC to form nanocapsules through hydrophobic self-assembly, effectively dissolving Alda-1.

[0018] 2. The nanoparticle micelles of the present invention have an average particle size of 110±8.48 nm; a water solubility of 6.34±0.23 mg / ml and a solubility in PBS of 5.12±0.36 mg / ml; an encapsulation efficiency of approximately 36.1±1.15% for Alda-1, and a drug loading efficiency of 41.92±0.78%. The complex also exhibits excellent thermal stability, broad-spectrum antibacterial properties, and biocompatibility. Specifically: (1) Enhanced antibacterial properties and biocompatibility Dual antibacterial mechanism: Chitosan and tannic acid self-assemble to form composite micelles through electrostatic and hydrogen bonding, exerting the cationic antibacterial activity of chitosan (destroying bacterial cell membranes) and the phenolic hydroxyl antibacterial effect of tannic acid (inhibiting enzyme activity), with an inhibition rate of ≥90% against Gram-positive and Gram-negative bacteria Biosafety: Natural macromolecular materials (chitosan, tannic acid) are non-cytotoxic and suitable for surface coating of implants in vivo, inhibiting postoperative infection and biofilm formation.

[0019] (2) Improve drug solubility and drug loading efficiency Solubilization of fat-soluble drugs: Through the electrostatic self-assembly of chitosan and γ-polyglutamic acid, fat-soluble drugs such as Tanshinone IIA are encapsulated in nanocapsules, with the water solubility increased to 6.34 mg / mL (the original drug is poorly soluble in water), the drug loading rate reaches 41.92%, and the encapsulation rate is ≥36%. pH / ion-responsive release: The nanocomplex is stable in a physiological environment (pH 7.4) and rapidly releases the drug in the slightly acidic environment of the lesion (pH 5.4), achieving targeted controlled release, with a cumulative release rate of ≤60% within 48 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the synthesis process of NCH in Example 1 of the present invention.

[0021] Figure 2 A. 1H NMR spectrum of NCH in Example 1 of the present invention; B. FTIR spectrum of NCH; C. X-ray photoelectron spectroscopy (XPS) spectrum of NCH.

[0022] Figure 3 A. Morphological characterization of NCH under SEM microscope in Example 1 of the present invention; B. Morphological characterization of NCH after encapsulating Alda-1 under SEM microscope.

[0023] Figure 4 This is the solubilization effect of NCH on Alda-1 in Example 1 of the present invention.

[0024] Figure 5 The cell compatibility of NCH in Example 2 of the present invention with human umbilical vein endothelial cells (HUVECs) and human renal proximal tubular cells (HK-2) is shown.

[0025] Figure 6 A. Intracellular distribution of fluorescein-labeled NCH in HK-2 cells; B. Immunofluorescence staining showing co-localization of NCH and mitochondria in Example 2 of the present invention.

[0026] Figure 7 This is the immunofluorescence staining of FITC, VDAC1 and DAPI of the kidney tissue in Example 3 of the present invention after 1 hour, 3 hours of HMP treatment and 1 day after kidney transplantation.

[0027] Figure 8 These are A. H&E staining and TUNEL assay and calculation of H&E injury scores based on the specific damage conditions of glomeruli and tubules in Example 3 of the present invention; B. Survival curve 7 days after kidney transplantation; and C. Dynamic statistical graph of serum creatinine after kidney transplantation. DETAILED DESCRIPTION

[0028] The following will be combined with specific implementation methods and examples to specifically describe embodiments of the present invention, and the advantages and various effects of the embodiments of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementation methods and examples are used to illustrate embodiments of the present invention, rather than to limit the embodiments of the present invention.

[0029] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the embodiments of the present invention belong. In the event of any conflict, the present specification shall take precedence.

[0030] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be purchased on the market or obtained through existing methods.

[0031] The water-soluble self-assembled chitosan nanocomposite and its preparation method of the present application will be described in detail below with reference to examples and experimental data.

