CN110812335A - Silk fibroin micro-nano particle sustained-release preparation loaded with hydrophobic drug and preparation method thereof - Google Patents
Silk fibroin micro-nano particle sustained-release preparation loaded with hydrophobic drug and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a hydrophobic drug loaded silk fibroin micro-nano particle sustained release preparation and a preparation method thereof, belonging to the technical field of biological medicines. The invention adopts the polyethylene glycol with small molecular weight to disperse the hydrophobic drug, and then adopts the polyethylene glycol with large molecular weight to induce the silk fibroin to form drug-loaded micro-nano particles, thereby improving the drug loading rate of the hydrophobic drug. The solubilizer is not needed when the solution is prepared, so that the side effect caused by the solubilizer is removed, and the safety of the preparation is improved. The silk fibroin micro-nano particle sustained release preparation loaded with the hydrophobic drug has high drug loading rate which is as high as 5.99 +/-0.28%, and can be suspended in water, normal saline or glucose solution to form suspension for injection. Therefore, a solubilizer is not needed when the solution is prepared, the side effect caused by the solubilizer is removed, and the safety of the preparation is improved.
Description
Technical Field
The invention relates to a silk fibroin micro-nano particle sustained release preparation loaded with hydrophobic drugs and a preparation method thereof, belonging to the technical field of biological medicines.
Background
The drug molecules are converted into the micro-nano particles by the aid of the carrier materials, so that the inherent defects of free drugs, particularly hydrophobic drugs, such as poor water solubility, large drug dosage and short half-life of in-vivo drugs, can be overcome. However, most of the existing micro-nano particles have the disadvantage of low drug loading (usually less than 1%), which is more related to carrier materials. In order to reduce the pain and potential safety threat brought by drug administration to patients, in recent years, the drug-loading rate of the high-drug-loading nano-drug exceeds 5%, and more attention is paid. According to the preparation strategy of the micro-nano medicine, the micro-nano medicine with drug loading capacity is divided into two categories: the carrier-free micro-nano structure medicine and the micro-nano medicine prepared by carrying the carrier through a complex processing process.
The silk fibroin is a natural biological material extracted from silk and is composed of 18 amino acids such as glycine, alanine and serine. The silk fibroin has excellent physical and chemical properties, is nontoxic, nonirritating, good in biocompatibility and biodegradable, can be processed into various material forms such as films, particles, porous scaffolds, hydrogel and the like, and is widely applied to the fields of tissue engineering and drug sustained and controlled release. Compared with the traditional medicine carrying material, the silk fibroin has unique advantages. The silk fibroin is used as a medicinal material, so that the silk fibroin can treat diseases through medicines, can supplement nutrition and condition the body, and achieves two purposes at one stroke.
The carrier type micro-nano medicine carrying the hydrophobic medicine needs to be added with compatibilization/cosolvent, such as organic solvent, surfactant and various oils in the process of preparing the mother liquor. Such as paclitaxel, has the formula C47H51NO24The paclitaxel injection has molecular weight of 853.9, poor water solubility and solubility in water of less than 0.004g/L, a large amount of solubilizer is required to be added for preparing the paclitaxel injection with clinical drug concentration, the paclitaxel injection (Taxol, Bristol-Myers Squibb company, USA) is sold in foreign markets and is prepared by dissolving paclitaxel in a mixed solvent of polyoxyethylene castor oil (Cremophor EL) and absolute ethyl alcohol (1:1), but Cremophor EL is easy to cause various toxic and side reactions, such as severe anaphylactic reaction and toxic reactionKidney damage, neurotoxicity, cardiovascular toxicity, etc., and the polyoxyethylene castor oil also leaches out plasticizers in the polyoxyethylene infusion set to cause toxic reactions. The new preparation of taxane drugs researched at home and abroad at present comprises a prodrug, a clathrate compound, a micelle, an emulsion, a microemulsion, a liposome, nanoparticles, a nanosuspension and the like. However, these new formulations inevitably present some drawbacks: the main problems in the research of the prodrug are that the bridging group for connecting the drug and the macromolecule is not easy to find, and the connection efficiency is low; after the inclusion compound is diluted in an aqueous medium, the medicine is easy to precipitate from the inclusion compound; the paclitaxel is distributed in the oil phase to prepare the emulsion, so that the polyoxyethylene castor oil causing anaphylactic reaction can be avoided, but the paclitaxel has poor water solubility and poor fat solubility in practice; in practical application, the liposome has a series of problems of increased administration volume, easy leakage of the medicament, poor storage stability and the like caused by low medicament loading rate.
The Chinese patent CN104592375 utilizes the blending liquid of polyethylene glycol 10,000 and silk fibroin to prepare the silk fibroin microsphere, the operation flow is simple and easy, no organic solvent is added, and the biological safety of the prepared silk fibroin microsphere is high. However, the micro-nano medicine prepared by the method has low drug loading rate, and the drug loading rate is only 1.18% when curcumin is taken as an example, so that the dosage is increased.
In order to improve the drug loading rate of the silk fibroin micro-nano particles and remove the side effect brought by the solubilizer, the safety of the preparation is improved. The invention adopts the polyethylene glycol with small molecular weight to disperse the hydrophobic drug, and then adopts the polyethylene glycol with large molecular weight to induce the silk fibroin to form drug-loaded micro-nano particles, thereby improving the drug loading rate of the hydrophobic drug. The silk fibroin micro-nano particle sustained-release preparation loaded with the hydrophobic drug has high drug loading rate which is as high as 5.99 +/-0.28%.
