CN110974979B

CN110974979B - Preparation method and application of functionalized calcium phosphate gene delivery system

Info

Publication number: CN110974979B
Application number: CN201911070574.5A
Authority: CN
Inventors: 张邦乐; 许经良; 何炜; 马茜茜; 王伟; 周四元; 宦梦蕾; 李晨
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2019-11-05
Filing date: 2019-11-05
Publication date: 2020-12-15
Anticipated expiration: 2039-11-05
Also published as: CN110974979A

Abstract

The invention discloses a preparation method and application of a functionalized calcium phosphate gene delivery system. The invention uses disulfide bond with redox response characteristic to connect alendronic acid and mannitol segment to obtain the mannitol alendronic acid derivative containing disulfide bond. The obtained mannitol alendronate derivative containing disulfide bonds is further modified on the surface of the calcium phosphate nanoparticle and loads genes, so that a stable functionalized calcium phosphate gene delivery system is formed. The functional calcium phosphate gene delivery system constructed by the invention has the advantages of simple preparation process, good stability and safety, high transfection efficiency and the like, compared with the traditional calcium phosphate nanoparticles, the stability is greatly improved, simultaneously, the redox responsiveness of disulfide bonds can quickly release the loaded gene, the high-efficiency gene transfection activity is shown, and the clinical application prospect is good.

Description

Preparation method and application of functionalized calcium phosphate gene delivery system

Technical Field

The invention relates to the field of pharmaceutical preparations and the technical field of biological medicines, in particular to a preparation method and application of a functionalized calcium phosphate gene delivery system.

Background

Gene therapy is a biomedical treatment method for delivering therapeutic genes into cells, effecting gene editing and correcting genetic mutations by affecting or replacing dysfunctional genes, thereby treating diseases. The key to gene therapy is the construction of a suitable vector system to achieve safe, stable and efficient delivery of therapeutic genes. In this process, gene delivery systems are of critical importance.

At present, the commonly used gene vector systems mainly comprise two main types of viral vectors and non-viral vectors. The virus vector has the greatest advantage of higher transfection efficiency. The two types of viral vectors most commonly used are adeno-associated viral vectors and retroviral vectors [ journal of Control Release, 2012, 161(2): 377-388; biochemistry (Mosc), 2016, 81(7): 700-. In practical applications, the immunogenicity of viral vectors and the risk of tumor induction may lead to serious clinical adverse events, which greatly limits their applications [ Current Gene Therapy, 2011, 11(4): 321- "330; biotechnology and Bioengineering, 2018, 115(1): 25-40 ]. In addition, the low gene load of viral vectors and the difficulty of large-scale production are also one of the reasons for their limited clinical applications. Non-viral vectors include mainly calcium phosphate, cationic liposomes and cationic polymers such as polyamide dendrimers, Polyethyleneimine (PEI), and the like. Non-viral vectors are less toxic and more biocompatible and are considered as the most promising delivery system in gene therapy [ Nature Reviews Genetics, 2014, 15(8): 541-; current Gene Therapy, 2017, 17(2): 147-153], but the relatively low transfection efficiency of non-viral vectors limits the practical application in the biomedical field.

Calcium phosphate nano delivery system is a commonly used non-viral transfection material, has the advantages of excellent biocompatibility, simple preparation method, low cost and the like, and has become a commonly used material for in vitro gene transfection [ Current Pharmaceutical Design, 2016, 22(11): 1529-1533 ]. But the stability is poor, the aggregation and the precipitation are very easy to occur after the preparation, and the transfection efficiency needs to be further improved. Therefore, many researchers have focused on constructing calcium phosphate-related systems to achieve efficient gene delivery. Huang et al [ ACS Nano, 2013, 7: 5376-.

