Application of low-esterification pectin in preparation of intelligent nanoparticles for oral delivery of insulin

Abstract

The invention discloses application of low-esterification pectin in preparation of intelligent nanoparticles for oral delivery of insulin. The application comprises preparation of pectin with different esterification degrees, subcritical water treatment of pectin and preparation of pectin-based intelligent nanoparticles. According to the invention, through esterification degree regulation and control of pectin, a subcritical water treatment process and use of appropriate auxiliary materials, the embedding efficiency of insulin and the stability of nanoparticles are remarkably improved, the degradation of insulin in a gastrointestinal tract environment is reduced, and the oral bioavailability of drugs is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug delivery, and in particular relates to the application of low-esterified pectin in the preparation of smart nanoparticles for oral delivery of insulin. Background Art

[0002] Insulin, a protein-based drug used to treat diabetes, is traditionally administered via subcutaneous injection. However, injections present numerous inconveniences, including pain, infection risks, poor patient compliance, and potential psychological rejection with long-term use. Therefore, developing non-invasive, patient-friendly oral delivery systems has become a key research area in diabetes treatment.

[0003] The ideal route for oral delivery is for insulin to be effectively absorbed in the small intestine to exert its blood sugar-lowering effect. However, insulin is easily degraded by gastric acid and proteases in the stomach, and its oral bioavailability is generally low. Therefore, how to protect insulin in the gastric environment to prevent premature degradation while achieving controlled release in specific parts of the intestine is a key challenge in the current research of oral delivery systems.

[0004] Nanoparticles, as drug delivery vehicles, have attracted widespread attention in oral delivery systems due to their advantages, including small particle size, large surface area, and tunable release behavior. Pectin, a naturally derived polysaccharide, exhibits excellent biocompatibility, biodegradability, and significant pH responsiveness, making it particularly suitable for constructing intelligent delivery systems targeting the gastrointestinal tract.

[0005] The physicochemical properties of pectin, such as solubility, gelling ability, cross-linking characteristics and pH responsiveness, are significantly affected by its structural characteristics. In particular, the degree of esterification and molecular weight are considered to be two key factors affecting its delivery performance. Pectins with different degrees of esterification can be delivered through different mechanisms (e.g., Ca 2+ Ionic crosslinking or hydrophobic interaction) forms a three-dimensional gel network of different strengths, thereby affecting the encapsulation efficiency and release behavior of the drug; while the molecular weight affects its gelation ability, dissolution rate and degradation behavior in the body.

[0006] Subcritical water treatment, a green and efficient polysaccharide modification technology, can precisely control the molecular structure and molecular weight of polysaccharides by controlling temperature and pressure without the need for additional chemical reagents, thereby changing their physicochemical properties. This method provides a new technical path for the development of high-performance oral delivery materials. Summary of the Invention

[0007] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0008] In view of the above-mentioned problem of lack of high-performance oral insulin delivery system, the present invention is proposed.

[0009] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing smart nanoparticles for oral delivery of insulin using low-esterified pectin.

[0010] To solve the above technical problems, the present invention provides the following technical solution: an insulin solution, a sodium alginate solution, a γ-polyglutamic acid solution, and a pectin solution are mixed and stirred, a calcium chloride solution is added, and the mixture is stirred at room temperature, ultrasonicated, and centrifuged. The supernatant is discarded, and the resulting precipitate is washed with distilled water. The washed precipitate is redispersed in distilled water to obtain the smart nanoparticles for oral delivery of insulin.

[0011] The pectin solution is obtained by mixing deesterified pectin with water, wherein the pectin has an esterification degree of 15 to 30% and a molecular weight of 695 kDa to 286 kDa;

[0012] The intelligent nanoparticles have an insulin encapsulation rate of 90% to 95%, an insulin release rate of 90% to 95% in simulated intestinal fluid, a HepG2 cell death rate of less than 2% when co-cultured with HepG2 cells, and a hemolysis rate of less than 2% when co-cultured with red blood cells.

[0013] As a preferred solution of the application of the present invention, the mass fraction of the pectin solution is 0.2-0.4%.

[0014] As a preferred embodiment of the application of the present invention, an insulin solution, a sodium alginate solution, a γ-polyglutamic acid solution and a pectin solution are mixed and stirred, and a calcium chloride solution is added, wherein the addition ratio of the insulin solution, the sodium alginate solution, the γ-polyglutamic acid solution, the pectin solution and the calcium chloride solution is 1-2:0.5-1:0.5-1:2-4:4-8.

[0015] As a preferred embodiment of the application of the present invention, the mass concentration of the insulin solution is 0.5 mg / mL and the pH is 3.

[0016] As a preferred embodiment of the application of the present invention, the mass concentration of the sodium alginate solution is 2 mg / mL and the pH is 4; the mass concentration of the γ-polyglutamic acid solution is 2 mg / mL and the pH is 4.

[0017] As a preferred embodiment of the application of the present invention, the mass concentration of the calcium chloride solution is 2 mg / mL.

[0018] As a preferred embodiment of the application of the present invention, the preparation method of the low-esterified pectin comprises:

[0019] Natural pectin raw material is dispersed in methanol. Sodium hydroxide solution is slowly added dropwise to the system under stirring. After the reaction is completed, the system is allowed to stand to allow the pectin to precipitate and separate. The supernatant is discarded, and the resulting precipitate is washed with methanol. The washed precipitate is dried to a constant weight to obtain low-esterification degree pectin.

[0020] As a preferred solution of the application of the present invention, the addition ratio of the natural pectin raw material to the sodium hydroxide solution is 1:5 to 1:10.

[0021] As a preferred embodiment of the application of the present invention, the molar concentration of the sodium hydroxide solution is 0.1 mol / L to 0.2 mol / L.

[0022] Beneficial effects of the present invention:

[0023] (1) The pectin-based smart nanoparticles prepared by the present invention have good pH responsiveness. In an acidic environment, the nanoparticles can effectively protect insulin from degradation by gastric acid and pepsin; while in a neutral pH environment, they promote the release of insulin, thereby achieving effective protection and controlled release of insulin.

[0024] (2) The pectin-based smart nanoparticles of the present invention exhibit excellent Caco-2 cell uptake efficiency at different degrees of esterification, enabling the nanoparticles to better interact with the target cell membrane, thereby improving drug delivery efficiency and having broad biomedical application potential.

[0025] (3) The pectin-based smart nanoparticles of the present invention have good biocompatibility and are therefore suitable for oral delivery systems, avoiding the pain and infection risks associated with traditional insulin injections and improving patient compliance.

