CN106729754A - Lyophilized formulations of ternary gene delivery system and preparation method thereof - Google Patents

Abstract

The invention discloses a freeze-dried preparation of a ternary gene transfer system and a preparation method thereof, comprising the following steps: using 3-aminopropyltriethoxysilane to aminate and modify mesoporous silica nanoparticles to obtain MSNs-NH ₂ ; MSNs-NH ₂ is incubated with DNA and a cationic polymer carrier solution to construct a ternary gene delivery system, and the lyoprotectant is lyophilized to obtain a lyophilized preparation of the ternary gene delivery system. The lyophilized preparation of the ternary gene delivery system of the present invention has higher transfection efficiency, which can reduce the risk of toxicity in vivo application; it presents significant antiserum transfection characteristics, and its lyophilized preparation also has better transfection activity , can realize the solidification of liquid formulations, and can be stored for a long time under non-cold chain conditions, and maintain stability and transfection activity.

Description

Freeze-dried formulation of ternary gene delivery system and preparation method thereof

technical field

The invention belongs to the technical field of pharmaceutical preparations. More specifically, the invention relates to a freeze-dried preparation of a three-component gene delivery system based on mesoporous silica and organic polymers and a preparation method thereof.

Background technique

Gene therapy refers to the transfer of exogenous genes with therapeutic effects into specific cells in a certain way to achieve the purpose of treatment. Among them, how to effectively deliver the target gene to cells and exert curative effect is the key to this therapy.

Gene delivery usually requires the help of vectors, and the current gene vectors are often divided into two types: viral vectors and non-viral vectors. Although viral vectors can achieve high transfection efficiency, high immunogenicity, possible carcinogenicity and other safety issues limit the wide application of such vectors. Non-viral vectors have attracted the attention of researchers due to their low immunogenicity, high safety, and convenient preparation. This type of vectors mainly includes liposomes, cationic polymers, and inorganic nanoparticles. Among them, liposome is a relatively mature in vitro transfection carrier, but its stability is poor; cationic polymer carrier usually has a good in vitro gene transfection effect, but this type of material has obvious toxicity, and its transfection ability under serum conditions Poor, which limits the prospect of its in vivo application; Inorganic nanomaterials have good stability and are easy to modify, etc., and can be used as gene delivery carriers, but the disadvantage is that the transfection efficiency is generally low.

A good gene preparation should have high transfection efficiency, biological safety and stability, and be able to resist serum transfection. Therefore, consider the joint use of different types of vectors to achieve complementarity. The combination of inorganic materials and organic materials is one of the strategies to achieve this goal.

Among inorganic nanocarriers, mesoporous silica nanoparticles (MSNs) have unique advantages such as low cytotoxicity, good stability, easy modification and large specific surface area, pore volume and ordered pore structure. It has received extensive attention in the field of drug and gene delivery. The disadvantage is that the surface potential of nanoparticles loaded with genes will decrease, which is not conducive to the uptake of cells, and after the system enters cells, lysosomes will interfere with its transfection, causing transfection Efficiency drops.

On the other hand, the cationic polymer carrier can cause the "proton sponge effect", which can avoid the damage of lysosomes to the gene, thereby improving the transfection efficiency, but its defects such as high toxicity and weak antiserum transfection ability cannot be ignored.

In addition, the long-term stable preservation of gene preparations is of great significance for storage, transportation and convenient use. The freeze-drying method is a method that can effectively maintain the long-term stability of biologically active substances and realize the solidification of liquid preparations. This method has been widely used in the preservation of proteins and probiotics, and it has also been used in the solidification of liposomes/genes and cationic polymers/genes. However, the method of inorganic nanoparticles/genes/organic polymers There are few studies on the freeze-drying preservation of the three-component gene delivery system.

Contents of the invention

Based on this, in order to overcome the defects of the above-mentioned prior art, the present invention provides a lyophilized preparation of a three-component gene delivery system based on mesoporous silica and an organic polymer and a preparation method thereof.