Disclosure of Invention
In order to solve the technical problem, the hydrophobic drug is firstly dispersed by adopting polyethylene glycol with small molecular weight, then the polyethylene glycol is mixed with the silk fibroin solution to form a mixed solution, and then the mixed solution is incubated with the polyethylene glycol solution with large molecular weight to form the micro-nano drug.
The invention aims to provide a preparation method of a hydrophobic drug loaded silk fibroin micro-nano particle sustained release preparation, which comprises the following steps:
(1) preparing a silk fibroin aqueous solution;
(2) dissolving a hydrophobic drug in a small molecular weight polyethylene glycol solution to form a solution;
(3) mixing the solutions obtained in the step (1) and the step (2) to obtain a blending solution of a hydrophobic drug, silk fibroin and low molecular weight polyethylene glycol;
(4) and (4) mixing the blending solution obtained in the step (3) with a high molecular weight polyethylene glycol solution, and incubating to obtain the high-drug-loading-rate silk fibroin micro-nano particle sustained release preparation loaded with the hydrophobic drug.
Further, the silk fibroin sustained release preparation is micro-nano particles.
Further, the volume ratio of the silk fibroin to the small molecular weight polyethylene glycol solution to the large molecular weight polyethylene glycol solution is 10:1: 10-5: 1: 5.
Further, in the step (1), the concentration of silk fibroin in the silk fibroin aqueous solution is: 1-30 wt%.
Furthermore, the molecular weight of the micromolecular polyethylene glycol is 200-600.
Further, the concentration of the small molecular weight polyethylene glycol solution is as follows: 50 to 90 percent.
Furthermore, the molecular weight of the macromolecular polyethylene glycol is 6,000-20,000.
Further, the concentration of the said solution of polyethylene glycol with large molecular weight is: 20 to 60 percent.
Further, the hydrophobic drug is one or more of paclitaxel, docetaxel, curcumin, risperidone, rifampin, felodipine, carbamazepine, indomethacin, furosemide, camptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, topotecan, irinotecan, 9-aminocamptothecin, teniposide, etoposide, cyclosporine-A, fenofibrate, sirolimus, aprepitant, megestrol, paliperidone, erlotinib, silymarin, quercetin, itraconazole, naproxen, dexamethasone, diosmin, icarin, oridonin, puerarin, nevirapine or ziprasidone.
Further, a preparation method of the silk fibroin micro-nano particle sustained release preparation loaded with the hydrophobic drug specifically comprises the following steps:
(1) preparing a silk fibroin aqueous solution with the concentration of 3-30% (wt/v);
(2) preparing polyethylene glycol with molecular weight of 200-600 into 50-90 wt% polyethylene glycol solution;
(3) dissolving a hydrophobic drug in the polyethylene glycol solution obtained in the step (2) to obtain a polyethylene glycol solution containing the hydrophobic drug, wherein the concentration of the hydrophobic drug is 0.1-20 mg/mL;
(4) mixing the hydrophobic drug-containing polyethylene glycol solution obtained in the step (3) with the silk fibroin aqueous solution obtained in the step (1) to obtain a blended solution;
(5) and (3) preparing polyethylene glycol with the molecular weight of 6000-20000 into 20-60 wt% of polyethylene glycol solution, mixing the polyethylene glycol solution with the blending solution obtained in the step (4), incubating, centrifuging, and freeze-drying to obtain the silk fibroin slow-release preparation loaded with the hydrophobic drugs.
After the low-molecular-weight polyethylene glycol is added, the silk fibroin hydrophobic segment is combined with the hydrophobic drug, and the silk fibroin induced by the low-molecular-weight polyethylene glycol is assembled to form more binding sites and physical traps, but the degree of forming gel is not reached.
The second purpose of the invention is to provide the hydrophobic drug loaded silk fibroin micro-nano particles prepared by the method.
The third purpose of the invention is to provide the application of the silk fibroin sustained-release preparation loaded with the hydrophobic drug in the sustained release of the hydrophobic drug.
The invention has the beneficial effects that:
according to the invention, the hydrophobic drug is firstly dispersed by adopting the polyethylene glycol with small molecular weight, then the polyethylene glycol is mixed with the silk fibroin solution to form a mixed solution, and then the mixed solution is incubated with the polyethylene glycol solution with large molecular weight to form the nano-particles or microspheres, so that the complex and complicated steps are avoided, the operation process is convenient, the production cost is reduced, the popularization is facilitated, no organic solvent is added, the prepared silk fibroin nano-particles or microspheres have high biological safety, and the silk fibroin nano-particles or microspheres can be directly used in clinic.
The fibroin particle loaded with the hydrophobic drug has high drug loading rate of 5.99 +/-0.28 percent, can be suspended in water, normal saline or glucose solution to form suspension for injection, so that a solubilizer is not required to be used in the preparation of the solution, the side effect brought by the solubilizer is removed, and the safety of the preparation is improved.
The drug release result of the nano-particles or microspheres loaded with the hydrophobic drugs shows that the silk fibroin can obviously improve the stability of the hydrophobic drugs and prolong the release time of the hydrophobic drugs.
Drawings
Fig. 1 is a scanning electron microscope image of paclitaxel-loaded silk fibroin nanoparticles according to the present invention.
Fig. 2 is a fourier infrared spectrum of paclitaxel loaded silk fibroin nanoparticles of the present invention.
Fig. 3 is an in vitro release profile of paclitaxel loaded silk fibroin nanoparticles of the present invention.
Fig. 4 is a scanning electron microscope image of paclitaxel loaded silk fibroin microspheres of the present invention.
Fig. 5 is a fourier infrared spectrum of paclitaxel loaded silk fibroin microspheres of the present invention.
