CN107157952B - Silk fibroin nanoparticle and drug-loaded silk fibroin nanoparticle - Google Patents
Silk fibroin nanoparticle and drug-loaded silk fibroin nanoparticle Download PDFInfo
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Abstract
The invention provides silk fibroin nanoparticles and drug-loaded silk fibroin nanoparticles, wherein the silk fibroin nanoparticles are prepared by the following method: dissolving silk fibroin in water, and standing for 2-8 h at 2-6 ℃ to obtain a silk fibroin solution; mixing silk fibroin solution and an organic solvent in a volume ratio of 1: 4-8, and stirring to obtain a nanoparticle suspension, wherein the organic solvent is selected from ethanol, isopropanol or acetone; carrying out centrifugal separation on the nanoparticle suspension, washing with deionized water, and centrifuging to obtain a nanoparticle precipitate; and dispersing the nanoparticle precipitate by deionized water to obtain the silk fibroin nanoparticle. The silk fibroin nanoparticles after drug loading can improve the intake of the drug in cells, prolong the retention time of the drug, have the advantages of high intracellular efficiency, long-acting effect and targeting, and are beneficial to improving the bioavailability of the drug; has good biological adhesion, no immunogenicity, no obvious inflammatory reaction and tissue fibrosis, good biocompatibility and safety.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to silk fibroin nanoparticles and drug-loaded silk fibroin nanoparticles.
Background
The posterior segment of the eye, which mainly comprises age-related macular degeneration, retinopathy of prematurity and diabetic retinopathy, is the main reason for the loss of vision and blindness of patients at present. Common approaches to the clinical treatment of posterior segment ocular neovascular disease include: systemic administration (such as oral administration and intravenous injection), periocular administration, and intravitreal administration. Wherein the compliance of the systemic administration route is high, but because of the obstruction of blood-retina barrier and blood brain barrier to the medicine, only 1 to 5 percent of the medicine can enter the vitreous body finally, the bioavailability of the eye is extremely low, and the repeated administration is needed, thereby causing large side effect of the whole body. The drugs of the periocular route are mostly cleared by the capillaries of the conjunctiva and choroid, and the physiological barriers of the sclera, Bruch's membrane and retinal pigment epithelium layer make it difficult for the drugs of the periocular route to reach the retina for therapeutic effect. Intravitreal administration is currently considered to be the most effective method for treating posterior segment eye disease compared to other modes of administration, and can break through multiple barriers such as the cornea, sclera, blood-retinal barrier, choroid, and conjunctival blood flow elimination, and achieve higher drug concentrations in the vitreous and retina. In particular, for large molecule protein drugs, intravitreal injection is the best route for drug molecules to penetrate the multiple barriers of the cornea, sclera, blood-retinal barrier, choroid, etc., and ultimately reach the retina and choroid for efficacy.
Frequent vitreous injection administration easily brings adverse reactions and wounds to patients, and a drug delivery system with a slow release effect can effectively reduce the administration frequency and increase the retention of drugs in eyes through vitreous injection, thereby finally achieving the purposes of improving the bioavailability of the drugs and the medication compliance of the patients.
The drug is encapsulated in a carrier material or connected to the surface of the carrier material in a chemical crosslinking or electrostatic adsorption mode to form colloidal particles with the particle size of 10-1000 nm, and the colloidal particles are nanoparticles, and both water-soluble and fat-soluble drugs can be used in a nanoparticle delivery system. Cells have phagocytosis on nanoparticles with the particle size of less than 250nm, and after vitreous injection, the nanoparticles are more easily gathered on retina, so that the uptake of medicine by retina is effectively improved; in addition, some specific groups can be connected to the surface of the nanoparticle, and the specific binding effect of the specific groups and proteins or other receptors on the surface of cells is utilized to improve the uptake of the cells to the nanoparticle. Therefore, compared with other ophthalmic drug delivery preparations such as in situ gel, microspheres and the like, the nanoparticles have unique advantages in the treatment of retinal diseases.
Disclosure of Invention
In view of the above, the present invention provides silk fibroin nanoparticles and drug-loaded silk fibroin nanoparticles, which can improve the bioavailability of drugs.
The invention provides silk fibroin nanoparticles, which are prepared by the following method:
s1) dissolving silk fibroin in water, and standing for 2-8 h at 2-6 ℃ to obtain a silk fibroin solution;
s2) mixing a silk fibroin solution with a volume ratio of 1: 4-8 with an organic solvent, and stirring to obtain a nanoparticle suspension, wherein the organic solvent is selected from ethanol, isopropanol or acetone;
s3) carrying out centrifugal separation on the nanoparticle suspension, re-dispersing and washing with deionized water, and centrifuging to obtain a nanoparticle precipitate;
s4) mixing the nanoparticle precipitate with water, and performing ultrasonic treatment to obtain the silk fibroin nanoparticles.
Preferably, the concentration of the fibroin solution in the step S1) is 10-50 mg/mL.
