Engineered lipoid transporter carrier for efficient hydrophobic drug delivery as well as preparation method and application of engineered lipoid transporter carrier

Abstract

The invention relates to an engineering lipoid transporter carrier for efficient hydrophobic drug delivery and a preparation method and application thereof, and belongs to the technical field of biological medicine. The carrier disclosed by the invention is a carrier based on Bla g 1 protein mutation, and the mutant protein has three repetitive units consisting of 100-180 amino acids, so that a spherical structure with a hydrophobic inner cavity is formed. By simulating the lipid binding and transporting mechanism of human apoA-I / MSP, efficient encapsulation of hydrophobic drugs is achieved, the solubility and bioavailability of the drugs are remarkably improved, and toxic and side effects are reduced. The B1a g 1 protein carrier has a simple preparation method, large-scale production can be realized through recombinant protein engineering, and an efficient and safe new strategy is provided for delivery and clinical application of indissolvable drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and relates to an innovative drug delivery carrier based on Bla g 1 protein, its preparation method and application, specifically to a carrier of engineered lipid transfer protein for efficient hydrophobic drug delivery, its preparation method and application. Background Art

[0002] With the continuous advancement of drug research and development, a large number of drugs with high potential therapeutic value are difficult to reach effective therapeutic concentrations in vivo due to their hydrophobicity or low solubility, thus significantly limiting their clinical applications. Traditional solubility improvement strategies mainly include chemical modification, addition of surfactants or co-solvents. Although these methods can improve the solubility of drugs, they often come at the cost of sacrificing drug activity or introducing toxic side effects. In addition, these methods may reduce the therapeutic effect due to the instability of the drug or the change of metabolic pathways.

[0003] In recent years, drug delivery systems such as liposomes, cyclodextrin inclusion complexes and lipid nanoparticles have received extensive attention. These systems partially solve the problems of insufficient drug solubility and stability by encapsulating hydrophobic drugs in nanoscale carriers. However, these delivery systems still face many challenges: for example, liposomes usually require complex preparation processes and are prone to size heterogeneity or structural dissociation in vivo; cyclodextrin inclusion complexes have the problem of limited encapsulation capacity; lipid nanoparticles may be limited by high production costs and difficulties in large-scale production. Therefore, there is an urgent need for a new drug delivery strategy that combines high efficiency, safety, simplicity and controllable cost.

[0004] Bla g 1 protein is a lipid transfer protein derived from Blattella germanica and belongs to the MAs (Major Allergen from Cockroach) family. Such proteins usually have specific lipid-binding domains and can bind and transport a variety of hydrophobic molecules. The Bla g 1 protein consists of two highly similar α-helical modules, each module containing approximately 100 amino acids. These modules fold to form a highly hydrophobic globular inner cavity, enabling it to stably bind hydrophobic ligands such as fatty acids and phospholipids. Research shows that the binding of Bla g 1 protein to hydrophobic ligands can not only enhance its own thermal stability and anti-enzymatic ability, but also significantly improve the bioavailability of the bound molecules.

[0005] Similar to the functions of human apolipoprotein apoA-I and MSP, the Bla g 1 protein has a unique hydrophobic core and can efficiently bind to a variety of hydrophobic small molecules. Different from them, Bla g 1 can stably bind drugs without introducing sodium cholate or other surfactants, avoiding the toxic and side effects introduced by organic solvents or solubilizers. Its preparation method is simple. Only by performing gentle incubation in phosphate buffer can efficient encapsulation of hydrophobic drugs be achieved. More importantly, through recombinant protein technology, the Bla g 1 protein can be flexibly engineered to meet the delivery requirements of different drugs. For example, studies have shown that when the Bla g 1 protein is used to deliver poorly soluble drugs such as Cur, GA, β-Lap, SN38, and CBX, not only is the encapsulation efficiency high, but the overall delivery system has strong stability. Therefore, as a drug delivery carrier with significant advantages, the Bla g 1 protein provides an innovative solution to solve the clinical application bottleneck of poorly soluble drugs and opens up a new direction for future drug delivery technologies. Summary of the Invention

[0006] In order to overcome the above-mentioned deficiencies of the prior art, the present invention proposes an innovative drug delivery system based on the Bla g 1 protein. By utilizing the unique structural and functional characteristics of the Bla g 1 protein, hydrophobic drugs are efficiently encapsulated in its hydrophobic cavity, providing a new solution for the delivery of poorly soluble drugs.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] By mimicking the lipid binding and transport mechanism of human apolipoprotein (apoA-I / MSP), the present invention realizes the efficient encapsulation of hydrophobic drugs, such as the proteins in delivering curcumin (Cur), geldanamycin (GA), β-lapachone (β-Lap), SN38 (7-ethyl-10-hydroxycamptothecin, SN38), and cabazitaxel (CBX), and significantly improves the solubility and bioavailability of the drugs while reducing the toxic and side effects.