[0032] Example 1. Synthesis and Characterization of N-5β-cholanoic Acid-O-HTCC (NCH) 1. Specific steps for NCH preparation and Alda-1 loading: Preparation of O-HTCC: Chitosan with an 85% degree of deacetylation was used as the starting material. The experimental procedure was as follows: First, 2g of N-trimethylchitosan powder was dissolved in acetic acid. An equal volume of ethanol was added, and the mixture was stirred and heated to 70°C. 10.3g of benzaldehyde was slowly added dropwise to the reaction system, and the mixture was heated for an additional 4 hours. Upon completion of the reaction, a transparent, pale yellow gel-like product was obtained. 300ml of double-distilled water was added to obtain a uniform, milky white suspension. The pH of the product was adjusted to 7.0 with 8% sodium hydroxide solution. The product was filtered and washed with ethanol to obtain a pale yellow powder, the chitosan Schiff base. This process protects the amino group at the C-2 position of the chitosan. Next, a quaternization reaction was performed, and 12.3g of ammonium chloride granules were added. The ammonium chloride and chitosan Schiff base were dissolved in isopropanol and heated for 16 hours. The product was filtered, washed, and dialyzed to obtain the O-HTCC solution, which was concentrated by rotary evaporation and freeze-dried for storage.

[0033] Preparation of NCH: Take 100 mg of O-HTCC freeze-dried product and dissolve it in double-distilled water; take 10 mg of cholanic acid and add anhydrous methanol, mix the two, and use NHS and EDC to catalyze the coupling reaction between the amino group and the carboxyl group. After 24 hours of reaction, dialyze with a methanol: water = 4:1 solution to remove unreacted drugs, and gradually reduce the proportion of methanol in the dialysate until it is completely replaced by double-distilled water. The obtained dialyzed product is rotary evaporated to obtain about 30 ml of light yellow solution, which is freeze-dried and stored in a -20°C refrigerator. The preparation process of NCH is shown in Figure 1 .

[0034] Alda-1 loading: First, prepare a standard solution of Alda-1 using DMSO, plot an absorbance-concentration standard curve, and calculate its equation. Next, prepare the drug-loaded sample. Dissolve 2 mg of sample in 0.5 ml of double-distilled water, then add approximately 5 ml of ethanol and 4 mg of Alda-1 powder. Ultrasonicate for 20 minutes, then place the sample in a 500D dialysis bag and dialyze against PBS for 3 days to obtain the drug-loaded solution.

[0035] After the material preparation is completed, the chemical structure, micromorphology, drug loading and release properties of the material are characterized.

[0036] 2. Characterization of NCH Chemical Structure The spectral changes of the samples were measured using the KBr pellet method on an FTIR instrument. The proton spectra of the samples were determined using NMR. The samples were dissolved in deuterated water. The elemental composition of NCH was analyzed using X-ray photoelectron spectroscopy. The morphological characteristics of N-Alkyl-O-HTCC and its Alda-1 loading were observed using scanning electron microscopy and transmission electron microscopy.

[0037] The results showed that the characteristic peaks of cholanic acid can be observed at 1.31ppm, 2.20ppm and 2.58ppm in the hydrogen spectrum of the sample detected by NMR ( Figure 2 .A); In the infrared spectrum of NCH, a characteristic peak consistent with 5β-cholanoic acid can be observed at 1729cm-1 ( Figure 2 .B); X-ray photoelectron spectroscopy analysis showed that the O1s of chitosan was fitted into two peaks, 531.87eV corresponding to CO, and 533.10eV corresponding to C=O in the amide group. The O1s of NCH was fitted into three peaks, located at 531.39eV, 532.82eV, and 534.09eV, respectively. This indicates that the peak corresponding to the binding energy of C=O was split into two, corresponding to C=O in acetylamino and cholanic acid, respectively. Figure 2 .C). These results indicate that cholanic acid was successfully grafted onto the amino groups of quaternary ammonium salt chitosan.

[0038] 3.NCH encapsulated Alda-1, morphology and release in water The morphology of NCH in the dry state before and after Alda-1 encapsulation was observed by SEM. It can be seen that spherical particles with regular shapes were formed before and after drug loading, and the diameter of the spherical particles increased after Alda-1 encapsulation ( Figure 3 .AB).

