Roles Of Polyethylene Glycol Coated Gold Nanoparticles Biology Essay

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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.

Eukaryotic cell transfection is a widely used method for gene therapy. With the help of carrier, the deliveration of nucleic acids to cell can be done. There are two groups of methods used in research. Using viral vectors reach highest efficiency. However, cytotoxicity and immune response triggered are major undesirable drawbacks. Another method is using electroporation, cationic liposomes and polymers and nanoparticles are also employed in research. Due to the limitation low transfection efficacy by cellular uptake, gene therapy by gene deliveration still faces great challenges.

1.2 Therapeutic effect of siRNA

1.2.1 Function of siRNA

Among transfection of nucleic acids to cells, siRNA is the one found to have considerable therapeutic effect. 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.

1.2.2 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, and disease caused by virus infection like chronic liver disease due to HCV infection. Yet, researches often encounter obstacles as siRNA deliverance varies from treatment to treatment.

1.2.3 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. 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 carriers are needed to assist siRNA being internalized by cell.

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.

2. Carriers used for improving transfection

2.1 Carriers used for nucleic acids

Transport of bare siRNA to eukaryotic cells is restricted by body elimination and cell membrane barrier. Carriers are usually nanoparticles, liposomes and polymers can protect nucleic acids from removal hence increase the chance reaching target site. Criteria of an excellent carrying vehicle include low toxic effect, increase cell membrane endocytosis, bind to particular receptor of target cell and load nucleic acid chemically and physiologically stable and small size…etc are essential.

2.2 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. Nucleic can not be released even they arrived to the target site. In addition, transfection efficacy is inversely proportional to molecular size of polymer.

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:

a). Polymer in nature including PLGA-PEG or PLA-PEG (Avgoustakis, 2004)

b). Metal in nature: Gold nanoparticle

c). Semiconductor: Iron oxide nanoparticle, Zinc oxide nanoparticle

Iron oxide (Fe) nanoparticles have magnetic property that is commonly used for cancer research. Magnetic resonance imaging can show delivery of Fe nanoparticles. Besides these groups, the nanoparticles often have chemical modification so to improve the carrier performance.

3. Cellular uptake of nanoparticles

Nanoparticles are nanometer sized molecules which are able internalized by cell through different mechanisms.

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. Uptake system also depends on surface charge of nanoparticles. Positive charged nanoparticles have better interaction with cell membrane. In addition, with the presence of ligand, nanoparticles can bind to surface receptor and undergo receptor mediated endocytosis. Fig.1 shows the some possible factors affecting cellular uptake.

Figure 1: Factors affecting nanoparticles uptake by cell

Figure 2. Structure of Au-PEG-siRNA

Figure 3: Structure of NMDA receptor

4. NMDA receptor function and NR2B

NMDA receptor is an ionotropic and glutamate receptor (shown in figure 3) which consist subunit NMDAR1 and 4 NMDAR2 subunits include NR2B subunit. 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 disease. Excitotoxicity occurs when the NMDA receptor is excessive stimulated. The outcome is high concentration of calcium ion influx to cell and cause death.

Neurodegenerative disease including Parkinson disease can be a potential healing target by lower neuronal cell death. Parkinson disease refers to death of dopaminergic neurons in substantial nigra and cause motor symptoms such as tremor. Drugs (e.g. L-DOPA) developed nowadays can only relief symptoms but not cure.

NR2B is the target protein in the project. NR2B-specific siRNA is used to suppress the NR2B formation. Once the subunit can not develop, the NMDA receptor is not functional. Therefore, it prohibits cell death due to over-stimulation of NMDA receptor. Neuroprotection to disease is achieved.

5. 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 also dopaminergic in nature. Therefore, treatment targeting to dopaminergic cells can use SH-SY5Y to undergo 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.

Reagents used in project

Gold nanoparticle coated with polyethylene glycol with siRNA embedded (Au-PEG-siRNA) were formed by mixing

Dulbecco's modi¬ed Eagle's medium /F12 (DMEM/F12) contains

a). 80ml of complete growth medium

b). 8ml Fetal bovine serum

c). 0.8ml Penicillin-Streptomycin-Neomycin (PSN)

3. Serum free Dulbecco's modi¬ed Eagle's medium (DMEM/SF) is same as DMEM/F12 except without 0.8ml PSN added.

4. Blocking agent (5% milk with TBST)

5. Primary antibody (NR2B binding) with rabbit NR2B and 2% milk in 1:500 ratio.

6. Secondary antibody contains Goat Anti-rabbit Ig HRP Conjugate with 2% milk (1:2000)

7. Primary antibody (β-actin binding) contains mouseβ-actin and 2% milk in 1:2000 ratio.

8. Secondary antibody (binds specifically to 7.) contains Goat Anti-mouse Ig HRP Conjugate with 2% milk (1:5000)

9. Westsave (western blot substrate) with Reagent A and Reagent B (500:1)


Confocal fluorescence microscopy

1.1 Treatment with drugs

On the day before treatment (Day 1), 5x104 of SH-SY5Y cells are seeded per well on microscopic glass slide in a 4 well plate with completed growth medium. On day 2, each well is treated with different drug overnight. The wells are set as follow:

a). 84μl of 6X Au-PEG-siRNA with 416μl DMEM SF free medium

Au concentration: 1.5nM and siRNA concentration: 160nM

b). 84μl of Au-PEG with 416μl DMEM SF (Au concentration: 1.5nM)

c). 84μl of siRNA with 416μl DMEM SF (siRNA concentration: 160nM)

d). Control set up with 500μl DMEM SF

On day 3, all drugs and medium are discarded. PBS is added to rinse remaining medium. The cells are fixed by fixative. Then primary antibody, rabbit NR2B with PBS trition X 0.1% containing 2% NGS in 1:500 dilution, is added to each well. The 4 well plate was then wrapped by parafilm and kept under 4 degree Celsius overnight.