[0009] In the first aspect of the present invention, there is provided a carrier of an engineered lipid transfer protein for efficient delivery of hydrophobic drugs. The carrier is a carrier based on the mutated Bla g 1 protein. This mutated protein has three repeating units consisting of 100-180 amino acids, forming a spherical structure with a hydrophobic inner cavity. The amino acid sequence of the Bla g 1 protein is as shown in SEQ ID No.1.

[0010] Further, the mutant protein is denoted as P1 protein, and its amino acid sequence is one of SEQ ID No. 2 to 4.

[0011] Further, the particle size distribution of the mutant protein ranges from 8 to 15 nm, the average particle size is 12.4 ± 0.99 nm, and the PDI is 0.249 ± 0.036 nm.

[0012] The second aspect of the present invention provides a preparation method of the engineered lipid transfer protein for use as a carrier for efficient hydrophobic drug delivery. The P1 protein is obtained by recombinant protein expression, expressed in Escherichia coli using a prokaryotic expression system, and purified by affinity chromatography.

[0013] The specific steps of the preparation method are as follows:

[0014] (1) Synthesize the P1 gene containing NdeI and XbaI restriction sites, obtain the P1 gene with the expected length by PCR amplification, the primer sequences are P1-a-pCold-NdeI-F and P1-a-pCold-XbaI-R, then double-digest the PCR product, and transform Escherichia coli after ligation;

[0015] (2) Culture in LB medium containing ampicillin until OD600 = 0.6 - 0.8, cool at 4°C, add 0.5 mM IPTG, induce expression at 15°C for 18 - 24 h, collect the bacterial cells and disrupt them by sonication;

[0016] (3) After lysing the bacterial cells, centrifuge to take the supernatant, purify by nickel column affinity chromatography, and elute the target protein stepwise with Elution buffer containing 20, 40, 60, 80, 100, 150, 300 mM imidazole, collect the 300 mM imidazole elution peak to obtain high-purity P1 protein.

[0017] Preferably, the PCR amplification conditions are: denaturation at 95°C, annealing at 55°C, and extension at 75°C.

[0018] Preferably, the preparation of the competent cells includes:

[0019] Treat BL21(DE3) bacterial cells with 100 mM MgCl2, 100 mM CaCl2, and 85 mM CaCl2 - 15% glycerol solution, and store in aliquots after ice bath.

[0020] Preferably, the molecular weight of the P1 protein is 25 - 27 kD, and the purity is verified by SDS-PAGE.

[0021] Preferably, the amino acid sequence of the target protein is one of the following: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.

[0022] Preferably, the LB liquid medium contains 100 μg / mL ampicillin.

[0023] Preferably, the conditions for ultrasonic disruption are: power 300 W, working time 3 s, intermittent time 7 s, and total duration 30 - 60 min.

[0024] Preferably, the buffer for elution is 20 mM Tris-HCl, 500 mM NaCl, pH 8.0.

[0025] The purity of the P1 protein prepared by the method of the present invention can reach more than 95%, and the expression level reaches 50 - 80 mg / L. In the above method, a hydrophobic drug is encapsulated into the hydrophobic cavity of the P1 protein by an incubation method, avoiding the use of organic solvents or surfactants.

[0026] In the above method, the drug is a hydrophobic compound; the hydrophobic compound is selected from curcumin, geldanamycin, β-lapachone, SN38 or cabazitaxel.

[0027] In the above method, the incubation conditions include: incubating in a phosphate buffer solution at pH 7 - 7.6 in the dark at room temperature for 2 - 4 h, and the stirring rate is 200 - 280 rpm.

[0028] In the third aspect of the present invention, the protein described in the first aspect can be used as a novel drug delivery system to achieve the delivery of poorly soluble drugs. The novel drug delivery system has the effect of improving the solubility and bioavailability of hydrophobic drugs.

[0029] Preferably, the poorly soluble drugs include curcumin (Curcumin, Cur), geldanamycin

[0030] (Geldanamycin, GA), paclitaxel (Paclitaxel, Pacl), docetaxel (Docetaxel, Dxtx), imatinib, β-lapachone (β-Lapachone, β-Lap), cabazitaxel (Cabazitaxel, CBX) and 7-ethyl-10-hydroxycamptothecin (7-Ethyl-10-hydroxycamptothecin, SN38).

[0031] Specifically, the poorly soluble drugs include curcumin, geldanamycin, β-lapachone, cabazitaxel and SN38.

