In the present study, NR2B subunit of the N-methyl-D-aspartate receptor (NMDAR) is the focus of research. NR2B plays important roles in neuroprotection. NR2B protein is one of NMDA receptor subunit. The incomplete glutamate gated NMDA receptor is not functional. Therefore, NR2B suppression prohibits high concentration calcium ion influx into neuronal cell. Excitotoxicity and cell damage should be reduced. However, transfection of genetic material to eukaryotic cells is difficult especially for well differentiated cell such as neuronal cells. In order to enhance transfection efficiency, polyethylene glycol coated gold nanoparticles (Au-PEG) was chosen as siRNA carrying vehicle. Transfection efficiency improves showing NR2B suppression by gene silencing. In the present study, thiol group attached polyethylene glycol (PEG-SH) was added to gold nanoparticles. Au-PEG was held together by strong sulphur bond. The PEG polymer shielded siRNA from degradation. siRNA was embedded near Au surface and release when PEG degrade after cellular uptake. NR2B specific siRNA had been found to undergo gene silencing successfully with the presence of Au-PEG. Having comparison between Au-PEG-siRNA and siRNA added SH-SY5Y cells under confocal microscope, NR2B expressions in SH-SY5Y cells decreased by fluorescent intensity. Au-PEG was found to have low cytotoxicity and biocompatibility and these facilitate NR2B transfection. Neuroprotection may be archieved due to the raise of transfection efficacy with Au-PEG.-siRNA.
Table of Contents
Declaration i
Acknowledgement ii
Abstract iii
Table of contents v
List of tables and figures vii
Table of Abbreviation viii
1. Introduction
1.1 Transfection P.1
1.1.1 Gene delivering methods in research P.1
1.1.2 Function of siRNA P.1
1.1.3 Possible therapeutic effect by RNA interference P.2
1.1.4 Challenges in siRNA transfection P.2
1.2 Carriers used for nucleic acids for transfection improvement P.3
1.3 Polymer conjugated nucleic acid delivery P.4
1.4 Cellular uptake of nanoparticles P.4
1.5 NMDA receptor function and NR2B P.7
1.6 SH-SY5Y neuroblastoma P.7
2. Objectives P.10
3. Methodology
3.1. Cell culture P.11
3.2 Lactate dehydrogenase cytotoxicity Assay P.11
3.3 Immunofluoresence
3.3.1 Treatment with drugs (160nM siRNA, 1.5nM Au) P.12
3.3.2 Treatment with drugs (160nM siRNA, 1.5nM Au) P.13
3.4 Western blotting P.14
3.5 Data Analysis P.15
4. Results and Observations
4.1 Immunofluoresence
4.1.1 Treatment with drugs (160nM siRNA, 1.5nM Au) P.16
4.1.2 Treatment with drugs (320nM siRNA, 3.0nM Au) P.17
4.2. Lactate dehydrogenase cytotoxicity Assay P.17
4.3. Western blotting P.18
5. Discussion P.27
6. Further investigation P.35
7. Conclusion P.37
8. References P.38
9. Appendix P.44
List of tables and figures
Figure 1: Factors affecting nanoparticles uptake by cell P.6
Figure 2: Structure of NMDA receptor P.8
Figure 3: Images from confocal fluorescence microscopy of SH-SY5Y cells incubated with treatment (160nM siRNA, 1.5nM Au) at 40x magnification P.19
Figure 4: Images from confocal fluorescence microscopy of SH-SY5Y cells incubated with treatment (160nM siRNA, 1.5nM Au) at 60x magnification P.20
Figure 5: NR2B expression level under different treatment P.21
Figure 6: Images from confocal fluorescence microscopy of SH-SY5Y cells incubated with treatment (320nM siRNA, 3.0nM Au) at lower magnification P.22
Figure 7: Images from confocal fluorescence microscopy of SH-SY5Y cells incubated with treatment (320nM siRNA, 3.0nM Au) at higher magnification P.23
Figure 8: NR2B expression level again versus Au-PEG-siRNA concentration P.24
Figure 9: Graph showing cytotoxicity to SH-SY5Y cells induced by different concentrations by Au-PEG P.25
Figure 10: Graph showing NR2B level under different treatment in western blot P.26
Figure 11. Structure of Au-PEG-siRNA P.30
Figure 12: Graph showing relationship between surface charge and cytotoxicity and transfection efficacy P.31
Au
Gold nanoparticles
Au-PEG
Gold nanoparticles coated with polyethylene glycol
Au-PEG-siRNA
Gold nanoparticles coated with polyethylene glycol with siRNA embedded
ALS
Amyotrophic lateral sclerosis
DMEM
Dulbecco's modiï¬ed Eagle's medium
MNPs@SiO2(RITC)
Silica-overcoated magnetic nanoparticles containing rhodamine B isothiocyanate
