WO2006015519A1 - A method of preparation of cetyltriethyl ammonium bromide-midified polybutylcyanoacrylate nanoparticles - Google Patents

Abstract

A method of preparation of cetyltriethyl ammonium bromide-­modified polybutylcyanoacrylate nanoparticles (CTAB-PBCA-NPs) consists of the following steps: dissolving Tween 80 in H2O, adding butyl α -cyanoacrylate (BCA) monomers under stirring at constant temperature, centrifuging and removing the supernatant; filtering and removing the supernatant to form the PBCA-NP colloid storing solution; diluting the PBCA-NP colloid storing solution and adding it into the cetyltriethyl ammonium bromide (CTAB) aqueous solution, stirring and removing the supernatant at room temperature and then drying to form the CTAB-­modified polybutylcyanoacrylate nanoparticles. This invention allows that the plasmid DNA is combined efficiently, and the DNA is protected from destroying and the nanoparticles have higher gene delivery efficiency in vitro. The PBCA nanoparticles gene delivery system can protect the loaded DNA from digestion by ribonuclease and sonication-­induced shearing.

Description

 Method for preparing polybutylcyanoacrylate nanoparticle

Technology climbing

 The invention relates to a method for preparing nanometer particles of liver cancer gene therapy medicament, in particular to a polybutylcyanoacrylate-modified n-butyl acrylate nanoparticle modified with hexadecanoyltriethylammonium bromide

Preparation method of (CTAB-PBCA-NP).

Background technique

 In recent years, with the maturity of gene transfer technology, gene therapy of liver cancer has become one of the research hotspots in bioscience and clinical medicine. The regulation of suicide gene-specific introduction into liver cancer cells, or the specific expression in liver cancer ft cells, in the maximum killing of liver cancer cells without damaging normal liver cells, is the key to determine its efficacy and feasibility. At present, viral vectors and non-viral vectors commonly used in gene therapy research and clinical applications have insurmountable limitations. The nanoparticle gene transporter is a non-viral gene transporter developed in recent years. It encapsulates DNA, RNA and other gene therapy in nanoparticle or adsorbs on its surface, and is specifically affected by target molecules and cell surface. Body combination is expected to achieve safe and effective targeted gene therapy. The choice of nanomaterials is the key to successful nano-based transport and treatment. The material selected must be biodegradable or easy to excrete in the body, produce no harmful degradation products, and be non-immunogenic, and do not cause immune rejection of the body.

Summary of the invention

 '' The object of the present invention is to provide a cetyltriethylammonium bromide modified polycyanoacrylate n-butyl nanoparticle having good biodegradability, biocompatibility and no immunogen' fe Preparation method of particles (CTAB-PBCA-NP).

 The preparation method of the invention discloses polyacrylonitrile n-butyl acrylate nanoparticles (PBCA-NP) by using an emulsion polymerization method, the steps of which are: 'dissolve Tween 80 in an appropriate amount of water, and add α-cyanoacrylic acid under constant temperature and stirring. Butadiene monomer, centrifuge, take the supernatant. After filtration, the supernatant was removed to prepare a PBCA-gel colloidal storage solution. The PBCA-NP colloidal solution was diluted with water, added to an aqueous solution of cetyltriethylammonium bromide (CTAB), stirred at room temperature, centrifuged to remove the supernatant, and dried to prepare CTAB-modified polycyanoacrylic acid. N-butyl fat nanoparticles.

The nanoparticle gene transport carrier prepared by using the α-cyanoacrylate n-butyl acrylate monomer as a raw material can not only effectively bind the plasmid DNA but also protect the DNA from damage, and has a high surface modification. In vitro gene transfer efficiency. Through experiments, the present invention has the following effects: 1. Adoption The emulsion polymerization method optimized the process conditions and successfully prepared poly-cyanoacrylate n-butyl acrylate nanoparticles with small particle size and uniform distribution. The surface of the nanoparticle was modified with CTAB to positively charge and reversibly bind to the gene therapy plasmid DNA, and assembled into a PBCA nanoparticle gene transport system, and its safe use concentration for in vitro gene transfection was determined to be low at lOOng/μ. 1. 2, PBCA nanoparticle gene transport system, can protect the DNA carried away from nuclease degradation and ultrasonic shear damage. 3. The green fluorescent reporter gene was used to confirm that the PBCA nanoparticle gene transport system could successfully transport foreign DNA into the cell, and the transfection efficiency reached 47%.

DRAWINGS

 Figure 1 is a comparison of particle size analysis results of PBCA-P prepared under nine reaction conditions; Figure 2 is a particle size analysis result of PBCA-NP prepared under 6# reaction conditions;

Figure 3 shows the size and surface morphology of PBCA-P prepared under 6 ^# reaction conditions by atomic force microscopy;

 Figure 4 shows the cytotoxic effects of PBCA-P (A) and CTAB-PBCA-P (B) by MTT assay;

 Figure 5 shows the binding of CTAB-PBCA-NP to DNA under different pH conditions; Figure 6 shows the gel retardation experiment of CTAB-PBCA-NP binding to DNA;

 Figure 7 shows the evaluation of the binding efficiency of CTAB-PBCA-NP and DNA;

