Intelligent alginate-chitosan oligosaccharide microsphere and application thereof

Abstract

The invention discloses an intelligent alginate-chitosan oligosaccharide microsphere which is prepared by the following method: (1) adding functional protein into a sodium alginate solution; (2) dropwise adding calcium salt or a solution thereof, and inducing ionic crosslinking to obtain alginate hydrogel; (3) adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, and fully activating the carboxyl group on the chain of the alginate; and (4) dropwise adding a chitosan oligosaccharide solution, so that amino groups of chitosan oligosaccharide and the activated carboxyl groups are subjected to covalent coupling, and the intelligent alginate-chitosan oligosaccharide microspheres are obtained. The invention further discloses application of the intelligent alginate-chitosan oligosaccharide microspheres in serving as or preparing an intestinal tract positioning release preparation and application in delivering functional protein. The intelligent alginate-chitosan oligosaccharide microspheres can significantly prolong the residence time of encapsulated protein in intestinal tracts, show excellent cytocompatibility and in-vivo biological safety, and provide safety guarantee for oral application of the microspheres.

Description

Technical Field

[0001] This invention relates to a smart alginate-chitosan oligosaccharide microsphere and its application, belonging to the field of protein delivery technology. Background Technology

[0002] Oral delivery of functional proteins has garnered significant attention in the food and nutrition industries due to its potential to improve health outcomes through convenient and non-invasive administration. Proteins, such as bioactive peptides, enzymes, and nutritional supplements, can provide therapeutic or health-promoting effects when effectively delivered to the gut and absorbed or exerting local activity. However, the gastrointestinal tract presents several physiological barriers, including the acidic gastric environment, enzymatic degradation, and limited permeability across the intestinal epithelium. These challenges significantly reduce the bioavailability of orally administered proteins. Therefore, developing efficient delivery systems that protect proteins from gastric transport and enable targeted release in the gut is crucial for functional food design and nutritional supplement applications.

[0003] Carbohydrates have become promising biomaterials for constructing oral delivery systems due to their biocompatibility, biodegradability, and tunable chemical functionality. In particular, alginate (ALG) and chitosan derivatives are widely used for hydrogel formation because they can form stable three-dimensional networks through ionic or covalent crosslinking. Alginate can readily gel in the presence of divalent cations, while chitosan oligosaccharide (COS), as a water-soluble derivative of chitosan, offers enhanced mucosal adhesion properties and potential responsiveness to intestinal conditions. Importantly, alginate contains abundant carboxyl groups, which undergo deprotonation under the weakly alkaline conditions of the intestine. This ionization increases electrostatic repulsion within the gel matrix, leading to matrix swelling and subsequent release of encapsulated proteins. This pH-responsive behavior endows microspheres with site-specific release capabilities, minimizing premature leakage in the stomach and maximizing protein delivery in the highly efficient intestinal environment. Furthermore, the mucosal adhesion properties of chitosan oligosaccharide can promote prolonged retention on the intestinal mucosal surface, potentially enhancing local protein absorption. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides agglutinate-chitosan oligosaccharide microspheres (ALG-COSsmart microspheres), which can be used as a smart delivery carrier for site-directed release to deliver functional proteins.

[0005] This invention is achieved through the following technical solution: A method for preparing intelligent alginate-chitosan oligosaccharide microspheres includes the following steps: (1) Add the functional protein to the sodium alginate solution; (2) Add calcium salt or its solution to induce ionic cross-linking and obtain alginate hydrogel loaded with functional protein; (3) Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the alginate hydrogel loaded with functional proteins to fully activate the carboxyl groups on the alginate chain; (4) Add chitosan oligosaccharide solution dropwise to covalently couple the amino group of chitosan oligosaccharide with the activated carboxyl group to form amide bond, thus obtaining intelligent alginate-chitosan oligosaccharide microspheres, which are collected by centrifugation.

[0006] Furthermore, the functional protein is selected from any one or more of bovine serum albumin (BSA), lactoferrin (LF), immunoglobulin (IgG), insulin (INS), epidermal growth factor (EGF), fibroblast growth factor (bFGF), and whey protein (WP).

