Albumin-loaded calcium phosphate nanoparticle and use thereof

Abstract

The application discloses albumin-loaded calcium phosphate type nanoparticles and application thereof, which are prepared from nucleic acid drugs, albumin, Ca 2+ , PO4 3‑ and CaCl2, wherein the nucleic acid drugs are DNA, siRNA or mRNA. The DMEM solution of albumin is co-incubated with the nucleic acid drugs, and then CaCl2 is added for co-incubation to grow the nanoparticles, so that the albumin-loaded calcium phosphate type nanoparticles with suitable and uniform particle sizes are prepared, the nucleic acid drugs are efficiently loaded, the gene transfection capacity is improved, and the disease treatment purpose is achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to albumin-loaded calcium phosphate nanoparticles and application thereof. BACKGROUND

[0002] Cancer seriously threatens human life and health. At present, with the development of genetic engineering, gene therapy has become a new means for treating cancer. Among them, nucleic acid drugs such as DNA, small interfering RNA (siRNA) and messenger RNA (mRNA) are common gene therapy drugs and have great potential in cancer treatment. However, nucleic acid drugs such as siRNA have poor stability in the body and are easily degraded by ribonuclease; moreover, it is difficult for them to independently cross the lipid cell membrane into the cytoplasm; and even it is difficult for them to escape from the lysosome to achieve cytoplasmic release of genes. Therefore, it is necessary to develop a delivery system for efficiently transfecting nucleic acid drugs.

[0003] At present, the carriers for delivering nucleic acid drugs mainly include viral vectors and non-viral vectors. Although the transfection efficiency of viral vectors is high, they can easily cause immunogenicity. Non-viral vectors can broaden the delivery route of nucleic acid drugs due to good biocompatibility. As one of non-viral vectors, inorganic materials can escape from the phagocytosis of the reticuloendothelial system by adjusting their morphology, size and other properties to avoid the decomposition of nucleic acid drugs. Calcium phosphate (CaP) as a classic non-viral vector is widely used in efficient transfection of nucleic acid drugs.

[0004] With the application of nanotechnology, nanodrugs have entered the clinic and opened up a new track for the treatment of human diseases. At present, the materials of nanodrugs mainly include organic nanomaterials, inorganic nanomaterials and metal nanomaterials. Among them, albumin nanoparticles prepared by taking albumin as a drug carrier are an organic nanomaterial with great application prospect. Compared with other nanocarriers, albumin as an endogenous substance has good biocompatibility, high drug loading capacity, long half-life and good targeting. It can be paired with the albumin binding receptor Gp60 on the surface of tumor cells to promote the enrichment of albumin nanoparticles to the tumor area. With the approval of Abraxane® for marketing, many companies have carried out research on albumin for delivering other drugs and are committed to developing new albumin nanoparticle preparation platforms. At present, a series of albumin nanoparticle preparations have entered clinical trials, so they have more clinical application value.

[0005] At present, CaP nanoparticles can be prepared by methods such as precipitation and stoichiometric titration. However, there are many influencing factors for these methods, and it is difficult to realize large-scale production. In addition, cation modification or change of the spatial structure of albumin can be used for gene delivery, but this modification will be accompanied by problems such as high toxicity and low stability of the preparation. SUMMARY

[0006] One of the purposes of the present application is to provide an albumin-loaded calcium phosphate nanoparticle, which is prepared from a nucleic acid drug, albumin, Ca 2+ , PO4 3- and CaCl2.

[0007] Further, the nucleic acid drug is DNA, siRNA or mRNA. Preferably, it is siRNA.

[0008] Further, the albumin is bovine serum albumin.

[0009] The second purpose of the present application is to provide a preparation method of the albumin-loaded calcium phosphate nanoparticle, which comprises the following steps: dissolving albumin in a medium solution containing Ca 2+ and PO4 3- , mixing the solution with an aqueous solution of a nucleic acid drug, incubating the mixed solution, adding a CaCl2 solution, and continuing to incubate to obtain the albumin-loaded calcium phosphate nanoparticle.

[0010] Further, the mass ratio of the albumin to the nucleic acid drug is (10-500):1. Preferably, it is (110-500):1.