[0026] (4) The present invention enhances the drug loading capacity and cross-linking ability of pectin-based smart nanoparticles by using auxiliary materials such as sodium alginate and γ-polyglutamic acid, thereby improving the encapsulation efficiency of insulin and ensuring that it will not be released prematurely during oral delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0028] Figure 1 Schematic diagram of infrared spectra of pectin with different esterification degrees and subcritical water treated pectin according to the present invention, wherein Figure 1 (a) is pectin with different esterification degrees, Figure 1 (b) Subcritical water treated pectin.

[0029] Figure 2 Schematic diagram of the nuclear magnetic resonance hydrogen spectrum of pectin with different esterification degrees (a) and subcritical water treated pectin (b) in the embodiment of the present invention; schematic diagram of the nuclear magnetic resonance carbon spectrum of pectin with different esterification degrees (c) and subcritical water treated pectin (d), wherein Figure 2 (a) is the H NMR spectrum of pectin with different esterification degrees, Figure 2 (b) is the H NMR spectrum of subcritical water treated pectin. Figure 2 (c) is the C NMR spectrum of pectin with different esterification degrees, Figure 2 (d) is the carbon NMR spectrum of subcritical water treated pectin.

[0030] Figure 3 Schematic diagram of rheological analysis of pectin (a) with different esterification degrees and subcritical water treated pectin (b) according to the embodiment of the present invention, wherein Figure 3 (a) is pectin with different esterification degrees, Figure 3 (b) Subcritical water treated pectin.

[0031] Figure 4 Schematic diagram of the particle size of pectin-based nanoparticles prepared from pectin with different esterification degrees (a) and subcritical water-treated pectin (b) in the embodiment of the present invention, wherein Figure 4 (a) is pectin with different esterification degrees, Figure 4 (b) Subcritical water treated pectin.

[0032] Figure 5 Schematic diagram of the zeta potential of pectin-based nanoparticles prepared from pectin with different esterification degrees (a) and subcritical water-treated pectin (b) in the embodiment of the present invention, wherein Figure 5 (a) is pectin with different esterification degrees, Figure 5 (b) Subcritical water treated pectin.

[0033] Figure 6 Schematic diagram of insulin encapsulation efficiency of pectin-based nanoparticles prepared from pectin with different esterification degrees (a) and subcritical water-treated pectin (b) in the embodiment of the present invention, wherein Figure 6(a) is pectin with different esterification degrees, Figure 6 (b) Subcritical water treated pectin.

[0034] Figure 7 Schematic diagram of the microstructure of pectin-based nanoparticles prepared from pectin with different esterification degrees and subcritical water-treated pectin in the embodiments of the present invention.

[0035] Figure 8 Schematic diagram of in vitro simulated insulin release of pectin-based nanoparticles prepared from pectin with different esterification degrees and subcritical water-treated pectin in accordance with the present invention.

[0036] Figure 9 Schematic diagram of live / dead cell fluorescence staining experiment of pectin-based nanoparticles prepared from pectin with different esterification degrees and subcritical water-treated pectin in an embodiment of the present invention.

[0037] Figure 10 Schematic diagram of the hemolysis rate of pectin-based nanoparticles prepared from pectin with different esterification degrees (a) and subcritical water-treated pectin (b) in the embodiment of the present invention, wherein Figure 10 (a) is pectin with different esterification degrees, Figure 10 (b) Subcritical water treated pectin.

[0038] Figure 11 Schematic diagram of laser confocal scanning microscopy observation and analysis of Caco-2 cell uptake of pectin-based nanoparticles prepared from pectin with different esterification degrees and subcritical water-treated pectin in examples of the present invention. DETAILED DESCRIPTION

[0039] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0040] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0041] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0042] Unless otherwise specified, all raw materials used in the examples of the present invention are commercially available. Details are shown in Table 1.

[0043] Table 1

[0044]

[0045] Natural pectin: A natural pectin raw material, derived from citrus peel, characterized by a molecular weight of 693kDa, a degree of esterification of 49.72%, a galacturonic acid content greater than 50%, and a purity of 99%.

[0046] The mass concentration of the insulin solution is 0.5 mg / mL and the pH is 3;

[0047] The mass concentration of sodium alginate solution is 2 mg / mL and the pH is 4;

[0048] The mass concentration of γ-polyglutamic acid solution was 2 mg / mL and the pH was 4;

[0049] The mass concentration of the pectin solution is 4.0 mg / mL, and the mass concentration of the calcium chloride (CaCl2) solution is 2.0 mg / mL.

[0050] Example 1

[0051] This embodiment provides a method for preparing pectin with different degrees of esterification, specifically:

[0052] (1) Preparation method of low-esterification pectin: natural pectin raw material is dispersed in methanol to form a uniform suspension system. Under stirring conditions, sodium hydroxide solution is slowly added dropwise to the system. The mass concentration of pectin in the system is 1%. The amount of sodium hydroxide solution added is 5 times the mass of the natural pectin raw material, and the addition rate is 1mL / 30min. When the concentration of the sodium hydroxide solution is 0.2mol / L, it is used to prepare low-esterification pectin. After the addition is completed, the reaction is continued to stir at room temperature of 25°C for 2 hours. After the reaction is completed, the system is allowed to stand, the resulting precipitate is separated, and washed three times with methanol, and then dried to constant weight at 37°C to obtain low-esterification pectin.

[0053] (2) Preparation method of slightly low-esterification pectin: natural pectin raw material is dispersed in methanol to form a uniform suspension system. Under stirring conditions, sodium hydroxide solution is slowly added dropwise to the system. The mass concentration of pectin in the system is 1%. The amount of sodium hydroxide solution added is 5 times the mass of the natural pectin raw material, and the addition rate is 1mL / 30min. When the concentration of the sodium hydroxide solution is 0.1mol / L, it is used to prepare low-esterification pectin. After the addition is completed, the reaction is continued to stir at room temperature of 25°C for 2 hours. After the reaction is completed, the system is allowed to stand, the resulting precipitate is separated, and washed three times with methanol, and then dried to constant weight at 37°C to obtain slightly low-esterification pectin.

