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WO2006015519A1 - Procede de preparation de nanoparticules de polybutylcyanoacrylate modifiees par un bromure de cetyltrietyl-ammonium - Google Patents

Procede de preparation de nanoparticules de polybutylcyanoacrylate modifiees par un bromure de cetyltrietyl-ammonium Download PDF

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WO2006015519A1
WO2006015519A1 PCT/CN2004/001089 CN2004001089W WO2006015519A1 WO 2006015519 A1 WO2006015519 A1 WO 2006015519A1 CN 2004001089 W CN2004001089 W CN 2004001089W WO 2006015519 A1 WO2006015519 A1 WO 2006015519A1
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pbca
cells
ctab
dna
afp
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Yangde Zhang
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D4/00—Coating compositions, e.g. paints, varnishes or lacquers, based on organic non-macromolecular compounds having at least one polymerisable carbon-to-carbon unsaturated bond ; Coating compositions, based on monomers of macromolecular compounds of groups C09D183/00 - C09D183/16
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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
  • 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.
  • 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.
  • 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 ⁇ -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.
  • PBCA-NP polyacrylonitrile n-butyl acrylate nanoparticles
  • 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.
  • CTAB cetyltriethylammonium bromide
  • the nanoparticle gene transport carrier prepared by using the ⁇ -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/ ⁇ .
  • CTAB CTAB
  • PBCA nanoparticle gene transport system can protect the DNA carried away from nuclease degradation and ultrasonic shear damage.
  • 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%.
  • 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
  • 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)
  • 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 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.
  • 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.
  • PBCA-NP (A) and CTAB-modified PBCA-NP (B) showed no significant cytotoxicity to HepG2 cells at concentrations below 100 ng/ ⁇ , below 200 ng/ ⁇ There was no significant cytotoxicity to 3T3 cells at the concentration.
  • concentration of nanoparticles continued to increase, the cytotoxic effect increased sharply (P ⁇ 0.05), resulting in a sharp decrease in cell viability.
  • cytotoxic effects of PBCA-NP and CTAB-PBCA-NP on cells There was no significant difference in the cytotoxic effects of PBCA-NP and CTAB-PBCA-NP on cells.
  • ⁇ 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 ⁇ , it has no obvious cytotoxicity to HepG2 cells, and it does not show obvious cytotoxicity to 3 ⁇ 3 cells at a concentration of 200 ⁇ ⁇ / ⁇ 1 or less. When the concentration of nanoparticles continues to increase, the cytotoxic effect is sharply increased, resulting in a sharp drop in cell viability. * ⁇ 0.05, ⁇ 6.
  • PBCA-NP is a surface-negatively charged high molecular polymer that is inherently difficult to bind to negatively charged DNA molecules.
  • 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.
  • CTAB 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • D is the expression of EGFP-N1 after transfection of 3T3 cells by SuperFect Transfection Reagent.
  • PBCA Polybutylcyanoacrylate
  • CTAB cationic surfactant
  • PBCA nanoparticles are non-cytotoxic and have good transfection efficiency, and can be used as effective gene transfection reagents.
  • CTAB-modified PBCA nanoparticle carrier system enhances the uptake of its foreign genes by cells, provides protection against carrying genes, and has good safety.
  • PBCA nanoparticle gene transporter-mediated pAFP-TK/GCV system for gene therapy of AFP-positive liver cancer
  • 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. '
  • 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.
  • HepG2 was positive for AFP cytoplasm and HeLa and 3T3 cells were negative for AFP expression.
  • AFP expression was positive, and AFP expression was negative in HeLa and 3T3 cells.
  • 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 ⁇ -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. ⁇
  • CTAB-modified PBCA nanoparticles can effectively transfect foreign genes into cells and be expressed normally in cells.
  • 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.
  • 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.
  • MTT method tetramethylazozolium salt microenzyme reaction colorimetric method
  • 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.
  • 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.
  • 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.
  • 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.
  • Trypan blue exclusion assay confirmed GCV time-dependent killing of AFP-positive HepG2 cells.
  • GCV has a significant killing effect on AFP-positive hepatoma cells transfected with TK gene ( ⁇ 0.05).
  • P ⁇ 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).
  • 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.
  • 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).
  • 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.
  • GCV can induce apoptosis in cells expressing K gene.
  • 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.
  • 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.
  • 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.
  • HVJ Japanese hemagglutinating virus
  • CMV promoter/enhancer-regulated HSV-TK/GCV system pcDNA3 -TK expression plasmid
  • CTAB-modified polymer-degrading biomaterial PBCA nanoparticles as a gene transport vector
  • 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.
  • 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 100 ⁇ ⁇ / ⁇ 1 concentration; at 100 ⁇ ⁇ / ⁇ 1 to 200 ⁇ Under the action of ⁇ / ⁇ 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.
  • 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 ⁇ 53 gene and up-regulation of Fas/FasL expression in cells.
  • the AFP-positive cells transfected with pAFP-TK plasmid were stained with Hochest 33258 after GCV treatment, and typical apoptosis occurred.
  • apoptosis is an important mechanism for killing AFP-positive liver cancer cells by the pAFP-TK/GCV system.
  • BSE Bystander effect
  • 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.
  • 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.
  • APC antigen-presenting cells
  • 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.
  • GM-CSF granulocyte-macrophage-colony stimulating factor
  • 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.