[0011] Further, the medium solution containing Ca 2+ and PO4 3- is high-sugar incomplete medium.

[0012] In one embodiment of the present application, the albumin DMEM solution is co-incubated with siRNA, DMEM is used as the mineralization medium, the albumin can mineralize calcium phosphate therein and load the nucleic acid drug, and then CaCl2 is added to promote the growth of the nanoparticles to obtain BSA / CaP / siRNA nanoparticles with appropriate particle size. Specifically, the albumin is dissolved in DMEM at a concentration of 2 mg / mL, and then mixed with an aqueous solution of the nucleic acid drug at a concentration of 1 μg / mL, so that the mass ratio of the albumin to the nucleic acid drug is (10-500):1. The mixed solution is incubated at 37℃ for 24-36 h, and then a CaCl2 solution at a Ca 2+ concentration of 2 mM is added, and the incubation is continued for 6-12 h to obtain the albumin-loaded calcium phosphate nanoparticle.

[0013] Further, the Ca 2+ concentration in the preparation after adding the CaCl2 solution is 2 mM.

[0014] The third purpose of the present application is to provide the use of the albumin-loaded calcium phosphate nanoparticle in the preparation of a tumor treatment drug.

[0015] The synthesis of the CaP carrier is usually prepared by a precipitation method, and the method has many influencing factors, and it is difficult to control the size and shape of the CaP nanoparticles. The BSA / CaP / siRNA nanoparticles prepared by using the widely clinically applied BSA as a biological template to mineralize the CaP inner core loaded with siRNA overcome the defects of poor stability and easy aggregation of CaP.

[0016] In the application, the CaP can efficiently load siRNA, the albumin is used as a biological template to mineralize and stabilize the CaP inner core loaded with siRNA, and the BSA / CaP / siRNA nanoparticles are prepared. The nanoparticle preparation is simple and has high stability, and the problems of difficult production of CaP nanoparticles and high toxicity of cation-modified albumin are solved. In addition, the albumin-loaded calcium phosphate nanoparticles can prevent the degradation of nucleic acid drugs, prolong the half-life of the drugs, increase the uptake of tumor cells by combining with the albumin receptor Gp60 on the surface of tumor cells, promote the endosome escape of siRNA, realize the controlled release of nucleic acid drugs, silence target genes and down-regulate proteins, and finally realize the antitumor effect.

[0017] Effects of the application

[0018] 1. The albumin-loaded calcium phosphate nanoparticles prepared by the biological mineralization method have the advantages of simple preparation process, high encapsulation rate, suitable and uniform particle size, high stability and good reproducibility, and are easy to be clinically converted.

[0019] 2. The albumin-loaded calcium phosphate nanoparticles prepared by the application can load siRNA and other nucleic acid drugs, increase the drug uptake of tumor cells, promote the escape of nucleic acid drugs from lysosomes, enhance the gene transfection capacity, realize the purpose of gene therapy, and have high clinical use value. DETAILED DESCRIPTION

[0020] Figure 1 It is a schematic diagram of the structure of the BSA / CaP / siRNA nanoparticles.

[0021] Figure 2 It is an electrophoresis diagram of the siRNA encapsulation rate under different BSA / siRNA mass ratios (w / w) in Example 1.

[0022] Figure 3 It is a transmission electron microscope diagram of BSA / CaP / siRNA in Example 1.

[0023] Figure 4 It is a diagram of the colloidal stability and plasma stability of BSA / CaP / siRNA in Example 1.

[0024] Figure 5 It is a luciferase gene silencing diagram mediated by BSA / CaP / siLuc in Example 2.

[0025] Figure 6 Survival plot of BSA / CaP / siBcl2 killing 4T1 tumor cells in Example 2. DETAILED DESCRIPTION

[0026] The present application first co-incubates the DMEM solution of albumin with the nucleic acid drug siRNA, then adds CaCl2 for co-incubation to grow the nanoparticles, to prepare albumin-loaded calcium phosphate nanoparticles with suitable and uniform particle size, to realize efficient loading of siRNA, to improve its gene transfection capacity, and to achieve the purpose of disease treatment. The preparation process of the albumin-loaded calcium phosphate nanoparticles is shown in Figure 1 .