 Figure 8 shows the DNA electrophoresis pattern of CTAB-PBCA-NP/DNA after Dnasel and serum digestion. Figure 9 shows the CT electrophoresis pattern of CTAB-; PBCA-NP/DNA after ultrasonic shearing; Figure 10 shows EGFP-N1 Transfection in CTAB-PBCA-P (A, C) and SuperFect

Transfection Reagent transfected (B, D) in HepG2 (A, B) and 3T3 (C, D) cells (200x);

 Figure 11 shows the expression of AFP in HepG2, HeLa and 3T3 cells by indirect immunofluorescence (400x);

 Figure 12 shows the expression of AFP in HepG2, HeLa and 3T3 cells by Western blot; Figure 13 shows the expression of TK in each cell line transfected with different plasmids by RT-PCR; Figure 14 shows the transfection of pAFP in HepG2 cells by MTT assay. — Sensitivity to different concentrations of GCV after TK.

Figure 15 is a trypan blue exclusion test demonstrating GCV time-dependent killing of AFP-positive HepG2 cells; Figure 16 is a graph showing the effect of recombinant TK expression plasmid on the proliferation of AFP-positive HepG2 and APP-negative HeLa and 3T3 cells;

 Figure 17 shows the killing effect of GCV on different proportions of TK-positive HepG2 mixed cells; Figure 18 shows the apoptosis of cells expressing GC-induced TK gene (400x)

detailed description

 The invention will be specifically described below in conjunction with the examples.

 Dissolve Tween 80 in an appropriate amount of double distilled water and adjust the pH of the solution to below 2.0 with 0. IN HCL. Under the action of a constant temperature magnetic stirrer, the n-cyanoacrylate n-butyl monomer was slowly added to the solution to make the BCA monomer concentration 0.1-20%, and stirring was continued for 5 hours at room temperature. After centrifugation at 5000 rpm for 15 min at room temperature, the supernatant was taken and filtered through a membrane having a diameter of 0.22 μm, and the product was PBCA-NP. After centrifugation at 40000 rpm for 1 hour, the supernatant was removed, washed twice with double distilled water, dried, and weighed, and the precipitate was resuspended in an appropriate amount of double distilled water to prepare a PBCA-NP colloidal stock solution. .

 The octadecyl group at a concentration of 0.25 ml is added to a concentration of 0.23 ml of a cetyl group. The aqueous solution of triethylammonium bromide (CTA3⁄4) was stirred at room temperature for 1 hour. After centrifugation at 40000 rpm for 30 min, the supernatant was removed, washed with double distilled water, dried and weighed to obtain CTAB-modified polybutylcyanoacrylate nanoparticles.

 The physical properties, chemical properties, and experimental data of the present invention:

1. Preparation of CTAB modified PBCA-F and results of transfection

1. Preparation of polybutylcyanoacrylate nanoparticle (PBCA-NP) and observation of morphological particle size PBCA-NP prepared by emulsion polymerization under the nine reaction conditions in orthogonal experimental design, using MasterSizer2000 laser particle size The analyzer performs particle size and distribution detection. The results show that the 6 ^# reaction conditions are the optimal conditions for the preparation of PBCA-NP, that is, the pH of the reaction system is 2.5, the concentration of BCA monomer is 1%, and the concentration of Tween 80 is 0.6%. The average particle diameter value was the smallest, 106 nm, the maximum particle diameter was 170 nm, the minimum particle diameter was 72 nm, and the specific surface area of the nanoparticles was 57.2872 m ² /g. The particle size distribution is normally distributed and the distribution is the most uniform (Fig. 1, 2). The surface morphology of PBCA-NP was observed using an AJ-III atomic force microscope. The nanoparticles are spherical, the surface is smooth and complete, well dispersed, and there is no adhesion agglomeration. See Figure 3.

See Figure 1 for comparison of particle size analysis results of PBCA-NP prepared under nine reaction conditions. See Figure 2 for particle size analysis results of PBCA-NP prepared under 6 ^# reaction conditions. The particle size distribution of PBCA-NP was normally distributed, with an average particle size of 106 nm, a maximum particle size of 170 nm, a minimum particle size of 72 nm, and a particle specific surface area of 57.2872 m ² /g.

Referring to Figure 3, the size and surface morphology of PBCA-NP prepared under 6 ^# reaction conditions were observed by the original force microscope. The nanoparticles were spherical, the surface was smooth and intact, well dispersed, and there was no adhesion agglomeration.

2. Cytotoxicity analysis of PBCA-NP and CTAB modified PBCA-NP transport system

 At a certain dose, the cytotoxicity exhibited by non-viral vectors such as liposomes is an important problem limiting gene transfection in vitro. The charge and surface modification molecules on the surface of the nanoparticles determine their biological characteristics. Therefore, the cytotoxicity of different concentrations of PBCA-NP and CTAB-modified PBCA-NP on HepG2 cells and 3T3 cells was determined by MTT assay to determine the feasibility and availability of gene transfection.

 As shown in Figure 4, PBCA-NP (A) and CTAB-modified PBCA-NP (B) showed no significant cytotoxicity to HepG2 cells at concentrations below 100 ng/μΐ, below 200 ng/μΐ There was no significant cytotoxicity to 3T3 cells at the concentration. When the concentration of nanoparticles continued to increase, the cytotoxic effect increased sharply (P<0.05), resulting in a sharp decrease in cell viability. There was no significant difference in the cytotoxic effects of PBCA-NP and CTAB-PBCA-NP on cells.