In order to realize the above-mentioned purpose of the invention, the present invention adopts the following technical solutions:

A method for preparing a freeze-dried preparation of a three-element gene delivery system, comprising the following steps:

(1) Using the cationic surfactant cetyltrimethylammonium bromide (CTAB) as the template and ethyl orthosilicate as the silicon source, mesoporous silica nanoparticles MSNs were synthesized under alkaline aqueous conditions , and use the acid reflux method to remove the template agent;

(2) Use 3-aminopropyltriethoxysilane (APTES) to modify the mesoporous silica nanoparticles from which the template agent has been removed to obtain MSNs-NH ₂ , and the mesoporous silica The mass volume ratio of nanoparticles to 3-aminopropyltriethoxysilane is 1:5-15;

(3) Ultrasonic disperse MSNs-NH ₂ with deionized water to obtain a suspension, mix it with DNA solution in equal volume, incubate at room temperature for 10 to 50 minutes, add cationic polymer carrier solution and continue to incubate for 10 to 50 minutes to construct Ternary gene transfer system; the mass ratio of MSNs-NH ₂ , DNA, and cationic polymer carrier is 5-30:1:0.1-2;

(4) Mix the prepared ternary gene transfer system with a lyoprotectant with a concentration of 3% to 10% by mass, pre-freeze at -80°C, and then freeze-dry to obtain the ternary gene transfer system. Freeze-dried formulations.

In some of these embodiments, the mass ratio of MSNs-NH ₂ , DNA, and cationic polymer carrier in step (3) is 5-15:1:0.5-2.

In some embodiments, the mass ratio of MSNs-NH ₂ , DNA, and cationic polymer carrier in step (3) is 10:1:1.32.

In some of these embodiments, the cationic polymer carrier described in step (3) is one or more of polyethyleneimine PEI, polyamide-amine dendrimer PAMAM, and polylysine PLL .

In some of these embodiments, the concentration of the alkaline aqueous solution in step (1) is 0.2-1 mg/mL.

In some of these embodiments, the concentration of 3-aminopropyltriethoxysilane (APTES) in step (2) is 0.05-0.5 mg/mL.

In some embodiments, the lyoprotectant in step (4) is one or more of trehalose, sucrose, lactose, maltose, glucose, fructose, sorbitol, mannitol, and xylitol.

The present invention also provides the freeze-dried preparation of the three-element gene transfer system prepared by the above preparation method.

The present invention uses mesoporous silica nanoparticles (inorganic materials) and cationic polymer carriers (organic materials) in combination to construct a novel ternary gene transfer of mesoporous silica nanoparticles-plasmid DNA-organic polymers system, using the stability and low toxicity of mesoporous silica nanoparticles, as well as the positive charge and "proton sponge effect" of cationic polymer amines, the two play a synergistic effect, making the antiseroconversion of the new three-way gene delivery system The transfection ability, biological safety, and gene transfection efficiency are all improved, and it has a certain antiserum transfection performance, which provides the possibility to improve the in vivo transfection effect.

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

1. Under the same organic polymer consumption (lower), the freeze-dried preparation of the mesoporous silica/plasmid DNA/organic polymer ternary gene delivery system of the present invention has a higher In the transfection of 293T cells, the lyophilized preparation of the mesoporous silica/plasmid DNA/organic polymer ternary gene delivery system of the present invention is better than the transfection of the plasmid DNA/organic polymer complex The efficiency is nearly doubled, which can reduce the risk of toxicity in vivo application;

2. During the transfection process of A549 cells, the mesoporous silica/plasmid DNA/organic polymer ternary gene delivery system of the present invention exhibits similar transfection rules and significant antiserum transfection properties , its freeze-dried preparation also has good transfection activity, and can be stored stably at near normal temperature for at least 4 months, and at the same time, it still shows good resistance to serum transfection, which provides the possibility for clinical application in vivo;

3. The lyophilized preparation of the mesoporous silica/plasmid DNA/organic polymer ternary gene delivery system of the present invention prepared by the lyophilization method can realize the solidification of the liquid preparation, and can be stored for a long time under non-cold chain conditions, and Maintain stability and transfection activity to lay a good foundation for convenient transportation and long-term storage of gene drug delivery systems.