Fig. 6 is an in vitro release profile of paclitaxel loaded silk fibroin microspheres of the present invention.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1: preparation of paclitaxel loaded silk fibroin microspheres
(1) Preparing a silk fibroin aqueous solution with the weight percentage of 3 percent;
(2) preparing 80% polyethylene glycol 400 solution, 80% methanol, corn oil, 1% tween 80 and 1% span 80;
(3) respectively dissolving paclitaxel in the solution obtained in step (2) to obtain paclitaxel solutions with different concentrations, as shown in Table 1;
table 1: solubility of paclitaxel in different cosolvents
| |
80% polyethylene glycol 400 | 80% methanol | Corn |
1% Tween 80 | 1% |
| Paclitaxel concentration | 10mg/ml | 10mg/ml | 20mg/ml | 1mg/ml | 1mg/ml |
(4) Adding the paclitaxel solution containing paclitaxel in the step (3) into the silk fibroin aqueous solution in the step (1), and lightly blowing and beating until uniform mixing is achieved to obtain a blended solution, wherein the final concentration of the silk fibroin and the final concentration of the paclitaxel in the blended solution are shown in table 2;
table 2: final concentrations of fibroin and paclitaxel in the blend
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Final concentration of silk fibroin | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | |
| Final concentration of paclitaxel | 1mg/ml | 1mg/ml | 2mg/ml | 0.1mg/ml | 0.1mg/ml |
(5) Preparing polyethylene glycol with the molecular weight of 10,000 into a polyethylene glycol solution with the weight percentage of 50%, mixing the polyethylene glycol solution and the blending solution obtained in the step (4) in equal volume, inverting and uniformly mixing the polyethylene glycol solution and the blending solution, incubating at room temperature, centrifuging, and freeze-drying the collected paclitaxel-silk fibroin microspheres to obtain powdery paclitaxel-loaded silk fibroin microspheres.
(6) Determination of drug loading rate of paclitaxel-loaded silk fibroin nanoparticles/microspheres: weighing 5mg of paclitaxel-loaded silk fibroin nanoparticles or microspheres in a centrifuge tube, adding 1mL of an extraction solution into the centrifuge tube, sealing, placing in a shaker at 37 ℃, shaking for 6 hours, taking out, centrifuging, and taking out supernatant for detection.
(7) (6) the extract and 1mL of the released solution taken out of the column were measured by High Performance Liquid Chromatography (HPLC): the mobile phase is (A) water and (B) methanol. The program flow is as follows: the flow rate was 250. mu.L/min for a period of 16 min. The first 1.5 minutes were 40% a and 60% B, mobile phase B rose from 60% to 90% from 1.5 minutes to 4.5 minutes, mobile phase B remained at 90% for 4.5-14 minutes, mobile phase B fell back to 50% for 14.5-16 minutes. The PDA detection wavelength was 228 nm.
The drug loading rate of the silk fibroin microspheres of example 1 is shown in table 3.
Table 3: drug loading rate of silk fibroin drug-loaded microspheres prepared by using different cosolvents
| Cosolvent | PEG400 | Ethanol | | Tween | 80 | |
| Drug loading rate | 5.99％±0.28％ | 1.03％±0.09％ | 0.99％±0.03％ | 0.87％±0.11％ | 1.1％±0.07％ |
Example 2: preparation of felodipine-loaded silk fibroin microspheres
(1) Preparing a silk fibroin aqueous solution with the weight percentage of 3 percent;
(2) preparing 80% polyethylene glycol 400 solution, 80% methanol, corn oil, 1 % tween 80 and 1% span 80;
(3) respectively dissolving felodipine in the solution obtained in the step (2) to obtain felodipine solutions with different concentrations, which is shown in table 4;
table 4: solubility of felodipine in different co-solvents
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Felodipine concentration | 10mg/ml | 10mg/ml | 20mg/ml | 1mg/ml | 1mg/ml |
(4) Adding the felodipine-containing solution obtained in the step (3) into the silk fibroin aqueous solution obtained in the step (1), and gently blowing and beating the mixture until the mixture is uniformly mixed to obtain a blending solution, wherein the final concentration of silk fibroin and the final concentration of felodipine in the blending solution are shown in a table 5;
table 5: final concentrations of fibroin and felodipine in the blend
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Final concentration of silk fibroin | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | |
| Final felodipine concentration | 1mg/ml | 1mg/ml | 2mg/ml | 0.1mg/ml | 0.1mg/ml |
(5) Preparing polyethylene glycol 10,000 with the molecular weight into a polyethylene glycol solution with the weight percentage of 50%, mixing the polyethylene glycol solution and the blending solution obtained in the step (4) in equal volume, inverting and uniformly mixing the polyethylene glycol solution and the blending solution, incubating at room temperature, centrifuging, and freeze-drying the collected felodipine-silk fibroin microspheres to obtain the felodipine-silk fibroin microspheres loaded with powder.
(6) And (3) determining the drug loading rate of the felodipine-loaded silk fibroin nanoparticles/microspheres: weighing 5mg of felodipine-loaded silk fibroin nanoparticles or microspheres in a centrifuge tube, adding 1mL of extraction liquid into the centrifuge tube, sealing, placing the centrifuge tube in a shaker at 37 ℃, shaking for 6 hours, taking out, centrifuging, and taking out supernatant for detection.
(7) (6) the extract and 1mL of the released solution taken out of the column were measured by High Performance Liquid Chromatography (HPLC): the mobile phase is (A) water and (B) methanol. The program flow is as follows: the flow rate was 400. mu.L/min for a period of 13 min. The first 2 minutes were 20% a and 80% B, mobile phase B rose from 80% to 90% from minute 2 to minute 4.5, mobile phase B remained at 90% for minutes 4.5-11.5, mobile phase B fell back to 50% for minutes 11.5-13. The PDA detection wavelength was 221 nm.
