CN114983970B - Biological film coated drug-loaded protein nanoparticle and preparation method thereof - Google Patents

Biological film coated drug-loaded protein nanoparticle and preparation method thereof Download PDF

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CN114983970B
CN114983970B CN202210797070.9A CN202210797070A CN114983970B CN 114983970 B CN114983970 B CN 114983970B CN 202210797070 A CN202210797070 A CN 202210797070A CN 114983970 B CN114983970 B CN 114983970B
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陈立江
郝俊旭
陶旭
李指航
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Liaoning University
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Abstract

The invention relates to a medicine carrying protein nanoparticle wrapped by a biological film and a preparation method thereof, belonging to the field of pharmaceutical preparations. The biological film-coated drug-loaded protein nanoparticle is prepared by self-assembling a natural high-molecular protein carrier which has good biocompatibility and no toxicity and is easy to surface modify and a hydrophobic anti-tumor drug into the drug-loaded protein nanoparticle, and further preparing the biological film-coated drug-loaded protein nanoparticle by adopting a high-pressure extrusion method. The drug-loaded protein nanoparticle wrapped by the biological film is adhered to the intestinal tract through the biological film and phagocytized by the epithelial cells of the small intestine, so that the oral administration of the hydrophobic antitumor drug is realized, the antitumor effect of the hydrophobic drug is improved, and the adverse reaction is reduced.

Description

Biological film coated drug-loaded protein nanoparticle and preparation method thereof
Technical Field
The invention belongs to the field of pharmaceutical preparations, and in particular relates to a biological film coated drug-loaded protein nanoparticle and a preparation method thereof.
Background
Chemotherapy is one of the main treatment methods of malignant tumors at present, but a large number of therapeutic drugs have poor absorption and lack of targeting, so that larger adverse reactions can be generated. Paclitaxel (PTX) is a widely used chemotherapeutic agent in clinic that induces aggregation of tubulin, thereby inhibiting division of cancer cells and causing apoptosis. However, taxol also has certain application limitations, such as extremely poor water solubility, the need of adding cosolvent, frequent hypersensitivity reaction, etc.; meanwhile, taxol is used as a broad-spectrum antitumor drug, and has poor selectivity, and can generate larger adverse reactions such as cardiotoxicity, bone marrow suppression, digestive tract symptoms and the like.
The silk fibroin is derived from mulberry silk, has the advantages of good biocompatibility, no toxicity, easy surface modification and the like, and is an ideal carrier for packaging medicines with hydrophobic properties. Silk fibroin provides a good basis for transformation and modification to realize other functions due to the abundant chemical groups such as hydroxyl, amino, carboxyl and disulfide bonds. Nanoparticles, membranes, microspheres, gels, fibers, 3D scaffolds, coatings, etc. based on silk fibroin are continually developed and achieved with a certain result.
The nano particles are easy to be phagocytized by macrophages after entering a human body and cleared by other human immune mechanisms, and the oral administration treatment of cancer has the advantages of not damaging local skin or mucous membrane of the human body in the use process, and the like, and is widely used for long-term administration in clinic, but is also limited by factors such as multiple absorption barriers of gastrointestinal tracts, liver clearance, narrow therapeutic index, poor solubility of partial medicines, poor stability or poor penetrability and the like. Therefore, the preparation of the bionic drug carrier by wrapping the nano-particles with the biological membrane is a new idea. Bacterial Outer Membrane Vesicles (OMVs) are a bacterial derivative that, upon oral administration into the digestive system, adhere to the intestinal tract and are phagocytosed by small intestinal epithelial cells, with the delivery of anticancer drugs.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a biological film coated drug-loaded protein nanoparticle and a preparation method thereof.
The invention adopts the technical scheme that:
a biological film coated drug-loaded protein nanoparticle is prepared by self-assembling natural high-molecular protein carrier with good biocompatibility, no toxicity and easy surface modification with hydrophobic antitumor drug to obtain drug-loaded protein nanoparticle, and coating the drug-loaded protein nanoparticle with biological film.
