Zein fibril powder, zein neogel and preparation method and application thereof

Abstract

The present application relates to a kind of zein fibril powder, zein new gel and its preparation method and application.The preparation method of zein fibril powder includes the following steps: water is added to the zein gel containing solvent to solidify it;Then freeze-drying solidified product, obtain the zein fibril powder rich in fibril.The preparation method of zein new gel is: zein raw material powder, zein fibril powder is dissolved in solvent, forms the zein solution with concentration of 10-60% (w / v), forms zein new gel after standing.Compared with prior art, the present application has the advantages of significantly different from the micro-morphology of zein spherical, different solvent system, highly repeatable, controllable performance and the like.

Description

Technical Field

[0001] This invention relates to the fields of food science, materials science, biomedical engineering, pharmaceuticals and biosciences, specifically to zein prolysin cellulose powder, zein novel gel, their preparation methods and applications. Background Technology

[0002] Under a scanning electron microscope, zein powder appears as irregular spheres, ranging in size from several micrometers to hundreds of micrometers; similarly, the surface structure of two-dimensional films prepared by coating with zein solution is composed of microspheres ranging in size from tens of nanometers to several micrometers. Therefore, the idea that the fundamental microscopic structure of zein is spherical is widely accepted. On the other hand, the gelation of zein solutions has long been considered a drawback in food processing.

[0003] For many years, several research groups have attempted to elucidate the gelation mechanism by tracking the gel formation process and to find highly reproducible gel generation methods, but have yielded conflicting results. For example, P. Nonthanum et al. reported that γ-zein is crucial for the gelation of zein solutions. In their first report, 27% (w / v) zein in 70% (v / v) ethanol (zein extracted using three different methods) gelled in approximately 3 hours. They suggested that the coexistence of γ-zein might be the cause of gelation in zein solutions, based on the fact that the addition of β-mercaptoethanol (BME) shortened the gelation time. In their second report, they prepared 20% zein solutions in 65-90% ethanol under different pH conditions. Solutions with high γ-zein content gelled in shorter times than those with low γ-zein content; solutions without γ-zein did not gel within 240 minutes. Because γ-zein contains a high concentration of -SH groups, which can form disulfide bonds, they proposed that disulfide bonds are important for gel formation. However, in another study reported by S. Uzun et al., 60% and 40% zein solutions in 90% ethanol only gelled after 4 months of storage, suggesting that zein self-assembly was the cause of gel formation. Meanwhile, Shi et al. reported that a 25% zein solution in 80% (v / v) ethanol began to gel in 1.54 hours.

[0004] These findings indicate that the gelation process of zein solutions is difficult to repeat and control. At present, the conditions for the application of zein gel as a useful substance are not yet met. It is necessary to find the key factors of gelation to make the preparation process repeatable and the performance controllable.

[0005] An emerging field in additive manufacturing is 4D printing, where time, as a fourth dimension, is combined with 3D printing. The design goal of smart materials is to respond to external environmental stimuli; among various stimuli, only a limited number contribute to achieving 4D printing properties. In the biomedical field, tissue engineering and drug delivery are major areas where 4D printing can be applied. D. Han et al. used 4D-printed microneedles to enhance tissue adhesion; these microneedles have backward-curving barbs that deform and lock into the tissue when inserted into the skin. In another study, the water-induced shape recovery properties of a polyvinyl alcohol (PVA) system prolonged gastric retention time. However, these fourth-dimensional changes require complex post-printing processing. Developing low-cost printing inks with simple printing methods (few post-printing steps) is challenging. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a highly reproducible and controllable zein fibrillary powder, a novel zein gel, and their preparation methods and applications that are significantly different from the spherical microstructure of zein and can be prepared in different solvent systems. This invention also provides a rapid preparation technology for zein gels in different solvent systems that is highly reproducible and has controllable performance, using zein fibrillary powder as a seed.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] This invention discovered that gel formation can be achieved relatively quickly with the aid of shear force, but continuous stirring for more than 4 hours is still time-consuming. Based on the hydrophobic properties of zein, it was found that phase separation occurs during gel solidification under the influence of water, causing other solvents in the gel system to dissolve into the water. Freeze-drying the solidified gel yields a zein powder rich in fibrils. Adding this powder as seed fiber to a freshly prepared zein solution significantly accelerates gel formation, and this operation is highly repeatable, solving the long-standing problem of uncontrollable gel formation.

