Construction Of The Vector Actively Targeting To Malignant Glioma Cells And The Research On Its In Vitro Transfection Effect

BackgroundThe gliomas account for45%of intracranial tumors, which are often malignant, hard to be excised completely and prone to recur. The life span of patients with malignant gliomas is always short, and may be just extended in couples of months or half a year even if the surgery, chemotherapy and radiotherapy are carried out, because the gliomas have always invaded the brain tissue before operation and the residue tumor is easy to recur. Nowadays, besides the surgery, chemotherapy and radiotherapy, the biotherapy and gene therapy have been considered as promosing strategies, although most of them are still in experiments or clinic trials. However, gene therapy has a big problem that how to import the genetic products into tumor cells specifically, without harming the normal cells.Recently, some targeting strategies were investigated and classified into two main aspects:one is focusing on the special gene or enzyme, et al. of tumors, and the other on the gene carrier which can specifically target tumor cells. One kind of carrier is viral vector such as lentivirus or adenovirus vector, which has a high transfection efficiency but also immunoreactivity and potential oncogenicity. The other kind is non-viral vector such as liposome and nanoparticles, which is safer but less effective. Many techniques are used to improve the ability of non-viral vector so that these carriers have being researched more widely. Polyethylenimine (PEI) is one kind of cationic polymer that can condense DNA into nanoparticle, which can be absorbed by the cells. But the absorption is not cell-specific and its efficiency is not so high.Some functional molecules can be linked with PEI to improve its nature. Some targeting molecules such as the antibody of vascular endothelial growth factor receptor (VEGFR) have being reported to increase enrichment of the carrier to glioma cells but not really target the gliomas. What these carriers depend on is the vessel feeding tumors is more plenty than that of normal tissues. Some other new ligands were found recently, such as V peptide with sequence of VTWTPQAWFQWV, which can target the malignant glioma cells. It is reported that V peptide can specifically bind with U87glioblastoma cells but not with the glial cell lines. Its targeting ability can help the gene carrier to locate the glioma cells, leaving the normal glial cells and neurons alone.In this work, some kinds of nanoparticles based on PEI, V peptide and DNA were constructed and used to transfect the malignant glioma cells, with the primary neural cells from the rats as control, to test if they can be used as the targeting gene carrier for gliomas.ObjectiveTo construct the nanoparticles based on PEI and V peptide and test their ability to transfect glioma cells.Methods1. Primary neural cell cultureThe neuron of rat cerebral cortex was obtained and cultured as previously described with a little difference.1-day-old neonatal Wistar rats were obtained from Lab Animal Center of Shandong University (China), euthanatized using inhalant isoflurane and then decapitated. After removal of cranium and meningeal, the cerebral cortex was dissected in D-hank’s buffer and dissociated with0.25%trypsin for10-15min at37℃. After the digestion termination, cells were seeded into24-well plate at1×105 cells/well in neurobasal medium supplemented with20ng/ml neuron growth factor (NGF),2%B27supplement,0.5mM L-glutamine, and1%(v/v) penicillin/streptomycin. After4or5days culture, the cells were used as control of transfection study.U87and T98G (Human glioblastoma), U251(Human glioma), HEK293(Human embryonic kidney293), A549(Human lung adenocarcinoma) and PC12(Rat adrenal gland pheochromocytoma) cell lines and primary glioma cells were seeded in24-well plate and cultured in MEM supplemented with10%fetal bovine serum (FBS),2mM L-glutamine,100units/ml penicillin and100μg/ml streptomycin. When the convergence of cells reached80%, the cells were used to be transfected with nanoparticles.2. ImmunofluorescenceNeural cells were fixed with4%paraformaldehyde (PFA) for15min and permeabilized with PBST for15min. After rinse with phosphate-buffered saline (PBS), cells were blocked with3%bovine serum albumin (BSA) for1h at room temperature (RT). Afterwards, cells