NMDA receptor
N-methyl-D-aspartate receptor
NR2B
NGS
N-methyl D-aspartate receptor subtype 2B
Normal Goat Serum
PVDF membrane
Polyvinylidene Fluoride
PEG
Polyethylene glycol
PLA
Polylactide
PLGA
poly(lactic-co-glycolic acid)
siRNA
Small interfereing RNATable of Abbreviation
Introduction
1.1 Transfection
1.1.1 Gene delivering methods in research
Eukaryotic cell transfection is a widely used method for gene therapy. With the help of carrier, the delivering of nucleic acids into cell can be done. There are two groups of methods can be used in research. Firstly, using viral vectors can lead to the highest efficiency. However, cytotoxicity and immune response triggered are the major undesirable drawbacks (Luo and Saltzman, 2000). Another method methods included, using electroporation, cationic liposomes and polymers (Tong et.al, 2009) or nanoparticles which were employed in present research. As low transfection efficacy by cellular uptake was one of the major limitations in gene silencing therapy, application of gene therapy by using gene delivering still facing a great challenge.
1.1.2 Function of siRNA
Among transfection of nucleic acids to cells, siRNA is the one found to have considerable therapeutic effect (Wu et. al., 2010). siRNA belongs to microRNA which is 20-22 nucleotide long double stranded RNA. RNA interference involves binding of siRNA to the complementary mRNA in cytoplasm and facilitates removal by enzyme. Translation processes of mRNA by tRNA fail to proceed. Consequently, siRNA indirectly reduce target protein formation (Hammond et.al., 2006).
1.1.3 Possible therapeutic effect by RNA interference
Base on the RNA interference mechanism, siRNA is applied to cure disease. The hypothesis of specific siRNA can cope with target genes and down regulate the gene expression. In gene therapy, the future medical application by transfection can involve genetic diseases such as ALS, cancer treatment (Alexiou et. al., 2006), and disease caused by virus infection like chronic liver disease due to HCV infection (Aagaard and Rossi, 2007). Yet, researches often encounter obstacles as siRNA deliverance varies from treatment to treatment.
1.1.4 Challenges in siRNA transfection
One of the major barriers of siRNA transfection is the initiation of immune response. According to Hornung's research, the siRNA is noticed by Toll-like receptor 7. Interferon-alpha was released due to TLR7 activation (Hornung et. al., 2005). TLR recognize siRNA as antigens so that they will be eliminated either by phagocytosis or enzymatic reaction.
Low rate in cellular uptake is also a concern of transfection efficacy in siRNA. Cellular uptake is not favored by the negative charged in nature of nucleic acid.
Chemical alteration or conjugated with carriers are needed to assist siRNA being internalized by cell (Gary et. al., 2007).
Cases of down regulation of non-target gene was reported which can bring out unwanted phenotype. Non specific interaction of siRNA to gene needs modification to increase target gene binding specifically (Fedorov et. al., 2006).
1.2 Carriers used for nucleic acids for transfection improvement
Transporting bare siRNA to eukaryotic cells is restricted by body elimination and cell membrane barrier. Carriers that usually used including nanoparticles, liposomes and polymers (Gary, 2007) can protect nucleic acids from removal. Hence increase the chance reaching the target site. Criteria of an excellent carrying vehicle include low toxic effect (Wu. 2010), increase cell membrane endocytosis, bind to particular receptor of target cell and load nucleic acid (Wakebayashi, 2004). Furthermore, chemically and physiologically stable and small in size are also essential.
1.3 Polymer conjugated nucleic acid delivery
Researches make use of cationic polymers as carriers as it favors ionic exchange between negative charged cell membrane. Electrostatic attraction links between RNA/DNA with polymer. On the other hand, the attraction force is also a hindrance of transfection which lead to the nucleic acid cannot be released even they arrived at the target site. (Gary, 2007) In addition, transfection efficacy is inversely proportional to molecular size of polymer (Abdallah, 1996).