[0054] (3) Preparation method of high-esterification pectin: natural pectin raw material is dispersed in methanol, concentrated hydrochloric acid is added to the system and mixed thoroughly. The mass concentration of pectin in the system is 2%. The amount of concentrated hydrochloric acid added is 0.5 times the mass of the natural pectin raw material, and the hydrochloric acid concentration is 12 mol / L. The reaction system is placed in a water bath, heated to 60°C and stirred for 30 minutes. After the reaction is completed, the pectin is allowed to stand and precipitate, the supernatant is discarded, and the precipitate is washed with methanol three times. The washed precipitate is placed in a 37°C forced air drying oven and dried to constant weight to obtain high-esterification pectin.

[0055] (4) Preparation method of slightly high-esterification pectin: natural pectin raw material is dispersed in methanol, concentrated hydrochloric acid is added to the system, and the mixture is thoroughly mixed. The mass concentration of pectin in the system is 2%. The amount of concentrated hydrochloric acid added is 0.5 times the mass of the natural pectin raw material, and the hydrochloric acid concentration is 12 mol / L. The reaction system is heated to 30°C in a water bath and stirred for 30 minutes. After the reaction is completed, the pectin is allowed to stand for precipitation and separation, the supernatant is discarded, and the precipitate is washed with methanol three times. The washed precipitate is placed in a 37°C forced air drying oven and dried to constant weight to obtain slightly high-esterification pectin.

[0056] (5) Preparation of subcritical water-treated pectin: Dissolve natural pectin raw material in distilled water, take 50 mL and place in a conical flask, seal and set aside. Subsequently, transfer the pectin solution to an autoclave and perform subcritical water treatment under high temperature and high pressure conditions. Specifically, subcritical water-treated pectin sample 1 was obtained by treating at 120°C for 10 minutes.

[0057] (6) Preparation of subcritical water-treated pectin: Dissolve natural pectin raw material in distilled water, take 50 mL and place in a conical flask, seal and set aside. Subsequently, transfer the pectin solution to an autoclave and perform subcritical water treatment under high temperature and high pressure conditions. Specifically, subcritical water-treated pectin sample 2 was obtained by treating at 120°C for 20 minutes.

[0058] (7) Preparation of subcritical water-treated pectin: Dissolve natural pectin raw material in distilled water, take 50 mL and place in a conical flask, seal and set aside. Subsequently, transfer the pectin solution to an autoclave and perform subcritical water treatment under high temperature and high pressure conditions. Specifically, subcritical water-treated pectin sample 3 was obtained by treating at 120°C for 30 minutes.

[0059] (8) Preparation of subcritical water-treated pectin: Dissolve natural pectin raw material in distilled water, take 50 mL and place in a conical flask, seal and set aside. Subsequently, transfer the pectin solution to an autoclave and perform subcritical water treatment under high temperature and high pressure conditions. Specifically, subcritical water-treated pectin sample 4 was obtained by treating at 120°C for 40 minutes.

[0060] The molecular weight of the modified pectin was determined using a Waters 1525 series high performance liquid chromatography (HPLC) system equipped with a differential refractive index detector and an Ultrahydrogel TM Linear 250 chromatography column (7.8 mm × 300 mm, particle size 6 μm).

[0061] 50 mg of pectin (native pectin, low-esterification pectin, slightly low-esterification pectin, high-esterification pectin, slightly high-esterification pectin, and pectin treated with subcritical water for 10, 20, 30, and 40 minutes) was dissolved in 10 mL of ultrapure water and filtered through a 0.22 μm membrane. HPLC analysis conditions were as follows: mobile phase: 0.1 mol / L sodium nitrate, flow rate: 0.5 mL / min, column temperature: 40°C, injection volume: 10 μL, and total analysis time: 30 minutes. Calibration was performed using dextran of varying molecular weights (2,000,000, 300,600, 135,030, 9,750, and 2,700 Da) and glucose (180 Da). The results are shown in Table 1.

[0062] Table 1

[0063]

[0064]

[0065] Under conditions of reduced pectin esterification, the molecular weight of low-esterified and slightly low-esterified pectins decreased slightly compared to native pectin. This is presumably due to the rapid addition of sodium hydroxide during the deesterification process, which resulted in an overly alkaline system, inducing a β-elimination reaction and causing pectin chain breakage. The molecular weights of slightly high-esterified and high-esterified pectins were similar to those of native pectin, indicating that conventional esterification modification has limited effects on molecular weight. In contrast, subcritical water treatment significantly affected pectin molecular weight, decreasing significantly with increasing treatment time. The molecular weight of the pectin sample treated with subcritical water for 20 minutes was 487,057 Da, and the molecular weight of the pectin sample treated with subcritical water for 40 minutes further decreased to 286,520 Da. This indicates that the solubility and reactivity of subcritical water under high temperature and pressure are enhanced, effectively catalyzing the hydrolysis of pectin, breaking glycosidic bonds, and promoting molecular degradation.

[0066] Freeze-dried pectin samples (native pectin, low-esterification pectin, slightly low-esterification pectin, high-esterification pectin, slightly high-esterification pectin, and pectin treated with subcritical water for 10, 20, 30, and 40 minutes) were analyzed using a Fourier transform infrared spectrometer (NicoletiS10, Thermo Fisher Scientific, USA) in attenuated total reflectance mode. The spectral scanning range was 650–4000 cm -1 , with a resolution of 4cm -1The number of scans was 32. The esterification degree of pectin was determined by Fourier transform infrared spectrometer. -1 (stretching vibration of esterified carboxyl group) and 1630 cm -1 The degree of esterification was calculated by the absorption intensity at (stretching vibration of the carboxylate group). The spectrum was baseline corrected and quantitatively analyzed using Omnic 9.2 software. The degree of esterification calculation formula (1) is as follows:

[0067] Degree of esterification=A 1740 / (A 1740 +A 1630 ) formula (1);

[0068] Among them A 1740 and A 1630 Respectively represent 1740cm -1 and 1630cm -1 The results are as follows Figure 1 and as shown in Table 1.

[0069] Infrared spectrum Figure 1 As shown, NP = natural pectin, MLEP = slightly low esterification degree pectin, LEP = low esterification degree pectin, MHEP = slightly high esterification degree pectin, HEP = high esterification degree pectin, SW10 = pectin treated with subcritical water for 10 minutes, SW20 = pectin treated with subcritical water for 20 minutes, SW30 = pectin treated with subcritical water for 30 minutes, SW40 = pectin treated with subcritical water for 40 minutes. Values ​​of ae with different letters indicate significant differences (P < 0.05). After the degree of esterification was adjusted, the 1740 cm -1 and 1630cm -1 The absorption peak area at [the area of ​​​​the esterification peak] changes significantly, indicating that the degree of esterification has been successfully controlled. Calculations show that the degree of esterification of natural pectin is 49.72%, that of low-esterification pectin is 24.87%, that of slightly low-esterification pectin is 35.32%, that of slightly high-esterification pectin is 66.61%, and that of high-esterification pectin is 80.65% (Table 1). Subcritical water treatment has little effect on the degree of esterification. The degree of esterification of pectin samples treated with subcritical water for 10 minutes and for 40 minutes is similar to that of natural pectin, remaining essentially at around 50%. This indicates that subcritical water treatment primarily affects the molecular structure of pectin, rather than the degree of esterification.