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Description

聚氰基丙烯酸正丁脂纳米颗粒的制备方法
技术攀
本发明涉及一种对肝癌基因治疗药物纳米颗粒的制备方法， 特别是一种 十六垸基三乙基溴化铵修饰的聚氰基丙烯酸正丁脂纳米颗粒
(CTAB-PBCA-NP) 的制备方法。
背景技术
近年来， 随着基因转移技术的日趋成熟， 肝癌的基因治疗已成为生物科 学和临床医学的研究热点之一。 而调控自杀基因特异性导入肝癌细胞， 或在 肝癌 ft胞中实现特异性表达， 在最大限度地杀伤肝癌细胞的同时不损害正常 肝细胞，： 是决定其疗效及可行性的关键。 目前， 基因治疗的研究和临床应用 中常用的病毒载体和非病毒载体， 都存在无法克服的局限性。 纳米粒基因转 运体， 是近年发展起来的一种非病毒基因转运载体， 它将 DNA、 RNA等基因 治疗分 Ϋ包裹在纳米颗粒中或吸附在其表面， 通过靶向分子与细胞表面特异 性受体结合， 可望实现安全有效的靶向性基因治疗。 纳米材料的选择， 是成 功 行纳米基¾转运和治疗的关键。 所选择的材料必须是生物可降解型或者 易于 A体内排泄， 不产生有害的降解产物， 且无免疫原性， 不引起机体的 免疫排斥反应。
发明内容
'' 本发明的目的，'是要提供一种具有良好生物降解性、 生物相容性、 无免 疫原' fe的十六烷基三乙基溴化铵修饰的聚氰基丙烯酸正丁脂纳米颗粒 (CTAB-PBCA-NP) 的制备方法。
发明聚氰基丙烯酸正丁脂纳米颗粒（PBCA-NP) 的制备方法选用乳化 聚合法， 其步骤是：'将吐温 80溶解于适量水中， 在恒温、 搅拌下， 加入 α - 氰基丙烯酸正丁脂单体， 离心， 取上清。 过滤， 除去上清， 制成 PBCA- ΝΡ胶 体储存液。 将 PBCA-NP胶体.储存液用水稀释， 加入到十六烷基三乙基溴化铵 (CTAB)水溶液中， 室温下搅拌、 离心去上清， 干燥后即制成 CTAB修饰的 聚氰基丙烯酸正丁脂纳米颗粒。
本发明以 α -氰基丙烯酸正丁脂单体为原料制成的纳米颗粒基因转运载 体， 经表面的改性启， 不仅可有效结合质粒 DNA， 而且可保护 DNA免受破 坏， 并具有较高的体外基因转运效率。 经实验， 本发明有以下效果： 1、 采用 乳化聚合法， 优化了工艺条件， 成功制备出粒径小、 分布均匀的聚氰基丙烯 酸正丁脂纳米颗粒。 用 CTAB修饰该纳米颗粒表面， 使其带正电荷从而可逆 的结合基因治疗质粒 DNA, 组装成 PBCA纳米颗粒基因转运体系， 确定其用 于体外基因转染的安全使用浓度为低'于 lOOng/ μ 1。 2、 PBCA纳米颗粒基因转 运体系， 可保护所携带 DNA免受核酸酶的降解和超声剪切作用的破坏。 3、 采用绿色荧光报道基因， 证实 PBCA纳米颗粒基因转运体系外可成功转运外 源 DNA进入细胞内， 转染效率达 47%。
附图说明
图 1为九种反应条件下制备的 PBCA— P的粒径分析结果比较； 图 2为在 6#反应条件下制备的 PBCA— NP的粒径分析结果；
图 3为原子力显微镜观察 6#反应条件下制备的 PBCA— P大小及表面形 貌；
图 4为 MTT法分析 PBCA— P (A)及 CTAB— PBCA— P (B)的细胞 毒性效应；
图 5为不同 PH值条件下 CTAB— PBCA— NP与 DNA的结合情况； 图 6为 CTAB— PBCA— NP与 DNA结合的凝胶阻滞实验；
图 7为 CTAB— PBCA— NP与 DNA的结合效率评价；
图 8为 CTAB— PBCA— NP/DNA经 DNasel及血清消化后的 DNA电泳图 並 - 图 9为 CTAB—; PBCA— NP/DNA经超声剪切作用后的 DNA电泳图谱； 图 10为 EGFP— N1 在 CTAB— PBCA— P (A， C) 转染及 SuperFect
Transfection Reagent转染 （B， D) 的 HepG2 (A, B)及 3T3 (C， D) 细胞 中的表达 (200x);
图 11为间接免疫荧光检测 HepG2、 HeLa及 3T3细胞内的 AFP表达情况 (400x);
图 12为 Western blot检测 HepG2、 HeLa及 3T3细胞中 AFP表达情况； 图 13为 RT— PCR检测转染不同质粒后的各细胞系中 TK的表达情况； 图 14为 MTT法检测 HepG2细胞转染 pAFP— TK后对不同浓度 GCV的 敏感性。
图 15为台盼兰拒染实验证明 GCV时间依赖性杀伤 AFP阳性 HepG2细胞; 图 16为重组 TK表达质粒对 AFP阳性 HepG2及 APP阴性 HeLa、 3T3细 胞增殖的影响；
图 17为 GCV对不同比例 TK阳性的 HepG2混合细胞的杀伤作用； 图 18为 GCV诱导表达 TK基因的细胞发生凋亡 （400x)
具体实施方式
下面结合实施例具体介绍本发明。
将吐温 80溶解于适量双蒸水中， 用 0. IN HCL调节溶液的 PH值至 2. 8 以下。 在恒温磁力搅拌器作用下， 向溶液中缓缓加入 α -氰基丙烯酸正丁脂单 体， 使 BCA单体浓度为 0.1— 20%， 室温下继续搅拌 5小时。 室温、 5000rpm 离心 15min，取上清，用直径 0. 22 μ m的滤膜过滤，产物为 PBCA- NP。 40000rpm 离心 1 小时， 除去上清， 用双蒸水洗涤两次， 干燥后称重， 用适量双蒸水重 悬沉淀， 制成 PBCA- NP胶体储存液。 .