The above findings lead to less likely use of polymer as the only carrying vehicle. Yet, polymer is non-immunogenic in nature is still a great advantage in research. Instead, polymer coated nanoparticles or complex polymer nanoparticles are widely utilized. Nanoparticles can be briefly divided into: polymer in nature including PLGA-PEG or PLA-PEG (Avgoustakis, 2004), metal in nature such as gold nanoparticles or semiconductor for example, iron oxide nanoparticles (Sun et.al, 2010) and zinc oxide nanoparticles.
Iron oxide (Fe) nanoparticles have magnetic property that is commonly used for cancer research. Magnetic resonance imaging can show delivery of Fe nanoparticles (Sun et.al, 2008). Besides these groups, the nanoparticles often have chemical modification so to improve the carrier performance.
1.4 Cellular uptake of nanoparticles
Nanoparticles are nanometer sized molecules which are able internalized by cell through different mechanisms (Bailey and Koleske, 1991).
There are various kinds of nanoparticles present. The size, overall surface charge, physical and chemical properties are different. The ability and mechanism internalized by cells also varies.
The smaller size the nanoparticle, the better will be the cellular uptake. For nanoparticles larger in size (>500nm mention by Rejman's research in studying B-16 cells), other energy consuming endocytosis pathway are involved (Rejman et. al., 2004). Uptake system also depends on surface charge of nanoparticles. Positive charged nanoparticles have better interaction with cell membrane (Lin et. al., 2010). In addition, with the presence of ligand, nanoparticles can bind to surface receptor and undergo receptor mediated endocytosis (Zheng et. al., 2005). Some possible factors that affecting cellular uptake were shown in figure 1.
Figure 1: Possible factors affecting nanoparticles uptake by cell. (A) Smaller nanoparticle size favors internalization. (B) Cationic nanoparticles take up by negatively charged cell membrane than anionic charged nanoparticles. (C) Receptor mediated endocytosis reaction by ligand-bound nanoparticles. (D) Protein transduction domain bound nanoparticles allow fast internalization. (E) Nanoparticles localized on particular size depends on complementary DNA (F) Cationic nanoparticles released more after endocytosis
1.5 NMDA receptor function and NR2B
NMDA receptor is an ionotropic and glutamate receptor (Figure 2) which consist different subunits including NMDAR1 and NMDAR2 subunits (Rauner and Kohr, 2011). Activation of NMDA receptor helps in long term potentiation which relates to learning. Also, NMDA receptor plays important role in developing memory and also diseases including stroke and Parkinson's disease (Longstaff, 2005). Excitotoxicity occurs when the NMDA receptor is excessive stimulated which lead to the influx of high concentration of calcium ion into cell and cause cell death.
Neurodegenerative disease including Parkinson's disease can be a potential healing target by lower neuronal cell death. Parkinson's disease refers to death of dopaminergic neurons in substantial nigra and cause motor symptoms such as resting tremor (Addy et. al., 2009). Drugs (e.g. L-DOPA) developed nowadays can only relief symptoms but not cure.
NR2B is the target protein in present project. NR2B-specific siRNA is used to suppress the formation of NR2B protein through a gene silencing mechanism. It was hypothesized that avoiding the development of the subunit of the receptor will lead to the reduction in functional NMDA receptors present in cell. Therefore, it can prohibit cell death that due to over-stimulation of NMDA receptors and potentially lead to the neuroprotection to the disease. (Bear and Connors, 2005)
Figure 2: Structure of NMDA receptor
1.6. SH-SY5Y neuroblastoma
SH-SY5Y neuroblastoma was originated from bone marrow of a cancer patient. The advantage of using SH-SY5Y cells in project as it is a neuronal like and dopaminergic in nature. Therefore, treatment targeting to dopaminergic cells can be applied on SH-SY5Y in the experiment. In addition, due to the properties of cancer cells, the proliferation rate is high. It facilitates the research work in which needs large amount of cells per treatment within very short interval.
SH-SY5Y cells are unlikely to differentiate into other types of cell. The biological stability also helps in research work.
Objectives
The objective in the project was to examine any improvements of siRNA transfection efficacy using Gold nanoparticles coated with polyethylene glycol (Au-PEG) as carrier. The result was obtained by comparing NR2B expression level of SH-SY5Y cells of treatment sets using confocal fluorescence microscopy and western blotting.
