Preparation method and application of a bone-targeted gene delivery system

Abstract

The present invention discloses a preparation method and application of a bone-targeted gene delivery system. The present invention uses polyethyleneimine, a common carrier for gene transfection, as a skeleton material, connects alendronic acid with good bone targeting, and prepares a bone-targeted gene delivery system loaded with therapeutic gene miR-34a. The constructed bone-targeted gene delivery system has good storage stability and bone targeting, can deliver therapeutic genes to bone tumor sites in a targeted manner, enhances the tumor treatment effect, and has good clinical application prospects.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of a bone-targeted gene delivery system. The delivery system can deliver genes to bone tumor sites in a targeted manner, and is expected to reduce toxic side effects while enhancing gene therapy. This type of nano drug delivery system can be used for gene therapy of bone tumors, especially bone metastases. Background Art

[0002] Bone metastasis is the most common malignancy in the skeletal system. Unlike primary bone tumors, which develop in the bones, bone metastasis originates from the extraskeletal system and metastasizes to the bones via the blood and lymph nodes. Bone is a common site of tumor metastasis, especially breast cancer and prostate cancer, and once cancer spreads to the bones, it is usually difficult to treat.

[0003] In recent years, the incidence of bone metastasis has been on the rise. The prevention and treatment of bone metastasis has received increasing attention in the treatment of cancer patients. However, current treatments for patients with bone metastatic disease are limited in efficacy and most current therapies are primarily palliative in nature. It is necessary to develop new treatments and develop innovative therapies (gene therapy and immunotherapy, etc.) with improved bone affinity, selectivity and efficacy and their bone tumor delivery systems to significantly improve patients' quality of life and clinical efficacy.

[0004] Gene therapy is considered an effective cancer treatment method. MicroRNAs (miRNAs) can be used as important targets for oncogenes or tumor suppressors because they can regulate gene expression by pairing with mRNAs and regulate a series of biological processes including cell differentiation, proliferation, apoptosis and movement. MiRNA therapy uses exogenous genes as therapeutic agents and has the potential to inhibit oncogenes and inhibit the proliferation of refractory tumors. However, the easy degradation in biological fluids and the low cellular uptake efficiency of nucleic acids have led to gene therapy being strongly restricted by the development of gene vectors. Although traditional viral gene vectors have shown good gene transfection efficiency, their high carcinogenicity, immunogenicity and susceptibility have greatly limited their application. Therefore, in recent years, people have been committed to finding more effective non-viral gene vectors. As the "gold standard" for gene transfection, polyethyleneimine (PEI) combines strong gene compression ability with excellent endosomal escape properties. Although PEI has extremely broad application prospects in the field of gene delivery, there are still problems that need to be solved, such as low in vivo transfection efficiency and poor targeted delivery. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a bone-targeted gene delivery system. The constructed bone-targeted nano-delivery system has good storage stability and bone targeting. The nano-delivery system can deliver genes to bone tumor sites in a targeted manner, significantly improve the therapeutic effect of bone tumors, and provide new ideas for the research of anti-bone tumor drugs.

[0006] The present invention uses polyethyleneimine containing a modifiable amino group as a skeleton, connects p-carboxybenzaldehyde (CBA) through an amidation reaction to obtain an intermediate product PC containing an aldehyde group, and then connects alendronic acid (ALN) through a reductive amination reaction to obtain a bone-targeted delivery carrier material PCA. The obtained PCA is electrostatically compounded with miR-34a to obtain a PCA / miR-34a gene delivery system with bone targeting effect.

[0007] The present invention provides a method for preparing a bone-targeted gene delivery system, which specifically comprises the following steps:

[0008] (1) CBA, NHS and EDAC were dissolved in DMSO in a certain ratio in turn. After stirring at room temperature for a certain period of time, a certain amount of polyethyleneimine was dissolved in grade III water and slowly added to the above reaction solution. After stirring at room temperature for a certain period of time, the reaction solution was transferred into a dialysis bag (MWCO 7000) and dialyzed in 2000 mL grade III water for 24 hours. The water was changed every 6 hours, and white solid PC was obtained by rotary evaporation and freeze drying.

[0009] (2) A certain amount of ALN was dissolved in 2 mL of grade III water, and a certain amount of PC was added. After the reaction was allowed to react at a specific temperature for a certain period of time, the mixture was cooled to room temperature. The above reaction system was placed in an ice bath, and a certain amount of NaBH4 was added. After the reaction was stable, the mixture was placed at a specific temperature for a certain period of time. After the reaction was completed, the pH of the reaction system was adjusted with a 6 M HCl solution, and the reaction was continued with stirring for 2 hours. The pH of the reaction system was then adjusted with a 6 M NaOH solution. The reaction solution was transferred into a dialysis bag (MWCO 7000), and dialyzed against 2000 mL of grade III water for 24 hours. The water was changed every 6 hours, and white solid PCA was obtained by rotary evaporation and freeze drying.