Preferably, the mixing temperature in the step S2) is 20-60 ℃.
Preferably, the rotation speed of the centrifugation in the step S3) is 8000-20000 rpm; the temperature of the centrifugation is 4 ℃; the centrifugation time is 25-35 min.
Preferably, the concentration of the fibroin nanoparticles in the step S4) is 18-22 mg/mL.
The invention provides a drug-loaded silk fibroin nanoparticle which is prepared by the following method:
mixing the silk fibroin nanoparticles and the protein medicine, adding a cross-linking agent for cross-linking reaction, adding a terminating agent for terminating reaction, centrifuging the reaction product, and separating to obtain a precipitate product;
and re-dispersing the precipitation product, performing centrifugal washing and separation for multiple times, and adding water for ultrasonic re-dispersion to obtain the drug-loaded silk fibroin nanoparticles.
Preferably, the crosslinking agent is selected from glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide or N-hydroxysuccinimide.
Preferably, the mass ratio of the silk fibroin nanoparticles to the protein drug is 5-30: 1.
Preferably, the centrifugal temperature of the reaction product is 2-5 ℃; centrifuging the reaction product for 28-33 min; the centrifugal rotating speed of the reaction product is 15500-16500 rpm.
Preferably, the temperature of the crosslinking reaction is 2-5 ℃; the time of the crosslinking reaction is 22-28 h.
The invention provides silk fibroin nanoparticles, which are prepared by the following method: s1) dissolving silk fibroin in water, and standing for 2-8 h at 2-6 ℃ to obtain a silk fibroin solution; s2) mixing a silk fibroin solution with a volume ratio of 1: 4-8 with an organic solvent, and stirring to obtain a nanoparticle suspension, wherein the organic solvent is selected from ethanol, isopropanol or acetone; s3) carrying out centrifugal separation on the nanoparticle suspension, re-dispersing and washing with deionized water, and centrifuging to obtain a nanoparticle precipitate; s4) mixing the nanoparticle precipitate with water, and performing ultrasonic treatment to obtain the silk fibroin nanoparticles. The silk fibroin nanoparticles provided by the invention can improve the intracellular intake of the drug, prolong the retention time of the drug, have the advantages of intracellular high efficiency, long-acting effect and targeting, and are beneficial to improving the bioavailability of the drug. In addition, the silk fibroin nanoparticle has good biological adhesion, no immunogenicity, no obvious inflammatory reaction and tissue fibrosis, and good biocompatibility and safety. The silk fibroin nanoparticles are prepared by an anti-solvent method, and the drug-loaded silk fibroin nanoparticles are obtained by a cross-linking method, so that the whole preparation process is simple to operate, mild in condition and good in reproducibility. The experimental results show that: the average grain diameter of the silk fibroin nano-particles is below 250 nm; the silk fibroin nano-particles are obviously electronegative; 1mg/mL drug-loaded silk fibroin nanoparticle can continuously and slowly release drug in vitro for about one week; the uptake rate of ARPE-19 cells to the drug-loaded silk fibroin nanoparticles within 1h can reach more than 97%.
Drawings
FIG. 1 is a graph showing the results of the average particle size and polydispersity index of silk fibroin nanoparticles prepared in examples 1 to 3 of the present invention;
FIG. 2 is a scanning electron microscope image of drug-loaded silk fibroin nanoparticles prepared in embodiments 1-3 of the present invention;
FIG. 3 shows the drug encapsulation efficiency results of drug-loaded silk fibroin nanoparticles prepared in embodiments 1-3 of the present invention;
FIG. 4 shows in vitro cumulative release rate results of drug-loaded silk fibroin nanoparticles prepared in embodiments 1-3 of the present invention;
FIG. 5 shows the results of the cell uptake rate of ARPE-19 cells at different times for the drug-loaded silk fibroin nanoparticles prepared in examples 1-3 and the FITC-BSA solution.
Detailed Description
The invention provides silk fibroin nanoparticles, which are prepared by the following method:
s1) dissolving silk fibroin in water, and standing for 2-8 h at 2-6 ℃ to obtain a silk fibroin solution;
s2) mixing a silk fibroin solution with a volume ratio of 1: 4-8 with an organic solvent, and stirring to obtain a nanoparticle suspension, wherein the organic solvent is selected from ethanol, isopropanol or acetone;
s3) carrying out centrifugal separation on the nanoparticle suspension, re-dispersing and washing with deionized water, and centrifuging to obtain a nanoparticle precipitate;
s4) mixing the nanoparticle precipitate with water, and performing ultrasonic treatment to obtain the silk fibroin nanoparticles.