Furthermore, the biological membrane wrapped drug-loaded protein nanoparticle is an erythrocyte membrane, an immune cell membrane or a bacterial outer membrane vesicle.
Furthermore, the natural high molecular protein carrier is silk fibroin or albumin.
Furthermore, the above-mentioned drug-loaded protein nanoparticle wrapped by a biological membrane, wherein the hydrophobic antitumor drug is paclitaxel, doxorubicin, camptothecin, curcumin, sorafenib or gemcitabine.
Preferably, the above-mentioned drug-loaded protein nanoparticle wrapped by a biological membrane, wherein the biological membrane is a bacterial outer membrane vesicle; the natural high molecular protein carrier is silk fibroin; the hydrophobic antitumor drug is paclitaxel.
The preparation method of the drug-loaded protein nanoparticle wrapped by the biological film comprises the following steps:
1) Extraction of silk fibroin: taking 10g of chopped cocoon shell, placing the cocoon shell in Na 2 CO 3 Heating and stirring in water solution at 100deg.C for 30min, washing with a large amount of distilled water, repeating the above treatment process for three times, oven drying at 60deg.C to obtain degummed silk, collecting 5g degummed silk, placing in 50mL LiBr water solution, heating and stirring at 65deg.C for 2 hr to obtain silk fibroin water solution, dialyzing, centrifuging, collecting supernatant to obtain silk fibroin solution, lyophilizing to obtain solid powder, and sealing at 4deg.C;
2) Preparation of paclitaxel silk fibroin nanoparticles: dissolving paclitaxel in absolute ethanol, dropwise adding into the silk fibroin solution obtained in the step 1), stirring, centrifuging at a lower centrifugal speed for 5min to remove floccules in the product, centrifuging at a higher centrifugal speed for 20min to collect paclitaxel silk fibroin nanoparticles, washing with distilled water for 3 times, and storing at-20deg.C;
3) Extraction of bacterial outer membrane vesicles: taking escherichia coli culture solution, centrifugally collecting supernatant, filtering by using a 0.45 mu m filter membrane, then placing filtrate into an ultrafiltration centrifuge tube, and continuously centrifuging until the volume becomes 20% of the original volume to obtain concentrated bacterial outer membrane vesicle suspension;
4) Preparation of bacterial outer membrane vesicle-coated paclitaxel silk fibroin nanoparticle: preparing taxol silk fibroin nanoparticles wrapped by bacterial outer membrane vesicles by adopting a high-pressure extrusion method, centrifuging at 20000rpm for 1h at 4 ℃ and collecting the precipitate to obtain a target product.
Further, in the preparation method of the biomembrane-coated drug-loaded protein nanoparticle, in the step 1), the Na is as follows 2 CO 3 The concentration of the aqueous solution was 0.2mol/L; the concentration of the LiBr aqueous solutionThe degree is 9.5mol/L; the dialysis is performed by using a dialysis bag with a molecular weight of 12000 kDa.
Further, in the preparation method of the biomembrane-coated drug-loaded protein nanoparticle, in the step 2), the sampling amount of the paclitaxel is 12.66mg; the concentration of the silk fibroin solution is 3.78mg/mL; the stirring time was 75min.
Further, in the above preparation method of the drug-loaded protein nanoparticle coated with a biological film, in the step 2), the lower centrifugal rotation speed is 6000rpm; the higher centrifugal speed was 13000rpm.
Further, in the preparation method of the drug-loaded protein nanoparticle wrapped by the biological film, in the step 4), the specific steps of the high-pressure extrusion method are as follows: taking taxol silk fibroin nanoparticle, re-suspending, uniformly mixing with concentrated bacterial outer membrane vesicle suspension, placing the mixed solution in a liposome extruder, extruding through a 200nm polycarbonate membrane, and repeating the extrusion process for 10 times.