[0010] (3) Prepare miR-34a and PCA to a certain concentration using DEPC water. Mix the two solutions in proportion according to the experimental dosage, mix them evenly with a pipette, and leave them at room temperature for 30 minutes to obtain PCA / miR-34a nanoparticles.

[0011] In the specific preparation, in step (1), the molar ratio of CBA, NHS and EDAC is 1:1:1 to 1:3:5, and the reaction time is 0.5 to 2 hours. The polyethyleneimine used is linear polyethyleneimine (LPEI) or branched polyethyleneimine (BPEI), with a molecular weight range of 600 to 50K Da, and the mass ratio of CBA to polyethyleneimine is 10:1 to 1:100.

[0012] In step (2), the mass ratio of ALN to PC is 20:1 to 1:100. After adding PC, the reaction temperature is 10°C to 100°C, and the reaction time is 2 to 12 hours. The molar ratio of NaBH4 to ALN is 10:1 to 1:5, and the reaction temperature is 0°C to 60°C after adding NaBH4, and the reaction time is 2 to 100 hours. After the reaction is completed, the pH of the reaction system is adjusted to 1 to 5 with 6M HCl solution, and the reaction time is continued to stir for 1 to 6 hours, and then the pH of the solution system is adjusted to 10 to 14 with 6M NaOH solution.

[0013] In step (3), the concentration of miR-34a is 0.1-1 mg / mL, the concentration of PCA is 0.5-2 mg / mL solution, and the mass ratio of miR-34a and PCA when mixed is 1:50-20:1.

[0014] The present invention has the following outstanding beneficial effects:

[0015] For the first time, a PCA gene delivery vector was synthesized using p-carboxybenzaldehyde (CBA) as a linker arm, and nanoparticles were obtained by loading the therapeutic gene miR-34a and characterized and evaluated for anti-tumor activity. The particle size of the gene-loaded nanoparticles constructed by the present invention is about 130nm, and the particle size and PDI did not change significantly within 14 days, indicating that the nanoparticles have good storage stability. In vivo imaging experiments after co-incubation of hydroxyapatite and mouse tibia with nanoparticles respectively proved that the PCA / miR-34a gene delivery system has good bone targeting. In vivo anti-tumor activity studies have shown that the gene delivery system has a significant therapeutic effect on bone tumors. In summary, the gene delivery system can deliver genes to bone tumor sites in a targeted manner, improve the therapeutic effect of bone tumors, and has a good prospect for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The particle size (A) and stability (B) distribution diagram of the bone-targeted gene delivery system.

[0017] Figure 2 This is the in vitro bone targeting result of the bone-targeted gene delivery system. n=3, *P<0.05, compared with the PEI / miR-34a group.

[0018] Figure 3 This is the in vivo bone targeting result of the bone-targeted gene delivery system. n=3, **P<0.01, compared with the PEI / miR-34a group.

[0019] Figure 4 The in vivo anti-tumor effect of the bone-targeted gene delivery system (A) Mouse tumor volume change curve, (B) Mouse body weight change curve, n = 5, *P<0.05, **P<0.01, compared with the Saline group, & P<0.05, compared with the PEI / miR-34a group. DETAILED DESCRIPTION

[0020] The specific implementation of the present invention is further described below by way of examples. The specific examples described below further illustrate the technical problems and technical solutions solved by the present invention. It should be understood that the following are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0021] Example 1

[0022] Preparation of bone-targeted gene delivery vector PCA

[0023] 29.6 mg CBA (0.20 mmol), 34.0 mg NHS (0.30 mmol) and 56.7 mg EDAC (0.30 mmol) were weighed and dissolved in 4 mL DMSO, and stirred at room temperature for 1 hour. 535.3 mg branched polyethyleneimine 25K Da (BPEI 25K) was weighed and dissolved in 4 mL grade III water, and slowly added to the above reaction solution, stirred at room temperature for 12 hours, and the reaction solution was transferred into a dialysis bag (MWCO 7000) and dialyzed in 2000 mL grade III water for 24 hours, with the water changed every 6 hours. After most of the water was removed by rotary evaporation, it was freeze-dried to obtain 364.6 mg white solid PC.

