Method for improving enrichment of nanoparticles in silk and silk product

Abstract

The invention belongs to the technical field of nano-material biological application and functional textile, and particularly relates to a method for improving enrichment of nano-particles in silk and a silk product, the method for enriching in the silk comprises the following steps: (1) coating the surfaces of the nano-particles with modified silk fibroin to obtain functional nano-particles; wherein the modified silk fibroin has a group which is combined with the nanoparticles in a covalent bond form; the mass ratio of the modified silk fibroin to the nanoparticles is not less than 5; (2) adding the functionalized nanoparticles into feed for silkworms to feed the silkworms, and breeding the silkworms according to specified standardized breeding parameters of different varieties of silkworms until cocooning; wherein the functionalized nano particles added in each gram of the feed do not exceed 0.1 g on average. The enrichment amount of the nanoparticles in the silk is increased by 1.67-11.9 times compared with that of the conventional process.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological applications of nanomaterials and functional textile technologies, and particularly relates to a method for enhancing the enrichment of nanoparticles in silk and silk products. Background Art

[0002] The nanoparticle feeding method adds functional nanoparticles to silkworm feed, and utilizes the natural digestion and silk gland synthesis process of silkworms to integrate the nanoparticles into the interior of silk; this method can endow silk with antibacterial, conductive and other properties without destroying the natural structure of silk, and has the advantages of simple operation, low cost, seamless connection with traditional sericulture processes, and the potential for large-scale production. In addition, by selecting different types or surface properties of nanoparticles, such as Ag, TiO2, etc., the functions of silk can be flexibly regulated, avoiding the ecological risks brought by chemical post-treatment or gene editing.

[0003] Despite the significant advantages of the feeding method, its core bottleneck lies in the insufficient compatibility between nanoparticles and the silkworm physiological system. Unmodified nanoparticles are difficult to penetrate the lipid barrier of the silk gland due to their strong surface hydrophilicity, resulting in low absorption efficiency, forcing a large increase in the addition amount to maintain the function, which in turn causes silkworm toxicity or material waste. In addition, nanoparticles in the traditional feeding method are prone to agglomeration in the silkworm intestine or silk gland, resulting in uneven distribution in silk and poor functional stability. Existing improvement measures, such as gene editing to enhance the absorption ability or complex chemical coating, although partially alleviating the problem, introduce high costs, high toxicity or biosafety disputes and are difficult to be practical.

[0004] Therefore, it is very necessary to study a method that adapts the surface characteristics of nanoparticles to the silkworm physiological absorption mechanism while retaining the process advantages of the feeding method; by optimizing the surface characteristics and size distribution of nanoparticles, enhancing their biocompatibility with the internal environment of the silk gland, thereby significantly improving the absorption efficiency and reducing material waste; combining a standardized feeding process to precisely control the transfer path of nanoparticles in the silkworm body, and finally achieving the efficient and stable preparation of high-performance functional silk on the premise of avoiding complex processes and biosafety risks. Summary of the Invention

[0005] Based on the above-mentioned disadvantages and deficiencies existing in the prior art, one of the purposes of the present invention is to solve at least one or more of the above-mentioned problems existing in the prior art. In other words, one of the purposes of the present invention is to provide a method for enhancing the enrichment of nanoparticles in silk and silk products that meets one or more of the foregoing requirements.

[0006] In order to achieve the above-mentioned invention purpose, the present invention adopts the following technical solutions:

[0007] A method for enhancing the enrichment of nanoparticles in silk, comprising the following steps:

[0008] (1) Coating the surface of nanoparticles with modified silk fibroin to obtain functionalized nanoparticles; wherein, the modified silk fibroin has a group that binds to the nanoparticles in a covalent bond form; the mass ratio of the modified silk fibroin to the nanoparticles is not less than 5;

[0009] (2) Adding the functionalized nanoparticles to the feed of silkworms for feeding, and culturing them to cocoon formation according to the specified standardized breeding parameters for different varieties of silkworms; wherein, the amount of functionalized nanoparticles added to each gram of feed does not exceed 0.1 g.