The drug loading rate of the silk fibroin microspheres of example 2 is shown in table 6.
Table 6: drug loading rate of felodipine-loaded silk fibroin microspheres prepared by using different cosolvents
| Cosolvent | PEG400 | Ethanol | | Tween | 80 | |
| Drug loading rate | 4.71％±0.18％ | 1.23％±0.21％ | 0.79％±0.07％ | 0.77％±0.31％ | 2.1％±0.07％ |
Example 3: preparation of naproxen-loaded silk fibroin microspheres
(1) Preparing a silk fibroin aqueous solution with the weight percentage of 3 percent;
(2) preparing 80% polyethylene glycol 400 solution, 80% methanol, corn oil, 1 % tween 80 and 1% span 80;
(3) respectively dissolving naproxen in the solution obtained in the step (2) to obtain naproxen solutions with different concentrations, which are shown in a table 7;
table 7: solubility of naproxen in different cosolvents
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Naproxen concentration | 20mg/ml | 10mg/ml | 20mg/ml | 1mg/ml | 1mg/ml |
(4) Adding the naproxen-containing solution obtained in the step (3) into the silk fibroin aqueous solution obtained in the step (1), and lightly blowing and beating the mixture until the mixture is uniformly mixed to obtain a blended solution, wherein the final concentration of the silk fibroin and the final concentration of the naproxen in the blended solution are shown in a table 8;
table 8: final concentrations of fibroin and naproxen in the blend
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Final concentration of silk fibroin | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | |
| Final concentration of naproxen | 2mg/ml | 1mg/ml | 2mg/ml | 0.1mg/ml | 0.1mg/ml |
(5) Preparing polyethylene glycol solution with the weight percentage of 10,000 polyethylene glycol with the molecular weight, mixing the polyethylene glycol solution and the blending solution obtained in the step (4) in equal volume, inverting and uniformly mixing the polyethylene glycol solution and the blending solution, incubating at room temperature, centrifuging, and freeze-drying the collected naproxen-silk fibroin microspheres to obtain powdery naproxen-loaded silk fibroin microspheres.
(6) And (3) determination of the drug loading rate of the naproxen-loaded silk fibroin nanoparticles/microspheres: weighing 5mg of the naproxen-loaded silk fibroin nanoparticles or microspheres in a centrifuge tube, adding 1mL of an extraction solution into the centrifuge tube, sealing the extraction solution, placing the sealed extraction solution in a 37 ℃ shaking table, shaking for 6 hours, taking out the extraction solution, centrifuging, and taking out the supernatant for detection.
(7) (6) the extract and 1mL of the released solution taken out of the column were measured by High Performance Liquid Chromatography (HPLC): the mobile phase is (A) water and (B) methanol. The program flow is as follows: the flow rate was 250. mu.L/min for a period of 12 min. The first 1.5 minutes were 40% a and 60% B, mobile phase B rose from 60% to 90% from 1.5 minutes to 3 minutes, mobile phase B remained at 90% for 3-10 minutes, 10.5-12 minutes, and mobile phase B fell back to 50%. The PDA detection wavelength was 217 nm.
The drug loading rate of the silk fibroin microspheres of example 3 is shown in table 9.
Table 9: drug loading rate of silk fibroin-loaded naproxen microspheres prepared by using different cosolvents
| Cosolvent | PEG400 | Ethanol | | Tween | 80 | |
| Drug loading rate | 5.56％±0.18％ | 1.02％±0.21％ | 0.74％±0.07％ | 0.53％±0.12％ | 1.1％±0.14％ |
Example 4: preparation of silk fibroin microsphere loaded with carbamazepine
(1) Preparing a silk fibroin aqueous solution with the weight percentage of 3 percent;
(2) preparing 80% polyethylene glycol 400 solution, 80% methanol, corn oil, 1 % tween 80 and 1% span 80; (3) respectively dissolving carbamazepine in the solution obtained in the step (2) to obtain carbamazepine solutions with different concentrations, which is shown in table 10;
table 10: solubility of carbamazepine in different cosolvents
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Concentration of carbamazepine | 10mg/ml | 10mg/ml | 20mg/ml | 1mg/ml | 1mg/ml |
(4) Adding the solution containing carbamazepine in the step (3) into the silk fibroin aqueous solution in the step (1), and lightly blowing and beating until uniform mixing is achieved to obtain a blending solution, wherein the final concentration of the silk fibroin and the final concentration of the carbamazepine in the blending solution are shown in a table 11;
table 11: final concentrations of fibroin and carbamazepine in the blend
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Final concentration of silk fibroin | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | |
| Final concentration of carbamazepine | 1mg/ml | 1mg/ml | 2mg/ml | 0.1mg/ml | 0.1mg/ml |
(5) Preparing polyethylene glycol solution with the weight percentage of 10,000 polyethylene glycol with the molecular weight, mixing the polyethylene glycol solution and the blending solution obtained in the step (4) in equal volume, inverting and uniformly mixing the polyethylene glycol solution and the blending solution, incubating at room temperature, centrifuging, and freeze-drying the collected carbamazepine-silk fibroin microspheres to obtain the powdery carbamazepine-loaded silk fibroin microspheres.
(6) And (3) determining the drug loading rate of the silk fibroin nanoparticles/microspheres loaded with carbamazepine: weighing 5mg of the silk fibroin nanoparticles or microspheres loaded with carbamazepine in a centrifuge tube, adding 1mL of extract into the centrifuge tube, sealing the extract with 100% methanol, placing the sealed extract in a shaking table at 37 ℃, shaking the sealed extract for 6 hours, taking out the sealed extract, centrifuging the sealed extract, and taking supernatant for detection.