The beneficial effects of the invention are as follows:
1. the invention discovers through the research of the interaction mechanism of paclitaxel and silk fibroin: the silk fibroin contains a large amount of tryptophan, and once taxol interacts with the silk fibroin, the microenvironment of a large amount of tryptophan residues in the silk fibroin can be changed correspondingly, so that the fluorescence intensity of the silk fibroin is changed, and the phenomenon of fluorescence quenching is generated.
2. The invention discovers through the research on the interaction force of paclitaxel and silk fibroin: the combination of taxol and silk fibroin can be carried out spontaneously, and the main effects are exerted in the hydrophobic acting force and hydrogen bonding process.
3. Silk fibroin is an ideal carrier for packaging PTX, and nanoparticles based on silk fibroin can effectively improve the problems in PTX treatment, and PTX and silk fibroin are combined through hydrogen bond and hydrophobic interaction, so that the preparation of self-assembled nanoparticles (PTX-SF-NPs) is proved to be successful.
4. Bacterial Outer Membrane Vesicles (OMVs) are naturally secreted by bacteria in the normal growth process, the structure of the bacterial Outer Membrane Vesicles (OMVs) is similar to that of a bacterial membrane, the bacterial outer membrane vesicles can be taken up by intestinal wall cells, the OMVs based on escherichia coli can be taken up by the intestinal wall cells and partially resist gastric acid, the high-pressure extrusion method is adopted to prepare OMVs-coated PTX silk fibroin nanoparticles (OMVs-NPs), and a drug carrying system is adhered to the intestinal tract through a biological membrane and phagocytized by small intestinal epithelial cells, so that the oral administration of the drug carrying protein nanoparticles is realized, the anti-tumor effect of a hydrophobic drug is improved, and adverse reactions are reduced.
Drawings
FIG. 1 is a graph showing fluorescence quenching of PTX and silk fibroin at various concentrations.
FIG. 2 is a graph showing particle size of PTX-SF-NPs.
FIG. 3 is a potential diagram of PTX-SF-NPs.
FIG. 4 is a transmission electron microscope image of PTX-SF-NPs.
FIG. 5 is an infrared spectrum of different substances, wherein (a) represents a PTX drug substance, (b) represents a silk fibroin lyophilized powder, (c) represents a physical mixture of a PTX drug substance and a silk fibroin lyophilized powder, and (d) represents PTX-SF-NPs.
FIG. 6 is a differential scanning calorimetry diagram of different substances, wherein (a) represents a PTX drug substance, (b) represents a silk fibroin lyophilized powder, (c) represents PTX-SF-NPs, and (d) represents a physical mixture of a PTX drug substance and a silk fibroin lyophilized powder.
FIG. 7 is an in vitro release profile of PTX-SF-NPs under various conditions.
FIG. 8 is a graph of particle size of OMV-NPs.
FIG. 9 is a potential diagram of OMV-NPs.
FIG. 10 is a transmission electron microscope image of OMV-NPs.
FIG. 11 is an in vitro release profile of PTX, PTX-SF-NPs and OMV-NPs in an artificial gastric juice environment.
FIG. 12 is an in vitro release profile of PTX, PTX-SF-NPs and OMV-NPs in artificial gastric juice and artificial intestinal juice environments.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
EXAMPLE 1 preparation of biofilm-coated nanoparticles of paclitaxel silk fibroin (OMV-NPs)
Extraction of silk fibroin
The method comprises the following steps:
1) 10g of chopped cocoon shell is taken and placed in 0.2mol/L Na 2 CO 3 Heating and stirring for 30min at 100 ℃ in the aqueous solution, and then flushing with a large amount of distilled water;
2) Repeating the treatment process of the cut cocoon shells in the step 1) for three times, drying at 60 ℃ to obtain degummed silk, preparing a LiBr aqueous solution with the concentration of 9.5mol/L, placing 5g degummed silk into 50mL LiBr aqueous solution, and heating and stirring at 65 ℃ for 2 hours to obtain a silk fibroin aqueous solution;
3) Dialyzing the aqueous solution of silk fibroin by using a dialysis bag (MW 12000 kDa), dialyzing in continuously-replaced distilled water for 3d, centrifuging at 13000rpm for 20min after dialysis, collecting supernatant, namely the silk fibroin solution, lyophilizing to obtain solid powder, and sealing and preserving at 4deg.C.