[0007] Furthermore, the weight ratio of the functional protein, sodium alginate, and chitosan oligosaccharide is 20:(70-80):(20-25), preferably 20:74.025:21.25.

[0008] Furthermore, in step (1), the concentration of the sodium alginate solution is 500–800 mg / L, preferably 630 mg / L.

[0009] Furthermore, in step (2), the calcium salt is selected from calcium chloride.

[0010] Further, in step (2), the amount of calcium salt added is: 1.5 to 2.0 mM of calcium salt is added for every 1 g of sodium alginate, preferably 1.8 mM.

[0011] Further, in step (3), the amount of EDC added is: 197 mmol of EDC per 1 g of sodium alginate; the amount of NHS added is: 141 mmol of NHS per 1 g of sodium alginate.

[0012] Furthermore, in step (4), the concentration of the chitosan oligosaccharide solution is 800-900 mg / L, preferably 850 mg / L.

[0013] Furthermore, in step (4), the reaction time for covalent coupling is 80 to 100 minutes, preferably 90 minutes.

[0014] The intelligent alginate-chitosan oligosaccharide microspheres prepared using the above method have a functional protein loading of 30%–35%.

[0015] The application of the intelligent alginate-chitosan oligosaccharide microspheres in the preparation of intestinal targeted release formulations, wherein the intestinal targeted release formulation refers to a formulation that can selectively deliver functional proteins to the intestine after oral administration.

[0016] Application of the intelligent alginate-chitosan oligosaccharide microspheres in the delivery of functional proteins.

[0017] The intelligent alginate-chitosan oligosaccharide microspheres of the present invention have the following advantages: (1) The intelligent alginate-chitosan oligosaccharide microspheres of the present invention enhance the mechanical stability and resistance to the gastric environment of the microspheres through a dual network strategy combining ionic crosslinking and carbodiimide-mediated covalent crosslinking, effectively preventing premature leakage of proteins in the stomach.

[0018] (2) The intelligent alginate-chitosan oligosaccharide microspheres of the present invention exhibit reversible pH response behavior: in simulated gastric juice, the network shrinks and compacts due to protonation, resulting in very little protein release; in simulated intestinal juice, the hydrogel swells and polymer network relaxes due to deprotonation, achieving rapid and continuous protein release.

[0019] (3) The intelligent alginate-chitosan oligosaccharide microspheres of the present invention can provide an effective protective microenvironment for the encapsulated proteins. Circular dichroism spectroscopy shows that the secondary structure of the proteins released from the microspheres is effectively preserved after simulated gastrointestinal transport, and the changes are negligible compared with natural proteins.

[0020] (4) The intelligent alginate-chitosan oligosaccharide microspheres of the present invention utilize the mucosal adhesion properties of chitosan oligosaccharide to significantly prolong the retention time of encapsulated proteins in the intestine, and in vivo imaging shows that strong fluorescence signals persist for up to 24 hours after administration.

[0021] (5) The intelligent alginate-chitosan oligosaccharide microspheres of the present invention exhibit excellent cell compatibility and in vivo biocompatibility, providing a safety guarantee for their oral application.

[0022] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0023] Figure 1 Fourier transform infrared spectra of sodium alginate, chitosan oligosaccharide, and smart ALG-COS microspheres.

[0024] Figure 2 : 1H NMR spectra of sodium alginate, chitosan oligosaccharide and smart ALG-COS microspheres.

[0025] Figure 3 Zeta potential changes of intelligent ALG-COS microspheres during incubation in simulated gastric and intestinal fluids.

[0026] Figure 4 Scanning electron microscope (SEM) images of calcium-crosslinked alginate microspheres, ALG-COS microspheres, microspheres incubated in simulated gastric fluid, and microspheres incubated in simulated intestinal fluid. The top left image is an SEM image of calcium-crosslinked alginate microspheres, the top right image is an SEM image of ALG-COS microspheres, the bottom left image is an SEM image of microspheres incubated in simulated gastric fluid, and the bottom right image is an SEM image of microspheres incubated in simulated intestinal fluid.