The method comprises the steps of dissolving silk fibroin in water, and standing for 2-8 hours at the temperature of 2-6 ℃ to obtain a silk fibroin solution. The silk fibroin is preferably dissolved in water under stirring. The present invention preferably employs deionized water for dissolution. In the specific embodiment of the invention, silk fibroin is dissolved in deionized water and is kept stand for 8 hours at 4 ℃; or standing for 4h at 4 ℃; or standing at 4 deg.C for 2 h. In the invention, the concentration of the silk fibroin solution is preferably 10-50 mg/mL, namely the concentration of the silk fibroin solution is preferably 1.0-5.0% (w/v); in particular embodiments of the invention, the concentration of the silk fibroin solution is 1.0%, 2.5%, or 5%.
The silk fibroin solution and an organic solvent in a volume ratio of 1: 4-8 are mixed and stirred to obtain a nanoparticle suspension, wherein the organic solvent is selected from ethanol, isopropanol or acetone. The silk fibroin solution is preferably extracted by an injector and then is quickly mixed with an organic solvent; in the embodiment of the present invention, it is preferable to rapidly inject the silk fibroin solution into the organic solvent. According to the invention, the organic solvent is preferably heated to a temperature required by mixing to obtain a constant-temperature organic solvent; the mixing temperature is preferably 20-60 ℃. The present invention preferably employs a magnetic stirrer well known to those skilled in the art for stirring; preferably, stirring is carried out until a milky white suspension of nanoparticles is formed.
After the nanoparticle suspension is obtained, the nanoparticle suspension is centrifugally separated, added with deionized water for redispersion and washing, and centrifuged to obtain a nanoparticle precipitate. In the invention, the rotation speed of the nanoparticle suspension liquid is preferably 8000-20000 rpm; the preferred temperature for the centrifugation is 4 ℃; the time for centrifugation is preferably 25-35 min, more preferably 28-32 min, and most preferably 30 min. The invention preferably centrifuges the nanoparticle suspension, discards the supernatant, redisperses and washes the precipitate with deionized water, centrifuges the nanoparticle precipitate again, and repeats the operation for 3 times.
After the nanoparticle precipitate is obtained, the silk fibroin nanoparticle is obtained by mixing the nanoparticle precipitate with water and carrying out ultrasonic treatment. In the invention, the silk fibroin nanoparticles exist in the form of silk fibroin nanoparticle suspension; the concentration of the silk fibroin nanoparticle suspension is preferably 18-22 mg/mL, namely the concentration of the silk fibroin nanoparticle suspension is preferably 1.8-2.2% (w/v), more preferably 1.9-2.1%, and most preferably 2.0%.
The silk fibroin nanoparticles prepared by the anti-solvent method have the average particle size of less than 250nm and uniform particle size. The silk fibroin nanoparticle has good biological adhesion, no immunogenicity, no obvious inflammatory reaction and tissue fibrosis, and good biocompatibility and safety.
The invention provides a drug-loaded silk fibroin nanoparticle which is prepared by the following method:
mixing the silk fibroin nanoparticles and the medicine in the technical scheme, adding a cross-linking agent for cross-linking reaction, adding a terminating agent for terminating reaction, centrifuging the reaction product, and separating to obtain a precipitate product;
and re-dispersing the precipitation product, performing centrifugal washing and separation for multiple times, and adding water for ultrasonic re-dispersion to obtain the drug-loaded silk fibroin nanoparticles.
The silk fibroin protein-loaded macromolecular protein drug nanoparticles prepared by the cross-linking method can obviously improve the intracellular intake of macromolecular protein drugs and prolong the retention time of the drugs, have the advantages of intracellular high efficiency, long-acting effect and targeting, and are beneficial to improving the bioavailability of the drugs.
The silk fibroin nanoparticles and the protein medicine are mixed, a cross-linking agent is added for cross-linking reaction, a terminating agent is added for terminating reaction, and a reaction product is centrifuged and separated to obtain a precipitate product.
In the present invention, the protein drug is specifically Bovine Serum Albumin (BSA); for the convenience of detection, bovine serum albumin is preferably subjected to fluorescein labeling; the present invention is preferably labeled with Fluorescein Isothiocyanate (FITC). In the invention, the mass ratio of the silk fibroin nanoparticles to the protein drug is preferably 5-30: 1, and preferably 5-15: 1; in a specific embodiment of the invention, the mass ratio of the silk fibroin nanoparticles to the protein drug is specifically 5:1, 15:1 or 30: 1.
In the present invention, the crosslinking agent is preferably selected from glutaraldehyde, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) or N-hydroxysuccinimide (NHS). The cross-linking agent enables the drug to be connected to the silk fibroin nanoparticles.
In the present invention, the terminator used in the crosslinking reaction is preferably glycine. The temperature of the crosslinking reaction is preferably 2-5 ℃, and more preferably 4 ℃; the time of the crosslinking reaction is preferably 22-28 h, and more preferably 24 h. The centrifugal temperature of the reaction product is preferably 2-5 ℃, and more preferably 4 ℃; the time for centrifuging the reaction product is preferably 28-33 min, and more preferably 30 min; the centrifugal rotating speed of the reaction product is 15500-16500 rpm, and is more preferably 16000 rpm.