Disulfide bonds are susceptible to cleavage under high concentrations of Glutathione (GSH). The concentration of GSH inside and outside the cell is very different, the concentration of GSH in cytoplasm is as high as 1-10 mmol/L, and the concentration of GSH outside the cell is only 1-10 μmol/L [ Advances in Clinical Chemistry, 2018, 87: 141-. The disulfide bond can be kept stable in an extracellular environment and is easy to break in cells, and the redox response characteristic of the disulfide bond is utilized to improve the blood circulation of the loaded gene and the degradation stability of extracellular anti-ribozyme, and meanwhile, the intracellular rapid and effective release can be realized, so that the efficient gene transfection effect is achieved. Mannitol is a polyhydroxy compound, has strong hydrophilicity, and has the effect of stabilizing nanoparticles like polyethylene glycol. Therefore, the invention modifies the mannitol alendronate derivative with redox response characteristic and containing disulfide bonds on the surface of the calcium phosphate nanoparticle for the first time and loads the gene, and the functional calcium phosphate gene delivery system with good stability can be prepared by simple mixing under the condition of not using organic solvent and surfactant, so that the rapid and effective release of the loaded gene is realized, and the gene delivery and transfection efficiency is improved.

Disclosure of Invention

The invention aims to provide a preparation method of a functionalized calcium phosphate gene delivery system, which has the advantages of simple process, good stability, good biocompatibility and high transfection efficiency.

The invention uses disulfide bond with redox response characteristic to connect alendronic acid and mannitol segment to obtain the mannitol alendronic acid derivative containing disulfide bond. The obtained mannitol alendronate derivative containing disulfide bonds is further modified on the surface of the calcium phosphate nanoparticles and carries genes, so that a stable functionalized calcium phosphate gene delivery system is formed, and a new idea is provided for promoting further application of gene therapy in medicine and pharmacology.

A preparation method of a functionalized calcium phosphate gene delivery system comprises the following steps:

(1) preparation of a disulfide bond-containing mannosylated alendronic acid derivative Man-SS-Aln

Alendronate sodium (341.6 mg, 1.26 mmol) was dissolved in 10 mL of distilled water at room temperature and the pH was adjusted to 8 using 1M aqueous NaOH solution. SPDP with the corresponding ratio is dissolved in 10 mL acetonitrile, added to the reaction solution in 4 times, and the pH is adjusted to 8 by 1M NaOH aqueous solution before each addition, and the time is 2 h. After the addition of the SPDP solution is finished, stirring and reacting for a certain time at a specific temperature. And distilling off acetonitrile in the reaction solution under reduced pressure, adding methanol with 4 times of volume, standing overnight at room temperature, performing suction filtration, and performing vacuum drying to obtain a white solid SPDP-Aln.

② dissolving SPDP-Aln (100.8 mg, 205.58 mu mol) in 5 mL of distilled water at room temperature, adding Man-Cys with corresponding proportion for 3 times, and stirring and reacting for a certain time at specific temperature. And (3) quickly dialyzing the reaction solution for 1 h by using 300 mL of III-grade water, and freeze-drying to obtain a white solid Man-SS-Aln.

(2) Preparation of gene delivery system preparation solution: the solution a is prepared from calcium salt and water, and the concentration of the calcium salt is 2.5M; the solution b is prepared from phosphate, sodium chloride, a buffering agent and water, wherein the concentration of the phosphate is 1.5 mM, the concentration of the sodium chloride is 0.28 mM, the concentration of the buffering agent is 0.05 mM, and the pH value is 7.05; the solution c is prepared from a mannized alendronic acid derivative Man-SS-Aln and water, and the concentration is 0.1-500 mu M.

(3) And (3) mixing the specific gene with the solution a, diluting the specific gene with double distilled water to be ten times of the volume of the solution a, uniformly mixing the diluted specific gene with the solution b in the same volume, and adding a solution c in the same volume with the solution b to prepare the gene-loaded functionalized calcium phosphate gene delivery system.

In the specific preparation process, in the step (1), the molar ratio of alendronate sodium to SPDP is 0.5: 2-2: 0.5; the reaction temperature is-20 ℃ to 100 ℃, and the stirring time is 0.1 to 48 hours.