[0070] 30 mg of freeze-dried pectin sample was weighed and dissolved in 0.5 mL of heavy water (D2O). After centrifugation, the supernatant was taken and freeze-dried. The above operation was repeated three times to achieve sufficient removal of exchangeable protons in the sample. Finally, the treated sample was dissolved in 0.5 mL of D2O for nuclear magnetic resonance (NMR) measurement. The NMR spectrometer (AVANCE NEO 600 MHz, Bruker, Germany) was used to record the pectin sample.1 H and 13 C nuclear magnetic resonance (NMR) spectrum. The results are as follows Figure 2 shown.

[0071] Pectin 1 The H NMR spectrum shows a significant signal at 3.744 ppm, which is attributed to the methoxy group (-OCH3) in galacturonic acid. As the degree of pectin esterification increases, the intensity of this peak increases, indicating that the content of methoxy groups increases with the increase of the degree of pectin esterification. 1 In the HNMR spectrum, most of the signal intensities increased, especially the H-1 (5.029ppm), H-4 (4.390ppm), H-3 (3.942ppm), H-2 (3.658ppm) and methoxyl group signals (3.744ppm) of galacturonic acid, which were significantly enhanced. This change indicates that subcritical water treatment reduces the molecular weight of pectin, exposing more hydrogen atoms, especially those that are relatively hidden in the polymer chain, thereby enhancing 1 H NMR signals. Furthermore, subcritical water treatment likely disrupts hydrogen bonds and other non-covalent interactions, increasing the accessibility of functional groups and further enhancing signal intensity. New signal peaks appeared at 4.882 ppm and 4.843 ppm, indicating that subcritical water treatment caused hydrolysis of glycosidic bonds, generating new oligosaccharides or free monosaccharides.

[0072] Pectin 13 The C NMR spectrum shows that with increasing esterification degree (high-esterification pectin), the C-6 signal shifts upward (172.333 ppm, compared to 174.015 ppm in native pectin). This shift is likely due to the methyl ester group donating electrons to C-6 through an inductive effect, resulting in a change in its electronic environment and, in turn, the upward shift. In contrast, low-esterification pectin has a lower content of methyl ester groups, and the C-6 signal shifts downward (175.082 ppm), primarily due to an increase in carboxyl content. After subcritical water treatment, the signals of the galacturonic acid unit, such as C-6 (173.92 ppm), C-1 (100.42 ppm), and C-4 (79.02 ppm), are significantly enhanced, indicating that subcritical water treatment changes the chemical environment of galacturonic acid and enhances the signals at the corresponding carbon sites.

[0073] Monosaccharide composition analysis was performed on modified pectin samples. 5 mg of pectin (native pectin, low-esterification pectin, slightly low-esterification pectin, high-esterification pectin, slightly high-esterification pectin, and pectin treated with subcritical water for 10, 20, 30, and 40 minutes) was added to 4 mL of 4 mol / L trifluoroacetic acid (TFA) and hydrolyzed at 121°C for 2 hours. After hydrolysis, TFA was removed by heating, and the sample was dissolved in 10 mL of ultrapure water to prepare a 0.5 mg / mL solution. Analyses were performed using a high-performance anion exchange chromatography system (ICS-5000+SP-5, ThermoFisher, USA) equipped with a pulsed amperometric detector and a Dionex TM A CarboPac PA20 column (3 mm × 150 mm) was used. Analytical conditions were as follows: flow rate 0.5 mL / min, column temperature 30°C, injection volume 10 μL, and total analysis time 30 minutes. The mobile phase gradient was as follows: 60% H₂O and 40% NaOH (250 mM) from 0–21 minutes; 98% H₂O and 2% NaOH (250 mM) from 21–30 minutes. Calibration was performed using 40 μg / mL commercial carbohydrate standards (L-rhamnose, L-arabinose, D-galactose, galacturonic acid, glucose, and glucuronic acid) as external standards. The results are shown in Table 2.

[0074] Table 2

[0075]

[0076] After esterification modification, the galacturonic acid content of native pectin, low-esterification pectin, and high-esterification pectin samples showed minimal changes, reaching 49.96%, 49.27%, and 51.45%, respectively, indicating that esterification modification has a limited effect on the galacturonic acid backbone of pectin. However, the arabinose and glucose contents decreased significantly after esterification modification, suggesting that esterification modification may lead to degradation or rearrangement of the branching regions of pectin, particularly the RG-I and RG-II regions.

[0077] Subcritical water treatment significantly altered the monosaccharide composition of pectin. After 20 and 40 minutes of treatment, the galacturonic acid content decreased from 49.96% (native pectin) to 45.75% (pectin treated with subcritical water for 20 minutes) and 42.69% (pectin treated with subcritical water for 40 minutes), respectively. Simultaneously, the rhamnose (Rha) content increased from 9.06% (native pectin) to 10.39% (pectin treated with subcritical water for 20 minutes) and 10.71% (pectin treated with subcritical water for 40 minutes), indicating that subcritical water treatment led to significant degradation of the HG region skeleton. In a subcritical water environment, the increased polarity of water accelerates hydrolysis reactions, particularly of the glycosidic bonds in the HG region, which are easily hydrolyzed under acid catalysis, thereby destroying the pectin skeleton. In contrast, the backbone of the RG-I region is composed of alternating galacturonic acid and rhamnose linked by α-1,2 glycosidic bonds, making this region less susceptible to degradation under subcritical water treatment than the HG region, resulting in a relative increase in rhamnose content. In pectin samples treated with subcritical water for 20 minutes and 40 minutes, galactose content increased significantly, while glucose and xylose contents decreased significantly, with xylose content decreasing most significantly. This suggests that subcritical water treatment has a selective effect on different neutral sugars in pectin branches, and that galactose-enriched branch structures are relatively stable under subcritical water treatment.