[0010] As a preferred embodiment, in the step (1), the modified silk fibroin has at least one of a sulfhydryl group, a carboxyl group, an amino group, a hydroxyl group, an epoxy group, and a silyl group.

[0011] As a preferred embodiment, in the step (1), the particle size of the functionalized nanoparticles is 10 - 100 nm.

[0012] As a preferred embodiment, in the step (1), the nanoparticles are metal or non-metal nanoparticles.

[0013] As a preferred embodiment, in the step (1), the nanoparticles are Ag, Au, TiO2, Cu, ZnO or C.

[0014] As a preferred embodiment, in the step (2), the functionalized nanoparticles are dispersed in water with a concentration of 1 - 10 mg / mL.

[0015] As a preferred embodiment, in the step (2), the feeding is carried out three times a day.

[0016] As a preferred embodiment, in the step (2), the functionalized nanoparticles are added to the feed of silkworms starting from the 5th instar.

[0017] As a preferred embodiment, in the step (2), the standardized breeding parameters include:

[0018] The hatching period temperature is 27 - 29 °C, and the relative humidity is 90 - 95%; the temperature in the 1st - 2nd instar is 26 - 28 °C, and the relative humidity is 85 - 90%; the temperature in the 3rd instar is 24 - 26 °C, and the relative humidity is 80 - 85%; the temperature from the 4th instar to the 5th instar is 22 - 24 °C, and the relative humidity is 60 - 70%; the temperature during the cocoon formation period is 20 - 27 °C, and the relative humidity is 40 - 60%.

[0019] The present invention also provides a silk product obtained by the method according to any one of the above - mentioned embodiments.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] (1) The present invention innovatively uses silk fibroin from silkworms as a functional carrier, constructs a covalently bonded nanocomposite system through chemical modification, conducts directional functional group modification on natural silk fibroin, enables the surface active sites to form stable covalent connections with nanoparticles, and thus prepares a silk fibroin-nanoparticle complex with a core-shell structure, namely functionalized nanoparticles; the following technical advantages are achieved through a biocompatible delivery mechanism: ① Utilize the biocompatibility of homologous silk fibroin to significantly improve the compatibility of nanoparticles during the digestion and absorption process in silkworms; ② Ensure the structural stability of the coating layer during biological transport through covalent bonding, and avoid biological rejection caused by the exposure of the nanoparticle surface; ③ Rely on the high compatibility between silk fibroin and the silk matrix to promote the directional enrichment of composite nanoparticles in the silk gland; experiments have confirmed that the present invention increases the enrichment amount of nanoparticles in silk by 1.67 to 11.9 times compared with traditional processes;

[0022] (2) The present invention establishes the optimal addition threshold of nanoparticles through a toxicological model, and its dosage window is strictly controlled within the safe range of silkworm metabolism; verified by standardized breeding, the experimental group of silkworms showed excellent biocompatibility during the complete life cycle, and the toxicological safety indicators were significantly better than the industry standards, and the final survival rate and cocoon formation rate were both stably maintained above 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a flow chart of the method for enhancing the enrichment of nanoparticles in silk of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] The following details the method for enhancing the enrichment of nanoparticles in silk and silk products of the present invention.

[0025] The method of the present invention for enhancing the enrichment of nanoparticles in silk based on covalently bonded silk fibroin-coated nanoparticles overcomes the defects such as the attenuation of the enrichment amount caused by insufficient interfacial compatibility, the material loss caused by non-targeted delivery, and the potential biological safety hazards caused by excessive addition in the prior art by precisely regulating the surface biocompatibility of nanoparticles.