[0039] 1 ml of drug-loaded solution was centrifuged at 5000 rpm for 10 minutes. 0.5 ml of the supernatant was collected and added to 1 ml of DMSO. The absorbance at 286 nm was measured using a UV spectrophotometer. The Alda-1 concentration was calculated using the standard curve. The drug loading ratio (DLR) and encapsulation efficiency (EE) of the material were calculated using formulas a and b, respectively. The calculated encapsulation efficiency of Alda-1 loaded in NCH was approximately 36.1 ± 1.15%, and the drug loading efficiency was 41.92 ± 0.78%.

[0040] Alda-1 aggregated visibly in both ddH2O and DMSO / ddH2O (volume ratio 1:4) solutions, and the white drug was insoluble and quickly precipitated. In contrast, the same dose of Alda-1 formed transparent solutions in DMSO and NCH. This indicates that NCH can significantly increase the solubility of Alda-1 in water ( Figure 4 ). According to calculation, the solubility of NCH in water is 6.34±0.23 mg / ml, in PBS is 5.12±0.36 mg / ml, and in ethanol is 1.89±0.24 mg / ml.

[0041] Example 2: Biocompatibility and intracellular distribution of NCH Because NCH will be directly applied to ex vivo kidney machine perfusion, we evaluated its biocompatibility in HUVECs and HK-2 cells. To test the toxicity of NCH to cells, we performed live / dead experiments. After 1 and 3 days of co-culture with NCH, the cell number increased significantly. Compared with live cells (green), few dead cells (red) were observed in the field of view ( Figure 5 To investigate the distribution of NCH in vitro, HUVECs were cultured in 0.5 mg / ml NCH for 3 and 6 hours. The results showed that NCH was rapidly taken up by cells within 1.5 hours, and the fluorescein-labeled nanoparticles were effectively taken up by HUVECs in a time-dependent manner ( Figure 6 To investigate the mitochondrial targeting ability of NCH, mitochondria were labeled in red by immunofluorescence microscopy. NCH colocalized with mitochondrial membrane proteins TOMM20 and VDAC1 ( Figure 6 .B) The results showed that NCH showed the ability to target mitochondria in just 3 hours, and the targeting effect increased over time.

[0042] Example 3: Application of Alda-1-NCH in isolated HMP of rat kidney 1. Rat Renal HMP and Allogeneic Kidney Transplantation Model Twelve-week-old male Sprague-Dawley rats (350-400 g) were fasted for 24 hours before the experiment. Each rat was weighed and injected with 30 mg / kg of 1% sodium pentobarbital. Cardiac death was then induced by diaphragmatic incision. During this period, the rat's body temperature was maintained at 37°C. Thirty-five minutes after death, the left kidney was removed. The isolated kidney was then subjected to SCS or HMP in cold KPS-1 solution for 3 hours.

[0043] The HMP system was established as follows: an indwelling needle in the renal artery was connected to a baroreceptor, which was then connected to a flexible tube connected to a peristaltic pump. A beaker containing 100 mL of KPS-1 was placed on an ice tray, and the kidney was placed in the beaker. Perfusate was pumped through the flexible tube into the renal artery and out through the renal vein. The flow rate parameters were regulated by the peristaltic pump, and the continuous perfusion pressure was recorded by a biosensor and monitored by a computer. The perfusate in the HMP + A-NCH group contained 5 mg of N-Alky-O-HTCC and 5 mg of Alda-1 (post-dialysis). After 1 and 3 hours of HMP, respectively, all kidneys were transplanted into other rats as previously described. Simultaneously, both recipient kidneys were removed. On day 3 after allotransplantation, renal tissue and whole blood were collected for further analysis.

