Preparation method and application of bovine serum albumin loaded camptothecin nanoparticles

Abstract

The invention relates to a preparation method of bovine serum albumin-loaded camptothecin nanoparticles, which is characterized in that BSA (bovine serum albumin) is modified by PEG (polyethylene glycol), so that the biocompatibility and the stability of the nanoparticles are enhanced, PEG-BSA-CPT-NPs is prepared, and the water solubility of CPT is increased by nearly 10 times. A freeze drying-ultrasonic method is adopted to replace high-pressure homogenization to realize an emulsification process, so that the defect that the secondary and tertiary structures of the protein are changed due to high-pressure homogenization to cause expansion or denaturation of the protein is overcome. In addition, a freeze-drying step is introduced on the basis of a traditional ultrasonic emulsification method, the drug-loaded nanoparticles are freeze-dried into powder, and then ultrasonic treatment is performed. The particle size of the prepared PEG-BSA-CPT-NPs nanoparticle meets the standard of the ideal size of a nano-drug, the nanoparticle can penetrate through a tumor microenvironment and is prevented from being rapidly removed, and the anti-tumor activity of CPT is improved.

Description

Technical Field

[0001] The invention relates to the field of medicines, and in particular to a preparation method and application of bovine serum albumin-loaded camptothecin nanoparticles. Background Art

[0002] Camptothecin (CPT) is a quinoline alkaloid isolated from Camptotheca acuminata, which inhibits the replication of cancer cells by inhibiting the action of DNA topoisomerase Ι (TopΙ). As a natural TopΙ inhibitor, CPT has good anti-tumor effects on a variety of cancers such as lung cancer, gastric cancer, bladder cancer, and breast cancer, and has broad application prospects in the treatment of cancer. However, CPT has problems such as poor water solubility, instability, short half-life, and serious toxic side effects, which greatly limits its application in clinical practice.

[0003] In response to various problems of CPT in cancer treatment, various drug formulations such as liposomes, polymer micelles, nanoparticles and microspheres have been developed, which have improved the solubility of CPT, reduced toxicity and increased bioavailability to varying degrees. However, due to the influence of carrier materials, the delivery of drugs in the body is limited. For example, the phospholipids in liposomes are easily oxidized to disintegrate the structure and may cause toxic side effects. The poor stability of polymer micelles can easily cause drug leakage and cause vascular blockage. Therefore, the selection of appropriate drug carriers to improve the solubility of CPT, prolong the circulation time in the body and enhance tumor targeting is still the key to improving its therapeutic effect.

[0004] In recent years, the development of nano drug delivery systems has provided new ideas for solving these problems. Among them, serum albumin (SA), as a natural, biodegradable carrier material, has attracted widespread attention due to its good biocompatibility and targeting. The currently isolated SA mainly includes bovine serum albumin (BSA), human serum albumin (HSA) and rat serum albumin (RSA). Among them, BSA and HSA are widely used in drug delivery research due to their advantages such as safety, non-toxicity, non-immunogenicity, biocompatibility and biodegradability. Compared with HSA, BSA has a wider source and low cost, and is often used as a substitute for HSA in preclinical model research. Combining camptothecin drugs with bovine serum albumin (BSA) to prepare nanoparticles can improve the water solubility and stability of drugs, improve therapeutic efficacy, and reduce drug toxicity.

[0005] Polyethylene glycol (PEG) is one of the very few synthetic polymers approved by the FDA for in vivo injection. The pH of PEG aqueous solution is neutral, it is easily soluble in water and polar solvents, non-toxic, non-immunogenic, and biocompatible. It can also be coupled to proteins, liposomes, etc. through covalent or non-covalent bonds, thereby changing the pharmacokinetic properties of drugs. After PEG modification, the properties of most proteins change as follows: (1) Reduced immunogenicity and antigenicity; (2) Extended circulation half-life up to 50 times; (3) Increased solubility; (4) Resistance to protease hydrolysis; (5) Reduced toxicity; (6) Increased stability. BSA was modified with PEG to prepare PEG-BSA-CPT nanoparticles. The modified nanoparticles can avoid clearance by the reticuloendothelial system, prolong the circulation time of the drug in the body, and further improve the solubility and stability of CPT.

[0006] At present, the preparation methods of serum albumin nanoparticle drug delivery systems mainly include desolvation method, albumin nanotechnology based on disulfide bond formation (nab TM -Technology) technology, self-assembly technology, etc. The self-assembly technology needs to use a reducing agent to prepare serum albumin nanoparticles, but the reducing agent has potential toxicity; the desolvation method needs to use a cross-linking agent glutaraldehyde to fix the nanoparticles, and the residual glutaraldehyde is easy to cause damage to the human body; TM The technology is a process of emulsifying the aqueous phase and the organic phase under high-pressure homogenization conditions, but high-pressure homogenization may change the secondary and tertiary structures of the protein, causing the protein to unfold or denature. Summary of the invention

[0007] The present invention overcomes the disadvantages of the preparation method of the serum albumin nanoparticle drug delivery system. TM The technology is changed based on the actual situation, and the emulsification process is realized by freeze drying-ultrasound method instead of high pressure homogenization. The serum albumin drug-loaded nanoparticles are prepared by freeze drying-ultrasound method to avoid the unfolding or denaturation of serum albumin. The process is simple, reproducible and environmentally friendly.