[0078] The steady-state shear rheological behavior of the modified pectin sample solution was tested using a rheometer (DHR-3, TA, USA) equipped with a 50 mm diameter cone-plate device. The shear rate range was set to 0.1 s - 1 to 100s - 1. Use logarithmic scanning, the total test time is 10 minutes, and the test temperature is 25℃. Be careful to avoid bubbles during the experiment to ensure data accuracy. Figure 3 shown.

[0079] The results showed that all pectin solutions exhibited non-Newtonian fluid behavior with increasing shear rate, resulting in decreased apparent viscosity. Highly esterified pectin exhibited enhanced hydrophobic interactions due to its higher methoxy group content, resulting in decreased fluidity and a significant increase in viscosity. In contrast, lowly esterified pectin exhibited stronger intermolecular electrostatic repulsion due to its higher carboxylate group content, increasing fluidity and decreasing apparent viscosity. The apparent viscosity of the pectin solution decreased significantly with increasing subcritical water treatment time, primarily due to molecular degradation, changes in molecular conformation, and the promotion of hydration. This technique can be used to manipulate the rheological properties of pectin solutions and optimize their application in materials development.

[0080] Example 2

[0081] (1) Preparation of low-esterification pectin-based smart nanoparticles: 5 mL of insulin solution, 2.5 mL of sodium alginate solution, and 2.5 mL of γ-polyglutamic acid solution were slowly added dropwise to 10 mL of pectin solution (the low-esterification pectin in step (1) of Example 1 was prepared with water at a concentration of 4 mg / mL) using a constant flow pump at a rate of 1 mL / min. Continuous stirring was maintained during the addition process to ensure uniform mixing. Subsequently, the resulting mixed solution was slowly added to 20 mL of calcium chloride solution at the same flow rate, and stirring was continued throughout the entire addition process. After the addition was completed, the reaction was continued to stir at room temperature of 25°C for 3 hours to promote the formation of pectin-based smart nanoparticles. The resulting nanoparticle suspension was ultrasonically treated for 2 minutes at a maximum power of 1200 W and an amplitude of 30%. After ultrasonic treatment, the suspension was centrifuged at 5000 g for 20 minutes at 4°C, the supernatant was discarded, and the resulting precipitate was washed twice with distilled water at pH 4. Finally, the washed precipitate was redispersed in an equal volume of pH 4 distilled water to obtain pectin-based smart nanoparticle samples.

[0082] Example 3

[0083] The difference from Example 2 is that the pectin solution in step (1) is the slightly low esterification degree pectin in step (2) in Example 1, and the rest is consistent with the example, to obtain slightly low esterification degree pectin-based smart nanoparticles.

[0084] Example 4

[0085] The difference from Example 2 is that the pectin solution in step (1) is the high-esterification degree pectin in step (3) in Example 1, and the rest is consistent with the example, obtaining a high-esterification degree pectin-based smart nanoparticle sample.

[0086] Example 5

[0087] The difference from Example 2 is that the pectin solution in step (1) is the slightly high esterification degree pectin in step (4) in Example 1, and the rest is consistent with the example, to obtain slightly high esterification degree pectin-based smart nanoparticles.

[0088] Example 6

[0089] The difference from Example 2 is that the pectin solution in step (1) is the subcritical water-treated pectin 1 in step (5) in Example 1, and the rest is consistent with the example, obtaining a subcritical water-treated pectin-based smart nanoparticle sample 1.

[0090] Example 7

[0091] The difference from Example 2 is that the pectin solution in step (1) is the subcritical water-treated pectin 2 in step (6) in Example 1, and the rest is consistent with the example, obtaining a subcritical water-treated pectin-based smart nanoparticle sample 2.

[0092] Example 8

[0093] The difference from Example 2 is that the pectin solution in step (1) is the subcritical water-treated pectin 3 in step (7) in Example 1, and the rest is consistent with the example, obtaining a subcritical water-treated pectin-based smart nanoparticle sample 3.

[0094] Example 9

[0095] The difference from Example 2 is that the pectin solution in step (1) is the subcritical water-treated pectin 4 in step (8) in Example 1, and the rest is consistent with the example, obtaining a subcritical water-treated pectin-based smart nanoparticle sample 4.

[0096] Comparative Example 1

[0097] Preparation of natural pectin-based smart nanoparticles (control group): 5 mL of insulin solution, 2.5 mL of sodium alginate solution, and 2.5 mL of γ-polyglutamic acid solution were slowly added dropwise to 10 mL of pectin solution (prepared from natural untreated pectin in water, at a concentration of 4 mg / mL) using a constant flow pump at a rate of 1 mL / min. Continuous stirring was maintained during the addition to ensure uniform mixing. The resulting mixed solution was then slowly added to 20 mL of calcium chloride solution at the same flow rate, with continuous stirring throughout the addition. After the addition was complete, the reaction was stirred at room temperature (25°C) for 3 hours to promote the formation of pectin-based smart nanoparticles. The resulting nanoparticle suspension was sonicated for 2 minutes at a maximum power of 1200 W and an amplitude of 30%. After sonication, the suspension was centrifuged at 5000 g for 20 minutes at 4°C. The supernatant was discarded, and the resulting precipitate was washed twice with distilled water (pH 4). Finally, the washed precipitate was redispersed in an equal volume of pH 4 distilled water to obtain a pectin-based smart nanoparticle control sample.

[0098] Example 10

[0099] The particle size of freshly prepared pectin-based smart nanoparticles was measured using a particle size analyzer (NanoBrook Omni, Brookhaven Instruments Corporation, USA). Dynamic light scattering (DLS) was used with a scattering angle of 90° and a refractive index of 1.46. The test was performed at 25°C with an equilibrium time of 2 minutes. Figure 4As shown in the figure, where NP = nanoparticles prepared from natural pectin, MLEP = nanoparticles prepared from slightly low esterification degree pectin, LEP = nanoparticles prepared from low esterification degree pectin, MHEP = nanoparticles prepared from slightly high esterification degree pectin, HEP = nanoparticles prepared from high esterification degree pectin, SW10 = nanoparticles prepared from pectin treated with subcritical water for 10 minutes, SW20 = nanoparticles prepared from pectin treated with subcritical water for 20 minutes, SW30 = nanoparticles prepared from pectin treated with subcritical water for 30 minutes, and SW40 = nanoparticles prepared from pectin treated with subcritical water for 40 minutes.