[0032] Through verification, the results of the present invention show that the drug-loading capacity of P1 protein can significantly improve the solubility of hydrophobic drugs in vitro and in vivo, and at the same time enhance the biological stability of the drugs. In addition, after loading the above hydrophobic drugs, the structural stability of P1 protein is further enhanced, indicating that it is suitable as an efficient nano-drug delivery platform.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] In the present invention, by recombinant expression and purification of P1 protein, uniform nanoparticles with a particle size of about 12 nm are formed, which have a high proportion of α-helix conformation and exhibit excellent thermal stability and hydrolysis resistance. Different from the existing lipid nanodiscs (NDs) or liposome drug delivery systems, P1 protein can efficiently encapsulate hydrophobic drugs in a simple aqueous environment without adding external phospholipids or surfactants. Therefore, due to its unique hydrophobic pocket and excellent biological properties, the P1 protein provided by the present invention has good application prospects in the field of poorly soluble drug delivery. Description of the Drawings

[0035] Figure 1 The pCold I-P1 plasmid map shown;

[0036] Figure 2 The results of purification and identification of P1 protein shown;

[0037] Figure 3 The structure prediction and characterization of P1 protein shown. Figure A is the predicted structure of P1 protein, and Figure B is the particle size distribution and circular dichroism spectrum of P1 protein;

[0038] Figure 4 The stability evaluation of P1 protein shown. Figure A is the circular dichroism spectrum of P1 protein at different pH values, Figure B is the temperature-variable circular dichroism spectrum of P1 protein in the temperature range of 20 - 94 °C, and Figure C is the circular dichroism spectrum of P1 protein before and after heating;

[0039] Figure 5 The standard curve of the drug solution shown, which are the drug standard curves of Cur, GA, β-Lap, SN38, and CBX measured in methanol solution respectively;

[0040] Figure 6 The comparison diagram of particle size and circular dichroism of P1 protein before and after drug loading shown. Figure A is the particle size distribution of P1 before and after drug loading, and Figure B is the circular dichroism spectrum of P1 before and after drug loading;

[0041] Figure 7 The safety evaluation of P1 blank carrier shown, which are the cytotoxicity evaluations of P1 blank carrier on MCF-7, SKOV3, HUVEC, and A549 respectively.

[0042] Figure 8The cytotoxic effects of the shown P1 drug delivery system on A549, SKOV3, and MCF-7 cells are represented by Cur and P1@Cur, GA and P1@GA, β-Lap and P1@β-Lap, SN38 and P1@SN38, CBX and P1@CBX, respectively;

[0043] Figure 9 The shown study on the in vitro cellular uptake of DiO and P1@DiO. Figure A shows the cellular uptake of free dye DiO or P1@DiO by SKOV3 cells at 0.5 h and 2 h, and Figure B shows the comparison of the average fluorescence intensities of DiO and P1@DiO at the same time;

[0044] Figure 10 The shown evaluation of the in vivo anti-tumor effect of P1@CBX. Figure A shows the subcutaneous tumor model of human ovarian cancer SKOV3 and the drug administration scheme, Figures B and C show the curves of the changes in mouse body weight and tumor size, respectively, and Figure D shows the tumor weights of mice in each group. Detailed implementation manners

[0045] The following further describes the detailed implementation manners of the present invention. It should be noted here that the description of these implementation manners is used to help understand the present invention, but does not limit the present invention. In addition, the technical features involved in the various implementation manners of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0046] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples can all be obtained through conventional commercial channels unless otherwise specified.

[0047] Example 1 Design of the protein after mutation of the Bla g 1 protein

[0048] 1. Construction of the protein expression vector

[0049] (1) In the present invention, codon optimization is used to optimize the nucleotide sequence of the Bla g 1 protein for E. coli codons, and NdeI and XbaI restriction enzyme sites are added to the 5' end and 3' end of the optimized target gene sequence, respectively.

[0050] (2) The optimized gene is cloned into the multiple cloning site of the pCold I vector.

[0051] (3) The designed pCold I-P1 plasmid map ( Figure 1)And the whole gene was synthesized and sequenced with the sequence file to obtain the target protein P1 after the mutation of the Bla g 1 protein. The amino acid sequence of the Bla g 1 protein is shown in SEQ ID No.1, and the sequence of the mutated protein P1 is one of the following: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4;

[0052] Table 1 Protein Amino Acid Sequence

[0053]

[0054]

[0055] Example 2 Preparation of P1 Protein

[0056] 1. Clone the P1 protein sequence into the expression vector:

[0057] (1) Synthesize the coding gene fragment of the P1 protein according to the design. The nucleotide sequence contains a specific multiple cloning site (MCS), and this fragment is inserted between the NdeI and XbaI restriction sites of the pCold I expression vector.