[0008] In view of this, the purpose of the present invention is to provide a method for preparing polyethylene glycol-modified bovine serum albumin-loaded camptothecin nanoparticles, using a freeze-drying-ultrasound method, and the preparation method is as follows:

[0009] Step 1: Dissolve mPEG-NHS in water and slowly add it to the BSA aqueous solution under stirring. The reaction is carried out at room temperature for 12 hours. The obtained conjugate (PEG-BSA) is dialyzed with deionized water to remove unreacted mPEG-NHS, lyophilized and stored at -20°C until use.

[0010] Step 2: Weigh CPT and dissolve it in a mixed solution of chloroform and anhydrous ethanol as the organic phase. Dissolve the PEG-BSA obtained in step 1 in water as the aqueous phase. Mix the two and stir evenly to obtain a PEG-BSA nanoparticle solution that initially encapsulates camptothecin.

[0011] Step 3: The camptothecin-encapsulated PEG-BSA nanoparticle solution obtained in step 2 is rapidly frozen to below -80°C to form a frozen solution, and freeze-dried to obtain PEG-BSA-CPT nanoparticles.

[0012] Step 4, the freeze-dried PEG-BSA-CPT nanoparticles obtained in step 3 are redissolved in an appropriate amount of deionized water or PBS buffer, ultrasonically treated, and freeze-dried in a dryer to obtain polyethylene glycol-modified bovine serum albumin loaded with camptothecin nanoparticles (PEG-BSA-CPT-NPs).

[0013] Preferably, in step 1, the concentration of BSA is 2-10 mg / mL, and the molar ratio of mPEG-NHS to BSA is 20:1.

[0014] Preferably, in step 2, the volume ratio of chloroform to anhydrous ethanol is (1-9):1.

[0015] Preferably, in step 2, the CPT concentration is 0.53-4.8 mg / mL.

[0016] Preferably, in step 2, the volume ratio of the aqueous phase to the organic phase is (5-30):1.

[0017] Preferably, in step 2, the pH of the aqueous solution is 5-8.

[0018] Preferably, in step 4, the ultrasonic time is 4 to 12 minutes, and the ultrasonic power is 45% to 75%.

[0019] The invention provides bovine serum albumin-loaded camptothecin nanoparticles prepared by the preparation method.

[0020] Another object of the present invention is to provide a use of bovine serum albumin-loaded camptothecin nanoparticles in the preparation of anticancer drugs.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention enhances the biocompatibility and stability of nanoparticles by modifying BSA with polyethylene glycol (PEG) to prepare PEG-BSA-CPT-NPs, which increases the water solubility of CPT by nearly 10 times while prolonging the circulation time of the drug, thereby improving the anti-tumor activity of CPT.

[0023] 2. The present invention is in nab TMIn order to avoid the disadvantages of high-pressure homogenization that changes the secondary and tertiary structures of proteins and causes protein unfolding or denaturation, freeze drying-ultrasound method is used instead of high-pressure homogenization to achieve the emulsification process. The freeze drying method avoids the destruction of drug activity by high temperature or organic solvents, and by adjusting the ultrasonic process parameters, the particle size of nanoparticles can be effectively controlled. The particle size of the obtained PEG-BSA-CPT-NPs nanoparticles meets the standard of the ideal size of nanomedicines, and can penetrate the tumor microenvironment and avoid being quickly cleared.

[0024] 3. The present invention introduces a freeze-drying step based on the traditional ultrasonic emulsification method, first freeze-drying the drug-loaded nanoparticles into powder, and then ultrasonically treating them. The first step of freeze-drying plays a key role in avoiding organic solvent residues, improving the stability of nanoparticles, facilitating subsequent processing, reducing pollution risks, and improving phase dispersion; at the same time, it can also maintain the integrity of the drug structure, improve the encapsulation efficiency and stability of the drug, especially for the poorly soluble drug camptothecin, this method can effectively reduce the loss of the drug during the preparation process and ensure its effective release at the target site. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Example 1 Schematic diagram of the preparation of PEG-BSA-CPT-NPs.

[0026] Figure 2 Example 6 Preliminary stability diagram of nanoparticles.

[0027] Figure 3 Example 8 In vitro drug release curve.