In the specific preparation process, in the step (1), the mol ratio of the SPDP-Aln to the Man-Cys is 0.5: 2-2: 0.5; the reaction temperature is 0-100 ℃, and the stirring time is 0.5-24 h.

In the specific preparation process, the calcium salt in the solution a in the step (2) is CaCl₂、Ca(NO₃)₂One or a combination of two in any proportion; the phosphate in the solution b is Na₃PO₄、Na₂HPO₄、NaH₂PO₄、K₃PO₄、K₂HPO₄、KH₂PO₄、(NH₄)₃PO₄、(NH₄)₂HPO₄One or a combination of two or more of them at any ratio; the buffer is one or a combination of more than two of HEPES, BES, PBS, PIPES and Tris in any proportion.

In a specific preparation process, the gene in the step (3) is any one or more of DNA, siRNA, miRNA, lncRNA, circRNA or shRNA; the ratio of the gene mass (mug) to the volume (mL) of the solution a when the gene is mixed with the solution a is 50-5000 mug/mL; the mass ratio of Man-SS-Aln in the solution c is 0.1-60%.

The evaluation result of the functionalized calcium phosphate gene delivery system shows that the functionalized calcium phosphate gene delivery system constructed by the invention has the advantages of simple preparation process, good stability and safety, high transfection efficiency and the like, compared with the traditional calcium phosphate nanoparticles, the functionalized calcium phosphate gene delivery system greatly improves the stability, simultaneously, the redox responsiveness of disulfide bonds can quickly release the loaded gene, shows high-efficiency gene transfection activity, and has good clinical application prospect.

Drawings

FIG. 1 is a synthetic scheme for the preparation of Man-SS-Aln (3).

FIG. 2 shows the particle size and PDI measurement of the nanoparticles of Cap and Cap-MSSA-1, Cap-MSSA-2, Cap-MSSA-4, and Cap-MSSA-15. A is CaP, B is CaP-MSSA-1, C is CaP-MSSA-2, D is CaP-MSSA-4, and E is CaP-MSSA-15. :, its strength is too small to be measured.

FIG. 3 shows the results of agarose gel electrophoresis of nanoparticles of Cap and Cap-MSSA-1, Cap-MSSA-2, Cap-MSSA-4, and Cap-MSSA-15.

FIG. 4 shows the degradation protection experiments of the nano-particle endonucleases of Cap and Cap-MSSA-1, Cap-MSSA-2, Cap-MSSA-4, and Cap-MSSA-15. (+) indicates the presence of the degrading enzyme DNase I, and (-) indicates the absence of DNase I.

Fig. 5 is a cytotoxicity assay of HEK 293T cells, a: Man-SS-Aln (3), B: CaP and CaP-MSSA-1, CaP-MSSA-2, CaP-MSSA-4, CaP-MSSA-15 nanoparticles, representing a survival rate for p <0.05 compared to the same concentration of CaP.

FIG. 6 shows the results of experiments on the hemolysis of nanoparticles of Cap and Cap-MSSA-1, Cap-MSSA-2, Cap-MSSA-4, and Cap-MSSA-15.

FIG. 7 shows the transfection efficiency of the CaP, CaP-MSSA-1, CaP-MSSA-2, CaP-MSSA-4, and CaP-MSSA-15 nanoparticles in HEK 293T cells. A, percentage of transfected cells; b, average fluorescence intensity. Represents p <0.01 compared to the positive rate or mean fluorescence intensity of transfection of the same concentration of DNA; # represents p <0.05 compared to the positive transfection efficiency or mean fluorescence intensity of the same concentration of Cap;

# represents p <0.01 compared to the positive transfection rate or mean fluorescence intensity of the same concentration of Cap; & represents p <0.05 compared with the transfection positive rate or the mean fluorescence intensity of the same concentration of Cap-MSSA-1; and & represents p <0.01 compared to the transfection positivity or mean fluorescence intensity of the same concentration of CaP-MSSA-1.