[0100] The particle size of pectin-based smart nanoparticles increased significantly with the increase in the degree of pectin esterification (p < 0.05). The high methoxyl content in high-ester pectin enhances the hydrophobic interaction between molecules, causing high-ester pectin molecules to aggregate and form larger nanoparticles. In addition, the low content of carboxylate groups (-COO-) in high-ester pectin reduces the interaction with Ca 2+ The cross-linking sites form a loose three-dimensional network structure and increase the particle size. In contrast, the content of carboxylate groups in low-ester pectin is higher, which increases the interaction with Ca 2+ The cross-linking sites of the pectin molecules were increased, resulting in a tighter three-dimensional network structure and a smaller particle size. In addition, the hydrophilicity of the low-ester pectin molecules was enhanced, which weakened the hydrophobic interaction and inhibited the excessive aggregation of the nanoparticles, thereby further reducing the particle size. As the subcritical water treatment time increased, the particle size of the pectin-based smart nanoparticles decreased significantly (p < 0.05). The extension of the treatment time resulted in a decrease in the molecular weight of the pectin, which enhanced its hydrophilicity, reduced the hydrophobic interaction, and inhibited excessive aggregation. At the same time, the reduction in molecular weight exposed more carboxylate groups, enhancing the interaction with Ca 2+ The cross-linking efficiency is improved, which promotes the formation of a tighter three-dimensional network structure and ultimately significantly reduces the particle size.

[0101] Example 11

[0102] The Zeta potential of freshly prepared pectin-based smart nanoparticles was measured using a particle size analyzer (NanoBrook Omni, Brookhaven Instruments Corporation, USA). Figure 5 shown.

[0103] The experimental results showed that the absolute value of the zeta potential of pectin-based smart nanoparticles increased significantly with decreasing pectin esterification degree (p < 0.05), indicating that low-esterification pectin imparts a higher surface negative charge density to the nanoparticles due to the increased content of carboxylate groups. Furthermore, nanoparticles constructed using pectin treated with subcritical water for different times (e.g., 20 minutes and 40 minutes) showed a trend of gradually increasing absolute zeta potential. This phenomenon is attributed to the degradation and structural rearrangement of pectin molecules during subcritical water treatment, which leads to a decrease in its molecular weight, exposing more carboxylate groups to the molecular surface, and significantly enhancing the negative charge density on the nanoparticle surface. This method can be used to regulate the surface electrical properties and stability of pectin nanoparticles and has great application potential.

[0104] Example 12

[0105] The pectin-based smart nanoparticle suspension (before washing) was centrifuged at 4°C and 15,000 g for 20 minutes, and the supernatant was collected. The insulin concentration in the supernatant was detected by measuring the absorbance at 450 nm using a human insulin ELISA kit (B75453-96T, MERYER, China). The encapsulation efficiency was calculated according to the following formula: Encapsulation efficiency (%) = (total insulin - free insulin) / total insulin × 100%. The results are shown in Figure 2. Figure 6 shown.

[0106] The results showed that low-esterification pectin nanoparticles contained more carboxylate groups, which enhanced the electrostatic interaction with insulin and made it easier to interact with Ca 2+ A dense cross-linked network was formed, which significantly improved the encapsulation efficiency of insulin. Its insulin encapsulation efficiency was the highest, reaching 92.29%. In contrast, high-esterification pectin has strong hydrophobicity. Although it can promote initial encapsulation through hydrophobic effect, its overall encapsulation efficiency is limited to 78.16% due to the small number of carboxylate groups and weak cross-linking ability. Furthermore, by treating pectin with subcritical water for 10 minutes, the molecular weight of pectin was moderately reduced, the flexibility of the molecular chain was increased, and more carboxylate groups were exposed. This change enhanced the interaction between pectin and Ca 2+ The cross-linking ability and electrostatic adsorption with insulin form a dense and stable three-dimensional network structure, achieving the highest encapsulation efficiency. If the subcritical water treatment time is further extended, the pectin will be excessively degraded, resulting in shorter molecular chains, a loose network structure, and increased hydrophilicity. The binding force with insulin is reduced, and the encapsulation efficiency of the formed nanoparticles for insulin is reduced.

[0107] Example 13

[0108] The pectin-based smart nanoparticle sample was ultrasonicated for 5 minutes to obtain a uniform dispersion. 10 μL was then dripped onto the surface of a 5 mm × 5 mm silicon wafer and dried in a vacuum oven at 40°C to quickly remove the solvent. A gold coating was then applied using a vacuum coater, and the microstructure was observed under a scanning electron microscope (S-4800-I, Hitachi, Japan). Figure 7 shown.

[0109] Nanoparticles prepared from different pectin compositions all exhibited a well-defined spherical structure. Compared with native pectin nanoparticles and high-esterification pectin nanoparticles, low-esterification pectin nanoparticles exhibited smaller particle size, less aggregation and particle adhesion, and better dispersibility. Nanoparticles from the subcritical water treatment group also exhibited smaller particle size and better dispersibility. These observations are consistent with the particle size and potential results discussed previously.

[0110] Example 14

[0111] The pectin-based smart nanoparticle sample was mixed with simulated gastric fluid (SGF) in a 1:1 volume ratio. The SGF formula was 0.2% (w / v) NaCl and 0.1% (w / v) pepsin (3000U / mg), and the pH was adjusted to 1.2 with HCl. The mixture was incubated at 37°C and 150rpm for 2 hours with constant temperature shaking, and 2 mL was sampled every 0.5 hours for analysis. The mixed solution was centrifuged at 10,000g for 10 minutes at 4°C to separate the supernatant. The precipitate was washed twice with pH 4 distilled water, followed by the addition of 2 mL of pH 9 standard buffer and continued incubation at 37°C and 150rpm for 4 hours until the precipitate was completely dissolved. The insulin concentration (unreleased portion) was determined using an ELISA kit, and the release rate was calculated according to the following formula:

[0112] Release rate (%) = (total insulin - unreleased insulin) / total insulin × 100%

[0113] After incubation for 2 hours, an equal volume of simulated intestinal fluid (SIF) was added to the above mixture and the pH was adjusted to 7.4. The SIF formula is 0.1% (w / v) CaCl2, 1.0% (w / v) bile salts, 0.2% (w / v) Tris and 0.1% (w / v) pancreatic enzyme (130U / mg), and the pH was adjusted to 7.4 with NaOH. Incubate for another 6 hours at 37°C and 150 rpm, and take 2 mL samples for analysis at 0.5, 1, 1.5, 2, 3, 4, and 6 hours. Subsequent sample processing, insulin concentration determination, and release rate calculation methods are the same as above. The results are shown in Figure 2. Figure 8 shown.