[0028] Figure 4 Effects of the measurement graph in Example 1, wherein a is the fluorescence titration curve of camptothecin and BSA (λex=295nm); b is the Stern-Volmer fitting graph; c is the double logarithmic graph of log(F0-F) / F versus log[Q].

[0029] Figure 5 a is the most stable camptothecin-BSA complex structure obtained by molecular docking method in Example 1; b is a two-dimensional visualization diagram.

[0030] Figure 6 Appearance of BSA-CPT-NPs and PEG-BSA-CPT-NPs, A is the BSA-CPT-NPs prepared in Example 2, and B is the PEG-BSA-CPT-NPs prepared in Example 1.

[0031] Figure 7Transmission electron microscopy images of BSA-CPT-NPs and PEG-BSA-CPT-NPs, A is BSA-CPT-NPs, B is PEG-BSA-CPT-NPs.

[0032] Figure 8 XRD patterns of CPT, BSA, physical mixture, and BSA-CPT-NPs.

[0033] Fig. 9 XRD patterns of PEG, PEG-BSA, and PEG-BSA-CPT-NPs.

[0034] Fig.10 Infrared spectra of CPT (A), BSA (B), physical mixture (C), and BSA-CPT-NPs (D).

[0035] Fig.11 Infrared spectra of PEG (A), PEG-BSA (B), and PEG-BSA-CPT-NPs (C).

[0036] Fig.12 Cytotoxicity of blank carrier BSA-NPs and PEG-BSA-NPs to Hela cells.

[0037] Fig.13 Toxicity of CPT, BSA-CPT-NPs and PEG-BSA-CPT-NPs on Hela cells incubated for 24 h.

[0038] Fig.14 Toxicity of CPT, BSA-CPT-NPs and PEG-BSA-CPT-NPs on Hela cells incubated for 48 h. DETAILED DESCRIPTION

[0039] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all should be included in the protection scope of the present invention.

[0040] Example 1 Preparation of PEG-BSA-CPT-NPs

[0041] mPEG-NHS was dissolved in water and slowly added to the BSA aqueous solution under stirring conditions. The BSA concentration was 5 mg / ml, the molar ratio of mPEG-NHS to BSA was 20:1, the solution pH was 6, and the reaction was carried out at room temperature for 12 hours. The obtained conjugate (PEG-BSA) was dialyzed with deionized water to remove unreacted mPEG-NHS as the aqueous phase; CPT was weighed and dissolved in a mixed solution of chloroform and anhydrous ethanol as the organic phase. The CPT concentration was 3.7 mg / ml, and the volume ratio of chloroform to anhydrous ethanol was 6:1; the organic phase was stirred. The aqueous phase was slowly injected, and the volume ratio of the aqueous phase to the organic phase was 20:1, to obtain a PEG-BSA nanoparticle solution that initially encapsulated camptothecin; the PEG-BSA nanoparticle solution that encapsulated camptothecin was quickly frozen to below -80°C to form a frozen solution, and freeze-dried to obtain PEG-BSA-CPT nanoparticles; the freeze-dried PEG-BSA-CPT nanoparticles were redissolved in an appropriate amount of deionized water, ultrasonically treated at an ultrasonic power of 60%, ultrasonically treated for 8 minutes, and freeze-dried in a dryer to obtain polyethylene glycol-modified bovine serum albumin camptothecin nanoparticles (PEG-BSA-CPT-NPs). The nanoparticle diameter was 191.7±4.87nm, the encapsulation efficiency was 75.87%, and the drug loading was 2.26%.

[0042] Example 2 Preparation of BSA-CPT-NPs

[0043] Weigh CPT and dissolve it in a mixed solution of chloroform and anhydrous ethanol as the organic phase, the CPT concentration is 3.7 mg / ml, and the volume ratio of chloroform to anhydrous ethanol is 6:1; weigh BSA and dissolve it in water as the aqueous phase, the BSA concentration is 5 mg / ml, and the solution pH is 6; the organic phase is slowly injected into the aqueous phase under stirring conditions, the volume ratio of the aqueous phase to the organic phase is 20:1, and the stirring is continued to form colostrum, and then placed in an ultrasonic cell crusher under ice bath conditions, the ultrasonic power is 60%, the ultrasonic power is 8 minutes, the organic solvent is removed by magnetic stirring, and then pre-frozen in a -80℃ refrigerator, and finally freeze-dried in a vacuum freeze dryer to obtain BSA-CPT-NPs. The nanoparticles have a diameter of 256.7±3.77nm, an encapsulation efficiency of 78.91%, and a drug loading of 2.85%.