Description of drawings

Fig. 1 is the scanning electron microscope result (A) and the transmission electron microscope result (B) of the mesoporous silica prepared in embodiment 1;

Fig. 2 is the adsorption-desorption isotherm of the mesoporous silica nanoparticles of the mesoporous silica prepared in Example 1;

Fig. 3 is the Fourier transform infrared absorption spectrogram of the mesoporous silica prepared in Example 1;

Fig. 4 is the transfection efficiency of the mesoporous silica/plasmid DNA/organic polymer ternary gene delivery system in test example 1 in the presence of serum in A549 cells;

Fig. 5 is the effect of the concentration of trehalose in Test Example 2 on the transfection activity of the lyophilized preparation of the mesoporous silica/plasmid DNA/organic polymer ternary gene delivery system (n=3).

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the parts not mentioned in the present invention are applicable to the prior art. The specific examples of the present invention are given below, but the examples are only for further describing the description in detail, and do not limit the claims of the present invention.

In the following examples, unless otherwise specified, the raw materials are all commercially available.

Embodiments 1-3 Freeze-dried preparation of the three-way gene delivery system and its preparation method

The preparation method of the freeze-dried preparation of the ternary gene delivery system of Examples 1-3 comprises the following steps:

(1) Synthesis of mesoporous silica nanoparticles MSNs

a. Dissolve 1.0 g of cationic surfactant cetyltrimethylammonium bromide (CTAB, used as a template) and 0.28 g of NaOH in 480 mL of deionized water, stir and heat at 600 rpm to 80° C. to dissolve CTAB. After CTAB is completely dissolved to form a homogeneous system, add 5 mL silicon source tetraethyl orthosilicate (TEOS) drop by drop, hydrolyze for 2 hours, centrifuge to obtain a white solid, wash with a large amount of water until neutral, and then wash with ethanol for 2-3 times; to obtain mesoporous silica nanoparticles MSNs;

b. Use the acid reflux method to remove the template agent, specifically: weigh 1g of MSNs, disperse in 100mL of absolute ethanol, add 2mL of concentrated hydrochloric acid, reflux at 78°C for 24h, centrifuge to remove the supernatant, wash with ethanol, and vacuum at 60°C dry;

(2), synthesis of aminated MSNs

Weigh 0.3 g of dry MSNs from which the template agent has been removed, place them in a 100 mL round-bottomed flask, add 30 mL of toluene and 3 mL of 3-aminopropyltriethoxysilane (APTES) for amination modification, and Reflux at 110°C for 24 hours under protection, centrifuge to remove the toluene solution, wash with absolute ethanol, and dry the product in vacuum at 60°C to obtain MSNs-NH ₂ ;

MSNs-NH ₂ was scanned by electron microscope, and the results are shown in Figure 1. From the scanning electron microscope results in Figure 1, it can be seen that the nanoparticles are monodisperse spheres with a uniform size and a diameter of about 100nm. Particles with periodic light and dark patterns can be seen in the transmission electron microscope in Figure 1, in which the bright field and slightly dark field correspond to the channels and walls of the mesoporous silica nanoparticles, indicating that the particles have an ordered channel structure.

Figure 2 shows the nitrogen adsorption isotherm of mesoporous silica nanoparticles. It can be seen that the isotherm has a sharp jump, indicating that the pore size is uniform. In addition, specific information about the comparative area, pore volume, and pore diameter were obtained through BET, BJH and other formula calculations, which were specific surface area 824.17m ² /g, pore volume 1.09cm ³ /g, and pore diameter 3.05nm. It shows that the nanoparticles have high specific surface area and pore volume, which provides a theoretical basis for drug and gene loading.