The invention preferably re-disperses the precipitation product with deionized water, washes and centrifuges, repeats three times, and finally adds deionized water to carry out ultrasonic re-dispersion to obtain the drug-loaded silk fibroin nanoparticles.
The method for preparing the silk fibroin protein-loaded macromolecular protein drug nanoparticles by the anti-solvent method and the crosslinking method has the advantages of simple operation, mild conditions and good reproducibility of the preparation process, does not influence the basic structure of the biological macromolecular protein drug, and has practical value of clinical application.
The prepared silk fibroin nanoparticle dispersion liquid is diluted by 10 times by deionized water, and a Malvern laser particle size analyzer (Malvern, Nano ZS90) is adopted to determine the average particle size and the polydispersity index (PDI) of the nanoparticles. The examples show that: the PDI of the silk fibroin nanoparticles is 0.22 +/-0.04; or 0.15 plus or minus 0.03; or 0.31 ± 0.06.
The invention observes the form of the prepared suspension of the drug-loaded silk fibroin nanoparticles: dropping the suspension carrying the fibroin protein nanoparticles on tinfoil, after the tinfoil is dried in the air, adhering the tinfoil on a copper plate, spraying gold, and observing the morphology of the nanoparticles by adopting a scanning electron microscope (JEOL Ltd., JSM-6330F) after vacuum drying.
After the cross-linking reaction of FITC-BSA and silk fibroin nanoparticles is finished, centrifuging reaction liquid for 30min at the rotation speed of 16000rpm at 4 ℃, collecting supernatant, washing the nanoparticles with quantitative deionized water, collecting washing liquid, repeating the operation for 3 times, combining the supernatant and the washing liquid, measuring the fluorescence intensity of the mixed solution by a multifunctional enzyme-linked immunosorbent assay (BIO TEK, Synergy H1), and calculating the amount of free drugs. The encapsulation efficiency is calculated by the following formula:
the results of the examples show that: the encapsulation efficiency is 67.11 +/-3.56 percent; or 66.92 +/-6.89%; or 58.78 ± 4.21%.
The invention dilutes drug-loaded silk fibroin nanoparticles with PBS (pH value 7.4) to obtain dispersion liquid with the concentration of 1mg/mL of the drug-loaded silk fibroin nanoparticles, respectively takes 1mL of the dispersion liquid to a centrifuge tube, carries out in-vitro release research in a constant-temperature air bath shaking table with the temperature of 37 ℃ and the rotating speed of 100rpm, takes out 3 tubes from each group for 20min at 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 168h and 192h, measures the fluorescence intensity of supernatant by adopting a multifunctional microplate reader, calculates the drug release amount of FITC-BSA and calculates the cumulative drug release rate.
The invention adopts flow cytometry to quantitatively determine the silk fibroin-loaded FITC-BSA nanoparticle taken by cells, and the method comprises the following steps:
taking acute retinal pigment epithelium-19 cells (ARPE-19) as an experimental object, adopting a flow cytometer (Beckman Coulter, EPICS XL) to quantitatively detect the uptake condition of the prepared silk fibroin FITC-BSA nanoparticle-loaded cells by the cells, and taking FITC-BSA culture solution with equal concentration as a control, wherein the specific experimental operation steps are as follows:
(1) dispersing the silk fibroin FITC-BSA nanoparticles prepared by DMEM/F12 culture solution containing 0.4% fetal calf serum to ensure that the concentration of the nanoparticles is 500 mu g/mL, and preparing culture solution with equal FITC-BSA concentration;
(2) ARPE-19 cells were cultured at 2.5X 104The density of each well was inoculated in 12-well plates at 37 ℃ with 5% CO2Culturing for 24h under the condition until the cells grow completely adherent;
(3) absorbing original culture solution, adding the prepared drug-loaded nanoparticles and FITC-BSA culture solution with equal concentration respectively, and culturing at 37 deg.C and 5% CO2Incubating for 1-24 h under the condition;
(4) the culture medium was aspirated, and the cells were washed 3 times with pre-cooled PBS, terminating uptake;
(5) adding trypsin to digest cells, centrifuging for 5min at 4 ℃ and 1000rpm, removing supernatant, and collecting cells;
(6) the cells were resuspended in 500. mu.L of PBS solution, and immediately the amount of cell uptake was measured by flow cytometry, and 1X 10 cells were counted4And (4) cells.
The results of the examples show that: the uptake rate of ARPE-19 cells to the drug-loaded silk fibroin nanoparticles within 1h reaches 96.64 +/-3.08%; or 97.48 + -2.22%; or 88.96 + -3.25%.
The silk fibroin nanoparticles prepared by the anti-solvent method have uniform particle size and good biocompatibility, can successfully entrap macromolecular protein drugs through a cross-linking agent, and can continuously release drugs in vitro for one week. Compared with the macromolecular solution, the silk fibroin nanoparticles can obviously improve the drug uptake rate of cells, and the drug-loaded silk fibroin nanoparticles provide a new method for the macromolecular protein drugs for treating the posterior segment of the eye neovascular diseases.
