Recombinant spider silk protein and application thereof

Abstract

The invention belongs to the technical field of protein, and aims to solve the problem that the application range of spider silk particles as a drug carrier is limited due to the fact that the spider silk particles are in negative net charge under a neutral condition and can only load drugs with positive charges or neutral charges at present. The novel recombinant spider silk protein is prepared on the basis of an N connection sequence of araneus ventricosus MaSp1, has positive net charge under a neutral pH value, can load a negatively charged drug, and expands the application range of the spider silk protein as a drug carrier; and a drug release test result shows that the recombinant spider silk protein is superior to MaSp2-based silk protein particles in the aspect of etoposide delivery.

Description

Technical Field

[0001] This invention belongs to the field of protein technology, specifically relating to a recombinant spider silk protein and its applications. Background Technology

[0002] The development of drug delivery systems is crucial for drug targeting, pharmacokinetics, improving solubility and biodispersibility, and reducing toxicity. Colloidal nanoparticle carriers have been widely used as drug delivery systems due to their degradability within months. However, currently used drug carriers of this type are made from synthetic polyesters or other naturally derived hydrophilic polymers, such as polylactic-co-glycolic acid copolymer (PLGA), chitosan, and gelatin, which have several drawbacks, including the use of organic solvents in the preparation process and high burst release rates. To overcome these limitations, an ideal carrier material is needed that possesses properties such as biocompatibility, biodegradability, non-toxicity, good stability, high drug loading efficiency, and sustained drug release.

[0003] Spider silk proteins, the main components of spider silk fibers, can be processed into various material types, such as nanoparticles, fibers, hydrogels, and films. Compared to polymers, spider silk proteins are promising candidates due to their non-cytotoxicity, customizability, and efficient drug binding and release capabilities. Furthermore, spider silk protein-based particles exhibit high mechanical strength and long-term stability due to their reversible swelling behavior. A variety of molecules, including peptides, model drugs, proteins, and small molecules, can also be loaded onto spider silk particles in varying amounts depending on their physical and chemical properties. However, most reported spider silk particles exhibit a negative net charge under neutral conditions, limiting their application as drug carriers to positively or neutrally charged drugs.

[0004] Currently, research on drug carriers for negatively charged molecules mainly focuses on cationic liposomes or cationic polymers. However, a major drawback of liposomes is that they often trigger rapid and intense immune responses and accumulate in large quantities in the liver, potentially leading to various adverse effects and limiting their effectiveness as therapeutic drug delivery systems. Although linear polyethyleneimine (PEI) and branched polyethyleneimine have been widely used in gene delivery, they still suffer from problems such as toxicity and non-biodegradability. Recent studies have shown that by genetically modifying individual amino acid residues, novel bioengineered spider silk with unique properties can be obtained, enabling the encapsulation of negatively charged drugs. Due to changes in the amino acid sequence, genetic modification of spider silk proteins may also result in the inability to self-assemble into spider silk particles. With the rapid development of sequencing technology, a large number of spider silk gene sequences have been identified. Given the diversity of spider silk proteins, it is necessary to explore new spider silk proteins as carriers for negatively charged drugs. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a recombinant spider silk protein and its applications.

[0006] The first object of the present invention is to provide a gene whose nucleotide sequence is shown in SEQ ID NO.1.

[0007] A second objective of the present invention is to provide a recombinant spider silk protein encoded by the above-mentioned gene, which is composed of two MaSp1 N-linked regions connected in series; The amino acid sequence of the recombinant spider silk protein is shown in SEQ ID NO.2.

[0008] A third objective of this invention is to provide a recombinant vector containing the aforementioned genes.

[0009] A fourth object of the present invention is to provide a silk fibroin particle comprising the above-described recombinant spider silk protein.

[0010] A fifth objective of this invention is to provide an application of the above-mentioned gene, recombinant spider silk protein, recombinant vector, or silk fibroin particles in the preparation of a drug carrier.

[0011] Preferably, the drug is a negatively charged drug.

[0012] Compared with the prior art, the advantages of this invention are as follows: The recombinant spider silk protein synthesized in this invention has a positive net charge at neutral pH, enabling it to load negatively charged drugs, thus expanding the application range of spider silk protein as a drug carrier. Furthermore, drug release test results show that the recombinant spider silk protein is superior to MaSp2-based silk protein particles in etoposide delivery. Attached Figure Description

