Natural high polymer and controlled-release three-dimensional gel material prepared by using natural high polymer as raw material

Abstract

The invention belongs to the technical field of biology, and particularly relates to a brand-new natural high polymer and a preparation method thereof, and natural high polymer gel obtained by carrying out cross-linking reaction on the natural high polymer. The natural high polymer is prepared from bletilla striata plants through a newly designed separation and purification path, and is characterized in that the peak molecular weight is 630 kDa, the natural high polymer serving as a material has remarkably enhanced tensile resistance and mechanical strength, and an aqueous solution of the natural high polymer has remarkably improved viscosity and higher shear force resistance. The natural high polymer gel prepared from the natural high polymer has the capacity of encapsulating loaded small molecules and the capacity of slowly releasing the small molecules, and has longer release time and a smoother release curve for the loaded small molecules. The natural high polymer is a raw material with excellent physical properties, and has wide prospects in the field of development of various biological and pharmaceutical materials.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel natural polymer and its preparation method, as well as the application of this polymer as a raw material in the preparation of controlled-release three-dimensional gel materials. Background Technology

[0002] Natural polymers are high-molecular-weight macromolecules synthesized by organisms such as animals, plants, and microorganisms. They function in biological processes as structural support (e.g., cellulose), energy storage (e.g., starch), and the preservation and transmission of genetic information (e.g., DNA) [Nair LS, Laurencin CT. Biodegradable polymers as biomaterials. Prog PolymSci. 2007; 32(8-9):762-98.]. Natural polymers are characterized by high plasticity and good biocompatibility. Their high plasticity makes them a raw material for a range of industrial end products [Gao D, Lv J, Lee PS. Natural Polymer in Soft Electronics: Opportunities, Challenges, and Future Prospects. Adv Mater. 2022;34(25):e2105020.]. Good biocompatibility makes natural polymers indispensable in the pharmaceutical field, including tissue engineering and drug delivery [Zhao L, Zhou Y, Zhang J, et al. Natural Polymer-Based Hydrogels: From Polymer to Biomedical Applications. Pharmaceutics. 2023;15(10):2514.]. Natural polymers are renewable resources, derived from cultivable plants, farmable animals, and culturable microorganisms, making their sources biologically renewable. For example, cellulose, derived from the cell walls of cultivable plants, is a key raw material for papermaking, textiles, and nanocellulose with ultra-high strength properties [Aziz T, Farid A, Haq F, et al. A Review on the Modification of Cellulose and Its Applications. Polymers(Basel). 2022;14(15):3206.]. Silk fibroin, derived from the silk spun by farmable silkworms and spiders, possesses extremely high tensile strength and toughness, making it an ideal material for high-performance textile products and biomedical scaffolds [Sun W, Gregory DA, TomehMA, et al. Silk Fibroin as a Functional Biomaterial for Tissue Engineering.Int J Mol Sci. 2021;22(3):1499.].Natural rubber, derived from the latex of cultivable rubber trees, is irreplaceable in the tire and medical device industries due to its unparalleled elasticity [Men X, Wang F, Chen GQ, et al. Biosynthesis of Natural Rubber: Current State and Perspectives. Int J MolSci. 2018;20(1):50.]. Research and applications of natural polymers mainly focus on developing unknown natural polymer macromolecules from natural raw materials, modifying and altering existing natural polymer macromolecules to endow them with new properties, and applying existing natural polymer macromolecules to new application areas. This patent relates to a research project focusing on developing unknown natural polymer macromolecules from natural plant raw materials.

[0003] In previous research projects, a polysaccharide component with a peak molecular weight of 135 kDa was prepared from the tuber of Bletilla striata using a combination of water immersion and alcohol extraction with Sevag protein removal [Wang C, Sun J, Luo Y, et al. Apolysaccharide isolated from the medicinal herb Bletilla striata inducesendothelial cell proliferation and vascular endothelial growth factor expression in vitro. Biotechnol Lett. 2006;28(8):539-43.]. During the preliminary experimental design for this project, it was discovered that a high molecular weight polymer much larger than 135 kDa could not be separated and purified by the experimental procedure and was instead removed as a passing peak impurity. This was due to the limited experimental conditions at the time, and the experimental methods could only capture the relatively small peak of 135 kDa. Based on a series of preliminary experimental attempts, this invention patent ultimately separated, purified, and enriched this unknown new high molecular weight macromolecule by adding two mild and effective separation and purification steps: impurity adsorption using a porous polystyrene-polyethylene monolithic column and filtration using a polypropylene cotton filter. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to disclose a natural polymer BstrNPM-630k with a peak molecular weight of 630 kDa. It is rich in hydroxyl groups and does not contain carboxyl, amino, cyano, alkynyl, or benzene ring groups.