Also, nanotoxicity of Au-PEG at different concentration to SH-SY5Y neuroblastoma was examined. By performing Lactate dehydrogenase (LDH) cytotoxicity Assay, the relationship between cell viability and Au-PEG concentration was studied. Hence, we can study whether Au-PEG was a potential good transfection carrier.
Methodology
3.1 Cell culture
Human neuroblastoma SH-SY5Y cell line was cultured in DMEM F12 completed medium (Appendix III). Cells were grown in a humidified cell incubator at 37℃ with 5% CO2 provided.
3.2 Lactate dehydrogenase (LDH) cytotoxicity Assay
SH-SY5Y cells were seeded onto 96-well plates with 5 x 104 cells per well with 200μl DMEM/F12 completed growth medium overnight allowing cell attachment. On day 2, different concentrations of Au-PEG were added to the SH-SY5Y cells (3nM, 1.5nM, 0.15nM and 0.025nM) with 200μl DMEM/F12 serum free medium and incubated at 37℃ with 5% CO2 overnight. High control, low control and background experiments were also set. High control contained SH-SY5Y cells with 2% Triton X treatment to kill all cells. Cytotoxicity of treatments was calculated base on comparison absorbance of treatment to high control. Low control contained SH-SY5Y cells with DMEM/F12 SF examine normal cell viability without treatment. Experimental result was needed to subtract the absorbance of low control to examine cell death due to drug treatment. Background only contained medium that need ed subtraction to eliminate background absorbance. The set up was in triplicate to increase reliability of the test. On day 3, supernatant of each concentration was added to new 96-well plate and triplicate after centrifugation. A freshly prepared reaction mixture according to the supplier's instructions (Cytotoxicity Detection Kit, Roche) was added to each well and incubates at room temperature for 30 minutes in dark. Absorbance of each well was measured at 490nm by using microplate reader (BioTek, ELx800TM). Cytotoxicity was calculated by this equation:
Cytotoxicity (%) = {[(Experimental result - background) - (Low control - background)] / [(High control - background) - (Low control - background)]} x 100%
3.3 Immunofluorescence
3.3.1 Treatment with drugs (160nM siRNA, 1.5nM Au)
SH-SY5Y cell were plated onto 4-well plates at a density of 5 x 104 cells/ml in medium in volume in 200µl per well. The day after this plating, each well was treated with different kind of drugs overnight. The wells were set as follow:
84μl of 6X Au-PEG-siRNA with 416μl DMEM/F12 SF medium
(Au concentration: 1.5nM and siRNA concentration: 160nM)
84μl of Au-PEG with 416μl DMEM/F12 SF (Au concentration: 1.5nM)
84μl of siRNA with 416μl DMEM/F12 SF (siRNA concentration: 160nM)
Control set up with 500μl DMEM/F12 SF
On day 3, all drugs and medium were discarded. PBS was added to rinse remaining medium. The cells were fixed by fixative for 30 minutes. Then primary antibody, rabbit anti-NR2B (Millipore; Appendix I) in PBS containing 0.1% Triton X (USB) and 2% NGS (Vector Labs) in 1:500 dilution, was added to each well. The 4-well plates were then wrapped by parafilm and kept under 4℃ overnight.
On day 4, primary antibody NR2B was discarded. PBS was used and washed for two times. Secondary antibody anti-rabbit Alexa 488 (Molecular Probe; Appendix II) in PBS in 1:500 concentrations was added to each well and incubated for two hours at room temperature. After that, all the cells were mounted on clean slides with mounting medium (DAKO) for confocal microscope (FluoViewTM FV1000, Olympus) examination using multi-Argon laser at 488nm. Signal intensity for each image was measured using Metamorph.
3.3.2 Treatment with drugs (160nM siRNA, 1.5nM Au)
The treatment steps were same as that mentioned in chapter 3.3.1 except the drugs were applied in higher concentration. For Au-PEG-siRNA, Au-PEG and siRNA were added in each well, the concentration of Au and siRNA were 3nM and 320nM respectively.