Figure 3 shows the absorption spectra of mesoporous silica nanoparticles before and after amination. The two kinds of nanoparticles have similar silicon framework structures, which show similar silicon-oxygen tetrahedral framework vibration bands in the range of 400-1400 cm ^-1 , and show strong Si-O-Si bending vibrations around 470 cm ^-1 There is a symmetrical stretching vibration peak of silicon-oxygen tetrahedron at about 800cm ^{-1 , and a broad and large absorption band around 1050cm -1 ,} corresponding to the antisymmetric stretching vibration peak of Si-O-Si. Around 1600cm ^-1 there are H-OH bending vibration peaks attributed to capillary pores and surface adsorbed water, and -NH ₂ also has vibration peaks near this. The main difference between the two curves is that at 2900-3600cm ^-1 , the spectrum of MSN has a strong and broad Si-OH vibration peak and a characteristic peak of adsorbed water molecules in the interval of 3000-3600cm ^-1 , which does not appear in MSN-NH ₂ spectrum. In the MSN-NH ₂ spectrum, there is an absorption peak around 2900cm ^-1 , which is analyzed as the CH bond bending vibration peak in the -CH ₂ CH ₂ CH ₂ NH ₂ group. The above results show that the basic silicon skeleton structure of mesoporous silica nanoparticles has not changed after amination modification, while -OH has been replaced by -NH ₂ , and amination modification has been successfully realized.

(3), extract the plasmid DNA according to the kit instructions, prepare the MSNs suspension and mix it with the DNA solution in equal volumes, so that the mass ratio of MSNs to DNA is 10:1 (Example 1), 20:1 (Example 2), 30:1 (embodiment 3), mixed with a pipette, incubated at room temperature for 30min, then added a certain amount of cationic polymer carrier PEI solution and continued to incubate for 30min, wherein the mass ratio of PEI to DNA was 0.32: 1 (the corresponding nitrogen-phosphorus ratio N/P is about 2.5), construct a mesoporous silica/plasmid DNA/organic polymer ternary gene delivery system with different composition ratios for gene transfection;

(4) Mix the prepared ternary gene delivery system with 6% lyoprotectant trehalose in mass percent concentration, pre-freeze at -80°C, and then freeze-dry to obtain the lyoprotectant trehalose of the ternary gene delivery system. dry preparation.

Embodiments 4-6 Freeze-dried preparation of ternary gene delivery system and its preparation method

The preparation method of the freeze-dried preparation of the ternary gene delivery system of Examples 4-6 comprises the following steps:

(1), the synthesis of mesoporous silica nanoparticles MSNs, the steps are the same as in Example 1;

(2), the synthesis of aminated MSNs, the steps are the same as in Example 1;

(3), extract the plasmid DNA according to the kit instructions, prepare the MSNs suspension and mix it with the DNA solution in equal volumes, so that the mass ratio of MSNs to DNA is 10:1 (Example 4), 20:1 (Example 5), 30:1 (Example 6), mixed with a pipette, incubated at room temperature for 30min, then added a certain amount of cationic polymer carrier PEI solution and continued to incubate for 30min, wherein the mass ratio of PEI to DNA was 0.65: 1 (the corresponding N/P is about 5), constructing a three-way gene delivery system of mesoporous silica/plasmid DNA/organic polymer with different composition ratios for gene transfection;

(4), freeze-drying, step is with embodiment 1.