[0024] Weigh 53.53 mg ALN (0.20 mmol) and dissolve it in 2 mL of grade III water, add purified PC, react in a 65 ° C oil bath for 6 hours, weigh 14.9 mg NaBH4 (0.40 mmol), slowly add the above reaction solution in an ice bath, and stir at room temperature for 72 hours. After the reaction is completed, adjust the pH of the reaction system to 3 with 6M HCl solution, continue to stir and react for 2 hours, and then adjust the pH of the solution system to 13 with 6M NaOH solution. Move the reaction solution into a dialysis bag (MWCO 7000), place it in 2000 mL of grade III water for 24 hours, change the water every 6 hours, remove most of the water by rotary evaporation, and freeze-dry to obtain 561.0 mg of white solid PCA, with a yield of 61.9%.

[0025] Example 2

[0026] Preparation and characterization of bone-targeted gene delivery system

[0027] MiR-34a and PCA were prepared into solutions with concentrations of 0.5 μg / μL and 1 mg / mL using DEPC water. The two solutions were mixed in proportion according to the required experimental concentration, pipetted and mixed, and placed at room temperature for 30 minutes to obtain PCA / miR-34a nanoparticles. The loading mass ratio of PCA to miR-34a was screened by agarose gel electrophoresis. The particle size, PDI and storage stability of PCA / miR-34a were investigated using a laser particle size analyzer.

[0028] Experimental results: According to the gel electrophoresis experiment, PCA and miR-34a were screened in a mass ratio of 5:1 to prepare nanoparticles, and the particle size, PDI and storage stability were investigated. Figure 1 The results showed that the particle size of PCA / miR-34a nanoparticles was 131.1±3.1nm, and the corresponding polydispersity index (PDI) was 0.193±0.015 ( Figure 1 A). The particle size and PDI of the nanoparticles were about 120 nm and 0.250 on the 1st, 7th and 14th days, respectively. Figure 1 B), indicating that the nanoparticles have good physical storage stability.

[0029] Example 3

[0030] In vitro bone targeting experiment of bone-targeted gene delivery system

[0031] The posterior tibiae of three healthy mice were taken and divided into two groups. They were placed in 5 mL centrifuge tubes, and 1 mL of PEI / FAM-miR-34a and PCA / FAM-miR-34a nanoparticle solution with a concentration of 20 μg / mL labeled with FAM fluorescence was added. After incubation at 37°C and shaking at 100 rpm for 3 hours, the supernatant was discarded after centrifugation. After washing three times with 4 mL of PBS, the supernatant was discarded, and the surface liquid was removed with filter paper for in vivo imaging. The fluorescence intensity was recorded and quantitative analysis was performed.

[0032] Experimental results: In vivo imaging was used to observe the adsorption of nanoparticles on mouse tibia and quantify the adsorption. Figure 2 The PCA / FAM-miR-34a nanoparticle group showed a higher fluorescence intensity than the PEI / FAM-miR-34a nanoparticle group with the corresponding concentration, which has clear bone targeting.

[0033] Example 4

[0034] Study on bone targeting of bone-targeted gene delivery system in vivo

[0035] 50 μL of 2 × 10 7 The bone tumor model was established by injecting 6- to 8-week-old BALB / c female nude mice with MDA-MB-231-luc cells / mL into the right tibia bone marrow. PEI / FAM-miR-34a and PCA / FAM-miR-34a were administered to the tumor-bearing mice via the tail vein. The distribution of FAM-miR-34a was observed by IVIS at 6, 9, 12, and 24 hours, and the fluorescence intensity of the bone tumor site was quantified.

[0036] Experimental results: The distribution of bone-targeted gene delivery system in vivo determines its therapeutic effect on breast cancer bone metastasis. After tail vein injection of PEI / FAM-miR-34a and PCA / FAM-miR-34a (FAM-miR 34a = 1 mg / kg), we observed their distribution in tumor-bearing nude mice by in vivo imaging and quantified the in vivo distribution of bone tumor sites. The results showed that the distribution of bone tumors in the PCA group was significantly more than that in the PEI group, and there was a statistical difference ( Figure 3 ), with clear bone-targeted delivery properties in vivo.

[0037] Example 5

[0038] Evaluation of the anti-tumor effect of bone-targeted gene delivery system in vivo

[0039] 50 μL of 2 × 10 7cells / mL MDA-MB-231-luc cells were injected into the right tibia bone marrow cavity of 6-8 week old BALB / c female nude mice to establish a bone tumor model. On the 26th day of modeling, normal saline, PEI / miR-34a and PCA / miR-34a were given, respectively, where the dose of miR-34a was 6 mg / kg. At the predetermined time points, the weight and tumor growth of the mice were recorded every 3 days. Tumor volume (V) = length × width 2 / 2.