[0009] This invention discloses a zein protofibrillary powder and its preparation method, using the protofibrillary as the core structure of a gel network to induce rapid gelation of zein solution, thus solving the key problem of uncontrollable gel formation conditions. This highly reproducible gel preparation method can be used for the preparation of biomedical gel materials with controllable performance, and can be implemented for 3D and 4D printing. The specific scheme is as follows:

[0010] A method for preparing zein prolysin fibrillary powder, the method comprising the following steps:

[0011] Water is added to the zein gel containing solvent to solidify the gel;

[0012] The solidified material was then freeze-dried to obtain zein-rich fibroin-based fibrous powder.

[0013] Furthermore, the zein gel is prepared by the following method: zein raw material powder is dissolved in a mixed solvent to form a zein solution with a concentration of 10-60% (w / v), and then shear stress is applied until a zein gel is formed. That is, the zein gel used to prepare zein fibrillary powder contains 10-60% (w / v) zein.

[0014] Furthermore, the amount of water added is 4-6 times that of the zein gel.

[0015] Further, the solvent is 60-95% (v / v) ethanol / water, dimethyl sulfoxide (DMSO), 60-95% (v / v) N-methylpyrrolidone (NMP) / water, 60-95% (v / v) glycerol formaldehyde (GF) / water, or 60-95% (v / v) acetic acid / water.

[0016] Furthermore, the stirring speed for shear stress is 1500-2500 rpm, and the time is 5-10 h.

[0017] A method for preparing a novel zein gel, comprising: dissolving the zein fibrillary powder and zein raw material powder as described above in the solvent described above to form a zein solution with a concentration of 10-60% (w / v), and allowing it to stand to form a novel zein gel.

[0018] Furthermore, the mass ratio of the amount of zein protocellulose powder added to the zein raw material powder is (10-100):(0-90).

[0019] Furthermore, during preparation, active substances and / or porogens are added to the zein solution to form a novel zein gel loaded with active substances; the active substances include ciprofloxacin hydrochloride, dexamethasone, glyceryl phosphate, and vitamin C; the porogens include menthol.

[0020] A novel zein gel prepared by the method described above.

[0021] An application of the novel zein gel described above is provided, which is used for 4D printing in a support bath with a gradient. Printing 3D structures in a support bath formed by different solvent gradients enables fourth-dimensional deformation, and consequently, changes in drug loading, drug release rate, degradation rate, etc., which is 4D printing.

[0022] Furthermore, the specific process of 4D printing is as follows: the support bath material is dissolved in a solvent-water solution system and layered to form a support bath with gradient changes; the new zein gel is 4D printed in the support bath; after printing is completed, it is dried to obtain a 3D printed structure.

[0023] Furthermore, the supporting bath material includes carbomer, diatomaceous earth, silicate, sodium hydroxide, or silicone oil; the solvent-aqueous solution system consists of 10-80% (v / v) water and 20-90% (v / v) ethanol, DMSO, NMP, GF, or acetic acid.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) This invention not only significantly shortens the gel preparation time, but also has high repeatability, providing quality assurance for the use of corn protein gel as a biomedical material;

[0026] (2) In this invention, the new gel generated by the first use can be 4D printed in a gradient-changing support medium. The printed material has environmentally responsive intelligent features, including but not limited to biomedical functions such as adjustable drug loading, release rate and degradation rate.

[0027] (3) This invention uses fibrin powder as a seed and provides a rapid preparation technology for zein gel with different solvent systems that is highly reproducible and has controllable performance. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating an embodiment of the present invention;

[0029] Figure 2 The experimental diagrams show the evidence of fibril formation; Figures (a), (b), (c), (d) and (e) show the fluorescence spectra of zein gel and solution in 80% (v / v) aqueous ethanol, 80% (v / v) aqueous NMP, 80% (v / v) aqueous acetic acid, DMSO and glycerol formaldehyde, respectively.

[0030] Figure 3 Scanning electron microscope images of zein raw material powder (left) and fibrin powder (right);

[0031] Figure 4 To observe the zein raw material powder solution (left) and the new zein gel induced by fibrils (right) under a fluorescence microscope;

[0032] Figure 5Gelation of zein with added fibrils (left) and the time required (right); 30% (w / v) zein in an 80% (w / v) ethanol-water system;

[0033] Figure 6 This invention provides a 4D printing solution for zein gel in a dedicated support bath.