[0027] Figure 5 Results of L929 cell viability assay after incubation with different concentrations of intelligent ALG-COS microspheres.

[0028] Figure 6 Images of live / dead staining fluorescence of L929 cells after incubation with different concentrations of smart ALG-COS microspheres for 1–3 days.

[0029] Figure 7 : A graph of hematological parameters of mice, in which, from left to right, the parameters of red blood cells, white blood cells, platelets, and hemoglobin are plotted.

[0030] Figure 8 H&E stained sections of major organs of a mouse.

[0031] Figure 9 : Cumulative release curve of BSA.

[0032] Figure 10 Circular dichroism spectra of natural BSA and BSA released from microspheres after being processed with simulated gastrointestinal fluid.

[0033] Figure 11 : Ex vivo fluorescence imaging of the gastrointestinal tract at different time points. Detailed Implementation

[0034] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0035] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0036] Example 1: Preparation of intelligent alginate-chitosan oligosaccharide microspheres Using bovine serum albumin (BSA) as a model protein, intelligent alginate-chitosan oligosaccharide microspheres loaded with functional proteins were prepared by the following method: (1) Accurately measure 117.5 mL of sodium alginate solution with a concentration of 630 mg / L, and add 20 mg of bovine serum albumin under stirring conditions; (2) 7.5 mL of calcium chloride solution (concentration of 18 mM) was slowly added dropwise at a stirring speed of 800 rpm. After the addition was completed, the reaction was stirred for 60 minutes to allow the ionic cross-linking to proceed fully, and a calcium alginate hydrogel loaded with BSA was obtained. (3) Add 0.35 g EDC and 0.15 g NHS to the above hydrogel, stir for 3 h to fully activate the carboxyl groups on the alginate chain; (4) Under continuous stirring, slowly add 25 mL of chitosan oligosaccharide solution with a concentration of 850 mg / L, and react for 90 minutes to allow the amino group of chitosan oligosaccharide to covalently couple with the activated carboxyl group to form an amide bond; (5) After the reaction was completed, the mixture was centrifuged at 4°C and 8000 rpm for 10 minutes, and the precipitate was collected to obtain BSA-loaded smart alginate-chitosan oligosaccharide (ALG-COS) microspheres. The calculated loading of BSA in the microspheres was approximately 32%. The microspheres were then added to ultrapure water to obtain a microsphere suspension.

[0037] Experiment 1 Physicochemical Characterization of Smart ALG-COS Microspheres The intelligent ALG-COS microspheres prepared in Example 1 were characterized as follows.

[0038] (1) Fourier transform infrared (FTIR) spectroscopy analysis: Sodium alginate, chitosan oligosaccharide, and intelligent ALG-COS microspheres were used as samples. The samples were thoroughly dried, mixed with potassium bromide at a mass ratio of 1:100, ground, and compressed into tablets. A Nicolet iS10 FTIR spectrometer was used to analyze the samples at 4000–400 cm⁻¹. -1 Record the spectrum within the range.

[0039] Fourier transform infrared spectra of sodium alginate, chitosan oligosaccharide, and intelligent ALG-COS microspheres are shown below. Figure 1 As shown. The results show that the intelligent ALG-COS microspheres at 1654 cm⁻¹ -1 A carbonyl peak shift occurs, and it is located at 1538 cm⁻¹. -1 and 1415 cm -1 The appearance of new peaks belonging to amide II and amide III bands indicates the formation of amide bonds, confirming the covalent connection between the carboxyl group of alginate and the amino group of chitosan oligosaccharide mediated by EDC / NHS.

[0040] (2) Nuclear magnetic resonance hydrogen spectrum (1 1H NMR analysis: Sodium alginate, chitosan oligosaccharide and smart ALG-COS microspheres were used as samples, and the spectra were recorded at 298 K using an AVANCE NEO 600 MHz NMR spectrometer.