[0013] Figure 1 Figure 1 shows the analytical results of recombinant spider silk protein; where A is the amino acid sequence of recombinant spider silk protein; and B is the 15% SDS-PAGE gel analysis result of recombinant spider silk protein. Figure 2 Figure 1 shows the characterization results of NL silk fibroin particles; where A is a scanning electron microscope image; B is a particle size distribution map; C is the particle size result of NL silk fibroin particles after storage in ultrapure water and under different pH conditions for 1 hour and 24 hours; D is the Zeta potential of NL silk fibroin particles under different pH values ​​and ionic strengths. Figure 3 The results of a comparative study on the cytotoxicity of NL silk fibroin particles and MaSp2 spider silk protein are shown; the error bars represent the standard deviation. Figure 4The loading efficiency results of Rho-ODN, CFI, and ETP on NL silk fibroin particles at room temperature are compared with the loading rate results of ETP on NL silk fibroin particles and MaSp2 particles; where A is the loading efficiency results of Rho-ODN, CFI, and ETP on NL silk fibroin particles; B is the comparison of the loading rate results of ETP on NL silk fibroin particles and MaSp2 particles. Figure 5 The results show the release efficiency of Rho-ODN, CFI, and ETP from NL silk fibroin particles in vitro; where A represents the release efficiency of Rho-ODN, CFI, and ETP from NL silk fibroin particles at different pH values ​​over 30 days; and B represents the release efficiency of Rho-ODN, CFI, and ETP from NL silk fibroin particles at different ionic strengths over 24 hours. Figure 6 Comparison of ETP release rates on NL silk fibroin particles and MaSp2 particles. Detailed Implementation

[0014] The following will be described in conjunction with embodiments of the present invention. Figures 1 to 6 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0015] I. Materials and Methods 1. Design, production, and purification of recombinant spider silk protein (NL) The gene encoding NL (containing two tandem MaSp1 N linker regions) was synthesized by Qingke Biotechnology Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO.1. The gene encoding NL was ligated into the expression vector pEHS to obtain a recombinant vector. This recombinant vector was transformed into *E. coli* BL21(DE3) to obtain recombinant expression cells. After confirming the DNA sequence in the plasmid of the recombinant expression cells, the recombinant expression cells were used for protein production.

[0016] Recombinant expression cells were cultured in LB medium containing 100 μg / mL ampicillin at 37°C until the OD600 value reached 0.6–0.8. Subsequently, protein induction experiments were performed at 20°C with 0.3 mM isopropyl-β-D-thiogalactoside (IPTG). After the protein induction experiment, the obtained cells were resuspended in 20 mM Tris buffer (pH 8.0) containing 300 mM NaCl and 10 mM imidazole, and then disrupted using a JN-3000 Plus pressure cell disruptor (JNBIO, China).

[0017] Next, the supernatant was bound to a Ni-NTA column on ice for 1 hour to purify the recombinant protein. The resin was then washed twice with Tris buffer containing 30 mM imidazole. Finally, the purified recombinant protein was eluted with Tris buffer containing 250 mM imidazole to obtain the purified protein (i.e., recombinant spider silk protein (NL)). The amino acid sequence of NL is shown in SEQ ID NO.2. The specific sequence results are as follows: Figure 1 As shown in A in the figure. Dialyzed with Tris buffer at 4°C and analyzed by 15% SDS-PAGE gel.

[0018] 2. Preparation of NL silk fibroin particles The purified protein was diluted to a final concentration of 0.5 mg / mL with 2 M potassium phosphate buffer (pH 7.0) and incubated overnight at room temperature. The mixture was then centrifuged at 12,000 rpm for 10 minutes to obtain precipitated NL silk fibroin particles. The obtained NL silk fibroin particles were washed three times with ultrapure water, resuspended in ultrapure water, and stored at 4°C. The particle concentration was determined by gravimetric analysis.

[0019] 3. Particle size and zeta potential analysis Prior to measurement, NL silk fibroin particle samples were diluted directly to 0.01 mg / mL in the appropriate buffer solution. Particle size and zeta potential of the NL silk fibroin particles were determined using a Zetasizer Nano ZS (Malvin Panasonic, UK). Each measurement was performed in triplicate at 25°C.

[0020] 4. Scanning electron microscopy (SEM) analysis NL silk fibroin particles were washed three times with ultrapure water, air-dried, and then coated with platinum. Scanning electron microscopy analysis was performed using a Hitachi SU8010 instrument (Japan) at 25°C and an accelerating voltage of 3 kV.

[0021] 5. Cytotoxicity studies In vitro cytotoxicity studies of NL particles were conducted using HeLa cells, specifically as follows: By seeding 1×10 in each well of a 96-well plate 5 We collected 10 HeLa cells and seeded them with purified protein, then cultured them at 37°C in 5% CO2. After 72 hours of culture, cell viability was determined using the MTT assay. The absorbance of the solution was measured at 560 nm using an Epoch 2 microplate reader (BioTek).

[0022] 6. Particle Loading NL silk fibroin particles were loaded using a diffusion method with three substances: carboxyfluorescein (CFI), rhodamine-labeled oligonucleotides (Rho-ODN), and etoposide (ETP).