[0005] This invention relates to a method for preparing this natural polymer.

[0006] This invention relates to the preparation of this natural polymer, comprising the following steps: heating commercially available plant Bletilla striata tuber powder in water at 80°C at a constant temperature, obtaining an aqueous phase which is then filtered through three layers of gauze, a porous polystyrene-polyethylene monolithic column, and a 1 μm pore size polypropylene cotton filter to obtain a filtrate; removing protein from the filtrate using the Sevag method, and collecting the aqueous phase; performing DEAE-cellulose ion exchange column chromatography on the aqueous phase; pouring the aqueous solution obtained from the chromatography into 95% ethanol and collecting the precipitate; using an air dryer, drying the precipitate at room temperature until it is dry and has no ethanol odor, to obtain the solid natural polymer BstrNPM-630k.

[0007] This invention relates to the application of this natural polymer as a raw material in the preparation of the controlled-release three-dimensional gel material BstrNPM-630kG.

[0008] This invention relates to a method for preparing the controlled-release three-dimensional gel material.

[0009] This invention relates to the preparation of the controlled-release three-dimensional gel material, comprising the following steps: dissolving the natural polymer BstrNPM-630k in deionized pure water; adjusting the pH to > 9 by adding sodium hydroxide aqueous solution; adding the crosslinking agent 1,4-butanediol diglycidyl ether and incubating in an oven at 40 ℃ for 4 h; pouring into a dialysis bag with a molecular weight cutoff of 14,000 Da and dialyzing against deionized pure water for 3 days until the pH is neutral; and pouring the product, namely BstrNPM-630kG gel, from the dialysis bag.

[0010] The advantage of this invention lies in the fact that, through a novel preparation process, a natural polymer with a peak molecular weight 4.67 times (630 kDa / 135 kDa) was isolated and purified from the tuber of *Bletilla striata*. This polymer is superior to the macromolecule obtained by the original preparation process. Furthermore, from an environmental and sustainability perspective, this invention relies on plant-based raw materials, which are renewable resources. Compared to non-renewable resources such as petroleum, this invention offers advantages in environmental protection and sustainable development. The natural polymer BstrNPM-630k described in this invention exhibits higher tensile strength and mechanical strength compared to the 135 kDa sample. This means that BstrNPM-630k is more suitable as a raw material for downstream product development requiring high mechanical strength. The advantages of the natural polymer BstrNPM-630k described in this invention are further demonstrated by the following: During testing at 21 shear rate points from low to high, compared to a 135 kDa aqueous sample at the same concentration, the shear stress driving the flow of the BstrNPM-630k aqueous solution at the same shear rate is 10.3 to 2151 times greater, indicating that the BstrNPM-630k aqueous solution has a significantly stronger resistance to flow than the 135 kDa sample aqueous solution. Furthermore, at the same shear rate, the viscosity of the BstrNPM-630k aqueous solution is 10 to 2170 times that of the 135 kDa sample aqueous solution. This indicates that compared to the 135 kDa sample aqueous solution, the BstrNPM-630k aqueous solution possesses higher internal fluid friction resistance to flow, exhibiting better liquid stability as a material, better suspending particles and maintaining shape. Moreover, the viscosity of the BstrNPM-630k aqueous solution is significantly higher than that of the 135 kDa sample aqueous solution. The significant increase in viscosity (by multiples of the 135 kDa sample aqueous solution) with increasing shear rate indicates that BstrNPM-630k aqueous solution also possesses superior resistance to the reduction in fluid viscosity due to high shear forces. The advantage of the natural polymer BstrNPM-630k described in this invention lies in its application as an excellent raw material for developing three-dimensional polymer materials such as gels. Three-dimensional gel BstrNPM-630kG can be prepared using BstrNPM-630kk as a raw material through hydroxyl coupling reactions. The advantage of the BstrNPM-630kG gel described in this invention is that its three-dimensional network framework possesses cavities for storing the aqueous phase, enabling it to load various small molecules. Compared to the 135 kDa sample gel, the BstrNPM-630kG gel exhibits better encapsulation ability for loaded molecules, a longer release time, and a smoother release curve, demonstrating significant controlled-release advantages. Other features, objectives, and advantages of this invention will become apparent from the following examples and accompanying drawings. Attached Figure Description

[0011] Figure 1 A is a picture of the actual BstrNPM-630k.