were incubated with anti-NSE or anti-GFAP overnight at4℃and subsequently with FITC-and TRITC-labeled secondary antibodies at RT in the dark. The nuclei were stained with DAPI. After washes cells were mounted and the images were taken with a fluorescent microscope.3. Plasmid DNA amplificationPlasmid EGFP (pEGFP) plasmid was kindly provided by the Medical School of Shandong University. The plasmid was transformed and amplified in DH5-a competent Escherichia coli. DNA purification was performed using an Endo-free Plasmid Kit following the manufacturer’s instruction. The concentration and purity of plasmid were measured by ultraviolet spectrophotometer.4. Preparation of polymer PVThere were three groups. For the first group, PEI (1umol) in1ml HBS (0.5M NaCl,20mM HEPES, pH7.4) was mixed with SPDP dissolved in DMF (100mM,100μl) under the protection of argon for2h at RT. Then the pyridyldithiol-activated PEI was got and named as PEI-PDP10. For the other two groups, the same protocol was carried out except for the volumes of SPDP (200μl and400μl respectively) and PEI-PDP20and PEI-PDP40were got. The PEI-PDP polymers were purified with PD-10column pre-equilibrated in HBS according to the manufacturer’s instruction and the filtrate was stored at-80℃. The concentration of PEI was measured by TNBS assay as previous reports. The level of SPDP-modification was determined by measuring the absorbance of pyridine-2-thione at343nm. For synthesis of PEI-VTW (PV), VTW peptide (1μmol) was dissolved in900μl GnHC1buffer (2M guanidine hydrochloride (GnHC1),0.5M NaCl,20mM HEPES, pH8.0) and100μl DMF, and then stirred with PEI-PDP10(0.1μol) in1ml GnHC1buffer under the protection of argon at RT for2h. For the other two groups, VTW peptide (2μmol and4μol) was respectively mixed with0.1μmol PEI-PDP20and PEI-PDP40solutions. The subsequent purification was performed using PD-10column. All polymer solutions were filtered through0.22μm Millipore membranes and were stored at-80℃.5. Preparation of nanoparticlesPV polymers were dissolved in HBG buffer (20mM HEPES,5%w/v glucose, pH7.4) to prepare stock solutions. Then, pDNA at different molar ratios of nitrogen in amino residues of PEI to DNA phosphates (N/P ratio), was added into the polymer solutions separately under vortex for30sec and incubated for30min’at RT. Then the pDNA was condensed with polymers and nanoparticles were formed. The characteristics of copolymers were examined with the methods of1H NMR, particle size, zeta potential, transmission electron microscopy (TEM) and agarose gel electrophoresis assay.6. In vitro transfection studyThe U87cells were seeded onto24-well plates at a density of1×105cells per well. When the cells grew to approximately70%confluence, the freshly prepared nanoparticles, with1.0μg of DNA (pEGFP) at an N/P ratio of6and10, were added to the cells and incubated in500μl serum-free DMEM without antibiotics, at37℃. Simultaneously, the same procedures were carried out at the N/P ratio of6except for adding10%FBS into transfection medium to evaluate the influence of serum on nanoparticle transfection. After5h, the transfection medium was replaced with DMEM (10%FBS) for further incubation for additional24h. The images were captured with a fluorescent microscope (Olympus BX-51, Japan). The number of GFP positive cells was counted in10randomly selected fields at200-fold magnification and normalized to the number of cells in the same10images to calculate transfection efficiency (expressed as a percentage, mean±standard error), and counted with FACS flow cytometry. T98G and U251glioma cells were also transfected with the nanoparticles to assess their transfection efficiency. Naked DNA, PEI/DNA nanoparticles and Lipofectamine2000were used as vector control. PC12, A549and HEK293and primary neural cells from rat cerebral cortex were used as cell control. The factors which may influence the results should be considered:concentration of pDNA, N/P, transfection time, transfection medium, serum. All the experiments were performed in triplicate.7. Mechanism of transfectionThe mechanism of transfection was investigated with binding assay, internalization assay and competition assay.