[0040] Experimental results: In vivo anti-tumor activity experiments showed that ( Figure 4 ), after treatment, compared with the mice in the saline group (Saline group) without drug treatment, the tumor volumes of the mice in the PEI / miR-34a group and the PCA / miR-34a group were inhibited, and the tumor volume of the mice in the PCA / miR-34a group was the smallest, with the best anti-tumor effect (e.g. Figure 4 A), the gene therapy effect is significantly better than the commercial non-viral gene vector polyethyleneimine PEI / miR-34a group. Moreover, Figure 4 B The results also showed that after administration, each group had little effect on the weight of mice, and the bone-targeted gene delivery system had low toxicity and side effects.

Claims

1. A method for preparing a bone-targeted gene delivery system, characterized in that: Using polyethyleneimine containing a modifiable amino group as the skeleton, carboxybenzaldehyde (CBA) was connected via an amidation reaction to obtain an intermediate product PC containing an aldehyde group, which was then connected to alendronic acid (ALN) via a reductive amination reaction to obtain a bone-targeted delivery carrier material PCA. The obtained PCA was electrostatically complexed with miR-34a to obtain a PCA / miR-34a gene delivery system with bone targeting effect. The specific steps include: Step (1) CBA, NHS and EDAC are dissolved in DMSO in a certain ratio in sequence, and reacted at room temperature with stirring for a certain time, then a certain amount of polyethyleneimine is dissolved in grade III water, and slowly added to the reaction solution, and then reacted at room temperature with stirring for a certain time, and the reaction solution is transferred into a dialysis bag, and dialyzed against 2000 mL grade III water for 24 hours, and the water is changed every 6 hours, and white solid PC is obtained by rotary evaporation and freeze drying, wherein the molar ratio of CBA, NHS and EDAC is 1:1:1-1:3:5, the polyethyleneimine is branched polyethyleneimine (BPEI) with a molecular weight range of 600-50K Da, and the mass ratio of CBA to polyethyleneimine is 10:1-1:100; Step (2) a certain amount of ALN is dissolved in 2 mL of grade III water, a certain amount of PC is added, the reaction is carried out at a specific temperature for a certain period of time, and then cooled to room temperature, the reaction system is placed in an ice bath, a certain amount of NaBH4 is added, and after the reaction is stable, the reaction is placed at a specific temperature for a certain period of time. After the reaction is completed, the pH of the reaction system is adjusted with a 6 M HCl solution, and the reaction is continued with stirring for 2 hours. The pH of the reaction system is then adjusted with a 6 M NaOH solution, and the reaction solution is transferred into a dialysis bag and dialyzed against 2000 mL of grade III water for 24 hours, with the water being changed every 6 hours. The white solid PCA is obtained by rotary evaporation and freeze drying, wherein the mass ratio of ALN to PC is 20:1 to 1:100, and the molar ratio of NaBH4 to ALN is 10:1 to 1:5; Step (3) miR-34a and PCA are prepared into a certain concentration with DEPC water, the two solutions are mixed in proportion according to the experimental amount, mixed by pipetting, and placed at room temperature for 30 minutes to obtain PCA / miR-34a nanoparticles, wherein the mass ratio of miR-34a and PCA when mixed is 1:50~20:

1.

2. The method for preparing the bone-targeted gene delivery system according to claim 1, characterized in that: In step (1), the reaction time is 0.5 to 2 hours.

3. The method for preparing the bone-targeted gene delivery system according to claim 1, characterized in that: In step (2), after adding PC, the reaction temperature is 10°C ~ 100°C, and the reaction time is 2~12 hours; after adding NaBH4, the reaction temperature is 0°C ~ 60°C, and the reaction time is 2~100 hours. After the reaction is completed, the pH of the reaction system is adjusted to 1~5 with 6 M HCl solution, and the reaction time is continued to be stirred for 1~6 hours, and the pH of the solution system is adjusted to 10~14 with 6 M NaOH solution.

4. The method for preparing the bone-targeted gene delivery system according to claim 1, characterized in that: In step (3), the concentration of miR-34a is 0.1-1 mg / mL, and the concentration of PCA is 0.5-2 mg / mL solution.

5. Use of the bone-targeted gene delivery system obtained by the preparation method according to any one of claims 1 to 4 in the preparation of drugs for treating bone tumor diseases.

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