将 PBCA- NP胶体储存液稀释为 0. 625%的水溶液， 在磁力搅拌器作用下， 取 PBCA- NP稀释液 16. 67ml缓漫加入到 33. 33ml浓度为 0. 25%的十六烷基三乙 基溴化铵（CTA¾)水溶液中， 室温， 搅拌 1小时。 40000rpm离心 30 min， 去 上清， ^用双蒸水洗涤， 干燥后称重， 即制得 CTAB修饰的聚氰基丙烯酸正丁 脂纳 颗粒。
本发明的物、 化性能、 药效及实验数据：
一、 CTAB修饰的 PBCA- F的制备及转染的结果
1、聚氰基丙烯酸正丁酯纳米颗粒（PBCA-NP)的制备及形态粒径的观察检测 将正交实验设计中 9种反应条件下用乳化聚合法制备的 PBCA-NP, 采用 MasterSizer2000激光粒度分析仪进行粒径及分布的检测。 结果显示， 6#反应 条件为制备 PBCA-NP的最优条件， 即反应体系的 PH为 2.5， BCA单体浓度 为 1%， Tween80浓度为 0.6%。 其平均粒径值最小， 为 106nm， 最大粒径为 170nm, 最小粒径为 72nm， 纳米颗粒的比表面积为 57.2872m2/g。粒径大小分 布呈正态分布， 分布最为均匀 （图 1、 2)。 PBCA-NP表面形貌采用 AJ-III原 子力显微镜进行观测。 纳米颗粒呈圆球形， 表面平滑完整， 分散良好， 无粘 附团聚现象， 见.图 3。
参见附图 1 九种反应条件下制备的 PBCA-NP的粒径分析结果比较 参见附图 2 在 6#反应条件下制备的 PBCA-NP 的粒径分析结果， PBCA-NP粒径大小分布呈正态分布， 平均粒径大小为 106nm, 最大粒径为 170nm， 最小粒径为 72nm, 颗粒比表面积为 57.2872m2/g。
参见附图 3 原亍力显微镜观察 6#反应条件下制备的 PBCA-NP大小及表 面形貌， 纳米颗粒呈圆球形， 表面平滑完整， 分散良好， 无粘附团聚现象。
2、 PBCA-NP与 CTAB修饰的 PBCA-NP转运体系的细胞毒性分析
在一定剂量下， 脂质体等非病毒载体表现出的细胞毒性是限制体外基因 转染的重要问题。 纳米颗粒表面所带电荷及表面修饰分子决定了其生物学特 性。.因此本实验应用 MTT 法检测不同浓度 PBCA-NP 及 CTAB 修饰的 PBCA-NP分别对 HepG2细胞和 3T3细胞的细胞毒性， 以确定其应用于基因 转染的可行性及可用 1量。
如附图 4所显示， PBCA-NP (A)和 CTAB修饰的 PBCA-NP (B) 在低 于 100 ng/μΐ的浓度下对 HepG2细胞无明显的细胞毒性， 在低于 200 ng/μΐ的 浓度下对 3T3细胞无明显的细胞毒性。 而当纳米粒浓度继续增加时， 细胞毒 性效应则急剧增强 （P<0.05 )， 导致细胞存活率急剧下降。 而 PBCA-NP 与 CTAB-PBCA-NP对细胞的毒性效应无明显差异。
见附图 4 · MTT法分析 PBCA-NP (A)及 CTAB-PBCA-NP (B) 的细 胞毒性 ¾应， PBCA-NP (A)及 CTAB-PBCA-NP (B)在浓度为 lOO ng/μΙ以 下时， 对 HepG2细胞无明显的细胞毒性， 在浓度为 200 η§/μ1以下时， 对 3Τ3 细胞不表现明显的细胞毒性。 而当纳米颗粒浓度继续增加时， 细胞毒性效应 则急剧增强， 导致细胞存活率急剧下降。 *Ρ<0.05, Ν=6.
3, DNA结合效率分析
PBCA-NP是一种表面带负电荷的高分子聚合物， 因而其本身很难与带负 电荷的 DNA分子结合。但是由于 PBCA纳米颗粒粒径小， 比表面积大， 具有 生物亲和性， 易于在其表面结合其他分子。 因此我们用阳离子表面活性剂 CTAB修饰 PBCA-NP, 使其表面带有正电荷， 从而获得结合 DNA的能力。 然而影响 CTAB表面电性的主要因素是溶液的 pH值条件。因此，我们选取不 同 pH值溶液状态， 将 CTAB-PBCA-NP与质粒 DNA混合， 室温下放置 lh以 上， 取产物进行琼脂糖凝胶电泳。
参见附图 5不同 pH值条件下 CTAB-PBCA-NP与 DNA的结合情况： 1. 对照质粒 DNA， 2. pH=5时纳米粒的 DNA结合情况， 3. pH=7时纳米粒 的 DNA结合情况， 4. pH=9时纳米粒的 DNA结合情况。 电泳显示， 在 pH=7 时， DNA泳动慢， 与纳米颗粒结合的 DNA未出孔， 游离的 DNA数量最少。 在 pH=5、 9时， 纳米粒的 DNA结合能力相对较弱。
结果显示，在不同 pH值条件下， CTAB修饰的 PBCA- P均能结合 DNA， 但结合能力不同。 在中性（pH=7)条件下， DNA结合能力最强， 电泳中显示 泳动速度慢或停留在点样孔中的 DNA量最多；而在酸性与碱性条件下， DNA 结合能力相对较弱。
CTAB修饰的 PBCA-NP与 DNA的结合效率， 我们可以间接采用 DNA 凝胶阻滞实验和 DNA沉淀后紫外光吸收度来进行测定。 根据上面实验结果， 我们选取 pH=7 作为纳米粒与 DNA 结合时的环境 pH 值条件。 将 CTAB-PBCA- P与质粒 DNA按照不同的质量比混合， 室温放置 lh以上， 进 行凝胶电泳观察结合情况 （图 6)。 另取相同反应体系， 20,000rpm/min 离心 15min, 取上清液测定 260nm处的吸光度， 计算 DNA的浓度（图 7)。 经高速 离心， CTAB修饰的 PBCA-KP结合的 DNA沉淀下来， 未被结合的 DNA存 在于上清， 取上清液测定 260nm的吸光度， 可检测体系中未被结合的 DNA。 根据公式 [总 DNA-未结合 DNA/总 DNAxl00%] 计算 DNA的结合效率， 确 定纳米粒与 DNA结合效率最高时的质量比。