FIG. 8 shows the results of the expression of green fluorescent protein of CaP and CaP-MSSA-1, CaP-MSSA-2, CaP-MSSA-4, and CaP-MSSA-15 nanoparticles in HEK 293T cells.

Detailed Description

The following examples further describe embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 Synthesis and characterization of a mannosylated alendronic acid derivative, Man-SS-Aln (3)

A specific synthetic route for preparing the mannitol alendronate derivative Man-SS-Aln (3) is shown in figure 1.

1. Preparation and characterization of SPDP-Aln (1)

Alendronate sodium (341.6 mg, 1.26 mmol) was dissolved in 10 mL of distilled water at room temperature and the pH was adjusted to 8 using 1M NaOH solution. SPDP (500 mg, 1.60 mmol) was dissolved in 10 mL acetonitrile at room temperature and added to the alendronate sodium solution 4 times, with pH adjusted to 8 with 1M NaOH solution before each addition, for 2 h. After the addition of the SPDP solution is finished, the reaction is continued to be stirred at room temperature for 4 h. Distilling off acetonitrile in the reaction solution under reduced pressure, adding methanol with 4 times of volume, standing at room temperature overnight, filtering, and vacuum drying to obtain white solid SPDP-Aln (1). The structure of SPDP-Aln (1) is carried out¹H-NMR、¹³C-NMR and mass spectrometric identification.

And (3) characterization results:¹H-NMR: _H (ppm): 8.34 (1H, d, J=4 Hz), 7.75 ~ 7.82 (2H, m), 7.25 (1H, t, J=6 Hz), 3.09 (1H, t, J=6 Hz), 3.00 (1H, t, J=6 Hz), 2.59 (1H, t, J=8 Hz), 1.82 ~ 1.93 (2H, m), 1.71 ~ 1.76 (2H, m)；¹³C-NMR: _C (ppm)：173.38, 158.77, 148.83, 138.44, 121.73, 120.97, 40.22, 34.62, 33.83, 31.29, 23.42; ESI^--MS：[M-Na]^-the actual measured value was 445 with [ M-Na ]]^-The theoretical value 445 is consistent. Warp beam¹H-NMR、¹³The results of the obtained compound and the target compound are consistent through identification of C-NMR and mass spectrum.

2. Preparation and characterization of manning gem diphosphonic acid derivative Man-SS-Aln (3) containing disulfide bond

SPDP-Aln (1) (100.8 mg, 205.58. mu. mol) was dissolved in 5 mL of distilled water at room temperature, and after the addition of Man-Cys (2, prepared in chem. Res. Toxicol, 1999, 12: 331-334) (58.10 mg, 203.64. mu. mol, added in 3 portions, each 10 min apart), the reaction was stirred for 3 h. And (3) quickly dialyzing the reaction solution for 1 h, and freeze-drying to obtain a white solid Man-SS-Aln (3). The structure of Man-SS-Aln (3) is carried out¹H-NMR、¹³C-NMR and mass spectrometric identification.

And (3) characterization results:¹H-NMR: _H (ppm): 3.91 ~ 3.97 (2H, m), 3.51 ~ 3.73 (5H, m), 3.39 ~ 3.43 (1H, dd, J ₁=12 Hz, J ₂=4 Hz), 3.20 ~ 3.25 (1H, m), 3.05 ~ 3.14 (4H, m), 2.86 ~ 2.91 (2H, m), 2.54 ~ 2.61 (2H, m), 1.77 ~ 1.88 (2H, m), 1.67 ~ 1.71 (2H，m); ¹³C-NMR: _C (ppm): 174.04, 171.55, 71.17, 70.59, 68.95, 66.59, 63.09, 61.05, 50.19, 40.10, 37.11, 35.05, 33.02, 31.10, 23.33; HRMS：[M+Cl]^-= 677.0355. Warp beam¹H-NMR、¹³The results of the obtained compound and the target compound are consistent through identification of C-NMR and mass spectrum.