[0026] As Figure 1 shown, the method for enhancing the enrichment of nanoparticles in silk of the present invention includes the following steps:

[0027] (1) Coating modified silk fibroin on the surface of nanoparticles to obtain functionalized nanoparticles; wherein, the modified silk fibroin has groups that can bind to nanoparticles in a covalent bond form;

[0028] Specifically, mix the nanoparticles with deionized water to make the concentration of the nanoparticles 0.1 - 1 mg / mL, add modified silk fibroin, and the concentration in the solution reaches at least 5 mg / mL, that is, the mass ratio of modified silk fibroin to nanoparticles is greater than 5. Place the solution on a magnetic stirrer and stir it evenly at a stirring speed of 100 - 1000 rpm, reaction temperature of 20 - 60 °C, and reaction time of 0.5 - 6 h. After the reaction is completed, centrifuge to obtain functionalized nanoparticles; in addition, other methods such as photocatalysis and ultrasound can be added on this basis to improve the reaction rate.

[0029] The synthesis process of the above-mentioned nanoparticles is as follows:

[0030] Mix the nanoparticle precursor with the solvent, add a reducing agent, and the reducing agent includes but is not limited to ascorbic acid and sodium borohydride. React at a temperature of 20 - 60 °C to generate nanoparticles with a particle size of 10 nm to 100 nm; the weight-to-volume ratio of the nanoparticle precursor to the solvent is: (1 - 2) mg : (5 - 10) L, and the solvent is at least one of water or ethanol, or a mixture of the two; place the solution on a magnetic stirrer and stir it evenly at a stirring speed of: 100 - 1000 rpm, add the reducing agent dropwise, and react for 5 - 30 min; centrifuge and wash 3 times to ensure that the solvent and excessive reducing agent are removed to obtain pure nanoparticles;

[0031] The above-mentioned nanoparticle precursors include but are not limited to silver nitrate AgNO3, chloroauric acid HAuCl4, copper nitrate Cu(NO3)2, zinc nitrate Zn(NO3)2, and chloroplatinic acid H2PtCl6.

[0032] The synthesis process of the above-mentioned modified silk fibroin is as follows:

[0033] Natural silk fibroin can be commercially purchased or extracted from silk cocoons, and the extraction method uses conventional technical means in the art, which will not be elaborated here;

[0034] Disperse natural silk fibroin in water at a ratio of 1-5% w / v, mix and activate it with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC / N-hydroxysuccinimide NHS. The molar ratio of EDC:NHS:silk fibroin is 5-20:5-20:1. Add phosphate buffer solution PBS to adjust the activation environment to pH 6.5. The activation temperature is 20-30°C and the activation time is 15-40 min. After activation, adjust the pH to 7.0-7.5 and add 1-5 mM tris(2-carboxyethyl)phosphine TCEP to prevent oxidation. Subsequently, add a reagent with a group that can graft to the activated silk fibroin and covalently bond to the nanoparticles. The molar ratio of the activated silk fibroin to the grafting reagent is 1:8-20. The reaction temperature is 20-27°C and the reaction time is 2-8 h. The grafting rate is controlled at 10-30% to avoid protein denaturation. After the reaction, purify it using a dialysis bag. The cut-off molecular weight of the dialysis bag used for dialysis is 4-14 KDa. The dialysis solution is PBS buffer solution, the temperature is 4°C, and the dialysis time is 72 hours with 3 buffer changes.

[0035] The above grafting reagents are cysteine, oleic acid, ethylenediamine, polyethylene glycol, 3-glycidoxypropyltrimethoxysilane, 3-aminopropyltriethoxysilane, which are specifically determined according to actual needs.

[0036] (2) Add the functionalized nanoparticles to the silkworm feed and feed them, and raise them to cocoon formation according to the specified standardized breeding parameters for different varieties of silkworms. Among them, the amount of functionalized nanoparticles added to each gram of feed does not exceed 0.1 g.

[0037] In one or more embodiments, in the above step (1), the modified silk fibroin has at least one of sulfhydryl, carboxyl, amino, hydroxyl, epoxy, and silyl groups.

[0038] In one or more embodiments, in the above step (1), the particle size of the functionalized nanoparticles is 10-100 nm.

[0039] In one or more embodiments, in the above step (1), the nanoparticles are metal or non-metal nanoparticles.