See Figure 4. · MTT assay for PBCA-NP (A) and CTAB-PBCA-NP (B) cytotoxicity, PBCA-NP (A) and CTAB-PBCA-NP (B) at a concentration of 100 ng / When it is below μΙ, it has no obvious cytotoxicity to HepG2 cells, and it does not show obvious cytotoxicity to 3Τ3 cells at a concentration of 200 η _§ /μ1 or less. When the concentration of nanoparticles continues to increase, the cytotoxic effect is sharply increased, resulting in a sharp drop in cell viability. *Ρ<0.05, Ν=6.

 3, DNA binding efficiency analysis

 PBCA-NP is a surface-negatively charged high molecular polymer that is inherently difficult to bind to negatively charged DNA molecules. However, since PBCA nanoparticles have a small particle size, a large specific surface area, and biological affinity, it is easy to bind other molecules on the surface. Therefore, we modified the PBCA-NP with the cationic surfactant CTAB to give a positive charge on its surface, thereby obtaining the ability to bind DNA. However, the main factor affecting the surface electrical properties of CTAB is the pH condition of the solution. Therefore, we chose different pH solution states, mixed CTAB-PBCA-NP with plasmid DNA, placed it at room temperature for more than 1 hour, and took the product for agarose gel electrophoresis.

See Figure 5 for the binding of CTAB-PBCA-NP to DNA under different pH conditions: 1. Control plasmid DNA, 2. DNA binding of nanoparticles at pH=5, 3. Nanoparticles at pH=7 DNA binding, 4. DNA binding of nanoparticles at pH=9. Electrophoresis showed that at pH=7, the DNA migration was slow, the DNA bound to the nanoparticles was not vented, and the amount of free DNA was the least. At pH=5, 9, the DNA binding ability of the nanoparticles is relatively weak.

 The results showed that CTAB-modified PBCA-P could bind to DNA at different pH values, but the binding ability was different. Under neutral (pH=7) conditions, the DNA binding ability is the strongest, and the amount of DNA in the electrophoresis shows that the migration speed is slow or stays in the spotted well; while under acidic and alkaline conditions, the DNA binding ability is relatively high. weak.

 The binding efficiency of CTAB-modified PBCA-NP to DNA can be determined indirectly by DNA gel retardation assay and UV absorbance after DNA precipitation. Based on the above experimental results, we chose pH=7 as the environmental pH condition for the binding of nanoparticles to DNA. CTAB-PBCA-P and plasmid DNA were mixed at different mass ratios, and allowed to stand at room temperature for more than 1 h, and gel electrophoresis was observed to observe the binding (Fig. 6). The same reaction system was taken, centrifuged at 20,000 rpm/min for 15 min, and the supernatant was taken for the absorbance at 260 nm to calculate the DNA concentration (Fig. 7). After high-speed centrifugation, the CTAB-modified PBCA-KP-bound DNA was precipitated, and the unbound DNA was present in the supernatant. The supernatant was taken to measure the absorbance at 260 nm, and the unbound DNA in the system was detected. The DNA binding efficiency was calculated according to the formula [total DNA - unbound DNA / total DNA x 100%], and the mass ratio at which the nanoparticles were most efficiently combined with DNA was determined.

 See Figure 6 for the gel retardation experiment of CTAB-PBCA-NP binding to DNA. Lanes 1-7 represent the mass ratio of nanoparticles to DNA: 1: 1, 5: 1, 10: 1, 15: 1, 20 : 1, 30: 1, 50: 1 when the DNA binding situation. As the ratio increases, the proportion of bound DNA increases. At a mass ratio of 10:1, the DNA is almost completely bound to the nanoparticles.

 Referring to Figure 7, the binding efficiency of CTAB-PBCA-NP to DNA was evaluated. The results showed that the amount of DNA bound to CTAB-PBCA-NP gradually increased as the ratio of the two increased. When the mass ratio of nanoparticles to DNA is 10:1, the binding efficiency is 93%, and then the binding rate decreases slowly.

 The above results show that the ratio of nanoparticles to DNA is different, and the binding efficiency is also different. As the ratio increases, the amount of DNA bound increases. When the ratio of nanoparticles to DNA was 10:1, DNA was almost completely bound to CTAB-PBCA-NP, and the binding efficiency was 93% by UV absorption. As the ratio continues to increase, the efficiency of the combination slowly decreases. Therefore, we used a nanoparticle-DNA of 10: 1 for subsequent studies.

From the above results, DNA is bound to PBCA-NP by cationic surfactant CTAB. s surface. With the change of pH value in the reaction system, the binding ability of DNA also shows different, suggesting that the number of charges on the surface of nanoparticles is different under different acid-base conditions. Electrostatic attraction is one of the mechanisms of nanoparticle binding to DNA.