On day 4, primary antibody is discarded. PBS was used and washed for two times. Secondary antibody anti-rabbit Alexa 488 with PBS in 1:500 concentration was added to each well for two hours at room temperature. Glass slide on each well are fixed to glass slide for confocal microscope examination using multi-Argon laser at 488nm. Signal intensity for each image was measured using metamorph.

1.2 Treatment with double concentration of drugs

The treatment steps are same as 1.1 except the drugs are in higher concentration. For Au-PEG-siRNA, Au-PEG and siRNA added in each well, the concentration of Au and siRNA are 3nM and 320nM respectivelty.

2. Lactate dehydrogenase (LDH) cytotoxicity Assay

SH-SY5Y cells were seeded into 96-well plate 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 SF overnight. High control, low control and background were also set. 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 pate and triplicate after centrifugation. A freshly prepared reaction mixture (catalyst and dye solution in 1:45 concentration) was added to each well and incubates at room temperature for 30 minutes in dark. Absorbance of each well was measured at 490nm by ELISA reader. Cytotoxicity was calculated by this equation:

Cytotoxicity (%) = {[(Experimental result - background) - (Low control - background)] / [(High control - background) - (Low control - background)]} x 100%

Western Blotting

8 x 105 SH-SY5Y cells were incubated in a 6-well plate with DMEM F12 overnight before treatment. On day 2, treatments were added overnight (Au-PEG-siRNA, Au-PEG, siRNA and control) with same concentrations with methodology 1.1 to each well 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. Diluted protein by MilliQ was added to 96-well plate (3 trial per sample) and react with Reagent A and Reagent B. Then protein analysis was done by measuring absorbance at 750nm.

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 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 was added overnight at 4 °C. Then the secondary antibody was added for 1 hour after rinsing all unbounded primary antibody.

After rinsing with TBST and TBS, 1ml of Westsave Up (substrate) was added to PVDF membrane and plastic wrap wrapped around the membrane. Expose the film 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.

4. 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 anova and Paired student's 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.


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. The predictable result is Au-PEG-siRNA got the lowest NR2B expression level.

Confocal fluorescent microscopy

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, 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, 1997). The hydrophilic property (Lee, 2009) can elevate solubility for the lipid and oligonucleotides conjugates. (Zalipsky, 1997). They are not classified as antigen so no elimination reaction triggered even introduced to body (Andrade, 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, 2010). Some study suggested that PEG can increase cellular uptake due to the ability to interact with cell membrane (Zhang, 2002).

Gold nanoparticles are chemically stable, allow surface alteration and ranges of molecular size can be prepared (Shah, 2010). The uptake mechanism depends on warmer temperature (Shukla, 2005), smaller size aid pinocytosis.

In the project, PEG is conjugated with Au and siRNA. The higher density of PEG added can form coil intense coated around Au. PEG has thoil group attached (PEG-SH). Disulphide bond is formed though -SH interact with gold nanoparticle surface. siRNA is trapped in between the polymer and nanoparticle. With the specific property of nanoparticles mentioned above, gold nanoparticle is used to carry siRNA to target cell. Gold nanoparticles can disrupt cell membrane when they have electrostatic attraction (showed in figure 2). The endocytosed nanoparticle enters 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 4. Graph showing relationship between surface charge and cytotoxicity and transfection efficacy. (Lin, 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 nanoparticle - [email protected](RITC) can across blood brain barrier and other organs. It also retains at body for a long time. (Kim, 2005). Instead of magnetic nanoparticle, gold nanoparticles were used as the reported cytotoxicity level is lower for transfection.

Titanium Oxide (TiO2) nanoparticle had showed triggering neutrophil production in rodent model. The increases in polymorphonuclear granulocyte indicate infection or inflammation in pulmonary area (Oberdörster, 2005).

Iron oxide nanoparticles nanotoxicity may due to generation of reactive oxygen species and cause oxidative stress to cells (Singh, 2010). The toxicity levels differ when the nanoparticles were introduced to cell with or without polymer coating (Mahmoudi, 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.

siRNA is frequently used in many research 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, 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 nanoparticle for transfection.

Au-PEG was found to lower NR2B formation in cells. Investigation of Au-PEG interaction with SH-SY5Y cells should be done.


Biocompatiblity, extremely small in size and surface modifiable to Polyethylene Glycol are reasons for gold nanoparticle employed as carrying molecule in project. Au-PEG-siRNA enhance NR2B speicifc 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 protin. 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 nanoparticle 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