[0044] Example 3 Preparation of PEG-BSA-CPT-NPs

[0045] mPEG-NHS was dissolved in water and slowly added to the BSA aqueous solution under stirring conditions. The BSA concentration was 2 mg / ml, the molar ratio of mPEG-NHS to BSA was 20:1, the solution pH was 5, and the reaction was carried out at room temperature for 12 h. The obtained conjugate (PEG-BSA) was dialyzed with deionized water to remove unreacted mPEG-NHS as the aqueous phase; CPT was weighed and dissolved in a mixed solution of chloroform and anhydrous ethanol as the organic phase. The CPT concentration was 0.53 mg / ml, and the volume ratio of chloroform to anhydrous ethanol was 1:1; the organic phase was stirred. The aqueous phase was slowly injected, and the volume ratio of the aqueous phase to the organic phase was 5:1, to obtain a PEG-BSA nanoparticle solution that initially encapsulated camptothecin; the PEG-BSA nanoparticle solution that encapsulated camptothecin was quickly frozen to below -80°C to form a frozen solution, and freeze-dried to obtain PEG-BSA-CPT nanoparticles; the freeze-dried PEG-BSA-CPT nanoparticles were redissolved in an appropriate amount of deionized water, ultrasonically treated at an ultrasonic power of 45%, ultrasonically treated for 12 minutes, and freeze-dried in a dryer to obtain polyethylene glycol-modified bovine serum albumin camptothecin nanoparticles (PEG-BSA-CPT-NPs). The nanoparticle diameter was 192.6±0.87nm, the encapsulation efficiency was 74.47%, and the drug loading was 2.08%.

[0046] Example 4 Preparation of PEG-BSA-CPT-NPs

[0047] mPEG-NHS was dissolved in water and slowly added to the BSA aqueous solution under stirring conditions. The BSA concentration was 10 mg / ml, the molar ratio of mPEG-NHS to BSA was 20:1, the solution pH was 8, and the reaction was carried out at room temperature for 12 hours. The obtained conjugate (PEG-BSA) was dialyzed with deionized water to remove unreacted mPEG-NHS as the aqueous phase; CPT was weighed and dissolved in a mixed solution of chloroform and anhydrous ethanol as the organic phase. The CPT concentration was 4.8 mg / ml, and the volume ratio of chloroform to anhydrous ethanol was 9:1; the organic phase was stirred. The aqueous phase was slowly injected, and the volume ratio of the aqueous phase to the organic phase was 30:1, to obtain a PEG-BSA nanoparticle solution that initially encapsulated camptothecin; the PEG-BSA nanoparticle solution that encapsulated camptothecin was quickly frozen to below -80°C to form a frozen solution, and freeze-dried to obtain PEG-BSA-CPT nanoparticles; the freeze-dried PEG-BSA-CPT nanoparticles were redissolved in an appropriate amount of deionized water, ultrasonically treated at an ultrasonic power of 75%, ultrasonically treated for 4 minutes, and freeze-dried in a dryer to obtain polyethylene glycol-modified bovine serum albumin camptothecin nanoparticles (PEG-BSA-CPT-NPs). The nanoparticle diameter was 194.2±3.48nm, the encapsulation rate was 73.88%, and the drug loading was 2.05%.

[0048] Example 5 Determination of Encapsulation Efficiency and Drug Loading

[0049] Encapsulation efficiency (EE) refers to the ratio of the mass of CPT encapsulated in BSA to the total CPT input when preparing nanoparticles. Drug-loading capacity (DL) refers to the mass of CPT encapsulated in nanoparticles as a percentage of the total mass of PEG-BSA-CPT-NPs, where the total mass includes the total mass of the drug and BSA. Encapsulation efficiency and drug loading are important evaluation indicators in the preparation of nanoparticles, which directly affect the therapeutic effect of drugs. In this experiment, high-speed centrifugation combined with ultraviolet spectrophotometry was used to determine the encapsulation efficiency and drug loading of drug-loaded nanoparticles.

[0050] 1. Determination of total CPT content in nanosuspensions

[0051] 0.1 mL of camptothecin bovine serum albumin nanosuspension was accurately drawn, and 0.9 mL of methanol solution was added. The mixture was vortexed for 10 min, ultrasonicated for 15 min, and centrifuged (4 °C, 15000 rpm, 20 min) to completely release CPT from the nanoparticles. 0.5 mL of the supernatant was drawn, and its absorbance value was measured at the maximum absorption wavelength of 376 nm. The total content of CPT in the nanosuspension was calculated by substituting it into the standard curve and used as the total drug amount (W total ).

[0052] (2) Determination of free CPT content in drug-loaded nanoparticles

[0053] Accurately measure 1.0 mL of camptothecin bovine serum albumin nanosuspension, centrifuge (4°C, 15000 rpm for 20 min), take an appropriate amount of solution and measure its absorbance at 376 nm to calculate the content of free CPT in the nanosuspension, that is, the free drug content (W free ). EE and DL are calculated according to formulas (1) and (2). Where, W n is the total weight of the nanoparticles.