Examples 7-9 Freeze-dried preparation of ternary gene delivery system and its preparation method

The preparation method of the freeze-dried preparation of the ternary gene delivery system of Examples 7-9 comprises the following steps:

(1), the synthesis of mesoporous silica nanoparticles MSNs, the steps are the same as in Example 1;

(2), the synthesis of aminated MSNs, the steps are the same as in Example 1;

(3), extract plasmid DNA according to the kit instructions, prepare MSNs suspension and mix it with DNA solution in equal volume, so that the mass ratio of MSNs to DNA is 10:1 (Example 7), 20:1 (Example 8), 30:1 (Example 9), mixed with a pipette, incubated at room temperature for 30min, then added a certain amount of cationic polymer carrier PEI solution and continued to incubate for 30min, wherein the mass ratio of PEI to DNA was 1.3: 1 (the corresponding N/P is about 10), constructing a three-way gene delivery system of mesoporous silica/plasmid DNA/organic polymer with different composition ratios for gene transfection;

(4), freeze-drying, step is with embodiment 1.

Test Example 1 In Vitro Transfection of Mesoporous Silica/Plasmid DNA/Organic Polymer Ternary Gene Delivery System

1. Extraction of Plasmid DNA

Prepared according to kit instructions.

2. Cell culture

Human lung cancer cell A549 cells were cultured in DMEM medium containing 10% fetal bovine serum and 100U penicillin-streptomycin double antibody after cell recovery, and the culture flask was placed in an incubator at 37°C and 5% CO ₂ , after a suitable cycle (3-4 days) for passage.

3. The mesoporous silica/plasmid DNA/organic polymer ternary gene delivery system prepared in Examples 1-9 transfected A549 cells under the condition of serum

(1) A549 cells in the logarithmic phase were selected, seeded in a 24-well plate at a density of 5*10 ⁵ cells/well, added 500 μL of medium containing 10% serum to each well, and cultured at 37°C and 5% CO ₂ for 24 hours. When the confluence of the cells reaches 60%-70%, it is used for transfection.

(2) Discard the original medium, add 400 μL medium and 50 μL serum, and then add 50 μL each of the mesoporous silica/plasmid DNA/organic polymer ternary gene delivery system prepared in Examples 1 to 3, so that each well The DNA content was 1 μg, and incubated in a 37°C, 5% CO ₂ incubator for 4h. And set the corresponding negative control group and positive control group. Three replicate wells were set up for each sample.

(3) The medium was discarded, and 10% serum-containing medium was added to continue incubation for 44 hours.

(4) Discard the medium, wash the cells twice with PBS solution, digest the cells with trypsin, and centrifuge at 1500rpm for 5 minutes to collect the cells, then wash twice with PBS, carefully suck out the supernatant, and add 400 μL polysaccharide containing 0.5% Resuspend the cells in PBS buffered solution of paraformaldehyde. The number of cells expressing green fluorescent protein per 10,000 cells was measured by flow cytometry to obtain the cell transfection efficiency.

Figure 4 shows the transfection of each sample to A549 cells in the presence of serum, where "PEI2.5" represents the nitrogen-to-phosphorus ratio of PEI to DNA is 2.5, and "MSN10" represents the mass ratio of aminated MSNs to DNA is 10, and so on. It can be seen intuitively from the figure that as the amount of PEI increases, the transfection efficiency of the experimental group will also increase. In addition to Example 9, the mesoporous silica/plasmid DNA/organic polymer of Examples 1-8 Compared with plasmid DNA/organic polymer, the three-component gene delivery system has better antiserum transfection ability, and in Example 9, the increase of the amount of organic polymer PEI can make it play a dominant role in transfection, while the two The increase of the total amount of vectors (PEI and MSNs) may decrease the transfection efficiency because it is not conducive to the release of genes.

Test Example 2 Concentration Screening of Lyoprotectant

1. Cell culture: Human renal epithelial cells 293T were cultured in DMEM medium containing 10% fetal bovine serum and 100U penicillin-streptomycin double antibody after cell resuscitation, and the culture flask was placed at 37°C, 5% CO ₂ in an incubator for passage through a suitable cycle (3-4 days).

2. Prepare trehalose solutions with different concentrations of 20%, 16%, 12%, 8%, 4% and 2% (w/v) as the freeze-drying protection agent.