(1) Binding assay:U87, A549and neural cells were separately seeded onto24-well plates with a sterile cover slip at a density of1×105cells/well. After24h, the cells were incubated with blocking buffer (1%(w/v) BSA in PBS) at RT for1h. All cells were then exposed to FITC-VTW (0.02mg/ml) at37℃for30min. Cells were rinsed and fixed in4%PFA for15min and then incubated with DAPI. The cells were mounted and observed under fluorescent microscope.(2) Internalization assay:For the internalization of nanoparticles, the cellular uptake of the nanoparticles loaded with FITC-labeled pDNA was performed.Fluorescent labeling of the pDNA was carried out using Label IT(?) TrackerTM Intracellular Nucleic Acid Localization Kit (Mirus, USA) as the manufacturer’s instructions. The transfection protocol of the U87cells was carried out as per the aforementioned procedure except for using labeled pDNA. After3h incubation, the cells were washed with PBS and new complete medium was refreshed. Images of the cells were captured under fluorescent microscope.(3) Competition assay:U87cell was pre-incubated with1000molar times excess of free V peptide compared with peptide conjugated to PV20nanoparticles at37℃for1 h. Then the transfection assay and internalization assay with PV20nanoparticle were performed as per the aforementioned procedure.8. Cytotoxicity assayCytotoxicity was determined using a CCK-8Assay Kit according to the manufacturer’s instruction. Transfection procedures were as previously described except for using the96-well plate. The absorbance at450nm was measured using a microplate reader.9. Statistical analysisData in this study was calculated as mean±standard deviation. One way analysis of variance (ANOVA) and Student t-test were used to investigate statistically significant differences. A p value less than0.05was considered statistically significant.Results1. Cell cultureThe neural cells grew very well after4-5day culture, with neurite and soma developed very well. Glial cells were spindle-shaped, crossovering with neurons like a network. The shape of glioma cells was irregular. Immunofluorescence confirmed the neurons with anti-NSE antibody, and confirmed the glial cells with anti-GFAP antibody.2. PEI conjugationThe yield rate of polymers was80.1±1.53%according to TNBS assay. The standard curve was:Y=0.0697X+0.0071(R2=0.9998). Y:absorbance; X:the concentration of samples (umol/ml). The conjugation rate of PEI-PDP was86.2±5.17%, determined by pyridine-2-thione assay. According to the different conjugation rate, new copolymers were classified into PV10, PV20and PV40.3. Characteristics of nanoparticles’H-NMR confirmed the successful conjugation of PEI and V peptide. The particle size of PEI nanoparticle was stable at69.8±4.15nm when the dropping speed was10s and the vortex speed was moderate. It was found that PEI, PV10, PV20, PV40 nanoparticles completely condensed DNA at the N/P of2,3,5,6via agarose gel electrophoresis. The particle size of PV10、PV20、PV4o nanoparticle was larger than that of PEI nanoparticle, and zeta potential was lower。All nanoparticles were spherical observed by transmission electron microscopy (TEM). Anti-enzyme assay confirmed that all nanoparticles could protect DNA from degradation by DNase Ⅰ.4. Transfection evaluationFirst the transfection conditions of PEI nanoparticle were optimized using HEK293cell as a model. The transfection efficiency to HEK293cell by PEI nanoparticle was improved best when the concentration of DNA was0.1mg/ml, transfection time was5h and transfection medium was serum-free DMEM. PV10, PV20and PV40nanoparticles could increase the transfection efficiency to U87cell, while PV20nanoparticle did it best. In addition, the DMEM with serum could reduce the efficiency of Lipofectamine and PEI nanoparticle notably, while almost did not affect PV nanoparticles. There was a little different among different glioma cells. The transfection efficiency to U87cell increased by PV20nanoparticle was higher than that of T98G and U251cells, while the efficiency to primary glioma cells were also high but not stable. PV20nanoparticle didn’t work on PC12, A549cell lines and primary neural cells.5. CytotoxicityThe cytotoxicity of PV10, PV20and PV40nanoparticles were all lower than that of PEI nanoparticle (P<0.01) and Lipofectamine (P<0.05). The more Ⅴ peptide/PEI ratio was, Lipofectamine the lower cytotoxicity was. The cytotoxicity of PV20was very low to neural cells compared with that of PEI nanoparticle.6. Mechanism of transfection by PV nanoparticlesBinding assay, internalization assay and competition assay. Binding assay