[0054] EE%=(W total -W free ) / W total ×100%(1)

[0055] DL%=(W total -W free ) / W n ×100%(2)

[0056] Example 6 Preliminary stability test of nanoparticles

[0057] Serum albumin nanoformulations are generally stored at low temperatures of 2 to 8°C. Therefore, the freeze-dried nanoparticles were placed in a 4°C refrigerator to observe whether their appearance, particle size, encapsulation efficiency and drug loading have changed, and to investigate the storage stability of the nanoparticles. The experimental observation time was 40 days (once every 5 days). The changes in the particle size and storage time of the BSA-CPT-NPs prepared in Implementation 2 and the PEG-BSA-CPT-NPs prepared in Example 1 at 4°C were measured to investigate their storage stability. The results are shown in the attached figure. Figure 2 As shown, the particle size of BSA-CPT-NPs and PEG-BSA-CPT-NPs did not change significantly within 40 days, indicating that the nanoparticles had good initial stability within 40 days after freeze-drying.

[0058] Example 7 Nanoparticle Solubility Determination

[0059] Take enough CPT, BSA-CPT-NPs and PEG-BSA-CPT-NPs respectively and place them in 15mL centrifuge tubes, add 5mL of ultrapure water, seal and place in a shaker, set the shaker parameters (temperature is 37℃, shaking speed is 100rpm), and shake for 48h. Centrifuge (12000r, 10min), take the supernatant and dilute it appropriately, filter it through a 0.45μm microporous filter membrane, measure its absorbance value, record the peak area, and calculate the injection concentration and solubility. Take the supernatant of the centrifuged sample and dilute it appropriately, measure its absorbance value and calculate the solubility. The result is that the solubility of CPT is 1.82±0.15μgmL -1 , the solubility of CPT in BSA-CPT-NPs was 14.05±0.39 μgmL -1 , the solubility of CPT in PEG-BSA-CPT-NPs was 19.38±0.22μgmL -1 The results show that the solubility of CPT increased by nearly 7 times after it was made into BSA-CPT-NPs, and the solubility of CPT increased by nearly 10 times after it was made into PEG-BSA-CPT-NPs.

[0060] Example 8 Determination of in vitro drug release

[0061] Three batches of BSA-CPT-NPs and PEG-BSA-CPT-NPs were prepared according to Examples 1 and 2, and the dynamic membrane dialysis method was used to simulate the in vitro drug release experiment. The steps are as follows: 2 mL of BSA-CPT-NPs and PEG-BSA-CPT-NPs were accurately pipetted and added to dialysis bags (molecular weight of 8000-14000 Da), respectively, the bag opening was clamped with a dialysis bag clamp, 50 mL of PBS solution with a pH of 7.4 was added to the container, and oscillated on a constant temperature oscillator at 37°C (100 rpm). The sampling time was 0.083, 0.17, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12, 18, 20, 24, 36, and 72 h, and the sampling volume was 1 mL each time. After low-temperature high-speed centrifugation (15000 rpm, 4°C) for 15 min, 100 μL of the supernatant was measured, the CPT content was determined, the cumulative release percentage (Q) was calculated, and the cumulative drug release curve was obtained.

[0062]

[0063] Where: Ci is the concentration of CPT at the i-th sampling, V is the volume of dialysate in the system. C1 is the concentration at the first sampling, V1 is the volume of the first sampling, and Ci-1, Vi-1 and so on are the amounts of CPT in the dialysis bag.

[0064] The solubility of CPT in water is very low, so in the in vitro release experiment, 5% ethanol needs to be added to the release medium to achieve drug sink conditions. The in vitro release curves of BSA-CPT-NPs and PEG-BSA-CPT-NPs in the release medium with a pH value of 7.4 are shown in the attached figure. Figure 3 As shown in the figure, it can be seen that after 72 hours of continuous release of the two drugs, the cumulative release of BSA-CPT-NPs was 63.59%, and the cumulative release of PEG-BSA-CPT-NPs was 73.44%. It can be seen that the drug release was faster in the early stage, which may be due to the adsorption of part of CPT on the surface of the nanoparticles. As the release time increases, it shows obvious sustained release characteristics. At this time, the CPT that is continuously released mainly comes from the release of drugs after the dissolution of the nanoparticle structure. In addition, it can be seen from the figure that the release of PEG-BSA-CPT-NPs is more gentle than that of BSA-CPT-NPs. Before 36 hours, the cumulative release of PEG-BSA-CPT-NPs is lower than that of BSA-CPT-NPs. After 36 hours, the release exceeds that of BSA-CPT-NPs, and the release reaches 73.44% at 72 hours. It may be due to the formation of a spatial hydrophilic barrier by PEG on the surface of the nanoparticles, which leads to the slow release of the modified nanoparticles. This sustained release effect can prolong the circulation time of CPT in the body after administration, improve the bioavailability of CPT, and reduce the number of dosing times. .