[0040] In one or more embodiments, in the above step (1), the nanoparticles are Ag, Au, TiO2, Cu, ZnO, or C.

[0041] In one or more embodiments, in the above step (1), optionally, the finally obtained modified silk fibroin solution (with PBS as the solvent) can remove PBS by dialysis or choose to retain it, which is specifically determined according to the nanoparticles selected in the subsequent steps to ensure that PBS itself does not affect the nanoparticles.

[0042] In one or more embodiments, in the above step (2), the functionalized nanoparticles are dispersed in water at a concentration of 1-10 mg / mL.

[0043] In one or more embodiments, in the above step (2), feed three times a day to efficiently transfer the nanoparticles into the silk.

[0044] In one or more embodiments, in the above step (2), start adding functionalized nanoparticles to the silkworm feed from the fifth instar.

[0045] In one or more embodiments, in the above step (2), the standardized breeding parameters include:

[0046] The incubation temperature is 27-29 °C and the relative humidity is 90-95%; the temperature for the first and second instars is 26-28 °C and the relative humidity is 85-90%; the temperature for the third instar is 24-26 °C and the relative humidity is 80-85%; the temperature for the fourth to fifth instars is 22-24 °C and the relative humidity is 60-70%; the temperature during the cocooning period is 20-27 °C and the relative humidity is 40-60%.

[0047] The present invention also provides a silk product obtained by the method as described above, which can endow it with antibacterial, conductive and other properties without destroying the natural structure of the silk.

[0048] The following further explains the method for enhancing the enrichment of nanoparticles in silk and the silk product of the present invention through specific examples.

[0049] Example 1:

[0050] This example specifically describes a method for enhancing the enrichment of silver nanoparticles in silk by coating them with thiolated silk fibroin, including the following steps:

[0051] (1) Select AgNO3 as the nanoparticle precursor and L-ascorbic acid as the reducing agent;

[0052] Add 0.05 mL of 0.1 mol / L AgNO3 solution to 70 mL of deionized water, keep the temperature at 27 °C, dissolve 0.03 g of L-ascorbic acid in 10 mL of deionized water, and then drop it into the AgNO3 solution at a rate of 2 drops per second. React for 5 minutes with a stirring speed of 300 rpm. Centrifuge and wash the obtained silver nanoparticle mixture three times to obtain a pure silver nanoparticle precipitate; the morphology of the silver nanoparticles is spherical-like with a particle size of about 100 nm;

[0053] (2) Disperse natural silk fibroin in water at a ratio of 3% w / v, mix it with EDC / NHS (the molar ratio of EDC:NHS:silk fibroin is 10:10:1) for activation, add phosphate buffer solution (PBS) to adjust the activation environment to pH 6.5, with an activation temperature of 27 °C and an activation time of 20 min; after activation, adjust the pH to 7.0 and add 3 mM TCEP to prevent oxidation;

[0054] Add cysteine (the molar ratio of activated silk fibroin:cysteine is 1:15), with a reaction time of 4 h and a reaction temperature of 27 °C;

[0055] After the reaction, purify it using a dialysis bag. The cut-off molecular weight of the dialysis bag used for dialysis is 7 KDa, the dialysis solution is PBS buffer solution, the temperature is 4 °C, and the dialysis time is 72 hours, with the solution changed 3 times to obtain a modified silk fibroin solution, and the solvent is PBS buffer solution;

[0056] (3) Add the above-mentioned silver nanometer precipitate to deionized water to make its concentration 0.5 mg / mL, and keep the temperature at 50 °C; add modified silk fibroin, with its concentration in the solution being 10 mg / mL, react for 1 h, and after the reaction, centrifuge to obtain a functionalized silver nanometer precipitate;

[0057] In addition, add deionized water to the functionalized silver nanometer precipitate to adjust its concentration to 1 mg / mL, store it in the refrigerator to reduce its aggregation, and ultrasonicate it in an ultrasonic machine for 10 min each time it is taken out for use;