 4. Protection of plasmid DNA by CTAB-modified PBCA-NP

 Nucleases and intracellular lysosomes in the interstitial tissues of the body have degradation of foreign DNA and are one of the obstacles that hinder the expression of foreign genes. A good gene transfer vector should have the function of protecting exogenous DNA from enzymatic digestion in the body. The protective effect of the nanoparticle gene transport vector on the DNA carried, we detected by DNasel digestion, serum digestion and ultrasonic destruction, and the product was analyzed by agarose gel electrophoresis. '

 See Figure 8 CT electrophoresis pattern of CTAB-PBCA-NP/DNA after Dnasel and serum digestion: 1. Naked plasmid DNA was digested with DNasel at 37 °C for 30 min; 2. Untreated naked plasmid DNA; 3.5. CTAB-PBCA -NP/DNA via DNasel 37. C digested the DNA extracted from the complex after 30 min, lh, 1.5 h; 6. Naked plasmid DNA was digested with fetal bovine serum at 37 ° C after 81 °; 7. 6TAB-PBCA-P/DNA transfeginal bovine serum 37. Figure 8 shows the DNA electrophoresis pattern of CTAB-PBCA-NP/DNA after ultrasonic shearing after digestion for 8 hours: 1. Untreated naked plasmid DNA; 2. Naked plasmid DNA by ultrasound After 15s of action; 3. DNA extracted from the complex after CTAB-PBCA-NP/DNA was sonicated for 15 s.

 From the above results, the CTAB-modified PBCA-NP can impart a resistance to nuclease degradation to the plasmid DNA carried. The DNA in CTAB-PBCA-NP/DNA remained structurally intact after digestion for several hours at 37 ° C, DNasel and serum, while naked DNA was completely degraded under the same conditions for 30 min. At the same time, CTAB-modified PBCA-NP can make the plasmid DNA contained in the band have the ability to resist ultrasonic shearing. The DNA in CTAB-PBCA-NP/DNA still maintains structural integrity after 15 s of ultrasound at 230 W, while naked DNA has degraded under the same conditions. It can be seen that the CTAB-modified PBCA nanoparticle gene transport system provides complete protection against foreign plasmid DNA, keeping the DNA structure intact, thus ensuring its normal expression in vivo.

5. Analysis of in vitro gene transfer efficiency of CTAB-modified PBCA nanoparticle gene transporter

The CTAB-modified PBCA-NP has a positive charge on its surface and can bind to plasmid DNA and induce cells to interact with it by interacting with the negatively charged glycoproteins and phospholipids on the cell membrane by the cations carried on the surface of the nanoparticle-DNA complex. Endocytosis, thus entering the cytoplasm. This experiment used a reporter gene system to detect the in vitro gene transfer efficiency of CTAB-PBCA-NP. The CTAB-PBCA-NP transport reporter plasmid pEGFP-N1 was introduced into HepG2 cells and 3T3 cells, and the transfection efficiency was calculated by observing the transfected cells with green fluorescence under a fluorescence microscope. Liposomal transfection efficiency was used as a control. As a result (Fig. 10), the CTAB-PBCA-NP system efficiently transported foreign plasmid DNA into HepG2 and 3T3 cells to express EGFP protein (Fig. 10 A, C). Its transport efficiency can reach about 47%, slightly higher than the transport efficiency of SuperFect Transfection Reagent transfection reagent (42%, Figure 10 B, D) o

 See Figure 10 for expression of EGFP-N1 in HepG2 (A, B) and 3T3 (C, D) cells transfected with CTAB-PBCA-NP (A, C) and SuperFect Transfection Reagent (B, D) ( 200 x): A is the expression of EGFP-N1 after transfection of HepG2 cells with CTAB-PBCA-KP; B is the expression of EGFP-N1 after transfection of HepG2 cells with SuperFect Transfection Reagent; C is transfected with CTT-PBCA-NPs after transfection of 3T3 cells Expression of EGFP-N1; D is the expression of EGFP-N1 after transfection of 3T3 cells by SuperFect Transfection Reagent.

 Polybutylcyanoacrylate (PBCA) nanoparticles act as a gene transport carrier because of its good biodegradability, biocompatibility, and non-immunogenicity. Since the surface of PBCA nanoparticles is negatively charged, we have surface modified with a cationic surfactant CTAB to give a positive charge on the surface to obtain the ability to bind negatively charged DNA and some colloidal properties. At the same time, experiments have confirmed that PBCA nanoparticles/DNA complexes protect the DNA carried from nuclease degradation and sonication.

This study shows that PBCA nanoparticles and CTAB-modified PBCA nanoparticles have no obvious cytotoxicity to hepatoma cells (HepG2) and normal fibroblasts (3T3) at appropriate concentrations, and only show high concentrations. Cytotoxic effects. This study also confirmed that in vitro, the nanoparticles can effectively transport EGFP reporter gene expression plasmid into HepG2 and 3T3 cells, and its transport efficiency is 47%, which is stronger than the transfection efficiency of liposomes under the same conditions, which is compared with previous scholars. The results of the report are consistent ^[31] . At the transfection concentration in this experiment, PBCA nanoparticles are non-cytotoxic and have good transfection efficiency, and can be used as effective gene transfection reagents. In conclusion, the CTAB-modified PBCA nanoparticle carrier system enhances the uptake of its foreign genes by cells, provides protection against carrying genes, and has good safety.

Although the PBCA nanoparticle gene transport system has good biological properties and high transfection efficiency, its preparation process needs to be further optimized to reduce its particle size and enhance DNA loading. The amount of the band, and explore appropriate transfection methods to further improve its gene transfer efficiency. In vitro study of PBCA nanoparticle gene transporter-mediated pAFP-TK/GCV system for gene therapy of AFP-positive liver cancer

 1. Indirect immunofluorescence confirmed that AFP expression was positive in HepG2 cells, while AFP expression was negative in HeLa and 3T3 cells.