[0012] Figure 1 B is determined by GPC of BstrNPM-630k.

[0013] Figure 2 Infrared spectroscopy measurements were performed on the BstrNPM-630k.

[0014] Figure 3 Stress-strain curves for BstrNPM-630k and the control group 135 kDa sample.

[0015] Figure 4 A represents the shear rate-shear stress curves of BstrNPM-630k aqueous solution and the control group 135 kDa sample aqueous solution.

[0016] Figure 4 B represents the shear rate-viscosity curves of BstrNPM-630k aqueous solution and the control group 135 kDa sample aqueous solution.

[0017] Figure 5 Image A shows the actual product of BstrNPM-630kG gel.

[0018] Figure 5 B represents the SEM analysis of BstrNPM-630kG lyophilized gel, 100 × 100.

[0019] Figure 5 C represents the SEM analysis of BstrNPM-630kG lyophilized gel, 500 × 10⁻⁶.

[0020] Figure 5 D is the result of SEM analysis of BstrNPM-630kG lyophilized gel, 2000 × 10⁻⁶.

[0021] Figure 6 Controlled release assay for BstrNPM-630kG gel. Detailed Implementation

[0022] 1. Preparation of the natural polymer BstrNPM-630k: (1) Take commercially available plant Bletilla striata tuber and crush it. Heat the Bletilla striata powder in water at 80 ℃ for 4 hours. Filter the solution through three layers of gauze. (2) The filtrate was added into a porous polystyrene-polyethylene monolithic column (Nanjing Saikeluton Biotechnology Co., Ltd.) at a water pressure of 1.0 MPa. After the impurities were adsorbed by the groups in the pores of the monolithic column, the clear filtrate was collected. (3) The clarified filtrate was filtered through a 1 μm pore size polypropylene cotton filter (Shanghai Anjing Environmental Protection Technology Co., Ltd.) to obtain the filtrate; (4) Remove protein from the filtrate using the Sevag method, repeating the process multiple times until no more denatured protein precipitates appear, and then collect the aqueous phase; (5) Perform DE-52 type DEAE-cellulose ion exchange column (Whatman) chromatography on the aqueous phase; (6) Pour the aqueous solution obtained by chromatography into 95% ethanol at a ratio of water phase: alcohol phase = 1:5 (v / v), and collect the precipitate; (7) Using an air dryer, the precipitate was dried at room temperature until it was dry and had no ethanol odor, yielding the solid natural polymer BstrNPM-630k, see Figure 1 A.

[0023] like Figure 1 As shown in A, BstrNPM-630k is a sheet-like, translucent, hard solid.

[0024] 2. Molecular weight determination of natural polymer BstrNPM-630k: Prepare a 10 mg / ml BstrNPM-630k aqueous solution, filter through a 0.45 μm micrometer needle filter (Millipore) to obtain the filtrate, and load 50 μl of the filtrate for GPC analysis (Agilent 1260, PL aquagel-OH MIXED column). The GPC molecular weight is shown in the figure. Figure 1 B. For example Figure 1 As shown in Figure B, the peak molecular weight of BstrNPM-630k is 630,000 Da.

[0025] 3. Detection of protein and nucleic acid impurities in the natural polymer BstrNPM-630k: A 10 mg / ml solution of BstrNPM-630k was prepared and tested using a Coomassie Brilliant Blue assay kit (Nanjing Jiancheng Bioengineering Institute). The result was negative, indicating the absence of protein. Using a visible-ultraviolet spectrophotometer (Tianjin Tester Instruments Co., Ltd.), the absorbance of the 10 mg / ml BstrNPM-630k solution was measured at OD 260 nm. The result was 0, indicating that the nucleic acid content was 0 and the solution contained no nucleic acid.