[0027] The preferred embodiments of the present application will be described in detail with reference to the following examples. It should be understood that the following examples are given only to illustrate the present application and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and replacements to the present application without departing from the spirit and principles of the present application.

[0028] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0029] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0030] The sequences of siRNA in the following examples are as follows:

[0031] siNC, sense, 5'-UUC UCC GAA CGU GUC ACG UTT-3'

[0032] siLuc, sense, 5'-GGA CGA GGA CGA GCA CUU CUU-3'

[0033] siBcl2, sense, 5'-GCA UGC GAC CUC UGU UUG AdTdT-3'. Example 1

[0034] BSA was dissolved in 1 mL DMEM, and 1 μg / mL siRNA solution (siNC was used in this example) was prepared with DEPC water without RNase and added to the BSA DMEM solution, shaken at 1500 rpm for 1 min, and incubated at 37°C for 24 h. Then 2 μL CaCl2(1 M) was added for 6 h of co-incubation to prepare BSA / CaP / siRNA nanoparticles. After incubation, the BSA / CaP / siRNA nanoparticles were separated and purified by an ultrafiltration tube (MWCO: 10 kDa), washed several times with a buffer solution (2 mM CaCl2, 1 mM Na2HPO4, 25 mM Tris, 140 mM NaCl, pH 7.4), freeze-dried, and stored at -20°C.

[0035] 1. To evaluate the loading capacity of BSA / CaP carriers for siRNA, BSA / CaP / siRNA nanoparticles were prepared at different ratios of BSA / siRNA (w / w) of 95, 100, 105, 110, 115, and 120 to evaluate the loading capacity of BSA / CaP carriers for siRNA. The optimal w / w of loaded siRNA was analyzed by 1% (w / v) agarose gel electrophoresis, 100 mV for 30 min, and imaged and analyzed using a high-sensitivity chemiluminescence imaging system Chemidoc XRS+.

[0036] The results are shown in Figure 2 When w / w≥110, there was no obvious leakage of the siRNA band, indicating that the BSA / CaP carrier could almost completely load siRNA and had strong siRNA loading capacity.

[0037] 2. BSA / siRNA (w / w) of 110 was selected, and BSA / CaP / siRNA nanoparticles were prepared according to the above method, and their hydrated particle size and potential were measured using a Malvern nanoparticle size and Zeta potential instrument. The BSA / CaP / siRNA nanoparticles were placed on a copper grid, negatively stained with 1 wt% sodium tungstate solution, and excess liquid was absorbed with filter paper, then dried with an infrared lamp, and then loaded and observed for morphology using a transmission electron microscope.

[0038] The results are shown in Figure 3 The BSA / CaP / siRNA particle size was 123 nm, and the polydispersity index (PDI) was 0.137, indicating that the particle size was appropriate and uniform. Transmission electron microscopy (TEM) observation showed that the BSA / CaP / siRNA was spherical particles, and the particle size was consistent with the dynamic light scattering results. In addition, the potential measurement results showed that the potential of BSA / CaP / siRNA was -6.83 mV.

[0039] 3. Using a BSA / siRNA mass ratio (w / w) of 1:10, BSA / CaP / siRNA nanoparticles were prepared according to the above method. The prepared BSA / CaP / siRNA nanoparticles were stored at 4°C, and their particle size was measured using a Malvern nanoparticle size analyzer at different time points (0, 2, 4, 6, 8 days) to evaluate their colloidal stability. Simultaneously, equal volumes of BSA / CaP / siRNA nanoparticles and mouse serum were mixed and incubated in a 37°C water bath with shaking. Samples were taken at different time points (0, 8, 16, 24, 32, 40, 48 h) to measure particle size and evaluate plasma stability. Equal volumes of BSA / CaP / siRNA nanoparticles and mouse serum were mixed and incubated in a 37°C water bath with shaking. Samples were taken at different time points (0, 2, 4, 8, 12, 24 h), and 2% SDS was added to replace the siRNA in the nanoparticles. The samples were then loaded onto agarose gel electrophoresis to analyze the plasma stability of the siRNA and photographed.