[0058] Table 2

[0059]

[0060]

[0061] (2) Use the primer pairs in Table 2 to perform PCR amplification on the optimized P1 gene of the gene Bla g 1-a. First, denature at 95 °C, then anneal at 55 °C, and finally extend at 75 °C. After amplification by this program, the P1 gene fragment with the expected length was successfully obtained, and this fragment contains the restriction enzyme sites of NdeI and XbaI.

[0062] (3) Perform double digestion on the above amplification product and the pCold I vector to ensure the accurate insertion site of the gene. The digested target gene fragment and the vector are ligated by DNA ligase to form the pCold I-P1 recombinant plasmid, and its nucleotide sequence correctness is confirmed by sequencing.

[0063] (4) The results show that the synthesized gene sequence is completely consistent with the designed target sequence and can be used for subsequent protein expression.

[0064] 2. Express the P1 protein in Escherichia coli:

[0065] (1) Preparation of Escherichia coli BL21(DE3) competent cells

[0066] 1) Resuscitate the strain

[0067] Take out the cryopreserved BL21(DE3) Escherichia coli strain from an -80 °C refrigerator, streak and resuscitate it on an LB plate, and incubate it overnight in a 37 °C incubator until single colonies grow.

[0068] 2) Cultivate single colonies

[0069] Pick single colonies from the LB plate and inoculate them into 10 mL of LB liquid medium, and shake and culture them on a shaker at 37 °C and 250 rpm for 12 - 16 h.

[0070] 3) Scale-up culture

[0071] Inoculate the overnight culture broth into 100 mL of fresh LB liquid medium at a ratio of 1:100, and continue to shake and culture it at 37 °C and 250 rpm until the bacteria enter the logarithmic growth phase.

[0072] 4) Prepare competent cells:

[0073] Aliquot 100 mL of the bacterial broth into two sterile centrifuge tubes and let it stand on ice for 20 min. Centrifuge the above bacterial broth at 4 °C and 3000×g for 15 min, and discard the supernatant culture medium;

[0074] Add 10 mL of 100 mM MgCl2 solution, 10 mL of 100 mM CaCl2 solution, and 7 mL of calcium chloride - glycerol mixture (85 mM CaCl2, 15% glycerol), all pre-cooled aseptically, to each tube in sequence, then gently resuspend the bacterial cells. After standing on ice for 20 min, centrifuge again and discard the supernatant. Subsequently, add 1.5 mL of sterile pre-cooled calcium chloride - glycerol mixture to each tube, gently resuspend the bacterial cells, and then combine to obtain competent cells.

[0075] 5) Store competent cells

[0076] Aliquot the prepared competent cells into pre-cooled centrifuge tubes.

[0077] (2) Transform the recombinant plasmid into Escherichia coli BL21(DE3)

[0078] 1) Plasmid transformation

[0079] Add 2 μL of pCold I-P1 plasmid solution with a concentration of 100 ng / μL to the thawed competent cells, gently mix, and then let the mixture stand on ice for 30 min.

[0080] 2) Heat shock treatment

[0081] Place the mixture in a 42 °C water bath for heat shock treatment for 90 s. After heat shock, place the mixture back on ice and let it stand for 2 - 3 min.

[0082] 3) Cultivate the transformed cells

[0083] Add 800 μL of LB liquid medium to the treated competent cells. Incubate on a shaker at 37 °C and 200 rpm for 1 h.

[0084] 4) Collect the transformed cells

[0085] Centrifuge at 4000 rpm for 5 min and discard most of the medium. Resuspend the cell pellet in the remaining 100 μL of medium.

[0086] 5) Plate culture

[0087] Aspirate 30 μL of the bacterial suspension onto an LB plate containing 100 μg / mL ampicillin. Spread the bacterial suspension evenly and then invert the plate. Incubate the plate in a 37 °C incubator overnight for 12 - 16 h.

[0088] (3) Small - scale expression of P1 protein

[0089] 1) Revive and culture the bacterial strain

[0090] Mix 5 μL of the ampicillin working solution with 5 mL of LB liquid medium. Pick a single colony from the pCold I - P1BL21(DE3) plate and inoculate it into the medium. Incubate overnight at 37 °C and 250 rpm for 14 h. Take 625 μL of 80% glycerol and mix it with 375 μL of the overnight culture. Store the glycerol stock at - 80 °C.

[0091] 2) Scale - up culture

[0092] Inoculate 100 μL of the overnight culture into 10 mL of ampicillin - resistant LB liquid medium. Incubate with shaking at 37 °C and 250 rpm until OD600 = 0.5 - 0.6.