[0034] Figure 7 The structural features printed in 40% and 75% ethanol support baths according to the present invention are: (a) ciprofloxacin loading, (b) ciprofloxacin release rate, and (c) degradation rate of the printed scaffold. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0036] Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0037] A zein prolysin fibrillary powder, a novel zein prolysin gel, their preparation methods, and applications, comprising the following steps:

[0038] (1) Dissolve the zein raw material powder in 60-95% (v / v) ethanol / water, and the zein protein content is 10-60% (w / v), preferably 20-50%.

[0039] (2) Apply shear stress to the solution at room temperature (20-30℃) using methods such as magnetic stirring until gelation occurs.

[0040] (3) Add excess water, preferably 5 times the volume of the gel, to the zein gel for solvent exchange. Repeat this operation to completely remove the solvent. The zein rich in protofibrils in the gel solidifies and precipitates out. Freeze-dry the solidified zein to obtain zein protofibril powder with a microstructure of fibrous morphology, with a yield of over 90%.

[0041] (4) The fibril powder and zein raw material powder are mixed at a mass ratio of 10:90 to 100:0. Solutions containing 10-60% (w / v) zein are prepared using 60-95% (v / v) ethanol / water, dimethyl sulfoxide (DMSO), 60-95% (v / v) N-methylpyrrolidone (NMP) / water, 60-100% (v / v) glycerol formaldehyde (GF) / water, or 60-95% (v / v) acetic acid / water, preferably 20-50%. The mixture is allowed to stand to obtain a new gel. The gelation time decreases with increasing fibril content, ranging from 3 minutes to 3 hours.

[0042] Drug-loaded zein gels can be prepared by adding drugs to a solution of zein, with the amount added not exceeding 30% (w / w) of the zein protein content.

[0043] 3D printed structures with adjustable pore structure and porosity can be obtained by adding a pore-forming agent to a solution of zein, with the amount varying from 10% to 50% of the mass of the new gel.

[0044] (5) Support baths were prepared by dissolving Carbopol 980 (0.25% w / v) dissolved in 0.01N sodium hydroxide in 40% and 75% aqueous solutions of the above solvents, respectively.

[0045] (6) Using pneumatic extrusion-based 3D printing technology, zein gel containing or without drugs is printed into a support medium to construct a 3D scaffold. The printed structure undergoes a fourth-dimensional change in the support bath, enabling intelligent biological functions.

[0046] (7) Wash in sodium chloride solution (0.9% (w / v) aqueous solution) for 30 minutes to remove Carbopol.

[0047] (8) After washing, the printed structure is dried and then sterilized and packaged.

[0048] Example 1 (F1)

[0049] A gel was prepared by dissolving 2 g of zein in 10 ml of 80% (v / v) ethanol aqueous solution. The resulting solution was sonicated in an ultrasonic bath for 1 hour to remove air bubbles. Then, the solution was stirred magnetically at 2000 rpm at 25°C. After 6 hours, gel solidification was observed.

[0050] Fibrous formation was evaluated using a ThT (thioflavone) binding assay. 100 mg of zein solution was dispersed in 4 mL of 80% (v / v) ethanol aqueous solution. The dispersion was sonicated in an ultrasonic bath for 1 hour. 25 μL of 25 mM ThT (dissolved in 80% (v / v) ethanol aqueous solution) was added. Excitation was performed at 450 nm, and fluorescence spectra were measured in the 460–560 nm range. Fibrouss exhibited higher fluorescence intensity due to ThT binding. Figure 2 a).

[0051] Add 5 times the volume of water to solidify the zein gel. Allow it to stand at room temperature for 24-36 hours until fully solidified. Freeze-dry the solidified gel and grind it into powder using a grinder to obtain zein powder rich in fibrils. Figure 3 (Right), used as a seed to induce rapid gelation of zein solution.