Example 2 preparation and characterization of Gene-loaded functionalized calcium phosphate nanoparticles CaP-MSSA

1. Preparation of functionalized calcium phosphate nanoparticle CaP-MSSA

(1) Preparation of gene delivery system preparation solution: solution a is prepared from CaCl₂Prepared with water, CaCl₂The concentration is 2.5M; solution b is composed of Na₂HPO₄NaCl, HEPES and water, Na₂HPO₄Concentration 1.5 mM, NaCl concentration 0.28 mM, HEPES concentration 0.05 mM, pH 7.05; solution c was prepared from Man-SS-Aln and water at concentrations of 4.3. mu.M, 8.7. mu.M, 17.6. mu.M and 75.2. mu.M, respectively.

(2) And (3) measuring 50 mu L of the solution a by using a pipette, uniformly mixing the solution a with 25 mu g of pEGFP plasmid solution (0.5 mu g/mu L, double distilled water for removing ribozyme is used as a solvent), adding the double distilled water to dilute to 500 mu L, slowly dripping the diluted solution into 500 mu L of the solution b, dripping 500 mu L of the solution c, and mixing to obtain the CaP-MSSA nanoparticles. Unmodified calcium phosphate (CaP) nanoparticles were prepared in the same way, with the addition of an equal volume of double distilled water without Man-SS-Aln.

2. Characterization of characteristics of functionalized calcium phosphate nanoparticles CaP-MSSA

Preparing the CaP-MSSA nanoparticles of different modification ratios of CaP and Man-SS-Aln according to the method described in the step 1, and determining the particle size and PDI of the nanoparticles by using a nano laser particle size analyzer.

The redox response drug release characteristics of disulfide bonds in the constructed functionalized calcium phosphate nanoparticles CaP-MSSA are verified by examining the drug release behavior of a delivery system in a simulated intracellular reducing level (10 mM GSH) and an extracellular reducing level (10 mu M GSH) in vitro. 20 mu L of calcium phosphate (CaP) nanoparticle or CaP-MSSA (1 mg. mL) loaded with gene^-1) Different mimic media (pH 7.4 PBS + 10. mu. mol/L GSH, pH 7.4 PBS + 10 mmol/L GSH) were added and incubated in a 37 ℃ water bath to mimic the release of intracellular and extracellular genes.

The experimental results are as follows: in the experiment, functionalized calcium phosphate nanoparticles CaP-MSSA-1, CaP-MSSA-2, CaP-MSSA-4 and CaP-MSSA-15 with different modification ratios are prepared by changing the concentration of Man-SS-Aln. The measurement result of the nanometer laser particle size analyzer shows that the average particle size of the obtained nanometer particles is 200-300 nm, the particle size is smaller, and the dispersion is uniform (PDI < 0.3) (Table 1). The redox response characteristic evaluation experiment result shows that compared with the low-concentration GSH (10 mu mol/L) outside the cell, in a release medium (10 mmol/L) simulating the high-concentration GSH in the cell, the rapid release of the loaded gene can be realized by the CaP-MSSA drug delivery system due to the breakage of disulfide bonds, and compared with unmodified calcium phosphate (CaP) nanoparticles, the release speed and the release amount are obviously improved. The results show that the nano delivery system has better oxidation-reduction sensitivity, and can quickly release the loaded gene under the condition of high GSH concentration in cells so as to achieve efficient transfection effect.