[0093] 3) Induce expression

[0094] Cool the bacterial suspension to 4 °C and induce expression with 0.5 mM IPTG at 15 °C for 20 h.

[0095] 4) Lyse the cells

[0096] After the induction is completed, centrifuge to discard the medium, resuspend the cell pellet in 1 mL of Binding buffer, sonicate for 20 min, and centrifuge at 4 °C and 12000 rpm for 30 min.

[0097] 5) Protein identification

[0098] After Coomassie brilliant blue staining and decolorization, detect the protein expression by SDS - PAGE electrophoresis.

[0099] The results of electrophoresis are as Figure 2 shown to have an obvious band at the position of 26.66 kD, and the target protein is mainly distributed in the supernatant.

[0100] (4) Mass expression

[0101] 1) Resuscitation of glycerol bacteria

[0102] Streak the glycerol bacteria on an LB plate with ampicillin resistance and culture at 37 °C for 12 - 14 h, then store temporarily at 4 °C.

[0103] 2) Primary culture

[0104] Pick a single colony and inoculate it into 12 mL of LB liquid medium containing 12 μL of ampicillin, and culture at 37 °C and 250 rpm for 14 - 16 h.

[0105] 3) Fermentation of engineering bacteria

[0106] Add the overnight culture obtained in step 2) to 1 L of LB liquid medium, culture at 37 °C and 200 rpm until OD600 = 0.6 - 0.8, cool to 4 °C, and adjust the shaker temperature to 15 °C.

[0107] 4) Induced expression

[0108] Add 500 μL of 1 M IPTG and induce expression at 15 °C and 200 rpm for 18 - 24 h.

[0109] 5) Collection of bacterial cells

[0110] After the induction is completed, aliquot the bacterial liquid into centrifuge tubes, centrifuge at 12000 rpm for 10 min, discard the culture medium, resuspend the bacterial cells, combine them into one tube, and repeat the washing.

[0111] 6) Disruption of bacterial cells

[0112] After thawing the bacterial cells on ice, add the corresponding amount of Binding buffer according to 10 mL / g of bacterial weight, fully resuspend the bacterial cells and add lysozyme solution (100×) to the working concentration of 0.3 mg / mL. Place the centrifuge tube on ice for ultrasonic disruption (select the 6th amplitude transformer rod, with a power of 300 W, turn on for 3 s and stop for 7 s, for a total of 30 - 60 min). The lysate is centrifuged at 4 °C and 12000 rpm in a refrigerated centrifuge for 30 min to separate the supernatant and the precipitate. The supernatant containing the target protein is filtered through a 0.22 μm PES filter membrane and stored temporarily at 4 °C for subsequent affinity chromatography purification.

[0113] 3. Separate and purify protein P1 by affinity chromatography:

[0114] (1) Preparation of cobalt column

[0115] Fill a 12 mL empty affinity chromatography column with 5 mL of cobalt column resin, release the storage buffer, and wash and equilibrate the resin with ultrapure water and Binding buffer respectively.

[0116] (2) Protein purification

[0117] Add the supernatant filtered in step 1) to the resin, collect the flow-through, repeat loading onto the column twice, then wash the resin with Binding buffer to remove impurity proteins until the A280 value approaches zero. Then elute the target protein with Elution buffer containing different concentrations of imidazole in a gradient manner, and monitor the A280 value to judge the elution situation. Subsequently, wash the resin with Elution buffer containing 300 mM imidazole and ultrapure water, and store it in 20% ethanol at 4 °C.

[0118] (3) Protein electrophoresis identification

[0119] Mix the cell lysate, flow-through, and eluate with loading buffer, heat at 98 °C for 15 min to denature the proteins, and then perform SDS-PAGE electrophoresis under the conditions of 120 V and 75 min. Subsequently, perform Coomassie Brilliant Blue staining and decolorization, and then image and analyze on a gel imager.

[0120] The electrophoresis results are as Figure 2 , in the 300 mM imidazole eluate, a single and relatively deep target band appears near 25 kD, indicating that the P1 protein has a high purity and a significant yield.

[0121] 4. Structural stability and characterization

[0122] (1) DLS particle size measurement

[0123] Dilute the protein sample to be measured and the drug-loaded protein sample to 1 mg / mL, then filter through a 0.22 μm filter membrane and degas by ultrasonic treatment for 10 min. Then clean the sample cell with pure water and PBS. After cleaning, inject the sample to be measured into the sample cell, and then perform particle size measurement with the measurement temperature set at 25 °C.