[0041] The proton NMR spectra of sodium alginate, chitosan oligosaccharide, and smart ALG-COS microspheres are shown below. Figure 2 As shown. The intelligent ALG-COS microspheres... 1 A new weak signal appeared in the 1H NMR spectrum at approximately δ=8.2 ppm. This signal was not present in the spectra of sodium alginate and chitosan oligosaccharide and was attributed to the -NH proton in the amide bond, further confirming the successful formation of the covalent amide bond.

[0042] (3) Zeta potential analysis: The surface charge changes of smart ALG-COS microspheres in simulated gastrointestinal fluid (SGF: 0-3 h; SIF: 4-12 h) were monitored using a Zetasizer Nano ZS90 potential analyzer.

[0043] The zeta potential changes of intelligent ALG-COS microspheres during incubation in simulated gastric and intestinal fluids are shown in the figure below. Figure 3 As shown, the potential in SGF is close to neutral or slightly positive (2–4 mV), but drops rapidly to about -25 mV after transfer to SIF and remains strongly negative until 6 hours later, demonstrating that the smart ALG-COS microspheres have significant pH-responsive characteristics, which is related to the deprotonation of alginate carboxyl groups in the intestinal environment.

[0044] (4) Scanning electron microscopy (SEM) observation: calcium-crosslinked alginate microspheres (Ca 2+ The samples were crosslinked alginate), intelligent ALG-COS microspheres, microspheres incubated in simulated gastric fluid in the above (3), and microspheres incubated again in simulated intestinal fluid in the above (3). After the samples were fixed and vacuum sputtered with gold, they were observed using a scanning electron microscope.

[0045] Scanning electron microscope images of calcium-crosslinked alginate microspheres, ALG-COS microspheres, microspheres incubated in simulated gastric fluid, and microspheres re-incubated in simulated intestinal fluid are shown below. Figure 4 As shown, after covalent cross-linking, the diameter of the microspheres increases, exhibiting a wrinkled, multilayered morphology; after incubation in SGF for 3 hours, the size of the microspheres decreases significantly, and the structure becomes more compact; after being transferred to SIF and incubated for another 9 hours, partial re-swelling occurs, demonstrating its reversible pH-responsive swelling behavior.

[0046] The calcium-crosslinked alginate microspheres were prepared by the following method: the calcium alginate hydrogel loaded with BSA in Example 1 was directly centrifuged to obtain the microspheres.

[0047] Experiment 2 Biocompatibility Evaluation of Smart ALG-COS Microspheres The biocompatibility of the intelligent ALG-COS microspheres was tested and evaluated as follows.

[0048] (1) Cytotoxicity assay (CCK-8 assay): L929 fibroblasts were cultured at 1.8 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 hours. Subsequently, the culture medium was replaced with fresh medium containing 0, 50, 100, and 200 μg / mL of intelligent ALG-COS microspheres, respectively, and incubated for another 24 hours. The sample solution was discarded, and fresh medium containing CCK-8 reagent was added. Incubation was continued for 1 hour, and the absorbance at 450 nm was measured to characterize the cell viability of L929 cells after incubation with intelligent ALG-COS microspheres.

[0049] The results of cell viability assays for L929 cells after incubation with different concentrations of intelligent ALG-COS microspheres are as follows: Figure 5 As shown in the figure. The results showed that the relative cell viability of each concentration group remained above 90%, and there was no significant difference from the control group (fresh culture medium without the addition of intelligent ALG-COS microspheres), indicating that the cytotoxicity of intelligent ALG-COS microspheres was negligible within this concentration range.

[0050] (2) Live / dead cell staining: L929 cells were stained at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 μg / mL in 6-well plates and cultured for 24 hours. They were then exposed to suspensions of 0, 50, 100, and 200 μg / mL of intelligent ALG-COS microspheres and cultured for another 3 days. Samples were taken on days 1, 2, and 3 of culture, and cells were treated with Calcein AM / PI staining working solution and observed using an inverted fluorescence microscope.