TABLE 1 measurement results of particle size and PDI of Cap and Cap-MSSA nanoparticles

Nano particle	Average particle diameter (nm)	PDI
			CaP	238.60±4.38	0.187±0.011
CaP-MSSA-1	235.50±2.83	0.203±0.013
			CaP-MSSA-2	233.20±1.27	0.208±0.001
CaP-MSSA-4	241.35±1.20	0.214±0.012
			CaP-MSSA-15	258.70±2.83	0.207±0.020

Example 3 stability study of functionalized calcium phosphate nanoparticles Cap-MSSA

CaP and CaP-MSSA nanoparticles were prepared as described in example 2, and the particle size of the nanoparticles was measured on

days

0, 1, 3, 5, and 7 using a nano laser particle size analyzer, respectively.

The experimental results are as follows: the invention adopts a nanometer laser particle size analyzer to monitor the change of the particle size and PDI to measure the stability of the nanoparticles. As shown in fig. 2, the particle size of the conventional unmodified CaP nanoparticles rapidly increased at 24 hours. After 48 hours, the strength of the nanosuspension was so greatly reduced as to be impossible to measure due to aggregation and sedimentation of CaP nanoparticles (fig. 2A). However, when Man-SS-Aln was functionally modified, the CaP-MSSA nanoparticles remained stable within 7 days (fig. 2B, C, D and E), which indicates that the stability of CaP-MSSA nanoparticles was greatly improved compared to conventional CaP nanoparticles after Man-SS-Aln modification with different mass ratios.

Example 4 determination of Gene Loading and protective Capacity of Cap-MSSA nanoparticles

1. Cap and Cap-MSSA nanoparticle gene load capacity determination

The preparation of the CaP and CaP-MSSA nanoparticles was carried out as described in example 2, the nanoparticles were centrifuged for 2 h using a high-speed low-temperature centrifuge, and 30. mu.L of the supernatant and 30. mu.L of an equal concentration of DNA solution were subjected to agarose gel electrophoresis (electrophoresis conditions: 100V, 80 min). The end of electrophoresis was observed using a fluorescence and chemiluminescence system.

2. Determination of gene protection capability of Cap and Cap-MSSA nanoparticles

CaP and CaP-MSSA nanoparticles were prepared as described in example 2, and 60. mu.L of each nanoparticle (containing 1. mu.g plasmid DNA) was incubated with DNase I (0.14U/. mu.g DNA) for 30 min at 37 ℃. The enzymatic reaction was then stopped by the addition of 5. mu.L of EDTA solution (250 mM). All samples were mixed well with 5. mu.L of 1% SDS, and after incubation at 37 ℃ for 1 h, 30. mu.L of the sample was subjected to agarose gel electrophoresis (electrophoresis conditions: 100V, 80 min). The end of electrophoresis was observed using a fluorescence and chemiluminescence system.

The experimental results are as follows: agarose gel electrophoresis results of the nanoparticle supernatant show that no DNA band appears in each nanoparticle group at the same position of the naked DNA group band (figure 3), and show that the modified ratio of the CaP-MSSA nanoparticles has high-efficiency gene loading capacity as the traditional CaP nanoparticles. Under the condition of DNase I, naked DNA is completely degraded, and both the CaP-MSSA nanoparticles and the CaP nanoparticles can effectively protect the DNA from being degraded (figure 4), which shows that the CaP-MSSA nanoparticles with different modification ratios have the ribozyme degradation protection capability on loaded genes like the traditional CaP nanoparticles.

Example 5 evaluation of safety of Man-SS-Aln and Cap-MSSA nanoparticles

1. Evaluation of cytotoxicity of Man-SS-Aln and Cap-MSSA nanoparticles

HEK 293T cells at 2X 10⁴The concentration of each well is inoculated into a 96-well plate, the culture solution in the well is removed after being cultured for 24 h by using DMEM culture solution containing 10% serum, and 200 mu L of DMEM culture solution containing different concentrations of compound Man-SS-Aln (the final concentrations are respectively 0, 2, 20, 40, 200, 400 and 800 mu g/mL) or nanoparticles (the final concentrations of CaP are respectively 0, 2.5, 5, 10, 20 and 40 mu g/mL) is respectively added. After 24 h incubation, 20. mu.L of MTT (5 mg/mL) solution was added to each well and incubated for an additional 4 hours. After removing the above culture medium, 150. mu.L of DMSO was added to each well, and after shaking for 15 min to completely dissolve the crystals, the absorbance value was measured at 490 nm.