Aiming at the problems of short half-life period, poor membrane permeability, low bioavailability, poor curative effect, various adverse reactions caused by frequent medication and the like of macromolecular protein medicines for treating eye diseases, the invention constructs a long-acting, high-efficiency and targeted drug delivery system of fibroin protein-loaded macromolecular protein medicine nanoparticles by using an anti-solvent method and a cross-linking method, and treats the posterior segment of the eye neovascular diseases by vitreous injection drug delivery. By means of the adhesiveness and the long-acting drug release property of silk fibroin, the tissue repair and reconstruction performance can be promoted, the retina targeting property of the nanoparticles is realized, the uptake of macromolecular drugs by the posterior segment of the eye, particularly the retina, is improved, the tissue repair and reconstruction of retinal pigment epithelial cells are promoted, the drug release time is prolonged, the purpose of efficiently, long-acting and targeted treatment of the neovascularization diseases at the posterior segment of the eye is realized, and the adverse reaction and the injury brought to patients by repeated drug administration are reduced.
For further illustration of the present invention, the following examples are provided to describe the silk fibroin nanoparticles and drug-loaded silk fibroin nanoparticles in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Preparation of Silk fibroin nanoparticles
Dissolving silk fibroin material in deionized water, stirring until silk fibroin is completely dissolved, preparing into silk fibroin solution with concentration of 1.0% (w/v), and standing at 4 deg.C for 2 hr. And (2) extracting the silk fibroin solution by using an injector, quickly injecting the silk fibroin solution into a constant-temperature ethanol solution at the temperature of 60 ℃, wherein the volume ratio (v/v) of the ethanol to the silk fibroin solution is 4:1, continuously stirring the mixed solution by using a magnetic stirrer until forming milky nanoparticle suspension, and continuously stirring for 30min at the constant temperature. And (3) when the nanoparticles are completely formed, transferring the nano suspension into a centrifuge tube, centrifuging for 30min at 4 ℃ and 16000rpm, removing supernatant, redispersing and washing the precipitate with deionized water, centrifuging again to separate the nanoparticles, and repeating the operation for 3 times. Finally, a certain amount of deionized water is added into the washed nanoparticles, the nanoparticles are uniformly dispersed into a suspension with the concentration of 2.0 percent (w/v) by ultrasonic treatment, and the suspension is stored for later use under the low-temperature condition.
The average particle size and the polydispersity of the silk fibroin nanoparticles are measured by the testing method of the technical scheme, the result is shown in figure 1, and figure 1 is a result graph of the average particle size and the polydispersity of the silk fibroin nanoparticles of the embodiments 1-3 of the invention. As can be seen from fig. 1: the average particle size of the silk fibroin nanoparticles prepared in example 1 is 168.8 +/-7.4 nm, and the PDI is 0.22 +/-0.04; the Zeta potential value is-25.57 +/-0.99 mV, which shows that the silk fibroin nano-particle has obvious electronegativity.
2. Preparation of silk fibroin-loaded fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) nanoparticles
Measuring 1mL of the silk fibroin nanoparticle dispersion liquid with the concentration of 2.0% (w/v) prepared in the step 1, adding 4mg of FITC-BSA (fluorescein isothiocyanate-bovine serum albumin), namely the mass ratio of the silk fibroin nanoparticles to the medicine is 5:1, after uniformly mixing, dripping 100 mu L of cross-linking agent glutaraldehyde with the concentration of 25% (w/v) into the mixed solution, and continuously stirring for 24 hours in a dark place at 4 ℃. Adding 500 mu L of glycine solution (200mg/mL) to terminate the reaction, centrifuging the reaction solution for 30min at the rotation speed of 16000rpm and 4 ℃, separating supernatant, redispersing the precipitate with deionized water, then centrifuging and washing free FITC-BSA, repeating the operation for 3 times, finally adding deionized water into the washed drug-loaded nanoparticles, and performing ultrasonic treatment to uniformly disperse the nanoparticles to obtain the silk fibroin nanoparticle suspension loaded with FITC-BSA.
The invention adopts the technical scheme that the test method is used for observing the form of the drug-loaded silk fibroin nanoparticles, the result is shown in figure 2, and figure 2 is a scanning electron microscope image of the drug-loaded silk fibroin nanoparticles of the embodiments 1-3 of the invention. As can be seen from fig. 2: the drug-loaded silk fibroin nanoparticles prepared in example 1 are smooth and spherical, and only a small amount of nanoparticles are adhered.
The invention carries out the measurement and calculation of the encapsulation efficiency according to the test method of the encapsulation efficiency of the technical proposal; the result is shown in fig. 3, and fig. 3 shows the result of the drug encapsulation efficiency of the drug-loaded silk fibroin nanoparticles prepared in embodiments 1-3 of the present invention; FIG. 3 shows: in the embodiment 1, when the mass ratio of the silk fibroin nanoparticles to the medicine is 5:1, the encapsulation efficiency is 58.78 +/-4.21%; the amount of silk fibroin nanoparticles may not be sufficient to completely load the drug, resulting in more free drug and a reduced encapsulation efficiency compared to example 2.