 HepG2 is a human hepatocellular carcinoma cell, and the data provided by ATCC is AFP positive. The HeLa-derived cells are poorly differentiated squamous cell carcinoma of the human cervix, and the data provided by ATCC are negative for AFP. 3T3 is a fibroblast derived from mouse embryos, and the information provided by ATCC is AFP negative. To confirm the expression status of AFP in these three cells, we used indirect immunofluorescence analysis of murine-derived anti-AFP monoclonal antibodies. '

 The results showed that HepG2 cells showed diffuse green fluorescence in the cytoplasm, while HeLa and 3T3 cells showed no green fluorescence, confirming that HepG2 cells were positive for AFP expression (Fig. 11A), while HeLa and 3T3 cells were negative for AFP expression (Fig. 11) 11 B, C).

 See Figure 11 Indirect immunofluorescence detection of AFP expression in HepG2, HeLa and 3T3 cells (400x): A is an indirect immunofluorescence analysis of HepG2 cells using a murine-derived anti-AFP monoclonal antibody. The results show that APP is expressed in the cytoplasm of cells. B was indirect immunofluorescence analysis of HeLa cells using murine-derived anti-APP monoclonal antibody, and the results showed that AFP expression was negative. Indirect immunofluorescence analysis was performed on CT 3T3 cells using murine-derived anti-AFP monoclonal antibody, and the results showed negative expression of AFP.

 2. Western blot confirmed that HepG2 cells were positive for AFP expression, and HeLa and 3T3 cells were negative for AFP expression.

 Based on the results of indirect immunofluorescence, it was confirmed that HepG2 was positive for AFP cytoplasm and HeLa and 3T3 cells were negative for AFP expression. We further used Western blot to detect the expression of AFP protein in these three cells, thus confirming the expression of AFP protein in HepG2 cells. AFP expression was positive, and AFP expression was negative in HeLa and 3T3 cells.

 See Figure 12 Western blot analysis of AFP expression in HepG2, HeLa and 3T3 cells - no AFP protein bands were detected in HeLa and 3T3 cells, and 70KD AFP protein bands were detected in HepG2 cells. In this experiment, Tubulin protein was used as a control for each lane loading, showing no significant difference in the amount of each lane. This result is a representative figure in multiple experiments.

Further confirmation of AFP in HepG2 cells by Western blotting at protein expression levels The expression was positive, while HeLa and 3T3 cells were negative for AFP expression. Therefore, we can select these three cells as experimental materials and introduce various plasmids for in vitro studies of AFP-positive tumor gene therapy.

 3. RT-PCR detection of TK expression in each cell line after transfection

The CTB-modified PBCA nanoparticles-mediated expression of each TK gene expression plasmid was transfected into HepG2, HeLa and 3T3 cells, and the expression of mRNA in each cell line was identified by RT-PCR. Using β-actin as an internal control, the results are shown in Figure 3-3. In the control group transfected with the blank plasmid pcDNA3.1, no expression of the TK gene was observed. The TK genes were expressed in different degrees in the cells transfected with p3.1-TK and pAFP-TK plasmids. ·

 See Figure 13 for RT-PCR detection of TK expression in each cell line transfected with different plasmids: 1. DL2000 DNA marker; 2-4. HepG2 cells were transfected with pAFP-TK, p3.1-TK: Expression of purine in cells after pcDNA3.1 plasmid; 5-7. Expression of TK in cells after transfection of pAFP-TK, p3.1-TK and pcDNA3.1 plasmids in HeLa cells; 8-10. TK expression in cells after transfection of pAFP-TK, p3.1-TK, pcDNA3.1 plasmid; β-actin was an internal control.

 As can be seen from the figure, CTAB-modified PBCA nanoparticles can effectively transfect foreign genes into cells and be expressed normally in cells. In AFP-positive HepG2 cells, the expression level of TK after transfection of pAFP-TK plasmid was significantly stronger than that of p3.1-TK plasmid after transfection. In AFP-negative HeLa and 3T3 cells, the expression level of TK after transfection of pAFP-TK plasmid was weaker than that of P3.1-TK plasmid after transfection. It is suggested that in AFP-positive cells, the expression of TK-based guanidine regulated by AFP enhancer is much stronger than that of its basic promoter CMV.

 4, MTT method to detect the sensitivity of cells to GCV

 MTT method (tetramethylazozolium salt microenzyme reaction colorimetric method) The principle is: mitochondrial succinate dehydrogenase in living cells can catalyze the formation of blue hyperthyroidism in MTT, and the amount of formation is positive with the number of living cells and functional status. Related. After the cells were taken up to catalyze MTT, DMSO was added to fully dissolve the dark blue crystals, and the OD value was read at 570 nm on a microplate reader, and the value reflected the overall functional state of the cells. This topic applies this method as an indirect indicator of cell death.

 In this experiment, we used this method to detect the sensitivity of AFP-positive cells to GCV after transfection of pAFP-TK plasmid.