参见附图 6 CTAB-PBCA-NP与 DNA结合的凝胶阻滞实验， 1-7泳道分 别代表纳米粒与 DNA的质量比为 1 : 1、 5： 1、 10： 1、 15： 1、 20： 1、 30： 1、 50： 1时的 DNA结合情况。 随着比例的增加， 结合的 DNA比例增加。 在 质量比达 10: 1时， DNA几乎完全结合到纳米颗粒上。
参见附图 7 CTAB-PBCA-NP与 DNA的结合效率评价， 结果显示， 与 CTAB-PBCA-NP结合的 DNA数量随着两者比例的增加而逐渐增加。 当纳米 粒与 DNA的质量比为 10: 1时，结合效率达 93%，然后结合率又有缓慢下降。
以上结果显示， 纳米粒与 DNA比例不同， 其结合效率也是不同的。 随着 比例的增加， DNA的结合量增加。 当纳米粒与 DNA比例达 10: 1时， DNA 几乎完全结合到 CTAB-PBCA-NP 上， 用紫外光吸收法定量可知结合效率达 93%。 当比例继续增加时， 结合效率又缓慢下降。 因此， 我们采用纳米粒- DNA为 10: 1进行后续的研究。
由以上结果可知， DNA是通过阳离子表面活性剂 CTAB结合到 PBCA-NP 的表面。 随着反应体系中 pH值的变化， DNA的结合能力也显示出不同， 提 示不同酸碱条件下纳米颗粒表面所带的电荷数不同， 静电吸引是纳米颗粒结 合 DNA的机制之一。
4、 CTAB修饰的 PBCA-NP对质粒 DNA的保护作用
体内组织间隙的核酸酶及细胞内溶酶体对于外源 DNA有降解作用，是妨 碍外源基因表达的障碍之一。良好的基因转运载体应该具备保护外源 DNA免 受机体内酶消化的功能。纳米颗粒基因转运载体对所携带 DNA具有的保护作 用， 我们采用 DNasel消化、血清消化及超声破坏作用来检测， 产物进行琼脂 糖凝胶电泳分析。 '
参见附图 8 CTAB-PBCA-NP/DNA经 DNasel及血清消化后的 DNA电泳 图谱： 1.裸质粒 DNA经 DNasel 37°C消化 30min; 2.未经处理的裸质粒 DNA; 3.5. CTAB-PBCA-NP/DNA经 DNasel 37。C分别消化 30min、 lh、 1.5h后从 复合物中抽提出的 DNA; 6. 裸质粒 DNA经胎牛血清 37°C消化 81ι后； 7. 6TAB-PBCA- P/DNA经胎牛血清 37。C消化 8h后从复合物中抽提出的 参见附图 9 CTAB-PBCA-NP/DNA经超声剪切作用后的 DNA电泳图谱： 1 . 未经处理的裸质粒 DNA ; 2 . 裸质粒 DNA 经超声作用 15s 后； 3. CTAB-PBCA-NP/DNA经超声作用 15s后从复合物中抽提出的 DNA。
由以上结果可知， CTAB修饰的 PBCA-NP可使所载带的质粒 DNA具有 抵抗核酸酶降解的能力。 CTAB-PBCA-NP/DNA中的 DNA在 37°C 、 DNasel 及血清存在下消化数小时后，仍然保持结构的完整，而裸 DNA在同样条件下 30min即被完全降解。 同时 CTAB修饰的 PBCA-NP可使所载带的质粒 DNA 具有抵抗超声剪切的能力。 CTAB-PBCA-NP/DNA中的 DNA在 230W功率下 超声 15s后仍然能保持结构的完整， 而裸 DNA在同样条件下已发生降解。可 见， CTAB修饰的 PBCA纳米颗粒基因转运系统对外源质粒 DNA可提供完全 的保护作用， 使 DNA结构保持完好， 从而确保其在体内可能得以正常表达。
5、 CTAB修饰的 PBCA纳米颗粒基因转运体的体外基因转运效率的分析
CTAB修饰的 PBCA-NP表面带有正电荷， 可以结合质粒 DNA， 并通过 纳米粒 -DNA复合物表面所携带的阳离子与细胞膜上的带负电荷的糖蛋白及 磷脂相互作用而引发细胞对其的内吞作用， 从而进入胞质。 本实验应用报道基因系统来检测 CTAB-PBCA-NP的体外基因转运效率。 CTAB-PBCA-NP转运报道基因质粒 pEGFP-Nl进入 HepG2细胞和 3T3细胞， 通过在荧光显微镜下观察具有绿色荧光的转染细胞计算其转染效率。 以脂质 体转染效率作为对照。 结果发现（图 10)， CTAB-PBCA-NP系统能有效转运 外源质粒 DNA进入 HepG2和 3T3细胞， 表达 EGFP蛋白（图 10 A， C)。其 转运效率可达约 47%， 略高于 SuperFect Transfection Reagent转染试剂的转运 效率（42%， 图 10 B， D) o
参见附图 10 EGFP-N1在 CTAB-PBCA-NP (A, C) 转染及 SuperFect Transfection Reagent转染（B， D) 的 HepG2 (A, B)及 3T3 (C, D)细胞中的 表达 (200 x): A为 CTAB-PBCA-KP转染 HepG2细胞后 EGFP-Nl的表达； B 为 SuperFect Transfection Reagent转染 HepG2细胞后 EGFP-Nl的表达； C为 CTAB-PBCA-NPs 转染 3T3 细胞后 EGFP-Nl 的表达； D 为 SuperFect Transfection Reagent转染 3T3细胞后 EGFP-Nl的表达。