[0040] The results are as follows Figure 4 As shown, the particle size of the nanoparticles remained within the range of 120-140 nm over 8 days, exhibiting minimal fluctuations and demonstrating good colloidal stability. Furthermore, after incubation with serum, the particle size only slightly decreased within 48 hours, confirming the good serum stability of albumin-loaded calcium phosphate nanoparticles under physiological conditions. Gel electrophoresis results showed that after incubation with serum, free siRNA was largely degraded by RNase after 2 hours; however, in BSA / CaP / siRNA nanoparticles with completely compressed siRNA, most of the siRNA remained intact within 24 hours, demonstrating that BSA / CaP effectively protects siRNA and prevents degradation. Example 2

[0041] A 40 μg / mL siLuc solution was prepared using RNase-free DEPC water, and a 25 kPa PEI cation solution was prepared using 10 mM HEPES (pH 7.4). The siLuc solution and the polycation solution were mixed at a ratio of 25 kPa PEI / siLuc (w / w = 2:1), vigorously rotated for 30 s, and incubated at room temperature for 30 min to prepare the 25 kPa PEI / siLuc nanocomposite.

[0042] 2 mg BSA was dissolved in 1 mL DMEM, and a 1 μg / mL siLuc solution was prepared with RNase-free DEPC water. The BSA / siLuc solution (w / w = 110:1) was added to the BSA DMEM solution, and the mixture was shaken at 1500 rpm for 1 min and incubated at 37 °C for 24 h. Then, 2 μL of CaCl2 (1 M) was added and incubated for another 6 h to obtain BSA / CaP / siLuc nanoparticles. After incubation, the nanoparticles were purified by ultrafiltration (MWCO: 10 kDa), washed several times with buffer solution (2 mM CaCl2, 1 mM Na2HPO4, 25 mM Tris, 140 mM NaCl, pH 7.4), freeze-dried, and stored at -20 °C for later use.

[0043] 4T1-Luc cells were divided into groups of 5 × 10 3 / wells were seeded in 96-well plates and incubated in a CO2 incubator for 24 h. After incubation, the culture medium was discarded, and fresh DMEM and DMEM containing 10% FBS were added to dilute free siLuc, 25k PEI / siLuc (w / w = 2:1), and BSA / CaP / siLuc (w / w = 110:1) to a siLuc concentration of 200 nM. After co-incubation of the drug and cells for a period of time, the drug-containing culture medium was aspirated, and fresh DMEM was added to continue incubation for 24 h. The culture medium was then discarded, and 100 μL of fresh DMEM and an equal volume of luciferase detection reagent were added. After co-incubation for 10 min, the luciferase activity of each well was analyzed using an MD ID5 microplate reader.

[0044] The results are as follows Figure 5 As shown in the figure. First, the luciferase silencing ability of the formulation in serum-free conditions was verified. After treatment of cells with free siLuc, there was no change in luciferase expression. The 25k PEI / siLuc group silenced nearly 55% of the luciferase protein by facilitating the escape of siRNA from lysosomes through the proton sponge effect. At the same time, compared with the blank control group, the BSA / CaP / siLuc group could silence nearly 45% of the luciferase gene, confirming that BSA / CaP / siRNA has good gene transfection ability. Example 3

[0045] A 40 μg / mL siBcl2 solution was prepared using RNase-free DEPC water, and a 25kkPEI cation solution was prepared using 10 mM HEPES (pH 7.4). The siLuc solution and the polycation solution were mixed at a ratio of 25kPEI / siBcl2 (w / w = 2:1), vigorously rotated for 30 s, and incubated at room temperature for 30 min to prepare the 25kPEI / siBcl2 nanocomposite.

[0046] 2 mg BSA was dissolved in 1 mL DMEM, and a 1 μg / mL siNC solution was prepared with RNase-free DEPC-treated water. The BSA / siNC solution (w / w = 110:1) was added to the BSA DMEM solution, and the mixture was shaken at 1500 rpm for 1 min and incubated at 37 °C for 24 h. Then, 2 μL of CaCl2 (1 M) was added and incubated for another 6 h to obtain BSA / CaP / siNC nanoparticles. After incubation, the nanoparticles were purified by ultrafiltration (MWCO: 10 kDa), washed several times with a buffer solution (2 mM CaCl2, 1 mM Na2HPO4, 25 mM Tris, 140 mM NaCl, pH 7.4), freeze-dried, and stored at -20 °C for later use.