[0065] Effect Example 1: Study on the Interaction between Camptothecin and BSA

[0066] 1. Determination of the bonding constant between camptothecin and BSA

[0067] Fluorescence spectroscopic titration was performed using a Hitachi F-7100 fluorescence spectrophotometer equipped with a xenon lamp light source and a 10 mm long quartz cuvette. The excitation and emission bandwidths were set to 10 nm, respectively, and the scanning speed was 1200 nm min -1 , voltage was 400 V. The excitation wavelength was 295 nm, and the spectral changes were recorded in the wavelength range of 300-400 nm. BSA was dissolved in phosphate buffer at pH 7.40, and its concentration was 2×10 -6 mol·L -1 , respectively add 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 × 10 -6 mol·L -1 The fluorescence quenching constant Kq was obtained by fitting and analyzing the fluorescence titration data, which was 29.41×10 11 L·mol -1 ·s -1 This indicates that the fluorescence quenching of BSA by camptothecin is static quenching, and a stable complex can be formed between the two. The bonding constant Kb is 4.23×10 4 L·mol -1 , indicating that there is a strong interaction between the two. ΔG is -25.62KJ·mol -1 This indicates that the process of camptothecin inclusion into the BSA cavity is spontaneous.

[0068] 2. Molecular docking of camptothecin and BSA

[0069] AutoDock Tools software was used to dock camptothecin and BSA molecules. The 3D structure of BSA was selected from the PDB (Protein Data Bank) database (PDB ID: 4f5t); the 3D structure of camptothecin was downloaded from the PubChem database (PubChem ID: 24360), and the downloaded sdf format was converted into pdb format using Open Babel GUI software. The pdb format file of BSA was imported into the AutoDock Tools software as a macromolecule, and the pdb format file of camptothecin was imported into the AutoDock Tools software as a small molecule to simulate molecular docking. Finally, Ligplot 2.2.4 was used for two-dimensional visualization to analyze the binding force between camptothecin and BSA. The molecular docking results were analyzed, as shown in Table 1. The bonding energy of each site between camptothecin and BSA shows that camptothecin has a stronger interaction with site I of BSA; and the two-dimensional visualization results were analyzed, as shown in the attached Figure 5 The driving force for camptothecin to enter the hydrophobic cavity of BSA is hydrophobic and hydrogen bonding.

[0070] Table 1

[0071]

[0072] Effect Example 2: Characterization of Nanoparticles

[0073] 1. Appearance and shape:

[0074] The solutions of PEG-BSA-CPT-NPs and BSA-CPT-NPs prepared in Examples 1 and 2 were translucent with a slight blue opalescence. By comparison, it was found that the light transmittance of PEG-BSA-CPT-NPs was enhanced after PEG modification. Figure 6 shown.

[0075] The microscopic morphology of BSA-CPT-NPs and PEG-BSA-CPT-NPs was observed by transmission electron microscopy (TEM). Figure 7 As shown, the morphology of BSA-CPT-NPs and PEG-BSA-CPT-NPs is relatively uniform, mostly spherical, with a particle size of about 200 nm and uniform size.

[0076] 2.X-ray diffraction analysis:

[0077] (1) X-ray diffraction analysis of BSA-CPT-NPs prepared in Example 2

[0078] XRD characterization was performed on CPT, BSA, physical mixture of CPT and BSA, and BSA-CPT-NPs. The results are shown in the attached figure. Figure 8As shown: CPT has sharp diffraction peaks at 5.9°, 13.3°, 17.7° and 25.6°, proving that the CPT raw material exists in the form of a crystal structure. BSA has no diffraction peaks due to its amorphous nature. The characteristic peaks of CPT are still retained in the physical mixture of CPT and BSA, indicating that CPT still exists in the form of a crystal structure in this mixed system, and the presence of BSA has no effect on the crystal form of CPT. However, in BSA-CPT-NPs, the characteristic peaks of CPT disappear, indicating that the dispersion state of CPT in BSA-CPT-NPs has changed, which may be caused by the formation of CPT-albumin inclusion complexes.

[0079] (2) X-ray diffraction analysis of PEG-BSA-CPT-NP prepared in Example 1

[0080] The crystal structures of PEG, PEG-BSA and PEG-BSA-CPT-NPs were analyzed by XRD. Fig. 9 As shown: PEG has sharp diffraction peaks at 19.4° and 23.6°, which indicates that PEG exists in a crystalline state at room temperature. PEG characteristic peaks can be seen in PEG, PEG-BSA, and PEG-BSA-CPT-NPs, but in PEG-BSA-CPT-NPs, the characteristic crystal peak of CPT disappears, and its peak shape is similar to that of PEG-BSA, indicating that CPT is successfully encapsulated in the nanoparticles. On the other hand, no CPT characteristic peak is seen in PEG-BSA-CPT-NPs, indicating that CPT is encapsulated in PEG-BSA in an amorphous or disordered state.