聚氰基丙烯酸正丁酯（PBCA)纳米颗粒作为基因转运载体， 因为它具有 良好的生物降解性、 生物相容性， 无免疫原性。 由于 PBCA纳米颗粒表面带 有负电性， 因此我们采用阳离子表面活性剂 CTAB对其进行表面改性， 以使 其表面带上正电荷而获得结合带负电 DNA的能力及某些胶体特性。 同时， 实 验也证实， PBCA纳米颗粒/ DNA复合物能保护所携带的 DNA免受核酸酶的 降解和超声剪切作用。
本研究表明， 在适当浓度下 PBCA纳米颗粒与 CTAB修饰的 PBCA纳米 颗粒对肝癌细胞（HepG2)与正常的成纤维细胞（3T3)均无明显的细胞毒性， 只在高浓度下才会表现出一定的细胞毒性作用。 本研究也证实， 在体外， 该 纳米颗粒可有效转运 EGFP报道基因表达质粒进入 HepG2及 3T3细胞， 其转 运效率达 47%， 强于相同条件下脂质体的转染效率， 这与先前的学者报道结 果一致 [31]。 在本实验中的转染浓度下， PBCA纳米颗粒无细胞毒性， 且转染 效率好， 可用作有效的基因转染试剂。 总之， CTAB修饰的 PBCA纳米颗粒 载体系统可增强细胞对其外源基因的摄取， 对携带基因可提供保护作用， 具 有良好的安全性。
尽管 PBCA纳米颗粒基因转运系统具有良好的生物学特性及高转染效 率， 但是其制备工艺仍需进一步优化， 以降低其颗粒粒径大小， 增强 DNA载 带量， 并探讨适当的转染方法， 从而进一步提高其基因转运效率。 二、 PBCA纳米粒基因转运体介导的 pAFP-TK/GCV系统对 AFP阳性肝癌基 因治疗的体外实验研究
1、 间接免疫荧光证实 HepG2细胞内 AFP表达阳性， 而 HeLa及 3T3细胞内 AFP表达阴性
HepG2为人肝细胞癌细胞， ATCC提供资料为 AFP阳性。 HeLa来源细胞 为人宫颈低分化鳞状上皮癌组织， ATCC提供资料为 AFP阴性。 3T3为鼠胚 胎来源的纤维原细胞， ATCC提供资料为 AFP阴性。 为确证 AFP在此三株细 胞中的表达状态， 我们应用鼠来源抗 AFP单克隆抗体对其进行间接免疫荧光 分析。 '
实验结果显示， HepG2细胞于胞浆中可见弥漫绿色荧光，而 HeLa及 3T3 细胞均未见绿色荧光，证实 HepG2细胞为 AFP表达阳性（图 11 A)，而 HeLa 及 3T3细胞为 AFP表达阴性 （图 11 B、 C)。
参见附图 11 间接免疫荧光检测 HepG2、 HeLa及 3T3细胞内的 AFP表 达情况（400x): A为 HepG2细胞应用鼠来源抗 AFP单克隆抗体进行间接免 疫荧光分析， 结果显示 APP表达于细胞胞浆。 B为 HeLa细胞应用鼠来源抗 APP单克隆抗体进行间接免疫荧光分析，结果显示为 AFP表达阴性。 C为 3T3 细胞应用鼠来源抗 AFP单克隆抗体进行间接免疫荧光分析， 结果显示为 AFP 表达阴性。
2、 Western blot证实 HepG2细胞为 AFP表达阳性， HeLa及 3T3细胞为 AFP 表达阴性
在间接免疫荧光结果证实 HepG2为 AFP胞浆阳性、 HeLa及 3T3细胞为 AFP表达阴性的基础上，我们进一步利用 Western blot的方法检测在此三株细 胞中 AFP蛋白的表达状态， 从而确证 HepG2细胞中 AFP表达阳性， HeLa及 3T3细胞中 AFP表达阴性。
参见附图 12 Western blot检测 HepG2、 HeLa及 3T3细胞中 AFP表达情 况- HeLa及 3T3细胞内无 AFP蛋白条带的检出， 而 HepG2细胞内则可检出 70KD大小的 AFP蛋白条带。本实验以 Tubulin蛋白作为各泳道上样量的对照， 显示各泳道上样量无明显差别。 本结果为多次实验中的一个代表图。
通过 Western blot方法进一步在蛋白质表达水平证实 HepG2细胞内 AFP 表达阳性， 而 HeLa及 3T3细胞则为 AFP表达阴性。 因此， 我们可以选择此 三株细胞作为实验材料， 分别导入各种质粒进行 AFP阳性肿瘤基因治疗的体 外研究。
3、 转染后各细胞系中 TK表达的 RT-PCR检测 '
CTAB修饰的 PBCA纳米颗粒介导的各 TK基因表达质粒转染 HepG2、 HeLa及 3T3细胞后， 以 RT-PCR方法鉴定其在各细胞系中 mRNA水平上的 表达状况。 以 β-actin作为内对照， 结果见图 3-3。 转染空白质粒 pcDNA3.1的 对照组各细胞中， 未见 TK基因的表达。 而转染 p3.1-TK与 pAFP-TK质粒的 各组细胞中 TK基因均有不同程度的表达。 ·
参见附图 13 RT-PCR检测转染不同质粒后的各细胞系中 TK的表达情 况： 1. DL2000 DNA marker; 2-4. HepG2细胞中分别转染 pAFP-TK、 p3.1-TK：、 pcDNA3.1质粒后细胞中 ΤΚ的表达； 5-7. HeLa细胞中分别转染 pAFP-TK、 p3.1-TK、 pcDNA3.1质粒后细胞中 TK的表达； 8-10. 3T3细胞中分别转染 pAFP-TK、 p3.1-TK、 pcDNA3.1质粒后细胞中 TK的表达； β-actin为内对照。