[0047] 2 mg BSA was dissolved in 1 mL DMEM, and a 1 μg / mL siBcl2 solution was prepared with RNase-free DEPC water. The BSA / siBcl2 solution (w / w = 110:1) was added to the BSA DMEM solution, and the mixture was shaken at 1500 rpm for 1 min and incubated at 37 °C for 24 h. Then, 2 μL CaCl2 (1 M) was added and incubated for another 6 h to obtain BSA / CaP / siBcl2 nanoparticles. After incubation, the nanoparticles were purified by ultrafiltration (MWCO: 10 kDa), washed several times with buffer solution (2 mM CaCl2, 1 mM Na2HPO4, 25 mM Tris, 140 mM NaCl, pH 7.4), freeze-dried, and stored at -20 °C for later use.

[0048] 4T1 cells were divided into 5×10 3 The cells were seeded in 96-well plates and incubated at 37°C for 24 h until the cell density reached 80%. The culture medium was then removed, and DMEM-prepared free siBcl2, BSA / CaP / siNC (300 nM), 25k PEI / siBcl2 (100 nM), and BSA / CaP / siBcl2 (100, 200, and 300 nM) were added. After co-incubation with the cells for a period of time, the drug-containing culture medium was removed, and fresh DMEM was added for further incubation for 24 h. 20 μL of MTT (5 mg / ml) was added to the drug-containing culture medium, and the cells were incubated at 37°C for 4 h. The culture medium was then removed, and 200 μL of DMSO was added to dissolve formazan. The absorbance of each well was measured at a detection wavelength of 492 nm using a microplate reader, and the cell viability was calculated based on the absorbance.

[0049] The results are as follows Figure 6The cell viability of free siBcl2 and BSA / CaP / siNC groups had no effect on the cell viability, the cell viability of 25k PEI / siBcl2 group was 75% of the control group, and the cell viability of BSA / CaP / siBcl2 group with the highest concentration could be reduced to 50%, which indicated that BSA / CaP / siBcl2 could inhibit the proliferation of 4T1 cells by silencing Bcl2 gene.

Claims

1. Albumin-loaded calcium phosphate-based nanoparticles, characterized in that, from nucleic acid drugs, albumin, Ca 2+ , PO4 3- and CaCl2; the preparation method is to dissolve albumin in a medium solution containing Ca 2+ and PO4 3- , then mix with an aqueous solution of nucleic acid drugs, incubate the mixed solution, then add a CaCl2 solution, continue to incubate, to obtain albumin-loaded calcium phosphate nanoparticles.

2. The albumin-loaded calcium phosphate-based nanoparticle according to claim 1, wherein, The nucleic acid drug is DNA, siRNA or mRNA.

3. The albumin-loaded calcium phosphate-based nanoparticle according to claim 2, wherein, The nucleic acid drug is siRNA.

4. The albumin-loaded calcium phosphate-based nanoparticle of claim 1, wherein, The albumin is bovine serum albumin.

5. The method of producing albumin-loaded calcium phosphate nanoparticle according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: Albumin is dissolved in a medium solution containing Ca 2+ and PO4 3- , and then mixed with an aqueous solution of a nucleic acid drug, the mixed solution is incubated, and then a CaCl2 solution is added and incubated to obtain albumin-loaded calcium phosphate nanoparticles.

6. The production method according to claim 5, wherein The mass ratio of the albumin and the nucleic acid drug is (10-500):

1.

7. The preparation method according to claim 5, characterized in that, The medium solution containing Ca 2+ and PO4 3- is high-sugar incomplete medium.

8. The production method according to claim 5, characterized by, Ca in the formulation after addition of CaCl2 solution 2+ 2 mM.

9. Use of the albumin-loaded calcium phosphate nanoparticle according to any one of claims 1 to 4 in the preparation of a tumor treatment drug.

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