[0124] The results are as Figure 3 , more than 80% of the P1 protein particle size is distributed in the range of 8 - 15 nm, the average particle size is 12.4 ± 0.99 nm, and the PDI is 0.249 ± 0.036 nm. The nanoscale size of the P1 protein is beneficial for it to penetrate biological barriers such as tumor tissue vascular endothelium, promote intracellular uptake, and improve the effective delivery of drug molecules at the tumor site.

[0125] (2) Circular dichroism

[0126] 1) Sample preparation

[0127] Absorb 300 μL of the sample to be tested into the sample dish and place it in the sample cell.

[0128] 2) CD Scanning Parameter Setting

[0129] Use the Chirascan-V100 circular dichroism spectrometer, set the light source as xenon lamp, temperature at 20 °C, wavelength range 180 - 260 nm, and sampling time 0.5 s.

[0130] 3) Baseline and Sample Scanning

[0131] First, scan the PB buffer to establish the baseline, and then scan the protein sample to be tested. If the protein concentration is too high, dilute it with PB buffer to 5 - 20 μM, and control the ultraviolet absorbance value (AU) at 0.5 - 2. Subsequently, each sample is scanned three times to obtain the average value, and the background signal of PB buffer is subtracted.

[0132] 4) Data Processing

[0133] Draw a circular dichroism spectrum with the wavelength as the abscissa and millidegree (mdeg) as the ordinate, and use the CDNN software to analyze the proportion of the protein secondary structure.

[0134] The results are shown in Table 3 and Figure 3 , showing that the proportion of the α-helix structure of P1 protein is 45.1%, the proportion of random coil is 25.6%, and the proportions of β-turn and β-sheet are relatively low.

[0135] Table 3 Proportion of Protein Secondary Structure Calculated by CDNN1

[0136]

[0137]

[0138] Note: Helix: α-helix; Parallel, Antiparallel: Parallel and antiparallel structures in β-sheet; Beta-Turn: β-turn; Rndm.Coli: Random coil; Total Sum: Total

[0139] (3) pH Stability Evaluation Dilute the protein to be tested with PB buffer of different pH values and scan the circular dichroism spectrum at 20 °C.

[0140] The results show that under all pH conditions, obvious negative peaks of P1 protein appear at 222 nm and 208 nm, a positive peak appears at 192 nm, and the proportions of the protein secondary structure are similar, indicating the good pH stability of P1.

[0141] (4) Tm Value Determination

[0142] Set the temperature range from 20 to 94 °C, and scan the circular dichroism spectrum every 2 °C. Subsequently, wait for the sample to return to 20 °C and scan again to evaluate the thermal renaturation ability of the protein.

[0143] The results are as Figure 4 shown. As the temperature increases, the negative peaks of P1 protein at 222 nm and 208 nm gradually shift upward, and the positive peak at 192 nm shifts downward, indicating the loss of α-helix structure of the protein and the increase in random coil structure. Use Global 3 software to fit the denaturation process, and calculate that the midpoint temperature of thermal denaturation (Tm value) of P1 protein is 73.5 ± 0.3 °C. The bio-CD spectrum of P1 protein after thermal denaturation is similar to that before denaturation, and the proportion of secondary structure returns to the state before thermal denaturation. This indicates that P1 protein has good thermal renaturation ability.

[0144] 5. Determination of Encapsulation Efficiency and Drug Loading

[0145] (1) Establishment of P1 Nanodrug Delivery System

[0146] 1) Preparation and addition of drug solution

[0147] Take 5 sample bottles and add 500 μL of P1 protein solution with a concentration of 10 mg / mL to each. According to the molar ratio of drug to P1 protein (P / D ratio) of 1:1, 1:3, 1:5, 1:1, 1:1, add 10 μL of different drug solutions dissolved in DMSO to each sample bottle respectively. The drugs used are: Cur, GA, β-Lap, SN38, CBX. Use PBS buffer solution with pH 7.4 to make up the solution volume to 1 mL.

[0148] 2) Label the drug-loaded system

[0149] Label the drug-loaded systems obtained in step 1) as: P1@Cur, P1@GA, P1@β-Lap, P1@SN38, P1@CBX.

[0150] 3) Preparation of control group

[0151] The control group does not contain P1 protein. Add an equal amount of drug solution to the PBS solution as the free drug control group.

[0152] 4) Incubation of samples

[0153] Incubate the samples obtained in step 1) in the dark at room temperature for 2 h, and set the stirring rate to 250 rpm.

[0154] 5) Ultrafiltration centrifugation of drugs

[0155] After incubation, the drug-loaded system obtained in steps 1) to 4) was transferred to an ultrafiltration tube with a molecular weight cutoff of 10 kDa for ultrafiltration. The centrifugation conditions were set at 4°C, 4000 rpm, and 30 minutes to remove free drugs.