由图中可见， CTAB修饰的 PBCA纳米颗粒能有效的转染外源基因进入 细胞， 并在细胞内得以正常表达。 在 AFP阳性的 HepG2细胞中， pAFP-TK 质粒转染后 TK的表达水平明显强于 p3.1-TK质粒转染后 TK的表达。 而在 AFP阴性的 HeLa及 3T3细胞中， pAFP-TK质粒转染后 TK的表达水平则弱 于 P3.1-TK质粒转染后 TK的表达。 提示， 在 AFP阳性细胞中， AFP增强子 调控的 TK基囟的表达远远强于其基础启动子 CMV的表达水平。
4、 MTT法检测细胞对 GCV的敏感性
MTT法（四甲基偶氮唑盐微量酶反应比色法） 原理为： 活细胞内线粒体 琥珀酸脱氢酶能催化 MTT形成蓝色甲肷，其形成的量与活细胞数和功能状态 呈正相关。细胞摄取催化 MTT后，加入 DMSO充分溶解深蓝色结晶，在 570nm 下于酶标仪读取 OD值， 值的高低反映了细胞总体的功能状态。 本课题应用 此方法作为反映细胞死亡的一个间接指标。
在本实验中，我们采用该 法来检测 AFP阳性细胞转染 pAFP-TK质粒后 对 GCV的敏感性。
参见附图 14 MTT法检测 HepG2细胞转染 pAFP-TK后对不同浓度 GCV 的敏感性， 细胞种于 96孔板中， pAFP-TK质粒转染后加入不同浓度 GCV， 培养 7天后， 加入 MTT， 继续培养 4h， 加入 DMSO， 于酶标仪 570nm检测 吸光度， 并以此吸光度与对照组吸光度的比值作图。 *P<0.05， n=3。
实验结果显示， 使用 lO g/ml GCV浓度时对细胞生存率影响不大， 使用 5(^g/ml GCV浓度时约 50% (PO.05) 的 AFP阳性细胞死亡； 使用 lOO g/ml GCV浓度时约 80%(P<0.05)的 AFP阳性细胞死亡；当 GCV浓度达到 20(^g/ml 时则只有少数 AFP阳性细胞存活 （PO.05); GCV浓度继续升高达 500μ /ιη1 时对细胞的杀伤作用增加不明显。 提示在 pAFP-TK/GCV系统中， GCV呈剂 量依赖性杀伤 AFP阳性肝癌细胞。
5、 GCV呈时间依赖性杀伤 AFP阳性肝癌细胞
我们应用台盼兰拒染实验来直接反映 pAFP-TK/GCV系统对 AFP阳性肝 癌细胞的杀伤作用。
参见附图 15 台盼兰拒染实验证实 GCV 时间依赖性杀伤 AFP 阳性 HepG2细胞， pAFP-TK质粒转染后的 HepG2细胞接种于 96孔板中， 12h后 加用' lOC^g/ml的 GCV处理， 于所示天数进行台盼兰染液染色后， 显微镜下 计数兰染细胞的百分比。 *P<0.05， n=3。
由图中可见 GCV对转染 TK基因的 AFP阳性肝癌细胞具有明显的杀伤效 应 （Ρ<0.05)。 在加用 GCV后的第二天（P<0.05) HepG2细胞即出现明显杀 伤作用， 在第八天对细胞的杀伤作用达约 80% (P<0.05) o
6、 MTT结果证实 pAFP-TK表达质粒特异性抑制 AFP阳性肝癌细胞 HepG2 的增殖， 而对 AFP阴性细胞抑制作用较小
MTT法是确定细胞增殖状态的有效方法。上述实验证实， pAFP-TK表达 质粒可呈时间与剂量依赖性杀伤 AFP阳性肝癌细胞。 AFP增强子在 AFP阳性 细胞中作为增强子， 而在 AFP阴性细胞中作为抑制子发挥作用。 因此， 我们 应用 AFP阴性的 HeLa、 3T3细胞及 AFP阳性的 HepG2细胞为实验材料， 采 用 MTT法进一步证实该自杀基因治疗系统对 AFP阳性肝癌细胞的特异杀伤 作用。
参见附图 16 重组 TK表达质粒对 AFP阳性 HepG2细胞及 AFP阴性 HeLa、 3T3细胞增殖的影响， 各组细胞转染所示质粒后， 接种于 96孔板内， GCV终浓度为 ΙΟΟμ^πύ,培养 Ί天后，加入 ΜΤΤ，继续培养 4h，加入 DMSO， 于酶标仪 570nm检测吸光度， 并以此吸光度绝对值作图。 以 pEGFP-Nl绿色 荧光表达质粒为转染效率的对照， 结果证实各组转染效率均为 47%左右， 各 组间无明显差异。 * p<0.05， n=3。
结果可见， 各组细胞转染 pcDNA3.1质粒与 GCV处理后对细胞 MTT分 析结果无明显影响。而在 AFP阳性细胞 HepG2内，转染 p3.1-TK与 pAFP-TK 质粒加用 GCV处理均明显抑制细胞活力， p3.1-TK质粒转染组对细胞活力的 抑制达 60%左右 （P<0.05)， 而 pAFP-TK质粒转染组对细胞活力的抑制作用 更强达 80%(P<0.05)。 而在 AFP阴性的 HeLa细胞及 3T3 细胞中均可见， pAFP-TK质粒转染并加用 GCV处理对细胞活力的抑制较 p3.1-TK质粒转染者 明显减弱 （P<0.05)。 由此可见， pAPP-TK/GCV系统抑制细胞增殖的作用明 显强于 p3.1-TK/GCV系统。 因此证实 pAFP-TK/GCV治疗体系为一种高效高 特异性的杀伤 AFP阳性肿瘤细胞的基因治疗方法。
7、 旁观者效应