showed FITC-V could specifically bind onto the U87cells but not onto PC12and neural cells. Internalization assay showed there was almost no co-localization between lysosome (stained with Lyso-tracker Red) and FITC-labled pDNA nanoparticle, which meaned the nanoparticle successfully escaping from lysosome. In competition assay, the transfection efficiency of PV20nanoparticle to U87cell was decreased obviously after pre-incubation with free V peptide (P<0.001), which confirmed that the specific uptake of PV nanoparticles was indeed mediated via V peptide.ConclusionsV peptide can bind onto the membrane of malignant glioma cells such as U87, while not bind to other cells such as neural cells. The targeting PV nanoparticle can specifically deliver the gene product into several kinds of glioma cells especially glioblastoma cells, making no harm to neural cells. Among these nanoparticles, PV20nanoparticle is the best. BackgroundThe gliomas account for nearly half of intracranial tumors, most of which are malignant, hard to be excised completely and prone to recur. The prognosis of patients with malignant gliomas is always poor, and the life span may be just extended a short period even if the surgery, chemotherapy and radiotherapy are carried out together. Nowadays, besides the conventional therapies, the biotherapy and gene therapy have been considered as promosing strategies. However, there are some urgent questions with regard to gene therapy that investigate the gene map of gliomas, found new genetic products and safe and effective vector.Gene vector are classified into two main types:viral vector and non-viral vector. The former can lead to high transfection efficiency but also immunoreactivity and potential oncogenicity. The latter is safer but less effective. It will be perfect if advantages of both kinds of vectors can be combined. Therefore, some techniques are used to transform non-viral vectors so that they can mimic the viral vectors. Nanoparticles made by cationic polymers are one big group of non-viral vectors. PEI, Poly-L-lysine (PLL), Poly-Arginine and chitosan, et al. belong to cationic polymer which can condense DNA into nanoparticle. PEI is one of the most used polymers, which was also used by us in Part Ⅰ and confirmed effective and low-toxic after modification. However its application is still limited because it eventually will not be degraded in body. PLL and chitosan are biodegradable and safer, but also inefficient due to less cationic amino in their structures. Even a small amount of PLL or chitosan nanoparticles enter into cells, they cannot escape from lysosome successfully. Melittin (M) is a novel peptide that can keep high membrane-penerating ability at acidic environment, which can be weakened a lot at neutral environment. This ability makes M peptide "intelligent". It was reported low-effective nanoparticles changed to hold the "lysosome escape" ability after they were modified by M peptide, which was more powerful than PEI.V peptide can target the malignant glioma cells. It was reported that V peptide can specifically bind with U87glioblastoma cells but not with the glial cell lines. In this part, V and M peptide were both used to polish the PLL nanoparticle, to make the nanoparticle possess glioma-targeting and lysosome-escaping ability, and become high-effective and safe.ObjectiveTo construct the nanoparticles based on PLL, V peptide and M peptide, and test if they can specifically transfect glioma cells.Methods1. Cell cultureThe protocols of primary neural cell, glioma cell culture and immunofluorescence were as described in Part I2. Plasmid DNA amplificationPlasmid DNA amplification was carried out as those described in Part I.3. Preparation of copolymer PLL-V and PLL-MFor the first group, PLL (1μmol) in1ml HBS (0.5M NaCl,20mM HEPES, pH7.4) was mixed with SPDP dissolved in DMF (100mM:50,100and200μl respectively) under the protection of argon for2h at RT. Then three kinds of pyridyldithiol-activated PLL was got and named as PLL-PDP5, PLL-PDP10and PLL-PDP20. The PLL-PDP copolymer was purified with PD-10column. The concentration of PLL was measured by TNBS assay. The level of SPDP-modification was determined by pyridine-2-thione assay. For synthesis of PLL-VTW (PLL-V) and PLL-Melittin (PLL-M) copolymers, VTW and melittin peptide (0.5,1and2μmol) were repectively dissolved in900μl GnHCl buffer (2M