2. Evaluation of haemolysis of Cap and Cap-MSSA nanoparticles

1 mL of rat erythrocyte suspension was mixed with physiological saline, CaP-MSSA-1, CaP-MSSA-2, CaP-MSSA-4, CaP-MSSA-15 or Triton, respectively (final concentration of DNA was the same as that used in transfection experiment), and incubated at 37 ℃ for 1 h. The mixture was then centrifuged for 10 min (2000 rpm) and the supernatant collected and the absorbance of the supernatant was measured at 405 nm using a microplate reader.

The experimental results are as follows: MTT assay results showed that Man-SS-Aln was negligible cytotoxic even at a concentration of 800. mu.g/mL (FIG. 5A); compared with the traditional unmodified CaP nanoparticles, the CaP-MSSA nanoparticles with different modification ratios have improved biocompatibility to a great extent, and have lower cytotoxicity than CaP nanoparticles (fig. 5B). The hemolysis experiment result shows that the hemolysis rate of the CaP-MSSA nanoparticles with different modification ratios is less than 5% (figure 6), and the requirements of safe materials are met. In conclusion, the Man-SS-Aln modified CaP-MSSA nanoparticle has good safety and meets the requirement of an excellent gene delivery system.

Example 6 study of Gene transfection Activity of Cap-MSSA nanoparticles

1. Flow cytometry for determination of Gene transfection

HEK 293T cells at 1X 10⁵The concentration per well was inoculated in a 24-well plate, and after culturing with 10% serum-containing DMEM for 24 hours, the original culture was removed, and 308. mu.L of DMEM was added. 12 μ L of sterile water, aqueous DNA solution, Cap-MSSA-1, Cap-MSSA-2, Cap-MSSA-4 or Cap-MSSA-15 nanoparticle suspension (0.2 μ g DNA per well) was added in this order. After incubation for 8 h, the culture medium was removed from each well, and 1 mL of 10% serum-containing DMEM was added to each well, followed by further incubation for 40 h. After 40 h the cells were trypsinized, centrifuged for 3 min (1000 rpm), the supernatant was discarded and resuspended in 500. mu.L PBS (pH 7.4) and the samples from different groups were analyzed for green fluorescent protein expression by flow cytometry.

2. Fluorescence microscope observation of Gene transfection

HEK 293T cells at 1X 10⁵The concentration per well was inoculated in a 24-well plate, and after culturing with 10% serum-containing DMEM for 24 hours, the original culture was removed, and 308. mu.L of DMEM was added. 12 μ L of sterile water, aqueous DNA solution, Cap-MSSA-1, Cap-MSSA-2, Cap-MSSA-4 or Cap-MSSA-15 nanoparticle suspension (0.2 μ g DNA per well) was added in this order. After incubation for 8 h, the medium was removed from each well, and 1 mL of DMEM containing 10% serum was added to each wellCulturing in nutrient solution, and culturing for 40 h. After 40 h, fluorescence microscopy was performed.

The experimental results are as follows: in the experiment, a gene encoding Green Fluorescent Protein (GFP) is used as a model gene, and the transfection activity of the functionalized nanoparticles is researched by flow cytometry and a fluorescence microscope. As shown in fig. 7, both CaP nanoparticles and CaP-MSSA nanoparticles can achieve gene delivery and transfection; the transfection efficiency of the CaP-MSSA nanoparticles is related to the modification proportion of Man-SS-Aln, wherein when the modification proportion of Man-SS-Aln is 1%, the prepared CaP-MSSA-1 nanoparticles have the highest gene transfection efficiency and average fluorescence intensity, and are obviously superior to unmodified traditional CaP nanoparticles. The results of fluorescence microscopy measurements of the expression of the encoded protein (FIG. 8) are consistent with the flow cytometry measurements.