3. As in Example 1, prepare a mesoporous silica/plasmid DNA/organic polymer ternary gene delivery system, the mass ratio of the three is 10:1:0.65, and add trehalose solutions of different concentrations above in equal volumes , mixed evenly so that the final concentrations of trehalose were 10%, 8%, 6%, 4%, 2% and 1%, pre-frozen and freeze-dried, used for transfection of 293T cells, and the transfection efficiency was calculated.

As shown in Figure 5, when the concentration of trehalose (w/v) was 6% to 10%, the gene transfection efficiency did not show a significant difference between the groups; when the concentration dropped to 4% and below, with The reduction of trehalose concentration also significantly decreased the transfection efficiency, and there was a significant difference between the 4% group and the 6% group (P<0.05). Trehalose is used to protect the mesoporous silica/plasmid DNA/organic polymer ternary gene delivery system, and for subsequent long-term stability evaluation, etc. Therefore, the concentration of 6% trehalose is selected as the optimal concentration. The preparation powder prepared at this concentration is loose and the particles are finer. When redispersed with sterile water, the trehalose can be dissolved quickly and the solution system is uniform.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (8)

1. A preparation method of a lyophilized preparation of a ternary gene delivery system, comprising the following steps: (1) Using the cationic surfactant cetyltrimethylammonium bromide as the template and ethyl orthosilicate as the silicon source, synthesize mesoporous silica nanoparticles MSNs under alkaline aqueous conditions, and adopt Acid reflux method to remove template agent; (2), use 3-aminopropyltriethoxysilane to carry out amination modification to the mesoporous silica nanoparticles that have removed the template agent, and obtain MSNs-NH ₂ , the mesoporous silica nanoparticles and The mass volume ratio of 3-aminopropyltriethoxysilane is 1:5~15; (3) Ultrasonic disperse MSNs-NH ₂ with deionized water to obtain a suspension, mix it with DNA solution, incubate at room temperature for 10-50 minutes, add cationic polymer carrier solution and continue to incubate for 10-50 minutes to construct a ternary Gene delivery system; the mass ratio of the MSNs-NH ₂ , DNA, and cationic polymer carrier is 5-30:1:0.1-2; (4) Mix the prepared ternary gene transfer system with a lyoprotectant with a concentration of 3% to 10% by mass, pre-freeze at -80°C, and then freeze-dry to obtain the ternary gene transfer system. Freeze-dried formulations. 2. the preparation method of the freeze-dried preparation of ternary gene transfer system according to claim 1, is characterized in that, described in step (3) MSNs-NH ₂ , DNA, and the mass ratio of cationic polymer type carrier are 5～15:1:0.5～2. 3. the preparation method of the lyophilized preparation of ternary gene delivery system according to claim 2, is characterized in that, described in step (3) MSNs-NH ₂ , DNA, and the mass ratio of cationic polymer type carrier are 10:1:1.32. 4. the preparation method of the freeze-dried preparation of ternary gene transfer system according to claim 1 is characterized in that, the cationic polymer type carrier described in step (3) is polyethyleneimine PEI, polyamide-amine type One or more of dendrimer PAMAM and polylysine PLL. 5. The preparation method of the freeze-dried preparation of the three-component gene delivery system according to any one of claims 1-4, characterized in that the concentration of the alkaline aqueous solution in step (1) is 0.2-1 mg/mL. 6. according to the preparation method of the freeze-dried preparation of ternary gene delivery system described in any one of claim 1-4, it is characterized in that, the concentration of 3-aminopropyltriethoxysilane described in step (2) 0.05~0.5mg/mL. 7. according to the preparation method of the freeze-dried preparation of the ternary gene delivery system described in any one of claim 1-4, it is characterized in that, the freeze-drying protective agent described in step (4) is trehalose, sucrose, lactose, One or more of maltose, glucose, fructose, sorbitol, mannitol, and xylitol. 8. The freeze-dried preparation of the ternary gene delivery system prepared by the preparation method described in any one of claims 1-7.