Western Blotting
8 x 105 SH-SY5Y cells were incubated in 6-well plates with DMEM/F12 overnight before treatment. On day 2, treatments were performed and the cells were incubated with different tested solution (Au-PEG-siRNA, Au-PEG, siRNA and control) with same concentrations as those mentioned in chapter 3.3.1, overnight respectively. On day 3, protein extraction was done by collecting cell lysate using 10μl lysis buffer and homogenizer. Protein extract was collected from supernatant after centrifugation at 14000rpm under 4°C for 30 minutes. Protein concentration was quantified by DC Protein Assay kit (Bio-Rad Laboratories, Inc.). Then protein analysis was done by measuring absorbance at 750nm using microplate reader (ELx800TM, BioTek).
Each treatment with same protein concentration was added to stacking gel and run along 6% resolving gel. Electrophoresis was run at 60V at room temperature until the marker band reached near the bottom of gel. Protein in gel was transferred onto PVDF membrane (Bio-Rad Laboratories, Inc.) by gel sandwich in Mini Tank contains Transfer buffer. The set up stayed overnight at 110mA. The membranes were washed in TBST for 10 minutes and TBS for three times. Membrane was incubated for 1 hour in blocking agent then rinsed by TBST and TBS. Primary antibody NR2B in 2% milk (see Appendix I) was added overnight at 4 °C. Then the secondary antibody (see Appendix II) was added for 1 hour after rinsing all unbounded primary antibody. After rinsing with TBST and TBS, 1ml of ECL western blot detection reagents (Amersham Biosciences, England) was added to PVDF membrane and plastic wrap wrapped around the membrane. Expose the film (Kodak) to the substrate for 30 seconds. Develop the film in developer for 30 seconds and then to fixer for 30 seconds. Rinse the film with tap water and allow air dry.
3.5 Data Analysis
Data in each treatment was plot in bar chart and mean with standard error of mean (S.E.M). Using SPSS, one way ANOVAs and Paired T-Test were used to prove level of significance in the result. Only the result which has p-value smaller than 0.05 will considered as significantly different. Stars were given to show level of significance. Three stars will be given for P< 0.001. Two stars will be given for P< 0.01. One star will be given for P<0.05.
Results and observations:
Immunofluoresence
Treatment with drugs (160nM siRNA, 1.5nM Au)
Both confocal images captured in 40x and 60x magnification showed similar trend of fluorescent signal generation (Figure 3 and 4). By comparing the immunoreactivity among the four treatments, Au-PEG-siRNA treated cell showed the lowest immunoreactivity for NR2B. Down-regulation of NR2B protein expression in terms of fluorescence signal intensity was shown in Au-PEG-siRNA treated SH-SY5Y cells. Au-PEG and siRNA treated SH-SY5Y cells express similar intensity level with control which without underwent any treatment. Statistical analyses of the comparison of immunoreactivity for NR2B in SH-SY5Y cells treated with different treatments were done. Each bar represents the mean of NR2B protein expression level expressed in terms of immunoreactivity in different treatments (Figure 5). Significant decrease in immunoreactivity for NR2B was observed in Au-PEG-siRNA, Au-PEG and siRNA treated cell when compared with control. Moreover, the NR2B protein expression was mostly down-regulated after the treatment with Au-PEG-siRNA.
Significant decrease in immunoreactivity for NR2B was observed for Au-PEG-siRNA treated SH-SY5Y cells compare with siRNA treated SH-SY5Y cells. NR2B protein expression was further down-regulated by Au-PEG-siRNA.
Treatment with drugs (320nM siRNA, 3.0nM Au)
Confocal images captured in 20x and 60x magnification showed similar trend of fluorescent signal generation like treatment with 160nM siRNA, 1.5nM Au in chapter 5.1.1.1 (Figure 6 and 7). Down-regulation of NR2B expression in terms of fluorescence signal intensity was shown in Au-PEG-siRNA treated SH-SY5Y cells. Au-PEG and siRNA treated SH-SY5Y cells express similar intensity level with control. Statistical analysis by Metamorph was done (Figure 8). The comparison between the mean of NR2B protein expression level in confocal fluorescence images of different Au-PEG-siRNA concentration (160nM siRNA, 1.5nM Au versus 320nM siRNA, 3.0nM Au) treatment to SH-SY5Y cells was done. However, no significant difference in the immunoreactivity for NR2B was found between two treatments.
Lactate dehydrogenase (LDH) cytotoxicity Assay
Figure 9 is a graph compares the cytotoxicity evoked by Au-PEG in different concentration. The cytotoxicity significantly increased for 1.5nM of Au-PEG and 3.0nM of Au-PEG when compare with the lowest concentration 0.025nM Au-PEG. The higher the concentration, the higher the cytotoxicity evoked.