Referring to Figure 14, MTT assay was used to detect the sensitivity of HepG2 cells transfected with pAFP-TK to different concentrations of GCV. The cells were seeded in 96-well plates, and pAFP-TK plasmids were transfected with different concentrations of GCV. After 7 days of culture, MTT was added, culture was continued for 4 hours, DMSO was added, and the absorbance was measured at 570 nm by a microplate reader, and the ratio of the absorbance to the absorbance of the control group was plotted. *P<0.05, n=3.

 The experimental results showed that the use of lO g/ml GCV concentration had little effect on cell viability, and about 50% (PO.05) of AFP-positive cells died at 5 (^g/ml GCV concentration; using 100 g/ml GCV) At the concentration, about 80% (P<0.05) of AFP-positive cells died; when the GCV concentration reached 20 (^g/ml, only a few AFP-positive cells survived (PO.05); when the GCV concentration continued to rise to 500 μ /ιη1 The killing effect of cells was not significantly increased. It is suggested that GCV dose-dependently kills AFP-positive hepatoma cells in the pAFP-TK/GCV system.

 5, GCV time-dependent killing AFP positive liver cancer cells

 We used the trypan blue exclusion test to directly reflect the killing effect of the pAFP-TK/GCV system on AFP-positive hepatic cancer cells.

 See Figure 15. Trypan blue exclusion assay confirmed GCV time-dependent killing of AFP-positive HepG2 cells. HepG2 cells transfected with pAFP-TK plasmid were seeded in 96-well plates, and 12 hours later, GCV with 'lOC^g/ml was added. Treatment, after the trypan blue staining was performed on the indicated days, the percentage of blue-stained cells was counted under a microscope. *P<0.05, n=3.

 It can be seen from the figure that GCV has a significant killing effect on AFP-positive hepatoma cells transfected with TK gene (Ρ<0.05). On the second day after the addition of GCV (P<0.05), HepG2 cells showed obvious killing effect, and the killing effect on cells on the eighth day was about 80% (P<0.05).

 6. MTT results confirmed that pAFP-TK expression plasmid specifically inhibited the proliferation of AFP-positive hepatoma cells HepG2, but inhibited AFP-negative cells.

 The MTT method is an effective method for determining the state of cell proliferation. The above experiments confirmed that the pAFP-TK expression plasmid can kill AFP-positive liver cancer cells in a time- and dose-dependent manner. The AFP enhancer acts as an enhancer in AFP-positive cells and as a suppressor in AFP-negative cells. Therefore, we used AFP-negative HeLa, 3T3 cells and AFP-positive HepG2 cells as experimental materials, and further confirmed the specific killing effect of the suicide gene therapy system on AFP-positive hepatoma cells by MTT assay.

See Figure 16 for the effect of recombinant TK expression plasmid on the proliferation of AFP-positive HepG2 cells and AFP-negative HeLa and 3T3 cells. The cells of each group were transfected into the 96-well plate, and the final concentration of GCV was ΙΟΟμ^πύ. After sputum, add sputum, continue to culture for 4 h, add DMSO, measure the absorbance at 570 nm on the microplate reader, and plot the absolute value of absorbance. With pEGFP-Nl green The fluorescent expression plasmid was used as a control for transfection efficiency. The results confirmed that the transfection efficiency of each group was about 47%, and there was no significant difference between the groups. *p<0.05, n=3.

 As a result, it was found that the transfection of pcDNA3.1 plasmid and GCV treatment of each group had no significant effect on the cell MTT assay. In AFP positive cells HepG2, transfection of p3.1-TK and pAFP-TK plasmid plus GCV treatment significantly inhibited cell viability, and p3.1-TK plasmid transfection group inhibited cell viability by about 60% (P <0.05), and the pAFP-TK plasmid transfection group inhibited cell viability by 80% (P<0.05). In AFP-negative HeLa cells and 3T3 cells, the inhibition of cell viability by pAFP-TK plasmid transfection and GCV treatment was significantly weaker than that of p3.1-TK plasmid transfectants (P<0.05). Thus, the pAPP-TK/GCV system inhibited cell proliferation significantly more strongly than the p3.1-TK/GCV system. Therefore, the pAFP-TK/GCV treatment system was confirmed to be a highly efficient and highly specific gene therapy method for killing AFP-positive tumor cells.

 7, bystander effect

 To investigate whether the pAFP-TK/GCV system has a bystander effect on cell killing, we used HepG2 cells as a material and tested by MTT assay. The results showed that when the proportion of cells transfected with TK was 40% (that is, when the proportion of TK positive cells was about 20%), the percentage of cell survival was about 40%; when the proportion of cells transfected with TK was 80% (ie, TK was positive). When the cell ratio is 40%, the cell survival rate is less than 20%. It is suggested that the pAPP-TK/GCV system has a significant bystander effect on cell killing.

 See Figure 17 for the killing effect of GCV on HepG2 mixed cells with different proportions of TK positive. The cells were mixed in 96-well plates according to the ratio shown in the figure. The final concentration of GCV was ΙΟΟμ^πιΙ, and after 7 days of culture, 'add ΜΤΤ, The culture was continued for 4 hours, DMSO was added, and the absorbance was measured at 570 nm by a microplate reader, and the absorbance was compared with the absorbance ratio of the control group. *P<0.05, n=3.