[0026] 4. Solid-state infrared detection and active group detection of natural polymer BstrNPM-630k: The BstrNPM-630k sheet-like solid sample was cut into 0.5 cm × 0.5 cm pieces and prepared for solid-state infrared spectroscopy (Bruker Verter 70 Fourier Transform Infrared Spectrometer, 450 - 4000 cm⁻¹ region). The infrared spectrum of BstrNPM-630k was obtained, see [link to relevant documentation]. Figure 2 .like Figure 2 The infrared spectrum shown reveals the available functional groups and their properties in the BstrNPM-630k molecular structure. BstrNPM-630k is rich in hydroxyl groups (OH stretching vibrations in the 3600–3200 cm⁻¹ region, CO stretching vibrations of primary alcohols in the 1085–1050 cm⁻¹ region, and CO stretching vibrations of secondary alcohols in the 1125–1100 cm⁻¹ region). BstrNPM-630k lacks carboxyl groups (lacking OH stretching vibrations of carboxyl groups in the 3300–2500 cm⁻¹ region and a strong C=O peak for carboxyl groups in the 1700 cm⁻¹ region), amino groups (lacking a sharp peak for NH stretching vibrations in the 3500–3200 cm⁻¹ region), cyano groups, and alkynyl groups (lacking sharp peaks for cyano-C≡N and alkynyl-C≡CH groups in the 2260–2200 cm⁻¹ region), and benzene ring groups (lacking a sharp peak for aromatic skeleton vibrations in the 1500 cm⁻¹ region). We performed a colorimetric reaction using periodic acid (Shanghai Maclean Biochemical Technology Co., Ltd.) to oxidize hydroxyl groups, and the result was positive, further confirming that BstrNPM-630k is rich in hydroxyl groups. We also performed colorimetric reactions using ferric sulfate (Shanghai Maclean Biochemical Technology Co., Ltd.) to oxidize carboxyl groups, ninhydrin (Shanghai Aladdin Biochemical Technology Co., Ltd.) to oxidize amino groups, anisaldehyde (Shanghai Aladdin Biochemical Technology Co., Ltd.) to oxidize cyano groups, and bromine in carbon tetrachloride solution (Shanghai Aladdin Biochemical Technology Co., Ltd.) to oxidize alkynyl groups; all of these reactions were negative, further confirming that BstrNPM-630k does not contain carboxyl, amino, cyano, or alkynyl groups.

[0027] 5. Tensile test of natural polymer BstrNPM-630k: A 1 mm thick sheet-like BstrNPM-630k sample and a control group sheet-like 135 kDa sample were cut into 150 mm × 10 mm strips and mounted on the testing platform of a universal testing machine (Zwick Roell Z050). The stress and corresponding strain applied to the material at each test point were automatically detected and recorded. A stress-strain curve of the material was obtained by plotting strain on the x-axis and stress per unit cross-sectional area on the y-axis, as shown below. Figure 3 As shown.

[0028] from Figure 3 As can be seen above, when the tested materials are stretched by the same number of millimeters, the force applied per unit area by the testing machine to the BstrNPM-630k sample is greater than the force applied per unit area to the 135 kDa sample. At the extreme points of the stress-strain curve, to stretch the material by 7 mm, the testing machine needs to apply a tensile force of 6.95 MPa per square millimeter to the BstrNPM-630k. For the control group 135 kDa sample, the testing machine only needs to apply a tensile force of 2.80 MPa per square millimeter to stretch the material by 7 mm. Compared to the relatively low molecular weight 135 kDa sample, the high molecular weight BstrNPM-630k exhibits significant advantages in tensile strength and mechanical strength, possessing greater tensile force and mechanical strength.