[0081] 3. Fourier transform infrared (FT-IR) spectroscopy:

[0082] (1) Infrared spectroscopy analysis of BSA-CPT-NPs prepared in Example 2

[0083] Determine the infrared spectra of the original drug CPT, BSA, physical mixture of CPT and BSA, and BSA-CPT-NPs, as shown in the attached Fig.10 As shown: In the infrared spectrum, 3255cm -1 The absorption peak at 1650cm is the characteristic vibration of primary hydroxyl group -OH. -1 and 1589cm -1 The characteristic peak at 1741 cm -1 The peak at 1150-1330 cm-1 is the stretching vibration of the carbonyl C=O of the cyclic lactone. -1 There are some relatively concentrated absorption peaks at 1660cm, which are the antisymmetric stretching vibrations of COC and are the infrared characteristic peaks of CPT (A). -1There is a characteristic absorption peak of NH (B) at 1099 cm -1 、1680cm -1 、2981cm -1 、3408cm -1 There are four absorption peaks at 2000~2500cm in BSA-CPT-NPs, which are CO bond, C=O bond, CH bond and OH bond. In the physical mixture of CPT and BSA, the characteristic absorption peaks of CPT and BSA are both present. Compared with CPT, the infrared spectrum of BSA-CPT-NPs shows a hydroxyl stretching vibration peak with strong intensity and wide high wave number region, which is due to the large number of hydroxyl and carboxyl groups. -1 The small characteristic peak at 690-960cm is the stretching vibration caused by a small amount of C=C double bonds in the nanoparticles. -1 The presence of CH bending vibration peaks of aromatics or olefins at 1664 cm-1 proves that BSA-CPT-NPs have benzene rings or C=C double bond structures. -1 The absorption peak at the 30° point becomes weaker, broader and blunter, which may be caused by encapsulating CPT in BSA. In summary, the infrared spectra of BSA-CPT-NPs and CPT are basically consistent, and no new absorption peaks appear, proving that after CPT is encapsulated by BSA, there is only physical interaction between it and other components.

[0084] (2) Infrared spectroscopy analysis of PEG-BSA-CPT-NP prepared in Example 1

[0085] The infrared spectra of PEG, PEG-BSA-NPs and PEG-BSA-CPT-NPs were measured. The results are shown in the attached figure. Fig.11 As shown, at 1740cm -1 The peak at 1785 cm-1 is the characteristic peak of ester carbonyl. -1 and 1813cm -1 The characteristic peak at 1000 cm is the characteristic peak of the succinimide group, and the COC asymmetric stretching vibration peak is at 1000 cm -1 ~1250cm -1 , which are the infrared characteristic peaks of PEG (A). In the spectra of PEG-BSA-NPs and PEG-BSA-CPT-NPs, 3302 cm -1 A characteristic absorption peak is formed at 1649cm -1 and 1549cm -1 Two characteristic absorption peaks appeared at , and the two absorption peaks were found to be the absorption peaks of amide II band. All these indicate that the residual amino groups on the surface of PEG and BSA were successfully coupled.

[0086] Effect Example 2: Evaluation of in vitro antitumor activity

[0087] 1. Cytotoxicity of Blank Vector

[0088] A) Adjust the cell concentration, inoculate 5000 Hela cells / well in a 96-well plate, add 100 μL of cell suspension to each well, and culture at 37°C, 5% CO2 for 24 h (surrounding with PBS).

[0089] B) Discard the spent culture medium, replace with fresh culture medium, and then add blank carrier BSA-NPs and PEG-BSA-NPs solutions, 100 μL per well. Five concentrations of blank nanoparticles were set, namely 0.1, 1, 10, 100, and 1000 μg mL -1 At the same time, a well without nanoparticles was set as a blank control, and three replicate wells were set for each group of experiments. Incubate in a CO2 incubator for 24 h.

[0090] C) MTT method was used to determine the cell survival rate after the addition of blank vector. First, MTT (5 mg mL -1 ) solution 20 μL, and then transfer the 96-well plate to an environment with a temperature of 37°C and a CO2 concentration of 5% and continue incubating for 4 hours before stopping the culture.

[0091] D) Pour the solution in the wells, add DMSO solution, 150 μL / well, and gently shake for 10 minutes at 37°C to completely dissolve the formazan formed in the cells. Measure the absorbance at 490 nm and calculate the cell survival rate. The cell growth inhibition rate is calculated according to the following formula:

[0092]

[0093] As attached Fig.12 As shown in the in vitro cytotoxicity test results, the blank carrier BSA-NPs and PEG-BSA-CPT-NPs had no significant effects on the cytotoxicity of the NPs in the concentration range of 0.1 to 1000 μg mL -1 There was no statistical difference in the survival rate of Hela cells compared with the control group, and no obvious cytotoxicity was observed, indicating that BSA-NPs and PEG-BSA-NPs, as drug carriers, have little toxicity to cells and are safe and reliable drug carriers.