The method for measuring the in vitro drug release effect of the technical scheme is used for measuring the cumulative drug release rate of the drug-loaded silk fibroin nanoparticles prepared in the embodiment 1, and the result is shown in fig. 4, wherein fig. 4 is a graph of the in vitro cumulative drug release rate of the drug-loaded silk fibroin nanoparticles prepared in the embodiments 1-3 of the invention; as can be seen from fig. 4: the drug-loaded silk fibroin nanoparticles prepared in example 1 can continuously and slowly release drug for about one week; with the increase of the mass ratio of the silk fibroin nanoparticles to the FITC-BSA, the drug release rate is gradually reduced.
The method for quantitatively determining FITC-BSA (FITC-bovine serum albumin) nanoparticle-loaded silk fibroin nanoparticle-loaded cell uptake by cells according to the technical scheme of the invention tests the uptake rate of the drug-loaded silk fibroin nanoparticle prepared in example 1, and the result is shown in FIG. 5, wherein FIG. 5 shows the cell uptake rate result of ARPE-19 cells to the drug-loaded silk fibroin nanoparticle prepared in examples 1-3 and the FITC-BSA solution at different time; FIG. 5 shows that: after the drug-loaded silk fibroin nanoparticles prepared in example 1 are incubated for 1h, the uptake rate of ARPE-19 cells to the drug-loaded silk fibroin nanoparticles reaches 88.96 +/-3.25%.
Example 2
1. Preparation of Silk fibroin nanoparticles
Dissolving silk fibroin material in deionized water, stirring until silk fibroin is completely dissolved, preparing into silk fibroin solution with concentration of 2.5% (w/v), and standing at 4 deg.C for 4 hr. And (2) extracting the silk fibroin solution by using an injector, quickly injecting the silk fibroin solution into a constant-temperature acetone solution at 40 ℃, wherein the volume ratio (v/v) of acetone to the silk fibroin solution is 6:1, continuously stirring the mixed solution by using a magnetic stirrer until forming milky nanoparticle suspension, and continuously stirring for 30min at constant temperature. And (3) when the nanoparticles are completely formed, transferring the nano suspension into a centrifugal tube, centrifuging for 30min at 4 ℃ and 20000rpm, removing supernatant, re-dispersing and washing the precipitate with deionized water, centrifuging again to separate the nanoparticles, and repeating the operation for 3 times. And finally, adding quantitative deionized water into the washed nanoparticles, performing ultrasonic treatment to uniformly disperse the nanoparticles into silk fibroin nanoparticle suspension with the concentration of 2.0% (w/v), and storing at low temperature for later use.
The invention adopts the testing method of the technical scheme to measure the average particle size and the polydispersity index of the silk fibroin nanoparticles, and the result is shown in figure 1. As can be seen from fig. 1: the average particle size of the silk fibroin nanoparticles prepared in example 2 is 207.9 ± 5.6nm, and PDI is 0.15 ± 0.03; the Zeta potential value is-27.50 +/-0.87 mV, which shows that the silk fibroin nano-particle has obvious electronegativity.
2. Preparation of Silk fibroin FITC-BSA nanoparticle
Weighing 3mL of the silk fibroin nanoparticle dispersion liquid with the concentration of 2.0% (w/v) prepared in the step 1, adding 4mg of FITC-BSA (fluorescein isothiocyanate-bovine serum albumin), namely the mass ratio of the silk fibroin nanoparticles to the medicine is 15:1, uniformly mixing, then dripping 100 mu L of cross-linking agent EDC with the concentration of 15% (w/v) into the mixed solution, and continuously stirring and reacting for 24 hours in a dark place at 4 ℃. Adding 500 mu L of glycine solution (200mg/mL) to terminate the reaction, centrifuging the reaction solution for 30min at the rotation speed of 16000rpm and 4 ℃, separating supernatant, redispersing the precipitate with deionized water, then centrifuging and washing free FITC-BSA, repeating the operation for 3 times, finally adding deionized water into the washed drug-loaded nanoparticles, and performing ultrasonic treatment to uniformly disperse the nanoparticles to obtain the silk fibroin nanoparticle suspension loaded with FITC-BSA.
The invention adopts the technical scheme to observe the form of the drug-loaded silk fibroin nanoparticles, and the result is shown in figure 2. As can be seen from fig. 2: the drug-loaded silk fibroin nanoparticles prepared in example 2 are smooth and spherical, have no adhesion of the nanoparticles, and have uniform particle size.
The invention carries out the measurement and calculation of the encapsulation efficiency according to the measurement method of the encapsulation efficiency in the technical scheme; the results are shown in FIG. 3. FIG. 3 shows: in example 2, when the mass ratio of the silk fibroin nanoparticles to the drug is 15:1, the encapsulation efficiency is 66.92 +/-6.89%.