[0029] 6. Rheological tests of the natural polymer BstrNPM-630k: Prepare 40 mg / ml BstrNPM-630k aqueous solution and 135 kDa sample aqueous solution of control group, take 10 ml, and perform rheological tests (TA DHR10 rheometer). Set 21 shear rate points in the range of 0.1-1000 1 / s, and test the shear stress of BstrNPM-630k aqueous solution and 135 kDa sample aqueous solution at each shear rate. Figure 4 A), and the viscosity of the BstrNPM-630k aqueous solution and the 135 kDa sample aqueous solution at each shear rate ( Figure 4 B). From Figure 4 As shown in Figure A, a significantly greater shear stress must be applied to the BstrNPM-630k aqueous solution compared to the 135 kDa sample aqueous solution to propel it at the same shear rate. At 21 shear rate points, the shear stress required to propel the BstrNPM-630k aqueous solution is 10.3 to 2151 times that required to propel the 135 kDa sample aqueous solution at the same shear rate. This indicates that the BstrNPM-630k aqueous solution exhibits a much stronger resistance to flow than the 135 kDa sample aqueous solution. Figure 4As shown in Figure B, at all 21 shear rate points, the viscosity of the BstrNPM-630k aqueous solution was significantly higher than that of the 135 kDa sample aqueous solution. At the same shear rate, the viscosity of the BstrNPM-630k aqueous solution was 10–2170 times that of the 135 kDa sample aqueous solution. This indicates that compared to the 135 kDa sample aqueous solution, the BstrNPM-630k aqueous solution possesses higher resistance to internal fluid friction during flow, better liquid stability, and is better able to suspend particles and maintain their shape. Similarly, as the shear rate increased from 0.1 1 / s to 1000 1 / s, the viscosity of the BstrNPM-630k aqueous solution became 1 / 7.5 of its initial value, while the viscosity of the 135 kDa sample aqueous solution became 1 / 1562 of its initial value. This demonstrates that for the same increase in shear rate, the BstrNPM-630k aqueous solution exhibits superior resistance to the reduction in fluid viscosity caused by large shear forces compared to the 135 kDa sample aqueous solution.

[0030] We used Origin 2019b software to perform a cross-linear fitting of the shear rate-viscosity, extrapolating the zero-shear viscosity η0. The zero-shear viscosity η0 for the BstrNPM-630k aqueous solution was found to be 2 ± 0.43058, with R² = 0.99135; the zero-shear viscosity η0 for the 135kDa sample aqueous solution was found to be 0.25 ± 0.10728, with R² = 0.99215. In this cross-linear fitting, the zero-shear viscosity of the BstrNPM-630k aqueous solution was 4.4 to 17 times that of the 135kDa sample aqueous solution.

[0031] 7. Preparation of BstrNPM-630kG gel: (1) Disperse and dissolve 4.0 - 10.0 g of natural polymer BstrNPM-630k in deionized pure water; (2) Adjust the pH to > 9 by adding 5 mol / L sodium hydroxide (Shandong Xiya Chemical Co., Ltd.) aqueous solution; (3) Add 200-500 μl of crosslinking agent 1,4-butanediol diglycidyl ether (Shanghai Aladdin Biochemical Technology Co., Ltd.). (4) Incubate in a 40 ℃ oven for 4 h; (5) Pour into a dialysis bag with a molecular weight cutoff of 14,000 Da (Shanghai Yuanye Biotechnology Co., Ltd.), and dialyze with deionized pure water for 3 days until pH is neutral; (6) Pour the product, namely BstrNPM-630kG gel, from the dialysis bag, see... Figure 5 A.

[0032] like Figure 5 As shown in A, BstrNPM-630kG is a milky white gel that has both a solid form and flowability.

[0033] 8. Scanning electron microscopy (SEM) analysis of BstrNPM-630kG gel: The BstrNPM-630kG gel was lyophilized (Ningbo Xinzhi, SCIENTZ-10N / A), sputter-coated with gold, and analyzed by SEM (Zeiss, G360). The results are as follows: Figure 5 As shown in B, 5C, and 5D. From... Figure 5 As can be seen from B and 5C, the backbone of the BstrNPM-630kG gel is distributed in a three-dimensional network, with most of the backbone sheets being 20-50 μm wide, and water-locking cavities existing between the backbone sheets. From Figure 5 As can be seen from D, there are small pores of about 2 μm on the skeleton sheet, which serve to connect the water-locking cavities on both sides of the skeleton sheet. Figure 5 The SEM results shown in B, 5C, and 5D indicate that the BstrNPM-630kG gel has an excellent three-dimensional network structure and the ability to store the aqueous phase within the gel.