[0058] (4) While preparing silver nanometers, silkworms are also being raised. The specific steps are as follows:

[0059] Place silkworm eggs in an artificial climate chamber, set the temperature to 28 °C, the relative humidity to 95%, and the light to alternate between light and dark for 12 hours;

[0060] After the newly hatched silkworm larvae emerge, set the temperature to 27 °C, the relative humidity to 85%, and the light to alternate between 16 hours (light) and 8 hours (dark);

[0061] The temperature, humidity, and light duration settings for raising silkworms in the second instar are the same as those in the first instar;

[0062] The temperature for raising silkworms in the third instar is 25 °C, the relative humidity is 80%, and the light duration is the same as that in the first instar;

[0063] The temperatures for raising silkworms in the fourth and fifth instars are both 23 °C, the relative humidity is 65%, and the light duration is the same as that in the first instar;

[0064] On the first day of the fifth instar stage, fresh mulberry leaves were fed to the silkworms at 6:00 am, 2:00 pm, and 10:00 pm every day. On average, each silkworm could be allocated 2 g of mulberry leaves each time. The functionalized silver nanoparticles were sprayed on the surface of the mulberry leaves using a spray bottle to make it more uniform, and each mulberry leaf was sprayed 10 times on average.

[0065] The temperature during the cocooning stage was 25 °C, the relative humidity was 60%, and the light duration was the same as that during the hatching stage.

[0066] The cocoons were dried in a forced-air drying oven. First, they were dried at 110 °C for 1 h, and then at 80 °C for 3 h.

[0067] After ICP-OES detection, the average silver content in the silk obtained by feeding the silkworms with silk fibroin-coated silver nanoparticles was about 5 mg / kg.

[0068] Comparative Example 1:

[0069] The difference between the method of this comparative example and that of Example 1 is that: the pure silver nanoparticle precipitate of Example 1 was directly used to replace the functionalized silver nanoparticle precipitate of Example 1 and sprayed on the surface of the mulberry leaves, and the dosage and the feeding process of the silkworms were the same as those of Example 1.

[0070] After ICP-OES detection, the silver content in the silk obtained by feeding the silkworms with uncoated silver nanoparticles was about 0.6 mg / kg.

[0071] Comparative Example 2:

[0072] The difference between the method of this comparative example and that of Example 1 is that: sodium citrate was used to replace the modified silk fibroin as the coating agent.

[0073] The pure silver nanoparticle precipitate of Example 1 was added to deionized water to make its concentration 0.5 mg / mL, and the temperature was kept at 50 °C; 0.05 g of sodium citrate was weighed and dissolved in 10 mL of deionized water, and then it was added dropwise to the silver nanoparticle solution and reacted for 30 min. After the reaction, it was centrifuged three times, and the detergent added each time was deionized water, aiming to wash away the excess sodium citrate to obtain functionalized silver nanoparticles.

[0074] In addition, the feeding process of the silkworms was the same as that of Example 1.

[0075] After ICP-OES detection, the silver content in the silk obtained by feeding the silkworms with hydrophilic silver nanoparticles was about 0.42 mg / kg.

[0076] Comparative Example 3:

[0077] The difference between the method of this comparative example and that of Example 1 is that: the silk fibroin was not modified and was directly coated on the surface of the silver nanoparticles for subsequent feeding.

[0078] The pure nano-silver precipitate of Example 1 was added to deionized water to make its concentration 0.5 mg / mL, and the temperature was kept at 50 °C; silk fibroin was added, and its concentration in the solution was 10 mg / mL. After reacting for 1 h, the functionalized nano-silver precipitate was obtained by centrifugation after the reaction;

[0079] In addition, the feeding process of the silkworms was the same as that in Specific Example 1;

[0080] Detected by ICP-OES, the silver content in the silk obtained by feeding the silkworms with the functionalized nano-silver was about 2.98 mg / kg.