[0023] For loading NL silk fibroin particles with CFI, CFI was dissolved in ethanol at a concentration of 25 mmol. The dye solution (CFI) was diluted in Tris buffer, and freshly washed silk fibroin particles were added to precipitate, followed by incubation at room temperature for 1 hour. The supernatant was collected by centrifugation and then analyzed using UV / Vis spectroscopy at a wavelength of 492 nm to determine the loading capacity and loading efficiency of NL silk fibroin particles.

[0024] For NL silk fibroin particles loaded with Rho-ODN (5'-gtg cca tct ggt gct aac g-3'), 200 μL of 100 μM Rho-ODN was mixed with NL silk fibroin particles, incubated at 25 °C for 1 hour, and then centrifuged for 10 minutes. The amount of Rho-ODN loaded in the particles was quantified by measuring the absorbance of the resulting supernatant at a wavelength of 560 nm.

[0025] In the ETP loading experiment, NL silk fibroin particles were resuspended in phosphate buffer, mixed with 2 mg / mL ETP solution, and incubated overnight at 25°C with shaking. The drug concentration in the supernatant was then determined by measuring absorbance at 230 nm. The loading experiment was conducted at a drug-to-NL silk fibroin particle mass ratio of 50%. The loading capacity and loading efficiency of NL silk fibroin particles were calculated using the following formula: Loading (%) = (Amount of substance in particles) / (Amount of NL silk fibroin particles) × 100 (1) Loading efficiency (%) = (amount of substance in the particles) / (amount of dye added) × 100 (2) 7. Substance release To analyze the release of substances from NL silk fibroin particles, the particles were resuspended in the appropriate release buffer and incubated at 37°C for 1 hour, followed by centrifugation at room temperature. The supernatant was then collected and analyzed using UV / Vis spectroscopy at absorption wavelengths specific to the loaded substances.

[0026] II. Results and Discussion 1. Production and purification of NL NL containing two MaSp1 N-linked regions was produced and purified in *E. coli*. The average amount of purified NL per liter of cell culture medium was approximately 10 mg. SDS-PAGE analysis confirmed the high quality and purity of NL (>90%), and its molecular weight was consistent with theoretical calculations, as shown in the results. Figure 1As shown in B in the figure. Unlike MaSp1 from other spider species, MaSp1 from *A. ventricosus* contains a long linker region connecting the N-terminus and the repeat region. Previous studies have shown that the proline- and glycine-rich MaSp1 N-linker region promotes MaSp1 self-assembly and has a higher isoelectric point (8.43). To further modify this protein, two MaSp1 N-linker region sequences were linked together by genetic engineering, resulting in an NL protein with a higher molecular weight and isoelectric point (9.4). This characteristic, compared to the MaSp1 N-linker region, makes it a potential candidate for positively charged silk fibroin particles for drug delivery.

[0027] 2. Preparation, morphology, and zeta potential of NL silk fibroin particles NL was dissolved in 2 M potassium phosphate buffer, and it rapidly assembled into NL silk fibroin particles with a size of 145 ± 9 nm, such as... Figure 2 As shown in Figures A and B, high molar concentrations of potassium phosphate enhance the hydrophobic interactions between silk fibroin particles, thereby inducing their assembly into particles. Furthermore, scanning electron microscopy (SEM) results indicate that these NL silk fibroin particles possess a robust, dense, and uniform structure, specifically as shown in Figures A and B. Figure 2 As shown in Figure A, the colloidal stability of NL silk fibroin particles was analyzed at pH values ​​of 4, 6, and 8, as well as in ultrapure water. The results are shown in Figure A. Figure 2 As shown in C. (Through) Figure 2 The results from C indicate that NL silk fibroin particles are colloidally stable within 24 hours under acidic and ultrapure water conditions, but significant aggregation of NL silk fibroin particles occurs as the pH value gradually increases towards the isoelectric point of NL.

[0028] Previous studies have shown that the stability of positively charged NL silk fibroin particles largely depends on the zeta potential, which provides the repulsive force between charges to inhibit particle aggregation. Since pH and ionic strength significantly affect the zeta potential of charged particles, the zeta potential variations of NL silk fibroin particles under different pH values ​​(5 and 7) and ionic strengths (50, 100, and 154 mmol / L NaCl) were investigated. The results are as follows: Figure 2 As shown in D. (Through) Figure 2 As can be seen from Figure D, under the same ionic strength, the zeta potential of NL silk fibroin particles decreases as the pH value approaches or exceeds the isoelectric point. Furthermore, under the same pH conditions, the zeta potential of NL silk fibroin particles decreases with increasing ionic strength. In summary, these results indicate that NL silk fibroin particles are stable in liquid formulations with low ionic strength and low pH. Moreover, the decrease in zeta potential caused by increasing pH significantly leads to the aggregation of NL silk fibroin particles.