[0051] Images of live / dead staining fluorescence of L929 cells after incubation with different concentrations of intelligent ALG-COS microspheres for 1–3 days are shown below. Figure 6 As shown in the figure, the vast majority of cells exhibited strong green fluorescence (live cells), with only a very small amount of red fluorescence (dead cells) observed, and no concentration-dependent cytotoxic reaction was observed. This indicates that the intelligent ALG-COS microspheres have excellent cell compatibility.

[0052] (3) In vivo hematological analysis and histopathological evaluation: BALB / c mice were randomly divided into two groups (control group and experimental group, 3 mice in each group). The control group was given oral saline (0.2 mL / mouse), and the experimental group (microsphere group) was given oral ALG-COS microsphere suspension (200 μg / mL / mouse) (0.2 mL / mouse). After 24 hours, blood samples were collected for complete blood cell analysis, and major organs such as heart, liver, spleen, lungs, kidneys and colon were collected for H&E staining and sectioning.

[0053] The hematological parameters of the mice are shown in the figure. Figure 7 As shown, the hematological parameters (red blood cells, white blood cells, platelets, and hemoglobin) were all within the normal physiological range and showed no significant difference from the control group.

[0054] H&E stained sections of major organs of mice are shown below. Figure 8 As shown in the image, all organ and tissue structures appear normal, with no inflammatory infiltration, necrosis, or structural damage observed. The results indicate that oral administration of the intelligent ALG-COS microspheres did not induce any detectable hematological or histological adverse reactions.

[0055] Experiment 3 Evaluation of protein release behavior and structural protection of intelligent ALG-COS microspheres (1) Protein release behavior study: 1 mL of the BSA-loaded smart ALG-COS microsphere suspension (2 mg / mL) prepared in Example 1 was sealed in a dialysis bag with a molecular weight cutoff of 100 kDa. The dialysis bag was first immersed in 4 mL of simulated gastric fluid (SGF, pH 2.0) for 3 hours, and then transferred to 4 mL of simulated intestinal fluid (SIF, pH 7.4) for further incubation for 9 hours. The entire process was carried out at 37°C and 100 rpm with shaking. Samples were taken at predetermined time points and an equal volume of fresh medium was added. The amount of BSA released was quantified using a BCA protein assay kit.

[0056] The cumulative release curve of BSA is shown below. Figure 9 As shown in the figure. The results indicate that BSA release is minimal in SGF (less than 20%), but increases significantly after conversion to SIF, with cumulative release rapidly increasing to about 70% within 3–6 hours, followed by a slower release phase, reaching about 80% at 12 hours.

[0057] (2) Evaluation of protein secondary structure protection: The BSA-loaded smart ALG-COS microsphere suspension (2 mg / mL) was placed in SGF and incubated at 37℃ and 100 rpm for 3 hours, followed by incubation in SIF for another 9 hours. Protein samples were collected at predetermined time points and scanned in the range of 190–260 nm using a circular dichroism spectroscopy (CDI). Natural BSA was used as a control.

[0058] The circular dichroism spectra of natural BSA and BSA released from microspheres after treatment with simulated gastrointestinal fluid are shown below. Figure 10 As shown in the figure. The results show that the CD spectral characteristics of BSA released from the microspheres (a strong positive peak near 192 nm and negative shoulder bands at approximately 210 nm and 222 nm) are negligible compared to native BSA, indicating that the secondary structure of the protein is effectively protected during simulated gastrointestinal transport.

[0059] Experiment 4 Evaluation of the intestinal retention of intelligent ALG-COS microspheres The effect of intelligent ALG-COS microspheres on the intestinal retention of encapsulated proteins was evaluated using an in vivo imaging system.

[0060] Male BALB / c mice were randomly divided into two groups (control group and experimental group, 12 mice in each group). After a 12-hour fast, the control group was administered free BSA solution (500 μg / mL, BSA labeled with FITC) (0.2 mL / mouse) by gavage, while the experimental group was administered BSA-loaded smart ALG-COS microsphere suspension (200 μg / mL, BSA labeled with FITC) (0.2 mL / mouse) by gavage. At 2, 4, 6, and 24 hours post-gavage, whole intestinal segments were collected and in vitro imaging was performed using an in vivo imaging system (excitation wavelength 480 nm, emission wavelength 520 nm). The distribution and retention of the formulation in the intestine were assessed by recording and analyzing fluorescence signals.