[0114] In simulated gastric fluid, the insulin release rate of all pectin-based smart nanoparticles was less than 10% in the first two hours. This result indicates that under acidic conditions, the carboxylate groups in the pectin molecules are protonated to carboxylic acid groups (-COOH), rendering the molecules neutral. This reduces electrostatic repulsion, allowing the nanoparticle structure to remain compact, thereby effectively protecting insulin from damage by pepsin and the acidic environment in gastric fluid. In simulated intestinal fluid, nanoparticles prepared from different pectins exhibited different insulin release behaviors. The insulin release rate of low-esterification pectin nanoparticles was 94.21%, significantly higher than the 59.87% (at 8 hours) of high-esterification pectin nanoparticles. The higher carboxyl groups in low-esterification pectin easily dissociate into carboxylate ions in an alkaline environment, enhancing electrostatic repulsion between molecules and weakening intermolecular forces, resulting in a loose structure and accelerated insulin release. At the same time, the hydrophilicity of low-esterification pectin also promotes the disaggregation of the nanoparticles. Highly esterified pectin nanoparticles, however, have a high methoxy content, strong hydrophobicity, few carboxyl groups, a stable structure, a large particle size, a low specific surface area, and a low insulin release rate. For the subcritical water treatment group, the release rate of pectin nanoparticles treated with subcritical water for 20 minutes in simulated intestinal fluid was low, while the release rate of pectin nanoparticles treated with subcritical water for 40 minutes increased significantly. The pectin sample treated with subcritical water for 20 minutes had a moderate molecular weight, many cross-linking points, a dense structure, and strong stability. As the treatment time was extended to 40 minutes, the molecular weight of the pectin decreased significantly, making it difficult to form a stable three-dimensional network structure. At the same time, the exposure of the carboxylate groups increased, enhancing the electrostatic repulsion between molecules, resulting in a loose structure and promoting rapid insulin release.

[0115] Example 15

[0116] HepG2 cells were seeded in a culture plate, and after they adhered to the wall, fresh culture medium containing pectin-based smart nanoparticle samples (concentration of 100 μg / mL) was replaced and cultured for 24 hours. Subsequently, the cells were stained using a Calcein-AM / PI double staining kit (BL130A, Biosharp, China) for 15 minutes. The stained cell images were observed using a laser confocal scanning microscope (LSM880, Carl Zeiss AG, Germany). Cell activity was analyzed by green (live cells) and red (dead cells) fluorescence intensity to evaluate the biocompatibility of the pectin-based smart nanoparticle samples. The results are shown in Figure 2. Figure 9 shown.

[0117] The results showed that the low-esterification pectin nanoparticle sample, due to the presence of a large number of carboxyl groups in its molecular structure, exhibited strong hydrophilicity, which weakened its interaction with the cell membrane, thereby ensuring the integrity of the cell membrane. Only a few cells in the sample died. In addition, the sample had a small particle size and a high absolute value of zeta potential, which further reduced its adsorption on the cell membrane surface and weakened its cytotoxicity. Experimental data showed that the cell mortality rate of the low-esterification pectin nanoparticle sample was far below 5%, demonstrating excellent biocompatibility and safety, demonstrating its good potential for in vivo application and suitability for drug delivery and other biomedical uses.

[0118] Example 16

[0119] Freshly collected rat blood was centrifuged at 4°C and 2000g for 10 minutes. After removing the supernatant, it was repeatedly washed with 0.01MPBS until the supernatant was clear. The red blood cell suspension was diluted to a concentration of 2%. The pectin-based smart nanoparticle sample was dispersed in PBS and prepared into a 1mg / mL suspension. 0.2mL of red blood cell suspension was taken respectively, added to 0.8mL PBS (negative control), 0.8mL deionized water (positive control) and 0.8mL pectin-based smart nanoparticle sample suspension, mixed and incubated in a 37°C water bath for 2 hours. After that, it was centrifuged at 4°C and 2000g for 10 minutes, the absorbance of the supernatant at 540nm was measured, and the hemolysis rate was calculated according to the following formula:

[0120] Hemolysis rate (%) = (A s -A n ) / (A p -A n )×100%

[0121] Among them, A s is the sample absorbance, A n is the absorbance of the negative control, A p is the absorbance of the positive control. Figure 10 shown.

[0122] The hemolysis rates of low-esterification pectin nanoparticles and slightly low-esterification pectin nanoparticles were 1.91% and 2.39%, respectively, significantly lower than those of high-esterification pectin nanoparticles (4.74%) and slightly high-esterification pectin nanoparticles (3.28%) (p < 0.05). Analysis suggests that the higher methoxyl content in high-esterification pectin enhances the hydrophobicity of the nanoparticles, allowing them to interact with the hydrophobic structures in the erythrocyte membrane, disrupting the membrane structure and inducing hemolysis. Low-esterification pectin nanoparticles, however, have a higher number of hydrophilic groups, resulting in weaker interaction with the erythrocyte membrane and a lower hemolysis rate. The hemolysis rates of native pectin nanoparticles, pectin nanoparticles treated with subcritical water for 20 minutes, and pectin nanoparticles treated with subcritical water for 40 minutes were 2.35%, 1.95%, and 1.86%, respectively (p > 0.05). Subcritical water treatment significantly reduces the molecular weight of pectin, reduces the particle size of the nanoparticles, and enhances their hydrophilicity, thereby weakening their interaction with erythrocytes. Comprehensive analysis showed that the hemolysis rates of pectin nanoparticles samples with low esterification degree and pectin nanoparticles samples treated with subcritical water for 40 minutes were the lowest, indicating that they exhibited good biocompatibility in the blood and were suitable for biomedical fields such as drug delivery.

[0123] Example 17

[0124] 50mg FITC was dissolved in 10mL DMSO and slowly added dropwise to 100mL NaCO3 buffer solution (0.1mol / L, 0.2mmol / L EDTA, pH 9.0) containing 250mg insulin, and stirred in the dark at room temperature for 12 hours. After the reaction, ammonium chloride solution was added to terminate the reaction, dialyzed until colorless, freeze-dried and stored at -20°C. FITC-insulin was used instead of insulin to prepare pectin-based smart nanoparticle samples. Caco-2 cells were inoculated in glass-bottomed culture dishes and cultured to 70% confluence. A mixture of nanoparticle samples and culture medium was added, and cultured for another 2 hours. The cells were washed with PBS and fixed with 4% paraformaldehyde. The cell nuclei were labeled with DAPI dye, washed with PBS, and observed using a laser confocal microscope with excitation wavelengths of 405nm and 488nm to record images. The results are shown in Figure 2. Figure 11 shown.