[0029] 3. Cytotoxicity Cytotoxicity studies have shown that NL silk fibroin particles are non-toxic, as shown in the results. Figure 3 As shown. (Through) Figure 3 As can be seen, a slight decrease in cell viability was observed at a particle concentration of 50 μg / mL. Compared with silk fibroin particles based on the MaSp2 spider silk protein common sequence, the NL silk fibroin particles of the present invention exhibit better biocompatibility at high concentrations.

[0030] The loading of model substances was tested using labeled double-stranded DNA (Rho-ODN), carboxyfluorescein (CFI), and ETP as models of nucleic acids, negatively charged substances, and neutrally charged substances, respectively. Because NL silk fibroin particles cannot load positively charged substances due to charge repulsion, positively charged drugs such as DOX and MTX were not used in this study. Figure 4 As shown in Figure A, the uptake efficiency of Rho-ODN in NL silk fibroin particles is approximately 28%, higher than the loading efficiency in modified eADF4(C16) particles. For CFI, previous experiments have shown that its loading efficiency decreases with increasing drug concentration. At a protein-to-substrate ratio of 2:1, the loading efficiency of CFI on NL silk fibroin particles is approximately 94%, slightly lower than the 98% loading efficiency of CFI on eADF4(K16) particles at a protein-to-substrate ratio of 1:1. Given the relationship between loading efficiency and drug concentration, the loading efficiency of CFI on NL silk fibroin particles may further decrease when the molar concentrations of CFI and NL silk fibroin particles are equal. Unlike the substances mentioned above, ETP is moderately hydrophobic, carries a neutral charge, and can penetrate negatively charged particle matrices. Figure 4 As can be seen from B, the efficiency of ETP loading into NL silk fibroin particles is about 40%, which is significantly higher than that of loading into MS2 particles. This indicates that the permeability of ETP into particles is independent of the charge of the particles.

[0031] 4. Release of model substances For release measurements, the incorporated substance was released in 20 mM phosphate buffer at room temperature for 30 days, and the release was performed at different pH values ​​and ionic strengths. The results are as follows: Figure 5 As shown in A in the diagram. Through Figure 5As shown in Figure A, regardless of the charge of the substances, the release of these three model substances from NL silk fibroin particles exhibits pH dependence, with the fastest and most efficient release at pH 9, and the slowest and least efficient release at pH 3. ETP release is gradual across all pH values, with a release efficiency of approximately 15% at pH 3 over 30 days. In contrast, even after 30 days, no significant amounts of Rho-ODN and CFI were detected at pH 3. Furthermore, increasing the pH from 3 to 5 had no effect on the release efficiency, indicating a strong electrostatic interaction between silk fibroin and Rho-ODN and CFI. However, a sudden release of Rho-ODN was observed at pH 9, while a sudden release of CFI occurred at pH 7, suggesting that the loading of Rho-ODN and CFI onto NL silk fibroin particles is primarily driven by electrostatic interactions.

[0032] During the study, the pH was set to 7, and the ionic strength was increased from 50 mmol / L to 154 mmol / L. The release results of the model substances were as follows: Figure 5 As shown in B, through Figure 5 As shown in Figure B, changes in ionic strength significantly affected the release profiles of these three substances. Specifically, increasing the ionic strength from 50 mmol / L to 154 mmol / L resulted in a substantial increase in the percentage of molecules released over 24 hours: Rho-ODN increased from 17% to 44%, CFI from 51% to 90%, and ETP from 45% to 75%. Previous studies have shown that MaSp2-based silk fibroin particles (MS2) are more suitable for ETP delivery than particles prepared from MaSp1 (MS1). Figure 6 It can be seen that the novel recombinant spider silk protein (NL) proposed in this invention is superior to MS2 particles in ETP delivery, with higher loading efficiency and higher drug release rate.

[0033] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A gene, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. The recombinant spider silk protein encoded by the gene according to claim 1, characterized in that, The recombinant spider silk protein consists of two MaSp1 N linker regions in series; The amino acid sequence of the recombinant spider silk protein is shown as SEQ ID NO.

2.

3. A recombinant vector, characterized in that, The gene of claim 1.

4. A silk fibroin particle, characterized by, The recombinant spider silk protein of claim 2.

5. Use of the gene of claim 1, the recombinant spider silk protein of claim 2, the recombinant vector of claim 3 or the silk fibroin particle of claim 4 in the preparation of a pharmaceutical carrier.

6. Use according to claim 5, characterized in that, The drug is a negatively charged drug.

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