The method for measuring the in-vitro drug release effect of the technical scheme is adopted to measure the cumulative drug release rate of the drug-loaded silk fibroin nanoparticles prepared in the example 2, and the result is shown in figure 4; as can be seen from fig. 4: the drug-loaded silk fibroin nanoparticle prepared in example 2 can continuously and slowly release drug for about one week.
The method for quantitatively measuring the silk fibroin FITC-BSA nanoparticle taken by cells by flow cytometry according to the technical scheme measures the uptake rate of the drug-loaded silk fibroin nanoparticles prepared in example 2, and the result is shown in figure 5 and shows that: after the drug-loaded silk fibroin nanoparticles prepared in the embodiment 2 are incubated for 1h, the uptake rate of ARPE-19 cells to the drug-loaded silk fibroin nanoparticles reaches 97.48 +/-2.22%; the uptake rate of the FITC-BSA solution group at this time was only 5.35. + -. 2.06%. With the increase of the incubation time, the uptake rate of the solution group gradually rises, the uptake rate of the drug-loaded silk fibroin nanoparticle group basically remains stable, and after incubation for 24 hours, the cell uptake rate of the drug-loaded silk fibroin nanoparticle is still far higher than that of the solution group, which shows that the uptake rate of the cells to the drugs can be obviously improved by loading the macromolecular protein drugs on the silk fibroin nanoparticles, so that the bioavailability of the drugs can be effectively improved.
Example 3
1. Preparation of Silk fibroin nanoparticles
Dissolving silk fibroin material in deionized water, stirring until silk fibroin is completely dissolved, preparing into 5.0% (w/v) silk fibroin solution, and standing at 4 deg.C for 8 hr. And (2) extracting the silk fibroin solution by using an injector, quickly injecting the silk fibroin solution into a constant-temperature isopropanol solution at the temperature of 20 ℃, wherein the volume ratio (v/v) of the isopropanol to the silk fibroin solution is 8:1, continuously stirring the mixed solution by using a magnetic stirrer until forming milky nanoparticle suspension, and continuously stirring for 30min at the constant temperature. And (3) when the nanoparticles are completely formed, transferring the nano suspension into a centrifugal tube, centrifuging for 30min at 4 ℃ and 8000rpm, discarding supernatant, re-dispersing and washing the precipitate with deionized water, centrifuging again to separate the nanoparticles, and repeating the operation for 3 times. Finally, a certain amount of deionized water is added into the washed nanoparticles, the nanoparticles are uniformly dispersed into a suspension with the concentration of 2.0 percent (w/v) by ultrasonic treatment, and the suspension is stored for later use under the low-temperature condition.
The invention adopts the determination method of the technical proposal to determine the average grain diameter and the polydispersity index of the silk fibroin nanoparticles, and the result is shown in figure 1. As can be seen from fig. 1: the average particle size of the silk fibroin nanoparticles prepared in example 3 is 239.4 +/-8.5 nm, and the PDI is 0.31 +/-0.06; the Zeta potential value is-24.17 +/-0.45 mV, which shows that the silk fibroin nano-particle has obvious electronegativity.
2. Preparation of Silk fibroin FITC-BSA nanoparticle
Weighing 6mL of the silk fibroin nanoparticle dispersion liquid with the concentration of 2.0% (w/v) prepared in the step 1, adding 4mg of FITC-BSA (fluorescein isothiocyanate-bovine serum albumin), namely the mass ratio of the silk fibroin nanoparticles to the medicine is 30:1, uniformly mixing, then dripping 100 mu L of cross-linking agent NHS with the concentration of 15% (w/v) into the mixed solution, and continuously stirring for 24 hours in a dark place at 4 ℃. Adding 500 mu L of glycine solution (200mg/mL) to terminate the reaction, centrifuging the reaction solution for 30min at the rotation speed of 16000rpm and 4 ℃, separating supernatant, redispersing the precipitate with deionized water, then centrifuging and washing free FITC-BSA, repeating the operation for 3 times, finally adding deionized water into the washed drug-loaded nanoparticles, and performing ultrasonic treatment to uniformly disperse the nanoparticles to obtain the silk fibroin nanoparticle suspension loaded with FITC-BSA.
The invention adopts the testing method of the technical scheme to observe the form of the drug-loaded silk fibroin nanoparticles, and the result is shown in figure 2. As can be seen from fig. 2: the drug-loaded silk fibroin nanoparticles prepared in example 3 are smooth and spherical, and only some nanoparticles with larger particle size exist.
The invention carries out the measurement and calculation of the encapsulation efficiency according to the test method of the encapsulation efficiency of the technical proposal; the results are shown in FIG. 3. FIG. 3 shows: in the embodiment 3, when the mass ratio of the silk fibroin nanoparticles to the medicine is 30:1, the encapsulation rate is 67.11 +/-3.56%; the encapsulation efficiency of the silk fibroin nanoparticle is not obviously improved compared with that of the silk fibroin nanoparticle with the ratio of 15:1, and the reason for the phenomenon is probably that the drug loading capacity of the silk fibroin nanoparticle has an upper limit.