[0125] FITC-insulin green fluorescence signals were observed around the cell nuclei in Caco-2 cells treated with all pectin nanoparticle samples, indicating that these nanoparticles can be effectively taken up by cells, which is beneficial to the transmembrane transport of insulin in intestinal epithelial cells and subsequent systemic absorption.

[0126] Finally, during the preparation of pectin-based smart nanoparticles, the choice of low-esterification pectin versus high-esterification pectin has a significant impact on the structure and properties of the nanoparticles. Low-esterification pectin, due to its high carboxylate content, can form a tighter three-dimensional network by cross-linking with calcium ions, which helps improve the stability of the nanoparticles and may affect their responsiveness under different pH conditions. High-esterification pectin, on the other hand, has a higher concentration of methyl ester groups and may exhibit different physicochemical properties, affecting its structural strength and interaction with drug molecules.

[0127] During the preparation process, subcritical water treatment technology can effectively modify the molecular structure, molecular weight, and hydrophilicity of pectin by adjusting the treatment time. With increasing treatment time, the molecular weight of pectin gradually decreases, which may lead to changes in physical properties such as nanoparticle size and surface charge. Shorter treatment times can maintain a high molecular weight of pectin and enhance its cross-linking ability, while longer treatment times may lead to molecular chain breakage, affecting the stability and drug-loading capacity of the nanoparticles.

[0128] The encapsulation efficiency of insulin is one of the key properties of pectin-based smart nanoparticles. The encapsulation efficiency can be effectively influenced by adjusting the degree of esterification of pectin and the subcritical water treatment conditions. Low-esterification pectin, due to its strong hydrophilicity and negative charge, may be conducive to the stable encapsulation of insulin in the nanoparticles it forms, and has the highest insulin encapsulation efficiency (92.29%); while high-esterification pectin combines with insulin through hydrophobic interactions to form different drug-loading structures, thereby changing the encapsulation efficiency and release behavior of the nanoparticles. Subcritical water treatment has a significant effect on the encapsulation efficiency of insulin. Moderate subcritical water treatment can enhance the interaction between pectin and insulin and improve the encapsulation efficiency; excessive treatment may lead to pectin degradation, reducing drug loading capacity and nanoparticle stability.

[0129] In an in vitro simulated release experiment, pectin-based smart nanoparticles demonstrated good pH responsiveness. In an acidic environment, the nanoparticles can effectively inhibit the release of insulin, thereby protecting it from degradation by gastric acid and pepsin; in a neutral environment, the structural changes of the nanoparticles promote the release of insulin. Different pectin treatment methods also have different effects on the release behavior. The specific release pattern is closely related to factors such as the esterification degree, treatment method, and molecular weight of the pectin. Low-esterification pectin has a high carboxyl content and is easily ionized into carboxylate ions in an alkaline environment, which enhances intermolecular repulsion, resulting in a loose structure and promoting insulin release. At the same time, its strong hydrophilicity also accelerates the disaggregation of the nanoparticles. Within 8 hours, the insulin release rate of low-esterification pectin nanoparticles reached 94.21%, significantly higher than the 59.87% of high-esterification pectin.

[0130] The low-esterification pectin-based smart nanoparticles prepared by this invention exhibit excellent biocompatibility, with a hemolysis rate far exceeding 5%, indicating relatively mild interactions in blood and good safety, making them suitable for oral drug delivery systems. Furthermore, in Caco-2 cell uptake experiments, the low-esterification pectin-based nanoparticles were able to effectively enter the cells. This demonstrates the potential of low-esterification pectin nanoparticles to enhance drug bioavailability in oral delivery systems, further demonstrating their promising application in the biomedical field.

[0131] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. Application of low-esterified pectin in the preparation of smart nanoparticles for oral delivery of insulin, characterized by: The insulin solution, sodium alginate solution, γ-polyglutamic acid solution and pectin solution are mixed and stirred, and a calcium chloride solution is added. After stirring at room temperature, the mixture is ultrasonicated and centrifuged, and the supernatant is discarded. The resulting precipitate is washed with distilled water, and the washed precipitate is redispersed in distilled water to obtain the smart nanoparticles for oral delivery of insulin; The pectin solution is obtained by mixing deesterified pectin with water, wherein the pectin has an esterification degree of 15 to 30% and a molecular weight of 695 kDa to 286 kDa; The intelligent nanoparticles have an insulin encapsulation rate of 90% to 95%, an insulin release rate of 90% to 95% in simulated intestinal fluid, a HepG2 cell death rate of less than 2% when co-cultured with HepG2 cells, and a hemolysis rate of less than 2% when co-cultured with red blood cells.

2. The use according to claim 1, characterized in that: The mass fraction of the pectin solution is 0.2-0.4%.

3. The use according to claim 1, characterized in that: The insulin solution, sodium alginate solution, γ-polyglutamic acid solution and pectin solution are mixed and stirred, and calcium chloride solution is added, wherein the addition ratio of the insulin solution, sodium alginate solution, γ-polyglutamic acid solution, pectin solution and calcium chloride solution is 1 to 2. ： 0.5~1 ： 0.5~1 ： 2-4 ： 4~8.

4. The use according to claim 3, characterized in that: The mass concentration of the insulin solution is 0.5 mg / mL and the pH is 3.

5. The use according to claim 3, characterized in that: The mass concentration of the sodium alginate solution is 2 mg / mL, and the pH is 4; the mass concentration of the γ-polyglutamic acid solution is 2 mg / mL, and the pH is 4.

6. The use according to claim 3, characterized in that: The mass concentration of the calcium chloride solution is 2 mg / mL.

7. The use according to claim 1, characterized in that: The preparation method of the low-esterified pectin comprises: Natural pectin raw material is dispersed in methanol, and sodium hydroxide solution is slowly added dropwise to the system under stirring conditions. After the reaction is completed, the system is allowed to stand to allow the pectin to precipitate and separate, the supernatant is discarded, and the resulting precipitate is washed with methanol. The washed precipitate is dried to constant weight to obtain low-esterification degree pectin.

8. The use according to claim 1, characterized in that: The addition ratio of the natural pectin raw material to the sodium hydroxide solution is 1 ： 5~1 ： 10.

9. The use according to claim 5, characterized in that: The molar concentration of the sodium hydroxide solution is 0.1 mol / L to 0.2 mol / L.

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