[0044] The results showed that A-NCH can be absorbed by renal tubular epithelial cells through the glomeruli within 1 day after transplantation. In addition, the overlap of FITC and VDAC1 fluorescence indicated that A-NCH can enter the mitochondria. Since ALDH2 is mainly expressed in mitochondria, this provides a prerequisite for the activation of ALDH2 ( Figure 7 To investigate the ameliorative effect of A-NCH on renal injury, we added A-NCH during HMP and evaluated its effects on survival, renal function, and tissue damage in recipient rats after renal transplantation. H&E staining and histological injury scores showed that the addition of A-NCH significantly reduced renal tubular damage ( Figure 8 .A). TUNEL staining was used to evaluate the apoptosis level of renal cells. The results showed that A-NCH significantly reduced the apoptosis level of renal cells ( Figure 8 Compared with the SCS group and HMP group, the addition of A-NCH during HMP significantly reduced the mortality rate of rats after transplantation ( Figure 8 .B). Rapid recovery of renal function is a key factor in reducing mortality. Serum creatinine measurements showed that renal function in rats in the HMP+A-NCH group recovered fastest, especially on the third day after transplantation, when serum creatinine levels were lowest ( Figure 8 These results suggest that adding A-NCH during HMP can further improve DCD renal injury and enhance the efficacy of renal transplantation.

[0045] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0046] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.

[0047] Obviously, those skilled in the art may make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Thus, if such changes and modifications fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are intended to include such changes and modifications.

Claims

1. A method for preparing a water-soluble self-assembled chitosan nanocomposite, characterized in that: The method comprises: N-trimethyl chitosan was used as the raw material, the C-2 amino group was protected by benzaldehyde, and the chitosan was quaternized with ammonium chloride in isopropanol to obtain O-hydroxypropyl trimethyl ammonium chloride chitosan, namely O-HTCC. O-hydroxypropyl trimethyl ammonium chloride chitosan was then coupled with 5β-cholanic acid under the catalysis of NHS / EDC to obtain a water-soluble self-assembled chitosan nanocomposite, referred to as NCH, after purification.

2. The method for preparing a water-soluble self-assembled chitosan nanocomposite according to claim 1, characterized in that: The method uses N-trimethyl chitosan as a raw material, protects the C-2 amino group with benzaldehyde, and reacts with ammonium chloride in isopropanol to form a quaternary ammonium salt to obtain O-hydroxypropyl trimethyl ammonium chloride chitosan, which specifically includes: Dissolve N-trimethyl chitosan powder in acetic acid solution, add an equal volume of ethanol, stir and heat to 68-72°C, slowly add benzaldehyde dropwise, and after the reaction is completed, purify to obtain chitosan Schiff base; Ammonium chloride and the chitosan Schiff base are dissolved in isopropyl alcohol to carry out quaternary ammonium salt reaction to obtain O-hydroxypropyltrimethylammonium chloride chitosan.

3. The method for preparing a water-soluble self-assembled chitosan nanocomposite according to claim 2, characterized in that: The temperature of the quaternary ammonium salt reaction is 68° C.-72° C., and the reaction time is 14-18 hours.

4. The method for preparing a water-soluble self-assembled chitosan nanocomposite according to claim 2, characterized in that: The mass ratio of the N-trimethyl chitosan powder to the ammonium chloride is 1:2-1:

4.

5. The method for preparing a water-soluble self-assembled chitosan nanocomposite according to claim 1, characterized in that: The molar feed ratio of the 5β-cholanoic acid to O-HTCC is 1:2-1:

5.

6. A water-soluble self-assembled chitosan nanocomposite prepared by the method according to any one of claims 1 to 5.

7. The water-soluble self-assembled chitosan nanocomposite according to claim 6, characterized in that: The average particle size of the nanoparticle micelles is 110±8.48 nm; the water solubility is 6.34±0.23 mg / ml, and the solubility in PBS is 5.12±0.36 mg / ml.

8. Use of the water-soluble self-assembled chitosan nanocomposite according to claim 6 or 7 in the preparation of nanocarriers for encapsulating Alda-1 drugs.

9. The use according to claim 8, characterized in that The water-soluble self-assembled chitosan nanocomplex improves the solubility of the Alda-1 drug.

10. A nanoparticle encapsulating an Alda-1 drug, characterized in that: The nanoparticles are obtained by ultrasonically treating the water-soluble self-assembled chitosan nanocomposite according to claim 6 or 7 with Alda-1 in an ethanol / water mixed solvent.

Images