Western blotting
Form the western blotting result, the Au-PEG-siRNA treated SH-SY5Y cells showed the lowest protein level of NR2B when compared with the control (Figure 10). β-actin (42 kDa) in treatment was used as control to show equal concentration of protein was loaded in each lane for electrophoresis. NR2B protein expression from the band was analyzed by Metamorph in terms of optical density. Each bar represents the NR2B protein expression level expressed in terms of optical density in different treatments. NR2B expression level of Au-PEG-siRNA expressed least NR2B. Au-PEG expressed less NR2B protein than siRNA and control. siRNA also showed lowering NR2B protein expression compare with control.
B
A
D
C
Figure 3: Images from confocal fluorescence microscopy of SH-SY5Y cells incubated with treatment (160nM siRNA, 1.5nM Au) at 40x magnification. (A): Au-PEG-siRNA; (B): Au-PEG; (C): siRNA; (D): control
B
A
D
C
Figure 4: Images from confocal fluorescence microscopy of SH-SY5Y cells incubated with treatment (160nM siRNA, 1.5nM Au) at 60x magnification. (A): Au-PEG-siRNA; (B): Au-PEG; (C): siRNA; (D): control
D
C
***
D
C
B
A
P<0.05 = * P<0.01 = ** P<0.001 = ***
Figure 5: NR2B expression level under different treatment. Each bar represents the mean of NR2B protein expression level expressed in terms of immunoreactivity in different treatments. (A): Au-PEG-siRNA; (B): Au-PEG; (C): siRNA; (D): control
B
A
D
C
Figure 6: Images from confocal fluorescence microscopy of SH-SY5Y cells incubated with treatment (320nM siRNA, 3.0nM Au) at 20x magnification (A): Au-PEG-siRNA; (B): Au-PEG; (C): siRNA; (D): control
B
A
D
C
Figure 7: Images from confocal fluorescence microscopy of SH-SY5Y cells incubated with treatment (320nM siRNA, 3.0nM Au) at 60x magnification (A): Au-PEG-siRNA; (B): Au-PEG; (C): siRNA; (D): control
Figure 8: NR2B expression level again versus Au-PEG-siRNA concentration. Left:160nM siRNA, 1.5nM Au; Right: 320nM siRNA, 3.0nM Au
***
***
P<0.05 = *
P<0.01 = **
P<0.001 = ***
3nM
1.5nM
0.15nM
0.025nM
Figure 9: Graph showing cytotoxicity to SH-SY5Y cells induced by different concentrations by Au-PEG. Blue: 3nM; Red; 1.5nM; Green: 0.15nM; Yellow: 0.025nM
NR2B (180kDa)
Au-PEG
siRNA
control
β-actin (42kDa)
A
B
C
D
Au-PEG-siRNA
Figure 10: Graph showing NR2B level under different treatment in western blot. (A): Au-PEG-siRNA; (B): Au-PEG; (C): siRNA; (D): control. NR2B protein expression and β-actin protein expression in western blot analysis.
Discussion:
The set up for confocal fluorescent microscopy and western blot both have four treatments for comparison. Control was set as reference for treatment comparison. Au-PEG-siRNA was the conjugate that is hoped to prove our project aim. However, free siRNA and free Au-PEG were inevitable in the Au-PEG-siRNA conjugate mixture. In order to prove the decline of NR2B expression level was enhanced by Au-PEG-siRNA, not the other two, set up of siRNA and Au-PEG were needed as negative control. The predictable result is the cell treated with Au-PEG-siRNA show the lowest expression of NR2B protein level.
Immunofluorescence
Transfection efficacy of each treatment was measured in terms of NR2B expression level in SH-SY5Y cells. In figure 1.1, comparison between different treatments was done. The control set up had highest NR2B expression level in terms of average gray value. This set was also used as reference for other treatments.
We compare treatment with siRNA and control, NR2B expression level was significantly lowered. siRNA was able to perform RNA interference so that NR2B protein synthesis decreased. The result showed free siRNA without transfection vehicle also transfect SH-SY5Y.