Claims

1. A preparation method of a functionalized calcium phosphate gene delivery system is characterized in that disulfide bonds with redox response characteristics are connected with alendronic acid and mannitol fragments to obtain a mannitol alendronic acid derivative containing disulfide bonds, and the obtained mannitol alendronic acid derivative containing disulfide bonds is further modified on the surface of calcium phosphate nanoparticles and carries genes to form a stable functionalized calcium phosphate gene delivery system; the preparation method specifically comprises the following steps:

Firstly, dissolving 1.26 mmol of alendronate sodium with the corresponding mass of 341.6 mg in 10 mL of distilled water at room temperature, and adjusting the pH value to 8 by using 1M NaOH aqueous solution; dissolving 1.60 mmol of SPDP with a mass of 500 mg in 10 mL of acetonitrile, adding into the reaction solution 4 times, adjusting pH to 8 with 1M NaOH aqueous solution before each addition, and taking for 2 h; after the dropwise addition of the SPDP solution is finished, stirring and reacting for a certain time at a specific temperature; distilling off acetonitrile in the reaction solution under reduced pressure, adding methanol with 4 times of volume, standing overnight at room temperature, performing suction filtration, and performing vacuum drying to obtain a white solid SPDP-Aln;

dissolving 205.58 mu mol of SPDP-Aln with the mass of 100.8 mg in 5 mL of distilled water at room temperature, adding 203.64 mu mol of Man-Cys with the mass of 58.10 mg, and stirring for reaction for 3 hours, wherein the adding mode of the Man-Cys is as follows: dividing into 3 times, and each time interval is 10 min; quickly dialyzing the reaction solution for 1 h by using 300 mL of III-grade water, and freeze-drying to obtain a white solid Man-SS-Aln;

(2) preparation of gene delivery system preparation solution: the solution a is prepared from calcium salt and water, and the concentration of the calcium salt is 2.5M; the solution b is prepared from phosphate, sodium chloride, a buffering agent and water, wherein the concentration of the phosphate is 1.5 mM, the concentration of the sodium chloride is 0.28 mM, the concentration of the buffering agent is 0.05 mM, and the pH value is 7.05; the solution c is prepared by manning alendronic acid derivative Man-SS-Aln and water, the concentration of Man-SS-Aln is 4.3 mu M,

wherein the structural formula of Man-Cys is shown as formula (1), the structural formula of SPDP-Aln is shown as formula (2), and the structural formula of Man-SS-Aln is shown as formula (3):

(3) and (3) mixing the target gene with the solution a, diluting the target gene with double distilled water to be ten times of the volume of the solution a, uniformly mixing the target gene with the solution b in the same volume, and adding a solution c in the same volume with the solution b to prepare the gene-loaded functionalized calcium phosphate gene delivery system.

2. The method for preparing a functionalized calcium phosphate gene delivery system according to claim 1, wherein: in the step (2), the calcium salt in the solution a is CaCl₂、Ca(NO₃)₂One or a combination of two in any proportion; the phosphate in the solution b is Na₃PO₄、Na₂HPO₄、NaH₂PO₄、K₃PO₄、K₂HPO₄、KH₂PO₄、(NH₄)₃PO₄、(NH₄)₂HPO₄One or a combination of two or more of them at any ratio; the buffer is one or a combination of more than two of HEPES, BES, PBS, PIPES and Tris in any proportion.

3. The method for preparing a functionalized calcium phosphate gene delivery system according to claim 1, wherein: in the step (3), the gene is any one or more of DNA, siRNA, miRNA, lncRNA, circRNA or shRNA; the ratio of the gene mass to the volume of the solution a when the gene is mixed with the solution a is 50-5000 [ mu ] g/mL.

4. Use of a functionalized calcium phosphate gene delivery system prepared according to claim 1 in gene transfection.