[0156] 6) Drug concentration determination

[0157] The filtrate obtained in the above steps was taken to measure the concentration series of drug solutions of the drug-carrying system.

[0158] (2) Preparation of drug standard curve

[0159] Scan the UV absorption spectrum of the drug solution to determine the maximum absorption wavelength of the drug. Using methanol as the solvent, prepare drug solutions of different concentrations, measure their absorbance, and draw a drug standard curve. Perform linear regression analysis to obtain the relationship equation between concentration and absorbance, such as Figure 5 shown.

[0160] (3) Determination of drug encapsulation efficiency and drug loading

[0161] 1) Ultrafiltration and centrifugation to remove free drugs

[0162] Use an ultrafiltration tube with a cutoff value of 10 kDa and centrifuge at 4°C and 4000 rpm for 30 minutes. Take the drug-loaded system and mix it thoroughly with methanol with a volume 9 times that of the drug-loaded system to release the entrapped drug.

[0163] 2) Calculation of encapsulation efficiency (EE%)

[0164] Methanol was used as a blank solvent. The absorbance of the sample was measured at the maximum absorption wavelength of the drug and substituted into the drug standard curve to calculate the drug concentration in the drug loading system. The encapsulation efficiency (EE%) was calculated according to the following formula (1):

[0165]

[0166] D e —Quality of the encapsulated drug

[0167] D t —Total drug quality

[0168] 3) Calculation of drug loading (DL%)

[0169] Take the above freeze-dried drug loading system, dissolve it in PBS solution, dilute it, measure the absorbance at the maximum absorption wavelength, and substitute it into the standard curve to calculate the drug concentration. Calculate the drug loading amount according to the following formula (2):

[0170] (DL%):

[0171]

[0172] W d — Mass of drug in the carrier

[0173] W p — Mass of protein in the carrier

[0174] The results showed that the encapsulation efficiencies of P1@Cur, P1@GA, P1@β-Lap, P1@SN38, and P1@CBX were 65.82%, 69.30%, 76.34%, 57.25%, and 67.82% respectively, and the drug loading amounts were 1.12%, 4.18%, 3.35%, 1.04%, and 2.14% respectively. This indicates that P1 protein has a high drug encapsulation efficiency, which helps to improve the drug delivery efficiency.

[0175] In this experiment, the particle size distribution and circular dichroism spectrum of P1 protein before and after drug loading were measured, and the results were as Figure 6 , the particle size distribution of P1 protein was concentrated in the range of 8 - 13 nm; the particle size distribution range of the protein after drug loading was 10 - 15 nm, indicating that the particle size changed little before and after drug loading, and the CD spectra of the protein before and after drug loading were similar, mainly in the α-helix conformation, indicating that the drug loading did not destroy the original secondary structure of the protein.

[0176] 6. Cell lines and cell culture

[0177] Human gastric cancer cell line (SGC7901) and breast cancer cell line (MCF-7) were selected as experimental models. The cells were inoculated in RPMI-1640 medium containing 10% fetal bovine serum (FBS), and the culture conditions were 37 °C and 5% CO2 incubator. Subculture was carried out when the cells reached 80% confluence, and the cells were digested with trypsin and then recultured.

[0178] 7. Evaluation of cell proliferation toxicity

[0179] (1) The cell proliferation inhibition experiment was carried out using a CCK-8 kit. After treating the cells with different concentrations of drugs for 24 h or 48 h, CCK-8 solution was added, and the optical density (OD value) of each group was measured.

[0180] (2) According to the CCK-8 results, a curve of drug concentration vs. OD value was plotted, and the IC50 value (half maximal inhibitory concentration) was calculated by nonlinear regression analysis.

[0181] The analysis results are shown in Table 4 and Figure 8 as shown, the P1 protein carrier can significantly improve the solubility and bioavailability of poorly soluble drugs, and significantly show a more superior tumor cell killing effect compared with free drugs.

[0182] Table 4 Changes in IC50 before and after drug loading

[0183]

[0184] 8. Cellular uptake experiment

[0185] After the drug is labeled with a fluorescent label or a radioactive label, it is added to the cell culture medium. Samples are taken regularly, and a flow cytometer or a fluorescence microscope is used to analyze the cellular uptake of the drug.

[0186] The results are as Figure 9 shown. After treatment for 0.5 h and 2 h, the mean fluorescence intensity of the P1@DiO group was significantly higher than that of the free DiO group (p < 0.0001), 12 times and 64 times that of the free group, respectively, indicating that P1@DiO can significantly improve the cellular uptake efficiency of hydrophobic molecules. It is speculated that the P1 carrier promotes the entry of the dye into the cell through the endocytosis mechanism, improving its solubility and the interaction efficiency with the cell membrane.