(7) (6) the extract and 1mL of the released solution taken out of the column were measured by High Performance Liquid Chromatography (HPLC): the mobile phase is (A) water and (B) acetonitrile. The program flow is as follows: the flow rate was 200. mu.L/min for a period of 19 min. The first 2 minutes were 40% a and 60% B, mobile phase B rose from 60% to 90% from minute 2 to minute 4, mobile phase B remained at 90% for minutes 4-18.5, mobile phase B fell back to 50% for 18.5-19 minutes. The PDA detection wavelength was 244 nm.
The drug loading rate of the silk fibroin microspheres of example four is shown in table 12.
Table 12: drug loading rate of silk fibroin carbamazepine microspheres prepared by using different cosolvents
| Cosolvent | PEG400 | Ethanol | | Tween | 80 | |
| Drug loading rate | 4.77％±0.18％ | 2.00％±0.31％ | 1.32％±0.12％ | 0.92％±0.32％ | 0.61％±0.04％ |
Example 5: preparation of rifampicin-loaded silk fibroin microsphere
(1) Preparing a silk fibroin aqueous solution with the weight percentage of 3 percent;
(2) preparing 80% polyethylene glycol 400 solution, 80% methanol, corn oil, 1 % tween 80 and 1% span 80;
(3) respectively dissolving rifampicin in the solution obtained in the step (2) to obtain rifampicin solutions with different concentrations, see table 13;
table 13: solubility of Rifampicin in different Co-solvents
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Rifampicin concentration | 50mg/ml | 10mg/ml | 20mg/ml | 1mg/ml | 1mg/ml |
(4) Adding the rifampicin-containing solution obtained in the step (3) into the silk fibroin aqueous solution obtained in the step (1), and lightly blowing and beating the mixture until the mixture is uniformly mixed to obtain a blending solution, wherein the final concentration of the silk fibroin and the final concentration of rifampicin in the blending solution are shown in a table 14;
table 14: final concentrations of fibroin and rifampicin in the blend
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Final concentration of silk fibroin | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | |
| Final concentration of rifampicin | 5mg/ml | 1mg/ml | 2mg/ml | 0.1mg/ml | 0.1mg/ml |
(5) Preparing polyethylene glycol solution with the weight percentage of 10,000 polyethylene glycol with the molecular weight, mixing the polyethylene glycol solution and the blending solution obtained in the step (4) in equal volume, inverting and uniformly mixing the polyethylene glycol solution and the blending solution, incubating at room temperature, centrifuging, and freeze-drying the collected rifampicin-silk fibroin microspheres to obtain the rifampicin-loaded silk fibroin microspheres in powder form.
(6) And (3) determining the rifampicin-loaded silk fibroin nanoparticle/microsphere drug loading rate: weighing 5mg of rifampicin-loaded silk fibroin nanoparticles or microspheres in a centrifuge tube, adding 1mL of extraction liquid into the centrifuge tube, sealing the extraction liquid, placing the centrifuge tube in a shaker at 37 ℃, shaking the centrifuge tube for 6 hours, taking out the centrifuge tube, centrifuging, and taking out the supernatant for detection.
(7) (6) the extract and 1mL of the released solution taken out of the column were measured by High Performance Liquid Chromatography (HPLC): the mobile phase is (A) water and (B) methanol. The program flow is as follows: the flow rate was 450. mu.L/min for a period of 16 min. The first 1.5 minutes were 40% a and 60% B, mobile phase B rose from 60% to 90% from 1.5 minutes to 4.5 minutes, mobile phase B remained at 90% for 4.5-14 minutes, mobile phase B fell back to 50% for 14.5-16 minutes. The PDA detection wavelength was 224 nm.
The drug loading rate of the silk fibroin microspheres of example 5 is shown in table 15.
Table 15: drug loading rate of silk fibroin rifampicin-loaded microspheres prepared by using different cosolvents
| Cosolvent | PEG400 | Ethanol | | Tween | 80 | |
| Drug loading rate | 6.65％±0.32％ | 1.98％±0.11％ | 1.02％±0.06％ | 0.86％±0.38％ | 0.49％±0.14％ |
Example 6: preparation of indometacin-loaded silk fibroin microspheres
(1) Preparing a silk fibroin aqueous solution with the weight percentage of 3 percent;
(2) preparing 80% polyethylene glycol 400 solution, 80% methanol, corn oil, 1 % tween 80 and 1% span 80;
(3) respectively dissolving indomethacin in the solution obtained in the step (2) to obtain indomethacin solutions with different concentrations, see table 16;
table 16: solubility of Indometacin in different cosolvents
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Indometacin concentration | 10mg/ml | 10mg/ml | 20mg/ml | 1mg/ml | 1mg/ml |
(4) Adding the indometacin-containing solution obtained in the step (3) into the silk fibroin aqueous solution obtained in the step (1), and lightly blowing and beating until the mixture is uniformly mixed to obtain a blending solution, wherein the final concentration of the silk fibroin and the final concentration of the indometacin in the blending solution are shown in a table 17;
table 17: final concentrations of fibroin and indomethacin in the blend
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Final concentration of silk fibroin | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | |
| Final indomethacin concentration | 1mg/ml | 1mg/ml | 2mg/ml | 0.1mg/ml | 0.1mg/ml |
(5) Preparing polyethylene glycol solution with the weight percentage of 10,000 polyethylene glycol with the molecular weight, mixing the polyethylene glycol solution and the blending solution obtained in the step (4) in equal volume, inverting and uniformly mixing the mixture, incubating at room temperature, centrifuging, and freeze-drying the collected indomethacin-silk fibroin microspheres to obtain the powdery indomethacin-loaded silk fibroin microspheres.