NR2B expression level of Au-PEG-siRNA was the lowest among all the treatments. Au-PEG-siRNA not only achieves gene silencing as free siRNA did, but also further down regulate NR2B protein formation. PEG (2 kDa), siRNA and Au were allowed fuse so that nanometer sized complex formed to transfect SH-SY5Y (Wu et.al., 2010). It is suggested that siRNA transfection efficacy increases with the aid of transfection vehicle. More siRNA can successfully deliver into cells so that NR2B expression reduced.
Polyethylene glycol (PEG) is a non-immunogenic polymer as well as biocompatible to cells. They are applied for molecule conjugation for different function. For instance, conjugate and modify the drug so as to facilitate discharge at target site (Zalipsky et.al., 1997). The hydrophilic property (Lee et. al., 2009) can elevate solubility for the lipid and oligonucleotides conjugate (Zalipsky and Harris, 1997). They are not classified as antigen so no elimination reaction triggered even introduced to body (Andrade et.al., 1996). By wrapping the gold nanoparticles, Retention time in body circulation should be lengthened. Shielding of the gold nanoparticles to the immune system can be achieved (Sun et. al., 2010). Some study suggested that PEG can increase cellular uptake due to the ability to interact with cell membrane (Zhang et. al., 2002).
Gold nanoparticles are chemically stable, allow surface alteration and ranges of molecular size can be prepared (Shah et. al., 2010). The uptake mechanism depends on warmer temperature (Shukla et. al., 2005), smaller size aid pinocytosis.
In the project, PEG is conjugated with Au and siRNA the structure was shown in figure 11. The higher density of PEG added can form coil intense coated around Au. PEG has thiol group attached (PEG-SH). Disulphide bond is formed though -SH interact with gold nanoparticles surface. The siRNA is trapped in between the polymer and nanoparticles. With the specific property of nanoparticles mentioned above, gold nanoparticles are used to carry siRNA to target cell. Gold nanoparticles can disrupt cell membrane when they have electrostatic attraction (showed in figure 12). The endocytosed nanoparticles enter cytosol then release siRNA to bond with target mRNA. If the polymer coated nanoparticles work, cell internalization will increase compare with bare siRNA. Also, more siRNA can reach target site undergo gene silencing of NR2B protein.
Au-PEG was expected to have same NR2B expression level as control set. As Au-PEG has no siRNA conjugated, no RNA interference occurs even Au-PEG enters SH-SY5Y cells. However, the NR2B expression decreased as well. The result was unexpected. As the result was consistent, Au-PEG may have other interaction with NR2B gene which was not studied. Further investigation of Au-PEG and SH-SY5Y interaction is needed to be done.
Figure 11. Structure of Au-PEG-siRNA. PEG coil warp around gold nanoparticles through disulphide linkage. Disulphide bond is formed by thiol group of PEG interact with gold nanoparticles' surfaces. The siRNA is trapped in between the polymer and nanoparticles bond formation process.
Figure 12. Graph showing relationship between surface charge and cytotoxicity and transfection efficacy. (Lin et. al., 2010)
Au-PEG cytotoxicity
Nanotoxicity is raised as a concern but the toxicity and working mechanism are not fully understood. Different kinds of nanoparticles have shown the potential toxicity to eukaryotic cells or in animal model. Reports showed rats were intraperitoneally injected magnetic nanoparticles - MNPs@SiO2(RITC) can across blood brain barrier and other organs. It also retains at body for a long time. (Kim et.al., 2005). Instead of magnetic nanoparticles, gold nanoparticles were used as the reported cytotoxicity level is lower for transfection.
Titanium Oxide (TiO2) nanoparticles had showed triggering neutrophil production in rodent model. The increases in polymorphonuclear granulocyte indicate infection or inflammation in pulmonary area (Oberdörster et. al., 2005).
Iron oxide nanoparticles nanotoxicity may due to generation of reactive oxygen species and cause oxidative stress to cells (Singh et. al., 2010). The toxicity levels differ when the nanoparticles were introduced to cell with or without polymer coating (Mahmoudi et. al., 2010).
One of our aims in the project is to examine whether Au-PEG has potential cytotoxicity. If it greatly decrease cell viability, it is not suitable for therapeutic drug even it bears transfection property.