为研究 pAFP-TK/GCV系统对细胞杀伤作用是否具有旁观者效应， 我们 以 HepG2细胞为材料，利用 MTT法进行检测。结果发现，在转染 TK的细胞 比例占 40%时（即 TK阳性细胞比例约为 20%时）， 细胞生存百分数为 40%左 右； 在转染 TK的细胞比例为 80%时（即 TK阳性细胞比例 ^为 40%时）， 细 胞生存率低于 20%。 提示 pAPP-TK/GCV系统对细胞的杀伤作用存在明显的 旁观者效应。
参见附图 17 GCV对不同比例 TK阳性的 HepG2混合细胞的杀伤作用， 细胞按照图中所示比例混合后接种于 96孔板中， GCV终浓度为 ΙΟΟμ^πιΙ, 培养 7天后， '加入 ΜΤΤ， 继续培养 4h， 加入 DMSO， 于酶标仪 570nm检测 吸光度， 并以此吸光度与对照组吸光度比值作图。 *P<0.05， n=3。
8、 GCV可诱导表达 K基因的细胞发生凋亡 '
有文献报道， HSV-TK/GCV系统的体外细胞杀伤作用存在细胞凋亡， 因 此我们采用 Hochest 33258染色的方法来确定 pAFP-TK/GCV系统诱导的杀伤 作用是否为凋亡。 经染色后的细胞于荧光显微镜下观察， 如核内为均勾弥散 荧光则为正常生活细胞； 而如果出现核浓缩、 核碎裂及核溶解等则证实细胞 发生了凋亡。 pAFP-TK质粒转染的 HepG2细胞经 GCV处理后行 Hochest 33258 染色， 结果见图 18。
参见附图 18 GCV诱导表达 TK基因的细胞发生凋亡（400x): A为转 染后未经 GCV处理的 HepG2细胞的荧光染色图， 核内荧光均匀弥散分布， 为正常细胞图像； B为转染后加用 GCV处理的 HepG2细胞的荧光染色图， 可见有明显的核浓缩， 证实细胞发生了凋亡。
由结果可知， 对照组细胞核染色均匀， 而加用 GCV处理后， 可见有核浓 缩的出现。因此证实应用 GCV处理后，表达 HSV-TK基因的细胞发生了凋亡， 从而确证 pAFP-TK/GCV系统诱导细胞的凋亡为细胞杀伤作用的机制之一。 讨论
HSV-TK/GCV系统在肝癌自杀基因治疗的研究中进展迅速。 自 1995年首 次被引入到肝癌的基因治疗中，并证明能以剂量依赖的方式诱导 GCV对肝癌 细胞的杀伤作用后， HSV-TK/GCV系统的抗肝癌效能在体内、 外实验中不断 得到确证， 并有研究探讨体内实验中给药途径的选择对治^ f作用的影响。 然 而， HSV-TK基因在转染中非特异性地进入正常肝细胞会导致严重的肝功能 损害， 因此采用各种基因转运载体及特异性转录调控元件提高转染效率及靶 向性的研究不断的深入展开。
有研究采用慢病毒载体介导肝癌细胞特异性 AFP启动子调控的 HSV-TK/ EGFP融合基因表达 ¾粒转染肝癌细胞、非肝脏来源的人肿瘤细胞及原代正常 肝细胞， 结果发现在肝癌细胞中融合基因高表达， 非肝来源的人肿瘤细胞中 表达量较低， 而正常肝细胞中无表达。 该研究小组进一步采用 HIV-衍生型慢 病毒载体介导 HSV-TK基因转染， 结果发现其在体内、 外均可有效转导该自 杀基因进入肝癌细胞中，从而在 GCV作用下发挥杀伤作用。体内动物实验中， 任何给药途径下， 未见 TK基因进入正常肝细胞， 无明显肝功能损害的发生。 另有学者采用日本血凝病毒（HVJ)脂质体作为基因转运载体， 进行 CMV启 动子 /增强子调控的 HSV-TK/GCV系统（pcDNA3 -TK表达质粒）治疗肝癌的 临床前实验研究。结果显示， HVJ脂质体体外肝癌细胞转导效率为 20%， GCV 的有效浓度为 100mg/ml， 即产生肿瘤细胞的完全杀伤作用； 在体内实验中， 其转导效率为 19.7%， GCV处理后明显抑制肿瘤的生长。
在研究中，我们采用 CTAB修饰的高分子降解生物材料 PBCA纳米颗粒作 为基因转运载体， 以 AFP增强子、 CMV启动子调控的 HSV-TK基因表达质 粒 pAFP-TK作为治疗基因， 并加用 GCV处理进行 AFP阳性肝癌的基因治疗 研究。 我们以 AFP阳性的 HepG2细胞与 AFP阴性的 HeLa及 3T3细胞为实 验材料， 将重组质粒 pAFP-TK转染入各组实验细胞。 结果发现， CTAB修饰 的 PBCA纳米颗粒的转染效率达 47%左右，较同等条件下脂质体转染效率高。 利用 RT-PCR方法检测三株细胞系中外源 TK的表达情况发现， 在 mR A的 表达水平上， AFP阴性的 HeLa及 3T3细胞转染 pAFP-TK质粒后， TK的表 达较无 AFP增强子调控的对照质粒 p3.1-TK明显降低；而在 AFP阳性的肝癌 细胞 HepG2中， AFP增强子调控的 TK基因表达较无 AFP增强子调控者显著 增强。 这与先前的文献报道相一致。
在已经确证 CTAB修饰的 PBCA纳米颗粒能有效转染 pAFP-TK质粒进入 细胞， 并在 AFP阳性细胞中高效表达的基础之上， 我们进一步采用台盼兰拒 染实验及 MTT方法研究发现， AFP阳性的细胞在转染 pAFP-TK后对 GCV的 敏感性大大提高， 细胞存活率明显下降。 实验还发现， pAFP-TK/GCV系统对 肿瘤细胞的杀伤作用呈 GCV剂量和时间依赖性， 低于 100μ§/ηι1浓度的 GCV 作用时对细胞的杀伤作用较弱；在 100μ§/ιη1至 200μ§/πύ浓度的 GCV作用下， 杀伤作用显著， 肿瘤细胞只有少数存活； 而当 GCV浓度继续增加时， 细胞基 本死亡。