 8. GCV can induce apoptosis in cells expressing K gene.

 It has been reported in the literature that in vitro cell killing of HSV-TK/GCV system has apoptosis, so we used Hochest 33258 staining to determine whether the killing effect induced by pAFP-TK/GCV system is apoptosis. The stained cells were observed under a fluorescence microscope. For example, if the nucleus is homogenized, the fluorescence is normal living cells; if nuclear condensation, nuclear fragmentation, and nuclear lysis occur, the cells are confirmed to have apoptosis. HepG2 cells transfected with pAFP-TK plasmid were stained with H. coli and subjected to GCV treatment. The results are shown in Figure 18.

See Figure 18. GCV induces apoptosis in cells expressing the TK gene (400x): A is transgenic Fluorescence staining of HepG2 cells treated with GCV after staining, nuclear fluorescence was evenly distributed and distributed as normal cell images; B was fluorescent staining of HepG2 cells treated with GCV after transfection, and obvious nuclear condensation was observed. It was confirmed that the cells had undergone apoptosis.

 From the results, it was found that the nuclei of the control group were uniformly stained, and the appearance of nuclear condensation was observed after treatment with GCV. Therefore, it was confirmed that the cells expressing HSV-TK gene were apoptotic after GCV treatment, thus confirming that the apoptosis of pAFP-TK/GCV system induced cell apoptosis is one of the mechanisms of cell killing. Discussion

 The HSV-TK/GCV system is progressing rapidly in the study of suicide gene therapy for liver cancer. Since the introduction of hepatocarcinoma gene therapy for the first time in 1995 and the ability to induce the killing effect of GCV on hepatocarcinoma cells in a dose-dependent manner, the anti-hepatocarcinoma efficacy of HSV-TK/GCV system has been continuously confirmed in vivo and in vitro. And have studied the effect of the choice of the route of administration in vivo on the effect of treatment. However, the non-specific entry of the HSV-TK gene into normal hepatocytes during transfection can lead to severe liver function damage. Therefore, various gene transfer vectors and specific transcriptional regulatory elements have been used to improve transfection efficiency and targeting. In-depth development.

 Studies have shown that lentiviral vector-mediated HSV-TK/EGFP fusion gene expression regulated by hepatoma cell-specific AFP promoter can be transfected into liver cancer cells, non-liver-derived human tumor cells and primary normal liver cells. High expression of fusion gene in cells, low expression in non-hepatic-derived human tumor cells, but no expression in normal liver cells. The research team further used HIV-derived lentiviral vector to mediate HSV-TK gene transfection, and found that it can effectively transduce the autocidal gene into liver cancer cells in vitro and in vivo, thereby exerting a killing effect under the action of GCV. In the in vivo animal experiment, no TK gene entered normal liver cells under any administration route, and no obvious liver function damage occurred. Another scholar used the Japanese hemagglutinating virus (HVJ) liposome as a gene transport vector to carry out CMV promoter/enhancer-regulated HSV-TK/GCV system (pcDNA3 -TK expression plasmid) for the preclinical study of liver cancer. The results showed that the transduction efficiency of HVJ liposome in vitro was 20%, and the effective concentration of GCV was 100 mg/ml, which resulted in complete killing of tumor cells. In vivo, the transduction efficiency was 19.7%, GCV treatment. After significantly inhibiting the growth of the tumor.

In the study, we used CTAB-modified polymer-degrading biomaterial PBCA nanoparticles as a gene transport vector, and the HSV-TK gene expression plasmid pAFP-TK regulated by AFP enhancer and CMV promoter was used as a therapeutic gene and treated with GCV. Gene therapy studies for AFP-positive liver cancer. We use AFP-positive HepG2 cells and AFP-negative HeLa and 3T3 cells. The recombinant plasmid pAFP-TK was transfected into each group of experimental cells. The results showed that the transfection efficiency of CTAB-modified PBCA nanoparticles was about 47%, and the lipofection efficiency was higher than that under the same conditions. RT-PCR was used to detect the expression of exogenous TK in three cell lines. It was found that the expression of TK was down-regulated by AFP-negative HeLa and 3T3 cells transfected with pAFP-TK plasmid at the expression level of mR A. The control plasmid p3.1-TK was significantly reduced; whereas in the AFP-positive hepatoma cell HepG2, the AFP enhancer-regulated TK gene expression was significantly enhanced compared with the AFP-free enhancer. This is consistent with previous literature reports.

After confirming that CTAB-modified PBCA nanoparticles can efficiently transfect pAFP-TK plasmid into cells and express them in AFP-positive cells, we further use the trypan blue exclusion test and MTT method to find that AFP is positive. The sensitivity of cells to GCV after transfection of pAFP-TK was greatly enhanced, and cell viability was significantly reduced. The experiment also found that the killing effect of pAFP-TK/GCV system on tumor cells was GCV dose- and time-dependent, and the killing effect on cells was lower than that of GCV at 100μ _§ /ηι1 concentration; at 100μ _§ /ιη1 to 200μ Under the action of _§ /πύ concentration of GCV, the killing effect was significant, and only a few tumor cells survived; while when the GCV concentration continued to increase, the cells basically died.