(II) investigation of the interaction of paclitaxel with silk fibroin
1. The experimental procedure was as follows:
1) Precisely weighing 25mg of silk fibroin freeze-dried powder, placing the powder into a 50mL volumetric flask, and fixing the volume to obtain 0.5mg/mL silk fibroin standard solution; precisely weighing 34mg of Paclitaxel (PTX), dissolving with absolute ethanol, placing into 50mL volumetric flask, and fixing volume to obtain 8×10 -4 PTX standard stock of M; taking a proper amount of PTX standard stock solution, and respectively diluting the stock solution into 1X 10 according to a proportion -4 M、8×10 -5 M、6×10 -5 M、4×10 -5 M and 2×10 -5 PTX solution of M;
2) Placing the PTX solutions with different concentrations and the silk fibroin standard solution at 293K for 1h, ensuring a fluorescence measurement environment to be 293K, respectively adding the PTX solutions with different concentrations into the silk fibroin standard solution, standing for 5min, measuring fluorescence intensity in a fluorescence chromatograph, wherein the excitation wavelength of the fluorescence spectrometer is 295nm, and the emission wavelength is 305-500nm; the ambient temperature was changed to 303K, other conditions were kept unchanged, and the above experimental steps were repeated.
2. The fluorescence spectra of the PTX solution and the silk fibroin standard solution with different concentrations are shown in the figure 1, the peak value of the fluorescence spectrum is 345.5nm, which is the tryptophan residue absorption peak, and the fluorescence intensity of the silk fibroin is obviously reduced along with the increase of the PTX concentration, so that the interaction occurs between the silk fibroin and the PTX. The research result of the interaction mechanism of paclitaxel and silk fibroin shows that: the silk fibroin contains a large amount of tryptophan, and once taxol interacts with the silk fibroin, the microenvironment of a large amount of tryptophan residues in the silk fibroin can be changed correspondingly, so that the fluorescence intensity of the silk fibroin is changed, and the phenomenon of fluorescence quenching is generated.
3. Thermodynamic parameters were calculated using the Van't Hoff formula:
ΔG＝ΔH-TΔS (2)
where Δh and Δs are the enthalpy change and entropy change, respectively, Δg is the gibbs free energy, R is the gas constant, T is the absolute temperature, and Δh, Δs, and Δg are calculated from the KA values in equation (1).
TABLE 1 thermodynamic parameters of PTX and silk fibroin
Researchers summarize thermodynamic rules to determine the binding mode of proteins and small molecule drugs, and the interaction forces between small molecule drugs and proteins mainly comprise van der waals forces, electrostatic forces, hydrogen bonds, hydrophobic forces and the like, and are mainly determined by thermodynamic parameters. The results are shown in Table 1, and when ΔG <0, it is shown that the reaction entropy increases and can proceed spontaneously; Δs >0 indicates that the hydrophobic forces play a critical role. Furthermore, since the structure of PTX contains a plurality of hydroxyl groups, Δh is negative due to hydrogen bonding. In conclusion, the binding of PTX to silk fibroin can proceed spontaneously, with major roles in hydrophobic forces and hydrogen bonding processes.
Preparation and characterization of (III) Taxol silk fibroin nanoparticles (PTX-SF-NPs)
1. Preparation of PTX-SF-NPs
The method comprises the following steps: dissolving 12.66mg of PTX in absolute ethanol, dropwise adding the absolute ethanol into 3.78mg/mL of silk fibroin solution, stirring, carrying out 30% amplitude ultrasound on the solution in an ice bath for 2min, placing the solution in a ventilation device, stirring for 75min, centrifuging at a lower centrifuging speed (6000 rpm) for 5min to remove floccules in the product, centrifuging at a higher centrifuging speed (13000 rpm) for 20min to collect PTX-SF-NPs, washing with distilled water for 3 times, and preserving in an environment of minus 20 ℃.