(6) And (3) determining the drug loading rate of the indometacin-loaded silk fibroin nanoparticles/microspheres: weighing 5mg of indometacin-loaded silk fibroin nanoparticles or microspheres in a centrifuge tube, adding 1mL of extraction liquid into the centrifuge tube, sealing, placing the centrifuge tube in a shaker at 37 ℃, shaking for 6 hours, taking out, centrifuging, and taking out supernatant for detection.
(7) (6) the extract and 1mL of the released solution taken out of the column were measured by High Performance Liquid Chromatography (HPLC): the mobile phase is (A) water and (B) methanol. The program flow is as follows: the flow rate was 250. mu.L/min for a period of 23 min. The first 3 minutes were 40% a and 60% B, mobile phase B rose from 60% to 90% from minute 3 to minute 5, mobile phase B remained at 90% for minutes 5-21, and mobile phase B fell back to 50% for 22-23 minutes. The PDA detection wavelength was 236 nm.
The drug loading rate of the silk fibroin microspheres of example 6 is shown in table 18.
Table 18: drug loading rate of silk fibroin indometacin-carried microsphere prepared by using different cosolvents
| Cosolvent | PEG400 | Ethanol | | Tween | 80 | |
| Drug loading rate | 5.02％±0.18％ | 1.49％±0.37％ | 1.45％±0.16％ | 0.79％±0.25％ | 0.92％±0.17％ |
Example 7: preparation of furosemide-loaded silk fibroin microspheres
(1) Preparing a silk fibroin aqueous solution with the weight percentage of 3 percent;
(2) preparing 80% polyethylene glycol 400 solution, 80% methanol, corn oil, 1 % tween 80 and 1% span 80;
(3) respectively dissolving furosemide in the solution obtained in the step (2) to obtain furosemide solutions with different concentrations, and the furosemide solutions are shown in a table 19;
table 19: solubility of Furosemide in different cosolvents
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Furosemide concentration | 10mg/ml | 10mg/ml | 20mg/ml | 1mg/ml | 1mg/ml |
(4) Adding the furosemide-containing solution obtained in the step (3) into the silk fibroin aqueous solution obtained in the step (1), and lightly blowing and beating until the mixture is uniformly mixed to obtain a blending solution, wherein the final concentration of the silk fibroin and the final concentration of the furosemide in the blending solution are shown in a table 20;
table 20: final concentrations of fibroin and furosemide in the blend
| Cosolvent | 80% polyethylene glycol 400 | 80% | Corn oil | 1 |
1 |
|
| Final concentration of silk fibroin | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | 2.7wt％ | |
| Final concentration of furosemide | 1mg/ml | 1mg/ml | 2mg/ml | 0.1mg/ml | 0.1mg/ml |
(5) Preparing polyethylene glycol solution with the weight percentage of 10,000 polyethylene glycol with the molecular weight, mixing the polyethylene glycol solution and the blending solution obtained in the step (4) in equal volume, inverting and uniformly mixing the polyethylene glycol solution and the blending solution, incubating at room temperature, centrifuging, and freeze-drying the collected furosemide-silk fibroin microspheres to obtain powdery furosemide-loaded silk fibroin microspheres.
(6) And (3) determining the drug loading rate of the silk fibroin nanoparticles/microspheres loaded with furosemide: weighing 5mg of silk fibroin nanoparticles or microspheres loaded with furosemide in a centrifuge tube, adding 1mL of extraction liquid into the centrifuge tube, sealing, placing in a shaking table at 37 ℃, shaking for 6 hours, taking out, centrifuging, and taking out supernatant for detection.
(7) (6) the extract and 1mL of the released solution taken out of the column were measured by High Performance Liquid Chromatography (HPLC): the mobile phase is (A) water and (B) methanol. The program flow is as follows: the flow rate was 300. mu.L/min for 16 min. The first 2 minutes were 40% a and 60% B, mobile phase B rose from 60% to 90% from minute 2 to minute 4.5, mobile phase B remained at 90% for minutes 4.5-14, mobile phase B fell back to 50% for minutes 14.5-16. The PDA detection wavelength was 245 nm.
The drug loading rate of the silk fibroin microspheres of example 7 is shown in table 21.
Table 21: drug loading rate of silk fibroin Furosemide microspheres prepared by using different cosolvents
| Cosolvent | PEG400 | Ethanol | | Tween | 80 | |
| Drug loading rate | 4.02％±0.09％ | 1.52％±0.27％ | 2.45％±0.06％ | 0.96％±0.55％ | 1.33％±0.26％ |
Example 8: preparation of paclitaxel-loaded silk fibroin microspheres
(1) Preparing a silk fibroin aqueous solution with the weight percentage of 3 percent;
(2) preparing 80% polyethylene glycol (MW200, 300, 400, 600);
(3) respectively dissolving paclitaxel in the solution obtained in the step (2) to obtain a solution with the paclitaxel concentration of 10 mg/mL;
(4) adding the paclitaxel solution containing paclitaxel in the step (3) into the silk fibroin aqueous solution in the step (1), and lightly blowing and beating until uniform mixing is achieved to obtain a blending solution, wherein the final concentration of the silk fibroin in the blending solution is 2.7 wt%, and the final concentration of paclitaxel is 1 mg/ml;
(5) preparing polyethylene glycol solution with the weight percentage of 10,000 polyethylene glycol with the molecular weight, mixing the polyethylene glycol solution and the blending solution obtained in the step (4) in equal volume, inverting and uniformly mixing the mixture, incubating at room temperature, centrifuging, and freeze-drying the collected paclitaxel-silk fibroin microspheres to obtain powdery paclitaxel-loaded silk fibroin microspheres.