[0061] In vitro fluorescence imaging of the gastrointestinal tract at different time points after oral administration of free FITC-protein and FITC-protein encapsulated in ALG-COS microspheres is shown below. Figure 11 As shown in the figure. The results showed that the control group exhibited rapid gastrointestinal transit, with weak fluorescence signal after 6 hours and almost complete clearance after 24 hours. In contrast, the experimental group significantly prolonged the intestinal retention time of the encapsulated protein, with strong fluorescence signal persisting for up to 24 hours after administration. This enhanced retention is attributed to the mucosal adhesion properties of the alginate and chitosan oligosaccharide matrix, which promotes its adhesion to the intestinal mucosa.

[0062] The method for labeling BSA with FITC is as follows: Accurately weigh 5 mg of FITC and dissolve it in 1 mL of DMSO; accurately weigh 100 mg of BSA and dissolve it in 20 mL of 0.1 mol / L Na₂CO₃ solution. Add the FITC solution slowly dropwise to the BSA solution and react at 4°C in the dark for 12 h; then add 7 mL of 0.2 mol / L NH₄Cl solution and stir slowly at 4°C in the dark for 2 h to terminate the reaction. Freeze-dry to obtain FITC-labeled BSA.

[0063] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for preparing intelligent alginate-chitosan oligosaccharide microspheres, characterized in that, Includes the following steps: (1) Add the functional protein to the sodium alginate solution; (2) Add calcium salt or its solution to induce ionic cross-linking and obtain alginate hydrogel loaded with functional protein; (3) Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the alginate hydrogel loaded with functional proteins to fully activate the carboxyl groups on the alginate chain; (4) Add chitosan oligosaccharide solution dropwise to covalently couple the amino groups of chitosan oligosaccharide with the activated carboxyl groups to form amide bonds, thus obtaining intelligent alginate-chitosan oligosaccharide microspheres.

2. The method for preparing intelligent alginate-chitosan oligosaccharide microspheres according to claim 1, characterized in that: The functional protein is selected from any one or more of bovine serum albumin, lactoferrin, immunoglobulin, insulin, epidermal growth factor, fibroblast growth factor, and whey protein.

3. The method for preparing intelligent alginate-chitosan oligosaccharide microspheres according to claim 1, characterized in that: The weight ratio of the functional protein, sodium alginate, and chitosan oligosaccharide is 20:(70-80):(20-25).

4. The method for preparing intelligent alginate-chitosan oligosaccharide microspheres according to claim 1, characterized in that: In step (1), the concentration of sodium alginate solution is 500–800 mg / L.

5. The method for preparing intelligent alginate-chitosan oligosaccharide microspheres according to claim 1, characterized in that: In step (2), the calcium salt is selected from calcium chloride.

6. The method for preparing intelligent alginate-chitosan oligosaccharide microspheres according to claim 1, characterized in that: In step (2), the amount of calcium salt added is: 1.5 to 2.0 mM of calcium salt is added for every 1 g of sodium alginate.

7. The method for preparing intelligent alginate-chitosan oligosaccharide microspheres according to claim 1, characterized in that: In step (3), the amount of EDC added is 197 mmol of EDC per 1 g of sodium alginate; the amount of NHS added is 141 mmol of NHS per 1 g of sodium alginate.

8. The method for preparing intelligent alginate-chitosan oligosaccharide microspheres according to claim 1, characterized in that: In step (4), the concentration of the chitosan oligosaccharide solution is 800–900 mg / L.

9. The intelligent alginate-chitosan oligosaccharide microspheres prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The loading of functional proteins is 30%–35%.

10. The use of the intelligent alginate-chitosan oligosaccharide microspheres of claim 9 in the preparation of an intestinal-targeted release formulation, or in the delivery of functional proteins.

Images