2. Particle size and Zeta potential investigation of PTX-SF-NPs
Taking 2mL of PTX-SF-NPs suspension, placing the suspension in a particle size measuring dish and a potential measuring dish, and operating a workstation to carry out particle size measurement. As shown in FIGS. 2 and 3, the PTX-SF-NPs had an average particle diameter of 164.8.+ -. 2.6nm, a particle diameter distribution of a single peak, a PDI of 0.135.+ -. 0.021 and a potential of-14.8.+ -. 1.74mV.
3. Morphological investigation of PTX-SF-NPs
As a result of morphological examination of PTX-SF-NPs, the PTX-SF-NPs were in a regular sphere shape and were free from aggregation, and the nanoparticle size was about 160nm, which was consistent with the particle size measurement results, as shown in FIG. 4.
4. Infrared Spectroscopy (IR) investigation of PTX-SF-NPs
The infrared spectrum of PTX-SF-NPs is shown in FIG. 5, and the main characteristic absorption peaks of the freeze-dried powder of silk fibroin are 1646, 1552 and 1236cm -1 Wherein 1646cm -1 Is amide I,1552cm -1 Is amide II,1236cm -1 Is amide III; PTX-SF-NPs are substantially consistent with the absorption peaks of the freeze-dried powder of Silk fibroin, so that the structure of Silk fibroin in the nanoparticle is deduced to be that of Silk I mainly comprising random coil and alpha-helix; 1720cm of PTX-SF-NPs -1 The absorption peak is a PTX characteristic peak, which shows that PTX-SF-NPs successfully encapsulate the drug, and the chemical bond is kept unchanged after drug encapsulation.
5. Differential Scanning Calorimetric (DSC) investigation of PTX-SF-NPs
Differential thermal analysis was performed on the PTX drug substance, the silk fibroin lyophilized powder, the physical mixture of the two, and the PTX-SF-NPs by using a differential scanning calorimeter, and as shown in FIG. 6, the endothermic peak and the exothermic peak of PTX (a) are respectively at 226.5 ℃ and 242.7 ℃, which proves that the PTX drug substance exists in a crystal form; the physical mixture of the PTX drug substance and the silk fibroin lyophilized powder (d) had the same peaks at 226.5 ℃ and 242.7 ℃; while PTX-SF-NPs (c) showed no PTX absorption peak, indicating that PTX binds to silk fibroin as a new phase, which is consistent with fluorescence quenching results.
In vitro Release investigation of paclitaxel silk fibroin nanoparticles (PTX-SF-NPs)
In order to examine the drug release condition of PTX-SF-NPs under different conditions, different release mediums were formulated, and a PTX drug substance suspension was formulated as a control. 2mL of a suspension containing 1mg of PTX drug substance is placed in a dialysis bag (MW 3000 kDa), and the dialysis bag is sealed and placed in 40mL of a release medium (PBS solution of pH 7.4,0.5% Tween-80); 4 parts of PTX-SF-NPs suspension (PTX content is 1 mg) with volume of 2mL are respectively placed in dialysis bags (MW 3000 kDa), the dialysis bags are respectively placed in 40mL of different release mediums (PBS solution with pH of 7.4, pH of 5.0, pH of 7.4+GSH (10 mM) and pH of 5.0+GSH (10 mM) after being sealed, 0.5% Tween-80 is added, the mixture is shaken in a shaking table at 37 ℃ and 100rpm, 0.5mL of release liquid is taken out from each of 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48h, then 0.5mL of fresh release medium is supplemented, and the release liquid is detected by using high performance liquid chromatography.