大量文献证实， HSV-TK/GCV系统可诱导肿瘤细胞的凋亡。 在肝癌的 HSV-TK/GCV 治疗研究中同样也发现凋亡的存在， 且这种凋亡与细胞中的 Ρ53基因的表达及 Fas/ FasL表达上调有关。在本实验中证实， pAFP-TK质粒 转染的 AFP阳性细胞 GCV处理后， 经 Hochest 33258染色， 可见典型的细胞 凋亡发生。 因而证实细胞凋亡是 pAFP-TK/GCV系统杀伤 AFP阳性肝癌细胞 的一个重要机制。
目前， 限制基因治疗广泛应用的关键之一就是基因转运效率较低， 不能 把目的基因导入每个受者细胞。而自杀基因治疗系统中除直接细胞毒作用外， 其旁观者效应（Bystander effect, BSE) 的存在为转染效率低下提供了一定程 度上的补充， 是自杀基因疗效的关键因素。 它通过转染细胞对周围未转染细 胞的杀伤作用， 实现肿瘤细胞的全部杀伤， 而无需将自杀基因导入每个受者 细胞。 旁观者效应的作用机制目前尚无定论， 但至少有下面几个因素参与旁 观者效应的过程： 1 ) 细胞凋亡机制。 GCV对 TK阳性肿瘤细胞的直接杀伤， 细胞出现收缩、 染色体浓縮、凋亡小体出现等现象。 由于凋亡小体的小泡 中包含自杀基因的一些毒性代谢产物如 TK酶或者毒性药物可以被邻近的细 胞吞噬摄取， 导致邻近细胞的继发凋亡。 2)缝隙连接机制。 缝隙连接机制是 HSV-TK/GCV自杀基因治疗系统旁观者效应的主要机制。研究发现，将 GCV 进行同位素标记， 结果 TK阳性细胞与正常的 TK阴性细胞接触后， 同位素掺 入到正常的 TK阴性细胞的核酸之中，表明自杀基因的旁观者效应与由缝隙连 接产生的代谢协同作用有关。研究也表明， 缝隙连接的主要成分接合素（Cx) 的表达量与缝隙连接信息交流程度密切相关。 完整的肿瘤组织内三个方向的 细胞接触比在体外培养接触更加广泛， 因此更有利于细胞之间形成缝隙连接， 有助于自杀基因杀伤作用中旁观者效应的发挥。 3 )机体免疫机制。 完整的免 疫功能对自杀基因的旁观者效应是必须的。研究表明 [19、 20], 自杀基因引起 肿瘤细胞死亡后， 释放出的肿瘤源性肽类可被抗原提呈细胞（APC)摄取加 工， 然后提呈给 CD4+ T淋巴细胞，进而激活 CD8+ T淋巴细胞并使其功能增 强， 进一步扩大对肿瘤的杀伤作用， 并且能够杀灭机体远处部位接种的肿瘤 细胞。 而局部粒细胞巨噬细胞-集落刺激因子 （GM-CSF) 的表达能增强这种 抗康提呈功能，从而增强旁观者效应。在我们的实验中，采用 TK阴性肝癌细 胞与 pAFP-TK质粒转染的 TK阳性肝癌细胞按照不同比例混合， 并用 GCV 处理，观察旁观者效应的存在。结果发现，在转染 TK的肝癌细胞比例占 40% (即 TK阳性肝癌细胞比例约为 20%) 时， 混合细胞的存活率为 40%左右； 当转染 TK的肝癌细胞比例达 80% (即 TK阳性肝癌细胞比例约为 40%) 时， 细胞的存活率则已低于 20%， 从而证实 pAFP-TK/GCV系统的杀伤作用中旁 观者效应起了重要作用。 因此， 由于在不同的 AFP阳性肿瘤中， AFP表达水 平存在差异， 即使在低水平表达 AFP的肿瘤中， pAFP-TK/GCV系统也可以 对肿瘤细胞进行有效的杀伤。

Claims

权利要求
1、 一种用十六垸基三乙基溴化铵修饰的聚氰基丙烯酸正丁脂纳米颗粒 (CTAB-PBCA- P) 的制备方法， 其步骤是： 将吐温 80溶解于适量水中， 在 恒温、 搅拌下， 加入 α -氰基丙烯酸正丁脂单体，'离心， 取上清， 过滤， 除去 上清， 制成 PBCA- ΝΡ胶体储存液； 将 PBCA- ΝΡ胶体储存液用水稀释， 加入到 十六垸基三乙基溴化铵 （CTAB) 水溶液中， 室温下搅拌、 离心去上清， 干燥 后即制成 CTAB修饰的聚氰基丙烯酸正丁脂纳米颗粒。
2、 按权利要求 1 所述的纳米颗粒（CTAB-PBCA-NP) 的制备方法， 其步 骤是：
a、将吐温 80溶解于适量双蒸水中，用 0. IN HCL调节溶液的 PH值至 2. 8 以下， 在恒温磁力搅拌器作用下， 向溶液中缓缓加入 α -氰基丙烯酸正丁脂单 体，室温下继续搅拌 5小时；室温、 5000rpm离心 15min，取上清，用直径 0. 22 μ πι的滤膜过滤， 产物为 PBCA- ΝΡ, 40000rpm离心 1小时， 除去上清， 用双蒸 水洗涤两次， 干燥后称重， 用适量双蒸水重悬沉淀， 制成 PBCA- NP胶体储存 液； '
b 每 - 胶-体細夜 -稀释 *Θ: 的 作 mr; 取 PBCA NP稀释液 16. 67ml缓漫加入到 33. 33ml浓度为 0. 25%的十六烷基三乙 基溴化铵（CTAB)水溶液中， 室温， 搅拌 1小时； 40000rpm离心 30 min， 去 上清， 用双蒸 '水洗涤， 干燥后称重， 即制得 CTAB修饰的聚氰基丙烯酸正丁 脂纳米颗粒。 '
PCT/CN2004/001089 2004-08-11 2004-09-24 Procede de preparation de nanoparticules de polybutylcyanoacrylate modifiees par un bromure de cetyltrietyl-ammonium Ceased WO2006015519A1 (fr)

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