The method for measuring the in-vitro drug release effect of the technical scheme is adopted to measure the cumulative drug release rate of the drug-loaded silk fibroin nanoparticles prepared in the example 3, and the result is shown in figure 4; as can be seen from fig. 4: the drug-loaded silk fibroin nanoparticles prepared in example 3 can continuously and slowly release drug for about one week.
The method for quantitatively determining the silk fibroin FITC-BSA nanoparticle taken by cells by flow cytometry according to the technical scheme of the invention determines the uptake rate of the drug-loaded silk fibroin nanoparticles prepared in example 3, and the result is shown in figure 5, and figure 5 shows that: after the drug-loaded silk fibroin nanoparticles prepared in example 3 are incubated for 1h, the uptake rate of ARPE-19 cells to the drug-loaded silk fibroin nanoparticles reaches 96.64 +/-3.08%.
From the above embodiments, the invention provides a silk fibroin nanoparticle, which is prepared by the following method: s1) dissolving silk fibroin in water, and standing for 2-8 h at 2-6 ℃ to obtain a silk fibroin solution; s2) mixing a silk fibroin solution with a volume ratio of 1: 4-8 with an organic solvent, and stirring to obtain a nanoparticle suspension, wherein the organic solvent is selected from ethanol, isopropanol or acetone; s3) carrying out centrifugal separation on the nanoparticle suspension, carrying out redispersion washing by using deionized water, and centrifuging to obtain a nanoparticle precipitate; s4) mixing the nanoparticle precipitate with water, and performing ultrasonic treatment to obtain the silk fibroin nanoparticles. The silk fibroin nanoparticles loaded with the drugs can improve the intracellular uptake of the drugs and prolong the retention time of the drugs, has the advantages of intracellular high efficiency, long-acting effect and targeting, and is beneficial to improving the bioavailability of the drugs. In addition, the silk fibroin nanoparticle has good biological adhesion, no immunogenicity, no obvious inflammatory reaction and tissue fibrosis, and good biocompatibility and safety. The silk fibroin nanoparticles are prepared by an anti-solvent method, and the drug-loaded silk fibroin nanoparticles are obtained by a cross-linking method, so that the whole preparation process is simple to operate, mild in condition and good in reproducibility. The experimental results show that: the average grain diameter of the silk fibroin nano-particles is below 250 nm; the silk fibroin nano-particles are obviously electronegative; the drug-loaded silk fibroin nanoparticle dispersion liquid with the concentration of 1mg/mL can continuously and slowly release the drug for about one week; the uptake rate of ARPE-19 cells to the drug-loaded silk fibroin nanoparticles within 1h can reach more than 97%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (1)
1. A drug-loaded silk fibroin nanoparticle is prepared by the following method:
mixing the silk fibroin nanoparticles and the protein medicine, adding a cross-linking agent for cross-linking reaction, adding a terminating agent for terminating the reaction, centrifuging the reaction product, and separating to obtain a precipitate product; the mass ratio of the silk fibroin nanoparticles to the protein medicine is 15-30: 1; the protein drug is bovine serum albumin; the centrifugation temperature of the reaction product is 2-5 ℃; centrifuging the reaction product for 28-33 min; the centrifugal rotating speed of the reaction product is 15500-16500 rpm; the cross-linking agent is selected from 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide or N-hydroxysuccinimide; the temperature of the crosslinking reaction is 2-5 ℃; the time of the crosslinking reaction is 22-28 h;
re-dispersing the precipitation product, performing centrifugal washing and separation for multiple times, and then adding water for ultrasonic re-dispersion to obtain drug-loaded silk fibroin nanoparticles;
the silk fibroin nanoparticle is prepared by the following method:
s1) dissolving silk fibroin in water, and standing for 2-8 h at 2-6 ℃ to obtain a silk fibroin solution; the concentration of the fibroin solution in the step S1) is 10-50 mg/mL;
s2) mixing a silk fibroin solution with a volume ratio of 1: 4-8 with an organic solvent, and stirring to obtain a nanoparticle suspension, wherein the organic solvent is selected from ethanol, isopropanol or acetone; the mixing temperature in the step S2) is 20-60 ℃;
s3) carrying out centrifugal separation on the nanoparticle suspension, re-dispersing and washing with deionized water, and centrifuging to obtain a nanoparticle precipitate; the rotating speed of the centrifugation in the step S3) is 8000-20000 rpm; the temperature of the centrifugation is 4 ℃; the centrifugation time is 25-35 min;
s4) mixing the nanoparticle precipitate with water, and performing ultrasonic treatment to obtain silk fibroin nanoparticles; the average grain size of the silk fibroin nano-particles is less than 250nm, and the grain size is uniform; the concentration of the fibroin nanoparticles in the step S4) is 18-22 mg/mL.
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