The results are shown in FIG. 7, in which PTX-SF-NPs were released faster in acidic release medium (pH 5.0) than in release medium at pH 7.4, and the cumulative amount reached 78.25%, indicating that PTX-SF-NPs had a certain degree of pH-sensitive drug release capacity. This is due to the acidic environment which causes damage to the silk fibroin and thus accelerates the release of the drug. Compared with a release medium without GSH, the PTX-SF-NPs have stronger drug release capability after GSH is added, and the GSH is presumed to break disulfide bonds of silk fibroin, so that the drug release is further accelerated. In conclusion, the in vitro release results show that PTX-SF-NPs have the capability of stimulating corresponding drug release, are easier to release drugs in simulated tumor environments, and are excellent anti-tumor drug carriers.
Preparation and characterization of bacterial outer membrane vesicle-encapsulated paclitaxel silk fibroin nanoparticles (OMV-NPs)
1. Extraction of bacterial outer membrane vesicles
Taking a proper amount of escherichia coli culture solution, centrifuging at 8000rpm for 12min at 4 ℃, collecting supernatant, filtering by using a 0.45 mu m filter membrane, and then placing the filtrate into an ultrafiltration centrifuge tube, and centrifuging at 4000rpm at 4 ℃ until the volume becomes 20% of the original volume, thus obtaining concentrated OMVs suspension.
2. Preparation of OMV-NPs
OMV-NPs were prepared by high pressure extrusion: taking PTX-SF-NPs, re-suspending, uniformly mixing with concentrated OMVs suspension, placing the mixed solution in a liposome extruder, extruding through a 200nm polycarbonate film, repeating the extrusion process for 10 times, centrifuging at 20000rpm for 1h in a 4 ℃ environment, and collecting the precipitate to obtain the OMV-NPs.
3. Particle size and Zeta potential investigation of OMV-NPs
The particle size and Zeta potential of OMV-NPs were examined, and the results are shown in FIGS. 8 and 9, wherein the particle size of OMV-NPs was 199.8.+ -. 2.8nm, the particle size distribution was unimodal, the PDI was 0.156.+ -. 0.081, and the potential was-17.8.+ -. 1.3mV.
4. Morphological investigation of OMV-NPs
The morphological examination of OMV-NPs showed that OMV-NPs were regular spheres and no aggregation, and film-like structure was observed, indicating successful outer membrane vesicle encapsulation, particle size around 200nm, consistent with particle size measurement results, as shown in FIG. 10.
5. Stability investigation of OMV-NPs
The OMV-NPs were placed in an environment at 4deg.C for 10 days, and stability test was conducted with particle size and PDI potential as the index, and the results are shown in Table 2, where there was no significant change in both particle size and PDI, demonstrating good stability of OMV-NPs.
TABLE 2 OMV-NPs stability evaluation
Six in vitro release investigation of OMV-NPs
In order to examine the release condition of OMV-NPs entering human gastrointestinal tract by oral administration, artificial gastric juice and artificial intestinal juice are respectively prepared as release mediums. In order to simulate the digestion and release process of the nano-particles in the gastrointestinal tract of a human body, the nano-particles are firstly placed in a gastric juice environment for two hours and then placed in an intestinal juice environment. Placing 2mL of each of PTX bulk drug, PTX-SF-NPs and OMV-NPs suspension (the PTX content is 1 mg) in a dialysis bag (MW 3000 kDa), sealing the dialysis bag, placing the dialysis bag in 40mL of artificial gastric juice release medium, placing the dialysis bag in a shaking table at 37 ℃ and 100rpm for shaking, taking out the dialysis bag after 2 hours, placing the dialysis bag in 40mL of artificial intestinal juice release medium, placing the dialysis bag in the shaking table for shaking, taking out 0.5mL of release liquid at the time points of 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36 and 48 hours of total time, and then supplementing 0.5mL of fresh release medium, wherein the release liquid is detected by using high performance liquid chromatography.