[0094] 2. Antitumor activity of BSA-CPT-NPs and PEG-BSA-CPT-NPs

[0095] The cytotoxicity experiments of BSA-CPT-NPs and PEG-BSA-CPT-NPs were consistent with the "Blank Vector Cytotoxicity" operation steps. After cell counting, the concentration of Hela cells was adjusted and inoculated in a 96-well plate at a density of 5000 cells / well, 100 μL per well, and cultured in an incubator at 37°C and 5% CO2 for 24 h. The culture medium was discarded and replaced with fresh culture medium, and then CPT drug solution containing different CPT concentrations, BSA-CPT-NPs and PEG-BSA-CPT-NPs were added respectively, and the drug concentration gradient was set to 0.001, 0.01, 0.1, 1, 10, 100 μg mL -1 The blank control was the well without drug addition, and 3 replicate wells were set for each group. The cells were then placed in a CO2 incubator for further incubation, and the MTT assay was performed at 24h and 48h, respectively, to calculate the cell survival rate.

[0096] As attached Fig.13 and 14 As shown in the cytotoxicity test results, when the drug concentration is between 0.001 and 100 μg mL -1 Within the range, as the CPT concentration continued to increase, the cell survival rate gradually decreased. When the CPT concentration reached 10 μg mL -1 , the cell survival rate was significantly reduced. In addition, the antitumor activity of the three preparations was also significantly enhanced over time, indicating that the antitumor activity of free CPT, BSA-CPT-NPs and PEG-BSA-CPT-NPs on Hela was concentration-dependent and time-dependent. By comparison, compared with the bulk drug CPT solution, BSA-CPT-NPs and PEG-BSA-CPT-NPs had higher antitumor activity at the same CPT concentration. Compared with BSA-CPT-NPs, PEG-BSA-CPT-NPs had stronger antitumor activity. In summary, after CPT was prepared into BSA-CPT-NPs, compared with CPT, when the CPT concentration was 100 μg mL -1 When the exposure time was 48h, the cell survival rate decreased from 29.9% to 19.3%. After CPT was prepared into PEG-BSA-CPT-NPs, when the CPT concentration was 100μgmL -1 When the action time was 48h, the cell survival rate decreased from 29.9% to 13.7%, indicating that the anti-tumor activity of PEG-BSA-CPT-NPs was significantly improved.

Claims

1. A method for preparing bovine serum albumin-loaded camptothecin nanoparticles, characterized in that: The following steps are involved: Step 1: Dissolve mPEG-NHS in water, slowly add it to the BSA aqueous solution under stirring, react at room temperature for 12 hours to obtain the conjugate PEG-BSA, remove the unreacted mPEG-NHS, lyophilize and store at -20°C until use; Step 2: Weigh CPT and dissolve it in a mixed solution of chloroform and anhydrous ethanol as an organic phase, dissolve the PEG-BSA obtained in step 1 in water as an aqueous phase, mix the two, and stir evenly to obtain a PEG-BSA nanoparticle solution that initially encapsulates camptothecin; Step 3: The camptothecin-encapsulated PEG-BSA nanoparticle solution obtained in step 2 is rapidly frozen to below -80°C to form a frozen solution, and freeze-dried to obtain PEG-BSA-CPT nanoparticles; Step 4: The freeze-dried PEG-BSA-CPT nanoparticles obtained in step 3 are redissolved in an appropriate amount of deionized water or PBS buffer, subjected to ultrasonic treatment, and freeze-dried in a dryer to obtain drug-loaded nanoparticles.

2. The preparation method according to claim 1, characterized in that: In step 1, the concentration of BSA is 2-10 mg / mL, and the molar ratio of mPEG-NHS to BSA is 20:

1.

3. The preparation method according to claim 1, characterized in that: In step 2, the volume ratio of chloroform to anhydrous ethanol is (1-9):

1.

4. The preparation method according to claim 1, characterized in that: In step 2, the CPT concentration is 0.53-4.8 mg / mL.

5. The preparation method according to claim 1, characterized in that: In step 2, the volume ratio of the aqueous phase to the organic phase is (5-30):

1.

6. The preparation method according to claim 1, characterized in that: In step 2, the pH of the aqueous solution is 5-8.

7. The preparation method according to claim 1, characterized in that: In step 4, the ultrasonic time is 4 to 12 minutes, and the ultrasonic power is 45% to 75%.

8. Bovine serum albumin-loaded camptothecin nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the bovine serum albumin-loaded camptothecin nanoparticles as claimed in claim 8 in the preparation of anticancer drugs.

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