[0034] 9. Controlled-release function test of BstrNPM-630kG gel: In the preparation of BstrNPM-630kG gel, Texas Red fluorescent material was added after adding sodium hydroxide and before adding the crosslinking agent. After incubation in a 40 ℃ oven for 4 h, the gel surface was repeatedly washed with deionized pure water until the washing water was colorless. The control group 135 kDa sample was prepared using the same procedure to obtain a 135 kDa sample gel loaded with Texas Red.

[0035] The BstrNPM-630kG gel loaded with Texas Red and the 135 kDa sample gel loaded with Texas Red were ultrasonically disrupted (using a Kexi ultrasonic disruptor, 1740QT). After complete disruption, the aqueous phase was collected, and fluorescence spectrophotometry was performed at excitation light of 561 nm and emission light of 605 nm (using a Hitachi F-7000 fluorescence spectrophotometer). The encapsulation efficiency of Texas Red in the BstrNPM-630kG gel was 94%, and that in the 135 kDa sample gel was 88%. The BstrNPM-630kG gel and the 135 kDa sample gel loaded with Texas Red were placed in deionized water, and the aqueous phase was collected at various time points for fluorescence spectrophotometric analysis. The controlled-release curves of the three-dimensional gel materials were obtained by plotting time on the x-axis and the percentage of fluorescent dye cumulatively released into the aqueous phase relative to the total loaded fluorescent dye on the y-axis. The results are shown below. Figure 6 As shown. From Figure 6 As can be seen, in the initial 0.5 h, the BstrNPM-630kG gel exhibited excellent encapsulation ability for Texas Red, releasing 16.6% of Texas Red. In contrast, the 135 kDa sample gel showed weaker encapsulation ability in the same initial 0.5 h, releasing only 35.2% of Texas Red. After 24 h, the 135 kDa sample gel had accumulated 95.3% of Texas Red release, and the controlled-release curve entered a plateau phase, indicating that release was essentially complete. At this point, the BstrNPM-630kG gel had only accumulated 68.5% of Texas Red release, with nearly one-third of the fluorescent molecule loading still within the gel. It wasn't until 96 h that the BstrNPM-630kG gel accumulated 94.2% of Texas Red release, and the controlled-release curve entered a plateau phase, indicating that release was essentially complete. The above results indicate that, compared with the 135 kDa sample gel, the BstrNPM-630kG gel has better encapsulation ability for the loaded molecules, a longer release time, and a smoother release curve. Its controlled release ability for the loaded molecules is far superior to that of the 135 kDa sample gel.

Claims

1. A natural polymer, designated BstrNPM-630k, characterized by: The peak molecular weight Mw detected by gel permeation chromatography (GPC) was 630 kDa.

2. A method for preparing the natural polymer BstrNPM-630k as described in claim 1, characterized in that: The following steps were performed: commercially available plant Bletilla striata tubers were crushed and sequentially subjected to water extraction, three-layer gauze filtration, porous polystyrene-polyethylene monolithic column filtration, 1 μm pore size polypropylene cotton filter cartridge filtration, Sevag method for protein removal, DE-52 type DEAE-cellulose ion exchange column chromatography filtration, and then the resulting aqueous solution was poured into 95% ethanol, the precipitate was collected, and dried with an air dryer to obtain solid natural polymer BstrNPM-630k.

3. A BstrNPM-630kG gel prepared from the natural polymer BstrNPM-630k as described in claim 1, characterized in that... It is composed of natural polymer BstrNPM-630k, crosslinking agent 1,4-butanediol diglycidyl ether and deionized pure water, wherein the mass percentage of natural polymer BstrNPM-630k is 4-10% and the mass percentage of deionized pure water is 90-96%.

4. A method for preparing the BstrNPM-630kG gel according to claim 3, characterized in that... The following steps were performed: The natural polymer BstrNPM-630k was dissolved in deionized pure water, and then sodium hydroxide aqueous solution was added dropwise, followed by the addition of crosslinking agent 1,4-butanediol diglycidyl ether. The crosslinking reaction was incubated in an oven at 40 °C. The product was poured into a dialysis bag and dialyzed against deionized pure water until the pH was neutral. The product was then poured out of the dialysis bag to obtain BstrNPM-630kG gel.

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