(6) Determination of drug loading rate of paclitaxel-loaded silk fibroin nanoparticles/microspheres: weighing 5mg of paclitaxel-loaded silk fibroin nanoparticles or microspheres in a centrifuge tube, adding 1mL of an extraction solution into the centrifuge tube, sealing, placing in a shaker at 37 ℃, shaking for 6 hours, taking out, centrifuging, and taking out supernatant for detection.
(7) (6) the extract and 1mL of the released solution taken out of the column were measured by High Performance Liquid Chromatography (HPLC): the mobile phase is (A) water and (B) methanol. The program flow is as follows: the flow rate was 250. mu.L/min for a period of 16 min. The first 1.5 minutes were 40% a and 60% B, mobile phase B rose from 60% to 90% from 1.5 minutes to 4.5 minutes, mobile phase B remained at 90% for 4.5-14 minutes, mobile phase B fell back to 50% for 14.5-16 minutes. The PDA detection wavelength was 228 nm.
The drug loading rate of the silk fibroin microspheres of example eight is shown in table 22.
Table 22: drug loading rate of silk fibroin paclitaxel-loaded microspheres prepared by using polyethylene glycol with different molecular weights
| PEG200 | PEG300 | PEG400 | PEG600 | |
| Drug loading rate | 3.02％±0.19％ | 3.92％±0.37％ | 5.99％±0.28％ | 4.26％±0.34％ |
Example 9: preparation of paclitaxel-loaded silk fibroin microspheres
(1) Preparing a silk fibroin aqueous solution with the weight percentage of 3 percent;
(2) preparing 80 wt% of polyethylene glycol 400 solution;
(3) respectively dissolving paclitaxel in the solution obtained in the step (2) to obtain a solution with the paclitaxel concentration of 10 mg/mL;
(4) adding the paclitaxel solution containing step (3) into the silk fibroin aqueous solution of step (1), and gently blowing and beating until uniform mixing is achieved to obtain a blending solution, wherein the final concentration of the silk fibroin in the blending solution is 2.7 wt%, and the final concentration of paclitaxel is 1 mg/mL;
(5) preparing polyethylene glycol with molecular weight (MW6000, 8000, 10000, 20000) into polyethylene glycol solution with weight percentage, mixing the polyethylene glycol solution and the blending solution in the step (5) with equal volume, inverting and uniformly mixing up and down, incubating at room temperature, centrifuging, and freeze-drying the collected paclitaxel-silk fibroin microspheres to obtain powdery paclitaxel-loaded silk fibroin microspheres.
(6) Determination of drug loading rate of paclitaxel-loaded silk fibroin nanoparticles/microspheres: weighing 5mg of paclitaxel-loaded silk fibroin nanoparticles or microspheres in a centrifuge tube, adding 1mL of an extraction solution into the centrifuge tube, sealing, placing in a shaker at 37 ℃, shaking for 6 hours, taking out, centrifuging, and taking out supernatant for detection.
(7) (6) the extract and 1mL of the released solution taken out of the column were measured by High Performance Liquid Chromatography (HPLC): the mobile phase is (A) water and (B) methanol. The program flow is as follows: the flow rate was 250. mu.L/min for a period of 16 min. The first 1.5 minutes were 40% a and 60% B, mobile phase B rose from 60% to 90% from 1.5 minutes to 4.5 minutes, mobile phase B remained at 90% for 4.5-14 minutes, mobile phase B fell back to 50% for 14.5-16 minutes. The PDA detection wavelength was 228 nm.
The drug loading rate of the silk fibroin microspheres of example 9 is shown in table 23.
Table 23: drug loading rate of silk fibroin paclitaxel-loaded microspheres prepared by using polyethylene glycol with different molecular weights
| PEG6000 | PEG8000 | PEG10000 | PEG20000 | |
| Drug loading rate | 1.72％±0.35％ | 1.99％±0.24％ | 5.99％±0.28％ | 2.26％±0.12％ |
Example 10: preparation of paclitaxel loaded silk fibroin nanoparticles
(1) Preparing 1 wt% silk fibroin aqueous solution from the silk fibroin freeze-dried powder by using deionized water;
(2) preparing polyethylene glycol with molecular weight of 400 into 80 wt% polyethylene glycol solution;
(3) dissolving paclitaxel in the polyethylene glycol solution obtained in the step (2) to obtain a polyethylene glycol solution with the paclitaxel concentration of 10 mg/ml;
(4) adding the polyethylene glycol solution containing paclitaxel in the step (3) into the silk fibroin aqueous solution in the step (1), and lightly blowing and beating until uniform mixing is achieved to obtain a blending solution, wherein the final concentration of silk fibroin in the blending solution is 0.9 wt%, and the final concentration of paclitaxel is 1 mg/ml;
(5) preparing polyethylene glycol with the molecular weight of 10000 into 50% by weight of polyethylene glycol solution, mixing the polyethylene glycol solution and the blending solution obtained in the step (4) in equal volume, inverting and uniformly mixing the polyethylene glycol solution and the blending solution, incubating at room temperature, centrifuging, and freeze-drying the collected paclitaxel-silk fibroin nanoparticles to obtain powdery paclitaxel-loaded silk fibroin nanoparticles.
The silk fibroin nanoparticles obtained in example 10 were subjected to electron microscope scanning and fourier infrared spectrogram analysis, and the results are shown in fig. 1 and 2.
The drug loading rate of the silk fibroin microsphere of example 10 is (5.87 ± 0.54)%, measured by drug loading rate.