[0187] Evaluation of antitumor effect in vivo

[0188] A tumor transplantation model was constructed by subcutaneous injection of the MCF-7 cell line. At 0, 4, 8, and 12 days, PBS solution, Tween-CBX solution (2 mg / kg), and P1@CBX solution (2 mg / kg) were injected via the tail vein, respectively. The body weight and tumor diameter of the mice were recorded every two days to monitor the change in tumor size. The experiment was terminated when the tumor volume reached 1000 mm 3 . After the experiment, the mice were sacrificed by cervical dislocation. The tumors and main organs (heart, liver, spleen, lung, kidney) were dissected and fixed with 4% paraformaldehyde. After sectioning, they were stained with H&E to evaluate the pathological changes of tissues and organs.

[0189] The results are as Figure 10 , the P1@CBX group significantly slowed down tumor growth from the first administration. Compared with the PBS control group, the increase in tumor volume was significantly slowed down (p < 0.05), and the tumor weight was significantly lower than that of the control group (p < 0.05), and the tumor inhibition rate reached 73.9%.

[0190] In summary, due to its unique hydrophobic pocket and excellent biological properties, P1 protein has potential application value in the field of poorly soluble drug delivery.

[0191] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions, and variations made to these embodiments still fall within the protection scope of the present invention.

Claims

1. A carrier for efficient hydrophobic drug delivery using an engineered lipid transfer protein, characterized in that, The vector is a vector after mutation of the Bla g 1 protein. The mutant protein has three repeating units composed of 100-180 amino acids and has a globular structure with a hydrophobic inner cavity.

2. The carrier for efficient hydrophobic drug delivery using the engineered lipid transfer protein according to claim 1, wherein The mutant protein is denoted as P1 protein, and its amino acid sequence is one of SEQ ID No.2-4.

3. The carrier for highly efficient hydrophobic drug delivery using the engineered lipid transfer protein according to claim 1, characterized in that, The particle size of the mutant protein is distributed between 8-15 nm, the average particle size is 12.4±0.99 nm, and the PDI is 0.249±0.036 nm.

4. The preparation method of the carrier for highly efficient hydrophobic drug delivery using the engineered lipid transfer protein according to claim 1 or 2, characterized in that, The P1 protein is obtained by recombinant protein expression, expressed in Escherichia coli using a prokaryotic expression system, and purified by affinity chromatography.

5. The preparation method according to claim 4, characterized in that, The hydrophobic drug is encapsulated into the hydrophobic cavity of the P1 protein by an incubation method, avoiding the use of organic solvents or surfactants.

6. The preparation method according to claim 5, wherein, The hydrophobic compound is selected from curcumin, geldanamycin, β-lapachone, SN38 or cabazitaxel.

7. The drug delivery system according to claim 5, wherein The incubation conditions include: in a phosphate buffer at pH 7-7.6, incubating in the dark at room temperature for 2-4 h, and the stirring rate is 200-280 rpm.

8. The preparation method according to claim 4, wherein, The specific steps are as follows: (1) Synthesize the target gene sequence encoding the P1 protein, insert the target gene into the pColdI expression vector through the NdeI and XbaI restriction enzyme sites to construct the recombinant expression plasmid pColdI-P1; the pColdI vector contains the cold shock promoter cspA and the 6×His tag sequence; (2) Transform the recombinant plasmid into Escherichia coli BL21(DE3) competent cells, coat it on an LB solid medium containing ampicillin, and culture overnight to screen positive clones; (3) Pick a single colony and inoculate it into an LB liquid medium. When the OD600 reaches 0.6-0.8 after shaking culture, add an IPTG inducer with a final concentration of 0.5 mM to induce expression for 18-24 h; after collecting the bacteria, ultrasonically disrupt them, centrifuge to take the supernatant for Ni-NTA affinity chromatography purification, and sequentially elute the target protein with Elution buffer containing 20, 40, 60, 80, 100, 150, 300 mM imidazole, and collect the 300 mM imidazole elution peak to obtain high-purity P1 protein.

9. Use of the vector according to claim 1 or 2 in the preparation of a drug for improving the solubility and delivery efficiency of a hydrophobic drug.

10. A pharmaceutical composition based on the pharmaceutical delivery system according to claim 1 or 2, characterized in that, The pharmaceutical composition includes the P1@drug nanodelivery system and pharmaceutical excipients; the composition is used for cancer drugs; the cancer is breast cancer, ovarian cancer, non-small cell lung cancer, cervical cancer, gastric cancer or pancreatic cancer.

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