[0081] Comparing Example 1 and Comparative Example 1 above, the nano-silver in the silk of the former was more than 8 times that of the latter. It can be seen that the nano-silver wrapped by the modified silk fibroin is easier to enter the silk gland and accumulate in the silk than the nano-silver without any coating;

[0082] Comparing Example 1 and Comparative Example 2 above, the nano-silver in the silk of the former was about 11.9 times that of the latter. It can be seen that the nano-silver wrapped by the modified silk fibroin is easier to enter the silk gland and accumulate in the silk than the nano-silver wrapped by other substances;

[0083] Comparing Example 1 and Comparative Example 3 above, the nano-silver in the silk of the former was 1.67 times that of the latter. It can be seen that the nano-silver covalently bonded with silk fibroin can enter the silk gland and accumulate in the silk better than the nano-silver non-covalently bonded with silk fibroin;

[0084] The nano-silver tightly bonded with the modified silk fibroin in covalent bond provided by the present invention has an enrichment amount in the silk increased by 1.67 - 11.9 times compared with the traditional nano-silver with a bare surface, coated with other reagents or non-covalently bonded with silk fibroin, significantly improving the enrichment efficiency of the silk for nano-particles; and the addition period and addition amount of the nano-particles in the silkworm feed ensure that the survival rate of the silkworm body > 90%, realizing safe and efficient production.

[0085] In view of the large number of embodiments of the present invention, the raw materials and dosages involved can be selected according to actual needs within the limited range. The experimental data of each embodiment are huge and numerous, and it is not suitable to list them one by one here. However, the contents to be verified and the final conclusions obtained in each embodiment are close. Therefore, the verification contents of each embodiment will not be described one by one here.

[0086] The above is only a detailed description of the preferred embodiments and principles of the present invention. For those of ordinary skill in the art, according to the idea provided by the present invention, there will be changes in the specific implementation manners, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A method for enhancing the enrichment of nanoparticles in silk, characterized in that: The following steps are involved: (1) coating the surface of nanoparticles with modified silk fibroin to obtain functionalized nanoparticles; wherein the modified silk fibroin has a group that is covalently bonded to the nanoparticles; and the mass ratio of the modified silk fibroin to the nanoparticles is not less than 5; (2) Adding the functionalized nanoparticles to the feed of the silkworms and feeding them, and breeding them according to standardized breeding parameters specified for different silkworm species until they form cocoons; wherein, the average amount of the functionalized nanoparticles added per gram of feed does not exceed 0.1 g.

2. The method according to claim 1, characterized in that: In the step (1), the modified silk fibroin has at least one of a sulfur group, a carboxyl group, an amino group, a hydroxyl group, an epoxy group, and a silane group.

3. The method according to claim 1, characterized in that In the step (1), the particle size of the functionalized nanoparticles is 10 to 100 nm.

4. The method according to claim 1, characterized in that: In the step (1), the nanoparticles are metal or non-metal nanoparticles.

5. The method according to claim 4, characterized in that In the step (1), the nanoparticles are Ag, Au, TiO2, Cu, ZnO or C.

6. The method according to claim 1, characterized in that In the step (2), the functionalized nanoparticles are dispersed in water at a concentration of 1 to 10 mg / mL.

7. The method according to claim 1, characterized in that In the step (2), feeding is performed three times a day.

8. The method according to claim 1, characterized in that In the step (2), functionalized nanoparticles are added to the feed of silkworms starting from the fifth instar.

9. The method according to claim 1, characterized in that: In the step (2), the standardized breeding parameters include: The temperature during the incubation period is 27-29°C, and the relative humidity is 90-95%; the temperature during the 1st to 2nd age is 26-28°C, and the relative humidity is 85-90%; the temperature during the 3rd age is 24-26°C, and the relative humidity is 80-85%; the temperature during the 4th to 5th age is 22-24°C, and the relative humidity is 60-70%; the temperature during the cocooning period is 20-27°C, and the relative humidity is 40-60%.

10. A silk product obtained by the method according to any one of claims 1 to 9.

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