 A large body of literature has confirmed that the HSV-TK/GCV system can induce apoptosis of tumor cells. Apoptosis was also found in HSV-TK/GCV treatment studies of liver cancer, and this apoptosis was associated with up-regulation of Ρ53 gene and up-regulation of Fas/FasL expression in cells. In this experiment, it was confirmed that the AFP-positive cells transfected with pAFP-TK plasmid were stained with Hochest 33258 after GCV treatment, and typical apoptosis occurred. Thus, it was confirmed that apoptosis is an important mechanism for killing AFP-positive liver cancer cells by the pAFP-TK/GCV system.

At present, one of the keys to limiting the widespread use of gene therapy is the low efficiency of gene transfer, which cannot be introduced into each recipient cell. In addition to direct cytotoxicity, the existence of the Bystander effect (BSE) in the suicide gene therapy system provides a certain degree of supplementation for the low transfection efficiency, and is a key factor in the efficacy of suicide gene. It achieves the full killing of tumor cells by transfecting cells against the killing of surrounding untransfected cells without introducing suicide genes into each recipient cell. The mechanism of the bystander effect is currently inconclusive, but at least the following factors are involved in the process of bystander effects: 1) Mechanism of apoptosis. Direct killing of TK-positive tumor cells by GCV, cell shrinkage, chromosome concentration, and apoptotic bodies appear. Due to the vesicles of apoptotic bodies, some toxic metabolites containing suicide genes, such as TK enzymes or toxic drugs, can be Endocytosis uptake leads to secondary apoptosis in adjacent cells. 2) Gap connection mechanism. The gap junction mechanism is the main mechanism of the bystander effect of the HSV-TK/GCV suicide gene therapy system. It was found that GCV was isotopically labeled. As a result, TK-positive cells were in contact with normal TK-negative cells, and the isotope was incorporated into the nucleic acid of normal TK-negative cells, indicating that the bystander effect of the suicide gene and the metabolism caused by gap junctions. Synergistically related. Studies have also shown that the expression level of the main component of gap junction (Cx) is closely related to the degree of gap junction information exchange. Cell contact in three directions in intact tumor tissue is more extensive than in vitro culture, so it is more conducive to the formation of gap junctions between cells, which contributes to the bystander effect in suicide gene killing. 3) The body's immune mechanism. Complete immune function is necessary for the bystander effect of suicide genes. Studies have shown that [19, 20], after the suicide gene causes tumor cell death, the tumor-derived peptides released can be taken up by antigen-presenting cells (APC) and then presented to CD4+ T lymphocytes, thereby activating CD8+ T lymphocytes. The cells enhance their function, further expand the killing effect on the tumor, and can kill tumor cells inoculated in distant parts of the body. The expression of local granulocyte-macrophage-colony stimulating factor (GM-CSF) enhances this anti-candidate function and enhances the bystander effect. In our experiments, TK-positive hepatoma cells transfected with TK-negative hepatoma cells and pAFP-TK plasmid were mixed in different proportions and treated with GCV to observe the presence of bystander effects. The results showed that when the proportion of liver cancer cells transfected with TK was 40% (that is, the proportion of TK-positive liver cancer cells was about 20%), the survival rate of mixed cells was about 40%; when the proportion of liver cancer cells transfected with TK was 80% ( When the proportion of TK-positive hepatoma cells is about 40%, the survival rate of the cells is less than 20%, which confirms that the bystander effect of the killing effect of the pAFP-TK/GCV system plays an important role. Therefore, due to differences in AFP expression levels in different AFP-positive tumors, the pAFP-TK/GCV system can effectively kill tumor cells even in tumors that express AFP at low levels.

Claims

Rights request  A method for preparing polybutylcyanoacrylate n-butyl acrylate (CTAB-PBCA-P) modified with hexadecanyltriethylammonium bromide, the method comprising: dissolving Tween 80 in an appropriate amount of water, Under constant temperature and stirring, add n-butyl cyanoacrylate monomer, 'centrifuged, take the supernatant, filter, remove the supernatant, and make PBCA-ΝΡ colloidal storage solution; dilute PBCA-ΝΡ colloidal storage solution with water, It was added to an aqueous solution of hexadecanyltriethylammonium bromide (CTAB), stirred at room temperature, and centrifuged to remove the supernatant. After drying, CTAB-modified polybutylcyanoacrylate nanoparticles were prepared.  2. A method of preparing a nanoparticle (CTAB-PBCA-NP) according to claim 1, wherein the steps are:  a, the Tween 80 is dissolved in an appropriate amount of double distilled water, using 0. IN HCL to adjust the pH of the solution to below 2. 8 below, under the action of a constant temperature magnetic stirrer, slowly add α-cyanoacrylate to the solution The lipid monomer was stirred at room temperature for 5 hours at room temperature; the mixture was centrifuged at 5,000 rpm for 15 minutes at room temperature, and the supernatant was taken and filtered through a membrane having a diameter of 0.22 μm. The product was PBCA-ΝΡ, centrifuged at 40000 rpm for 1 hour, and the supernatant was removed. Wash twice with distilled water, dry and weigh, resuspend the precipitate with appropriate amount of double distilled water to make PBCA-NP colloidal storage solution;  5%的含含乙乙乙乙乙() 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 CTAB) aqueous solution, stirred at room temperature for 1 hour; centrifuged at 40000 rpm for 30 min, the supernatant was removed, washed with double-distilled water, dried and weighed to obtain CTAB-modified polybutylcyanoacrylate nanoparticles. '