Example 11: in vitro release assay of paclitaxel loaded silk fibroin nanoparticles
5mg of paclitaxel-loaded silk fibroin nanoparticles prepared according to the method of example 10 above were weighed into a centrifuge tube, 2mL of release solution was added to the centrifuge tube, and the centrifuge tube was sealed and then placed on a shaker at 37 ℃ for shaking. The release solution was phosphate buffered saline (PBS, pH 7.4) containing 0.5% tween 80 and 4% polyethylene glycol 400. At a specific time point, 1mL of the release solution is taken out for testing, then 1mL of fresh release solution is added into the centrifuge tube, and the centrifuge tube is sealed and then continuously placed in a shaking table at 37 ℃ for shaking.
In vitro release assay shows that the in vitro release curve of the silk fibroin nanoparticles of example 10 is shown in fig. 3, and compared with free hydrophobic drugs, the hydrophobic drugs loaded on the silk fibroin nanoparticles show good sustained release performance.
Example 12: preparation of paclitaxel loaded silk fibroin microspheres
(1) Preparing silk fibroin freeze-dried powder into a 10 wt% silk fibroin aqueous solution by using deionized water;
(2) preparing polyethylene glycol with molecular weight of 400 into 80 wt% polyethylene glycol solution;
(3) dissolving paclitaxel in the polyethylene glycol solution obtained in the step (2) to obtain a polyethylene glycol solution with the paclitaxel concentration of 10 mg/ml;
(4) adding the polyethylene glycol solution containing paclitaxel in the step (3) into the silk fibroin aqueous solution in the step (1), and lightly blowing and beating until uniform mixing is achieved to obtain a blending solution, wherein the final concentration of silk fibroin in the blending solution is 9 wt%, and the final concentration of paclitaxel is 1 mg/ml;
(5) preparing polyethylene glycol with the molecular weight of 10000 into a 50% polyethylene glycol solution by weight percentage, mixing the polyethylene glycol solution and the blending solution obtained in the step (4) in equal volume, inverting and uniformly mixing the polyethylene glycol solution and the blending solution, incubating at room temperature, centrifuging, and freeze-drying the collected paclitaxel-silk fibroin microspheres to obtain powdery paclitaxel-loaded silk fibroin microspheres.
The silk fibroin microspheres obtained in example 12 were subjected to electron microscopy scanning and fourier infrared spectrogram analysis, and the results are shown in fig. 4 and 5.
The drug loading rate of the silk fibroin microsphere of example 12 is (5.99 ± 0.28)%, measured by drug loading rate. Example 13: in vitro release assay of paclitaxel loaded silk fibroin microspheres
Weighing 5mg of paclitaxel-loaded silk fibroin microspheres prepared by the method in a centrifuge tube, adding 2mL of release solution into the centrifuge tube, sealing, and placing in a shaking table at 37 ℃ for shaking. The release solution was phosphate buffered saline (PBS, pH 7.4) containing 0.5% tween 80 and 4% polyethylene glycol 400. At a specific time point, 1mL of the release solution is taken out for testing, then 1mL of fresh release solution is added into the centrifuge tube, and the centrifuge tube is sealed and then continuously placed in a shaking table at 37 ℃ for shaking.
In vitro release assay shows that the in vitro release curve of the silk fibroin microsphere of example 12 is shown in fig. 6, and compared with free hydrophobic drugs, the hydrophobic drugs loaded on the silk fibroin microsphere show good sustained release performance.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A preparation method of a silk fibroin micro-nano particle sustained release preparation loaded with a hydrophobic drug is characterized by comprising the following steps:
(1) preparing a silk fibroin aqueous solution;
(2) dissolving a hydrophobic drug in a small molecular weight polyethylene glycol solution to form a solution;
(3) mixing the solutions obtained in the step (1) and the step (2) to obtain a blending solution of a hydrophobic drug, silk fibroin and low molecular weight polyethylene glycol;
(4) and (4) mixing the blending solution obtained in the step (3) with a high molecular weight polyethylene glycol solution, and incubating to obtain the high-drug-loading-rate silk fibroin micro-nano particle sustained release preparation loaded with the hydrophobic drug.
2. The method as claimed in claim 1, wherein the volume ratio of the silk fibroin to the small molecular weight polyethylene glycol solution to the large molecular weight polyethylene glycol solution is 10:1: 10-5: 1: 5.
3. The method as claimed in claim 1, wherein in step (1), the concentration of silk fibroin in the aqueous silk fibroin solution is: 1-30 wt%.
4. The method according to claim 1, wherein the molecular weight of the small-molecule polyethylene glycol is 200-600.
5. The method according to claim 1, wherein the concentration of the small molecular weight polyethylene glycol solution is: 50 to 90 percent.
6. The method of claim 1, wherein the molecular weight of the macrogol is 6,000-20,000.
7. The method according to claim 1, wherein the concentration of the solution of high molecular weight polyethylene glycol is: 20 to 60 percent.
8. The method of claim 1, wherein the hydrophobic drug is one or more of paclitaxel, docetaxel, curcumin, risperidone, rifampin, felodipine, carbamazepine, indomethacin, furosemide, camptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, topotecan, irinotecan, 9-aminocamptothecin, teniposide, etoposide, cyclosporine-A, fenofibrate, sirolimus, aprepitant, megestrol, paliperidone, erlotinib, silymarin, quercetin, itraconazole, naproxen, dexamethasone, diosmin, icaritin, oridonin, puerarin, nevirapine, or ziprasidone.
9. The hydrophobic drug loaded silk fibroin micro-nano particle sustained release preparation prepared by the method of any one of claims 1-8.
10. The hydrophobic drug loaded silk fibroin micro-nano particle sustained release preparation of claim 9, and its application in hydrophobic drug sustained release.
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