[0052] To prepare a zein gel loaded with active material, 0.08 g of ciprofloxacin hydrochloride was added to a zein solution as an active material model, and gelation was performed under the aforementioned conditions. Carbomer (0.25% w / v) dissolved in 0.01 N sodium hydroxide was dissolved in 40% and 75% (v / v) ethanol aqueous solutions to prepare two support baths with different water contents. The zein gel loaded with ciprofloxacin was used as printing ink to print in the Carbomer support bath. After the printed structure remained in the support bath for 12 hours, it was washed with a 0.9% (w / v) sodium chloride aqueous solution for 30 minutes to remove the Carbomer, and then dried in a dry heat oven at 37°C for 12 hours to obtain the finished product. Figure 6 Compared to structures printed in a 75% aqueous solution support bath, structures printed in a 40% aqueous solution support bath exhibited lower drug loading, slower drug release rates, and slower degradation rates. Figure 7 ).

[0053] Example 2 (F2)

[0054] A zein solution was obtained by dissolving 0.6 g of zein fibrillary powder and 0.4 g of zein raw material powder in 10 ml of 80% (v / v) N-methylpyrrolidone (NMP) aqueous solution. Under fibrillary induction, the gelation time was shortened from 4 days to 40 minutes. (See Example 1 and...) Figure 2 b. The gel formation process was tracked through ThT binding experiments.

[0055] Carbomer (1% w / v) was dissolved in aqueous solutions containing 10%, 50%, and 90% (v / v) NMP, respectively, to prepare three support baths with different water contents. Zein gels loaded with three active substances (dexamethasone, β-glycerol phosphate, and vitamin C, which induce bone marrow stem cells to differentiate into osteoblasts) were printed in these support baths. The printed structures were allowed to remain in the support baths for 6 hours. After washing with phosphate buffer to remove carbomer, the printed 3D structures were freeze-dried.

[0056] Example 3 (F3)

[0057] 0.4 g of zein fibrillary powder and 3.6 g of zein raw material powder were dissolved in 10 mL of dimethyl sulfoxide (DMSO) to obtain a zein solution, and then 50% menthol (w / w) was added as a porogen. Under fibrillary induction, the gelation time was shortened from 4 days to 4 hours. (See Example 1 and...) Figure 2 d. The gel formation process was tracked through ThT binding experiments.

[0058] Layered silicate Laponite (1-4% w / v) was dissolved in 20% and 60% (v / v) water-ethanol systems, respectively, and allowed to stand to form a gel, thus preparing two support baths. The gel containing the pore-forming agent was used as printing ink in the Laponite support bath for printing. After the printed 3D structure was cured in an aqueous environment, it was removed and subjected to menthol sublimation in a 90°C dry heat oven. Depending on the amount of menthol added, a 3D scaffold with controllable pore structure could be obtained. Compared to structures printed in a support bath containing 60% aqueous solution, structures printed in a support bath containing 20% ​​aqueous solution exhibited higher porosity and a shorter degradation period after implantation. Figure 7 c).

[0059] Example 4 (F4)

[0060] A gel was prepared by dissolving 2 g of zein in 10 mL of glycerol formaldehyde. The resulting mixture was stirred for 6 hours to dissolve the zein, and then sonicated in an ultrasonic bath for 2 hours to remove air bubbles. The mixture was then stirred magnetically at 2000 rpm at 25°C. Gelation was observed after 36–48 hours. The viscosity of the gel at 0.1% strain was approximately 1500 Pa·s. This gel is ready for its intended application.

[0061] As in Example 1 and Figure 2e. Fiber formation was evaluated using ThT binding experiments. To prepare ciprofloxacin-loaded zein gels, 0.08 g of ciprofloxacin hydrochloride was added during the preparation of the zein gel. Carbomer 980 (0.25% w / v) dissolved in 0.01 N sodium hydroxide was dissolved in 40% and 75% (v / v) glycerol formal aqueous solutions to prepare two support baths. The ciprofloxacin-loaded zein gel was printed in the support baths. The printed structures were allowed to remain in the support baths for 12 hours. Carbomer was removed by washing with sodium chloride solution (0.9% (w / v) aqueous solution) for 30 minutes. The washed printed structures were dried in a dry heat oven at 37°C for 12 hours. Compared with structures printed in a 75% aqueous solution support bath, structures printed in a 40% aqueous solution support bath exhibited lower drug loading, slower drug release rate, and slower degradation rate.

[0062] Example 5 (F5)

[0063] A new gel was prepared by dissolving 1.0 g of zein fibrillary powder in 10 mL of 80% (v / v) acetic acid aqueous solution. The gelation time was reduced from 4 days to 3 minutes. (See Example 1 and...) Figure 2 c. The formation of fibrils was evaluated through ThT binding experiments.