The release of PTX drug substance, PTX-SF-NPs and OMV-NPs suspensions under various conditions was tested and the results are shown in FIGS. 11 and 12. To simulate the human condition as much as possible, the first two hours of nanoparticle release were performed in artificial gastric juice and then in artificial intestinal juice. From the experimental results, the PTX-SF-NPs observe obvious abrupt release phenomenon at the initial stage of release, but OMV-NPs have no abrupt release phenomenon, the release is stable and slow, the biofilm encapsulation solves the problem of abrupt release of the drug by nanoparticles, the drug release capacity is better, 36.2% of the drug is released by the PTX-SF-NPs in an artificial gastric juice environment for two hours, the OMV-NPs release only 16.7%, which indicates that gastric acid can damage the structure of the silk fibroin, so that the drug in the PTX-SF-NPs is released rapidly, and OMVs protect the silk fibroin from being damaged to a certain extent, and the release of the drug in the stomach is slowed down. In conclusion, the in vitro release result shows that the OMV-NPs has better slow release performance, has certain capacity of resisting the damage of gastric acid, and can protect the silk fibroin nano particles from reaching the small intestine.

Claims (5)

1. The preparation method of the drug-loaded protein nanoparticle wrapped by the biological film is characterized by comprising the following steps:
1) Extraction of silk fibroin: taking 10g cut cocoon shells, placing the cocoon shells in Na 2 CO 3 Heating and stirring in water solution at 100deg.C for 30min, washing with a large amount of distilled water, repeating the above treatment process for three times, oven drying at 60deg.C to obtain degummed silk, collecting 5g degummed silk, placing in 50mL LiBr water solution, heating and stirring at 65deg.C for 2h to obtain silk fibroin water solution, dialyzing, centrifuging, collecting supernatant to obtain silk fibroin solution, lyophilizing to obtain solid powder, and sealing at 4deg.C;
2) Preparation of paclitaxel silk fibroin nanoparticles: dissolving paclitaxel in absolute ethanol, dropwise adding into the silk fibroin solution obtained in the step 1), stirring, centrifuging at a lower centrifugal speed for 5min to remove floccules in the product, centrifuging at a higher centrifugal speed for 20min to collect paclitaxel silk fibroin nanoparticles, washing with distilled water for 3 times, and storing at-20deg.C;
3) Extraction of bacterial outer membrane vesicles: taking escherichia coli culture solution, centrifugally collecting supernatant, filtering by using a 0.45 mu m filter membrane, then placing filtrate into an ultrafiltration centrifuge tube, and continuously centrifuging until the volume becomes 20% of the original volume to obtain concentrated bacterial outer membrane vesicle suspension;
4) Preparation of bacterial outer membrane vesicle-coated paclitaxel silk fibroin nanoparticle: the taxol silk fibroin nanoparticle wrapped by the bacterial outer membrane vesicle is prepared by adopting a high-pressure extrusion method, and then is centrifuged at 20000rpm for 1h at the temperature of 4 ℃ to collect the precipitate, thus obtaining the target product.
2. The method for preparing a biomembrane coated nanoparticle of a drug-loaded protein as claimed in claim 1, wherein in step 1), the Na is as follows 2 CO 3 The concentration of the aqueous solution was 0.2mol/L; the concentration of the LiBr aqueous solution is 9.5mol/L; the dialysis is performed by using a dialysis bag with a molecular weight of 12000 kDa.
3. The method of claim 1, wherein in step 2), the paclitaxel is sampled in an amount of 12.66 and mg; the concentration of the silk fibroin solution is 3.78mg/mL; the stirring time was 75min.
4. The method of claim 1, wherein in step 2), the lower centrifugation speed is 6000rpm; the higher centrifugal speed was 13000rpm.
5. The method for preparing the biomembrane coated nanoparticle of the present invention as set forth in claim 1, wherein in the step 4), the high-pressure extrusion method comprises the following specific steps: taking taxol silk fibroin nanoparticle, re-suspending, uniformly mixing with concentrated bacterial outer membrane vesicle suspension, placing the mixed solution in a liposome extruder, extruding through a 200nm polycarbonate membrane, and repeating the extrusion process for 10 times.
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