guanidine hydrochloride (GnHC1),0.5M NaCl,20mM HEPES, pH8.0) and100μl DMF, and then stirred with PLL-PDP5, PLL-PDP10and PLL-PDP20respectively (0.1μmol) in1ml GnHC1 buffer under the protection of argon at RT for2h. The subsequent purification were performed using PD-10column. All polymer solutions were filtered through0.22μm Millipore membranes and were stored at-80℃.5. Preparation of nanoparticlesDifferent kinds of PLL-V and PLL-M copolymers were dissolved in HBG buffer (20mM HEPES,5%w/v glucose, pH7.4) and mixed with each other in equal volume to make stock solutions. Then, pDNA solution with different N/P ratio was added into the polymer solutions separately under vortex for30sec and incubated for30min at RT. Then the PLL-V^PLL-M and PLL-VM nanoparticles were formed. The characteristics of nanoparticles were examined with the methods of1H NMR, particle size, zeta potential, transmission electron microscopy (TEM) and agarose gel electrophoresis assay.6. In vitro transfection studyThe basal conditions of transfection included:N/P=10/l, DNA=0.1mg/ml, transfection time=5h, transfection medium=serum-free DMEM. The U87, T98G, U251and primary glioma cells were transfected with all the nanoparticles to assess transfection efficiency. Naked DNA, PLL/DNA nanoparticle and Lipofectamine2000were used as vector control. PC12, A549and primary neural cells from rat cerebral cortex were used as cell control. All the experiments were performed in triplicate.7. Mechanism of transfectionThe mechanism of transfection was investigated via competition assay. Glioma cells were pre-incubated with1000molar times excess of free V peptide compared with V peptide conjugated to nanoparticles at37℃for1h. Then the transfection assay with different nanoparticles was performed as mentioned in Part I.8. Cytotoxicity assayCytotoxicity was determined using a CCK-8Assay Kit according to the manufacturer’s instruction.9. Statistical analysisData analysis methods were the same with those in Part I Results1. PLL conjugationThe yield rates of PLL-V and PLL-M polymers were78.3±1.69%and79.2±2.37%respectively according to TNBS assay. The conjugation rate of PLL-PDP was89.3±2.27%according to pyridine-2-thione assay.3. Characteristics of nanoparticlesconfirmed the successful conjugation of PLL and V peptide. The particle size of PLL nanoparticle was stable at72.3±3.61nm when the dropping speed was10s and the vortex speed was moderate. It was found that PLL、PLL-V5or-M5、 PLL-V10or-M10、PLL-V20or-M20nanoparticles completely condensed DNA at the N/P of3,4,6,8via agarose gel electrophoresis. The particle size of all the modified nanoparticles was larger than that of PEI nanoparticle, and zeta potential was lower。 All nanoparticles were spherical observed by transmission electron microscopy (TEM). Anti-enzyme assay confirmed that all nanoparticles could protect DNA from degradation by DNase I4. In vitro transfection evaluationThe transfection efficiency to U87cell by all kinds of modified PLL nanoparticles was increased. PLL-V20M20nanoparticle led to the highest transfection efficiency to U87cell. In addition, the DMEM with serum could reduce the efficiency of Lipofectamine and PEI nanoparticle notably, while almost did not affect PLL-V20M20nanoparticle. To other glioma cells, including T98G, U251and primary glioma cells, the efficiency was improved best by PLL-V20M20nanoparticle. All nanoparticles didn’t work on primary neural cells.5. CytotoxicityThe cytotoxicity of PLL-V20M20nanoparticle was lower than that of PLL nanoparticle (P<0.01) and Lipofectamine (P<0.05). The cytotoxicity of PLL-V20M20nanoparticles were very low to neural cells compared with that of PLL nanoparticle and Lipofectamine.6. Mechanism of transfection by PV nanoparticles Competition assay showed that the transfection efficiency of PLL-V20M20nanoparticles to glioma cells were decreased obviously after pre-incubation with free V peptide (P<0.001), which confirmed that the specific uplifting transfection efficiency was indeed mediated via V peptide and M peptide. The efficiency improvement of PLL-M20nanoparticle could not be inhibited by the free V peptide showed it was not mediated via V peptide but only via M peptide.ConclusionsThe targeting PLL-V20M20nanoparticles can specifically and effectively deliver pDNA into several kinds of glioma cells especially glioblastoma cells, withour harming neural cells.