[0064] To prepare a zein gel loaded with ciprofloxacin (the drug), 0.08 g of ciprofloxacin hydrochloride was added during the preparation of the novel zein gel. Carbopol 980 (0.25% w / v) dissolved in 0.01 N sodium hydroxide was dissolved in 40% and 75% (v / v) aqueous acetic acid solutions to prepare two support baths. The ciprofloxacin-loaded zein gel was printed in the Carbopol support bath. The printed structure was allowed to remain in the support bath for 12 hours. The Carbopol was removed by washing with sodium chloride solution (0.9% (w / v) aqueous solution) for 30 minutes. The washed printed structure was then dried in a dry heat oven at 37°C for 12 hours. Compared to the structure printed in the 75% aqueous solution support bath, the structure printed in the 40% aqueous solution support bath exhibited a lower drug loading, a slower drug release rate, and a slower degradation rate.

[0065] In summary, this invention relates to a zein protofibrillary powder and its preparation method, the use of this protofibrillary as a seed fiber to induce gel formation, and its biomedical application as a 3D / 4D printing ink. First, a zein gel is prepared using conventional methods. After solidification in an aqueous medium to release the solvent, the solidified material is freeze-dried to obtain a powder rich in protofibrillary fibers. This powder, along with zein raw material powder, is dissolved in various solvent systems to induce rapid gel formation. This method not only significantly shortens the gel preparation time but also has high reproducibility, providing quality assurance for the use of zein gel as a biomedical material. The novel gel induced using the first application of this invention can be 4D printed in a gradient-changing support medium. The printed material exhibits environmentally responsive intelligent features, including but not limited to biomedical functions such as adjustable drug loading, release rate, and degradation rate.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing zein gel, characterized in that, The method is as follows: dissolve zein protocellulose powder and zein raw material powder in a solvent to form a zein solution with a concentration of 10-60% by mass-volume ratio, and let it stand to form a zein gel. The solvent is an aqueous solution of ethanol with a volume fraction of 60-95%, dimethyl sulfoxide, an aqueous solution of N-methylpyrrolidone with a volume fraction of 60-95%, an aqueous solution of glycerol formaldehyde with a volume fraction of 60-95%, or an aqueous solution of acetic acid with a volume fraction of 60-95%.

2. The method for preparing a zein gel according to claim 1, characterized in that, The mass ratio of the amount of corn gliadin fibrin powder added to the mass of corn gliadin raw material powder is (10-100): (0-90), and the mass of the corn gliadin raw material powder is not 0.

3. The method for preparing a zein gel according to claim 1, characterized in that, In preparation, active substances and / or porogens are added to a zein solution to form a zein gel loaded with active substances; the active substances include ciprofloxacin hydrochloride, dexamethasone, etc. β - Glyceryl phosphate or vitamin C; the pore-forming agent includes menthol.

4. The method for preparing a zein gel according to claim 1, characterized in that, The preparation method of the zein prolysin fibrillary powder includes the following steps: Water is added to the zein gel containing solvent to solidify the gel; The solidified material was then freeze-dried to obtain zein-rich fibroin-based fibrous powder.

5. The method for preparing a zein gel according to claim 4, characterized in that, The zein gel is prepared by the following method: zein raw material powder is dissolved in a solvent to form a zein solution with a mass-volume concentration of 10-60%, and then shear stress is applied until a zein gel is formed.

6. A zein gel prepared by the method according to any one of claims 1-5.

7. An application of the zein gel as described in claim 6, characterized in that, This zein gel is used to perform 4D printing in a support bath with gradient changes.

8. The application of the zein gel according to claim 7, characterized in that, The specific process of 4D printing is as follows: the support bath material is dissolved in a solvent-water solution system and layered to form a support bath with gradient changes; zein gel is 4D printed in the support bath; after printing is completed, it is dried to obtain a 3D printed structure.

9. The application of a zein gel according to claim 7, characterized in that, The supporting bath material includes Carbopol 980, diatomaceous earth, silicate, sodium hydroxide, or silicone oil; the solvent-aqueous solution system consists of 10-80% water by volume and 20-90% ethanol, dimethyl sulfoxide, N-methylpyrrolidone, glycerol formaldehyde, or acetic acid by volume.

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