The siRNA was frequently used in many researches due to the low cytotoxicity. In the project, only Au-PEG was used to verify level of toxicity to eukaryotic cells. In figure 2.1, the cytotoxicity of Au-PEG increases with concentration added to SH-SY5Y. 3.0nM of Au-PEG lead to 19% cytotoxicity which indicated toxic effect at high Au-PEG concentration. 1.5nM Au-PEG had lower cytotoxicity (8.7%). For 0.15nM and 0.025nM of Au-PEG, negative results (-17.6% and -23.8% respectively) were reached. The minimum cytotoxicity should be zero instead of negative percentage. The results were due to higher absorbance in low control than treatment which was a technical error. The 0.15nM and 0.025nM treatment should be considered as very low cytotoxicity.
By grouping the result of confocal fluorescent microscopy and cytotoxicity test, concentration of Au-PEG-siRNA used for research should depend on transfection efficacy and cytotoxicity raised. The best concentration must bring maximum transfection efficiency but not elicit high toxic consequence to cells.
Western blot
Confocal fluorescent microscopy only inspects the signal intensity of parts of SH-SY5Y cells. The result can not fully demonstrate the effect of Au-PEG-siRNA to cells in general. Therefore, western blot was employed to examine overall NR2B expression in SH-SY5Y cells in different treatments. β-actin (42 kDa) in treatment were later tested (Khaitlina et. al. 2001). β-actin is one of the isoform of actin which found in most cells except muscle cells. The test is used as control to show equal concentration of protein was loaded in each lane for electrophoresis. In the result, the band in each lane for each lane has no considerable difference in terms of optical density. The difference of NR2B level in each treatment is due to drug effect.
The NR2B expression levels of Au-PEG-siRNA, Au-PEG, siRNA were compared with control. The trend had a great resemblance to the result of confocal fluorescence microscopy. siRNA restrain NR2B expression but the level was not obvious compare with Au-PEG-siRNA. The level of NR2B was the lowest in the Au-PEG-siRNA treated SH-SY5Y cells. Similar unanticipated result of Au-PEG was reached. The NR2B expressed was even less than the treatment with siRNA.
As the experiment only did once due to time limitation, we should repeat the experiment to achieve consistent result.
Further investigation
One of the project aims is to evaluate whether Au-PEG-siRNA has neuroprotective effect to the SH-SY5Y cells. The experiment requires the LDH assay. Four treaments (Au-PEG-siRNA, Au-PEG, siRNA and control incubate with 5 x 104 SH-SY5Y cells) have 6-hydroxydopamine (6-OHDA) added to the treatment on the next day. Absorbance will show the viable cell remained in each treatment. If the Au-PEG-siRNA has lowered amount of cell death than control, neuroprotective effect can be proved.
Other than nanoparticles, there are other kinds of technique for transfection still under investigation. Electroporation is another method which administers electric pulse to generate pores on cell membrane. The infinitesimal pores provide a channel for siRNA uptake by eukaryotic cells. The transfection efficacy improvement by various methods can be used for assessment.
In the project, the siRNA with polyethylene glycol coated gold nanoparticle had higher NR2B repression effect. Only 3nM and 1.5nM of Au-PEG-siRNA were used in the project. For further investigation, different concentration of Au-PEG-siRNA can be applied to examine NR2B expression level and cytotoxicity. Hence, we can determine the most suitable for transfection. In addition, there are other kinds of nanoparticles and polymers can be selected to look for the best polymer coated nanoparticles for transfection.
Au-PEG was found to lower NR2B formation in cells. Investigation of Au-PEG interaction with SH-SY5Y cells should be done. For further study, we can examine the localization of Au-PEG in cells using Transmission electron microscopy. May be we can see special patterns of transfection or grouping of nanoparticles that relates to the NR2B lowering.
Conclusion
Biocompatiblity, extremely small in size and surface modifiable to Polyethylene Glycol are reasons for gold nanoparticles employed as carrying molecule in project. Au-PEG-siRNA enhance NR2B specific siRNA transfection by decrease NR2B expression significantly which can be observed from confocal fluorescence microscopy and western blot. Compare with siRNA, Au-PEG-siRNA further repress NR2B coding gene to form NR2B protein. The transfection efficacy improves also helped by PEG shielding effect.
NR2B suppression effect has no significant difference even Au-PEG-siRNA doubled concentration. The result may due to saturation of cellular uptake or inhibition at high concentration.
Increase cytotoxicity can be observed for 3.0nM Au (19%) but level of toxicity is also acceptable for lower gold nanoparticles concentration.
Unpredicted lowering NR2B level was found in Au-PEG. Interactions of Au-PEG to SH-SY5Y cells or other mechanisms involved still need further investigation
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