It has been highlighted as a genetic disease with a growing need for the development of innovative gene therapy strategies and, thus, the development of safe and effective means of gene delivery. In this study, the unique properties of AuNPs, cationic polymers, and mRNA molecules were exploited and optimized to provide effective gene delivery vehicles for intracellular delivery of mRNA for future cancer gene therapy applications, an area of research which until recently has not been investigated.
PLAGIARISM
PUBLICATIONS
Aim and Objectives
Functionalization of AuNPs will be performed using cationic polymers, chitosan and poly-l-lysine. To evaluate the binding and protective ability of AuNPs, CS and PLL functionalized to Fluc-mRNA using gel retardation, dye displacement and nuclease protection assays.
Outline of thesis
To ultimately investigate the biological activity of these nanocomplexes with respect to cytotoxicity using the MTT assay gene expression using the Luciferase reporter gene assay, and apoptosis induction using the acridine-orange-ethidium bromide (AO/EB) double staining method in vitro.
Cancer
- The burden of cancer, causes and risk factors
- The development and genetics of cancer
Mutations of cellular genetic elements are indicated as one of the causes of cancer development (Lodish et al., 2000). These genes can be proto-oncogenes that are activated or tumor suppressor genes that are inactivated, giving rise to the development of cancer.
Gene therapy
- Viral gene delivery methods…
- Non-viral gene delivery methods
- Physical methods
- Chemical methods
- Chitosan
- Poly-l-lysine
These vesicles spontaneously self-assemble when lipids are dispersed in aqueous solutions (Ibraheem et al., 2014). PLL is derived from the polymerization of the N-carboxyanhydride component of lysine (Gascón et al., 2013).
Inorganic Nanoparticles : Gold nanoparticles (AuNPs)
- Properties of AuNPs
- AuNP Synthesis
- Chemical methods
- Physico-Chemical methods
- Biological methods
- Functionalisation of AuNPs with Cationic Polymers (CPs)
- Gene delivery and Gold Nanoparticles
- Intracellular Trafficking
- Cellular uptake
- Transfection Efficiency
A simple one-pot synthesis method of AuNPs using N-(2-hydroxyethyl)-N-methylmorpholinium tetrafluoroborate has been developed (Herizchi et al., 2016). Biological methods involve the use of microorganisms, enzymes, plants and plant extracts (Mohanpuria et al., 2008).
Introduction
Protein expression is promoter-independent and suitable for cells that are difficult to transfect (Matsui et al., 2015). The size of the genetic material that can be inserted is limited, difficult to prepare in large quantities, and very expensive (Ibraheem et al., 2013). In particular, gold nanoparticles (AuNPs) are excellent candidates for nucleic acid delivery (Ding et al., 2014).
This is due to their many desirable properties, such as biocompatibility, safety and relatively inert core (Rana et al., 2011). It has been reported that more than 100 ligands can be conjugated to a 2 nm AuNP core (Hostetler et al., 1998). Carriers containing cationic polymers are attractive because of their ease of synthesis and their ability to integrate different functional groups within the same molecule without compromising nucleic acid binding capacity (Zhou et al., 2008).
Materials and methods
- General materials and reagents
- Experimental methods
- Colloidal AuNP synthesis by citrate reduction
- Functionalisation of AuNPs with chitosan
- Elemental verification of synthesized nanoparticle solutions
- Formation of mRNA:NP complexes
- Characterization of nanoparticle and nanocomplexes
- Assessment of CS and CS-AuNP binding to Fluc-mRNA
- Nuclease Protection assay
- In Vitro cell culture studies
- Cell culture materials and reagents
- Reconstitution of frozen cells and routine cell culture maintenance
- Trypsinization of cells and cell counts
- Cryopreservation
- MTT cytotoxicity assay
- Luciferase reporter gene assay
- Acridine Orange/Ethidium Bromide (AO/EB) apoptosis assay
- Statistical analysis
Dialysis of the CS-AuNP solution was performed to remove any unbound polymer from the solution and determine the binding efficiency of AuNPs to CS. A simple test to validate the successful functionalization and verify the presence of CS via its amine groups in the CS-AuNP solution was performed using the qualitative ninhydrin reaction. Complexes were prepared by adding Fluc-mRNA (0.25 µg/µl) to increasing amounts of CS-AuNPs and CS, respectively, to generate different CS-AuNP/CS:Fluc-mRNA mass ratios (w/w).
The cells were monitored under an inverted microscope to observe the activity of the enzyme and. Spent medium was removed from each well and replaced with fresh complete medium, followed by the addition of complexes to the appropriate wells. The cells were then incubated at 37 °C for 24 h, followed by removal of the spent medium and washing of the cells with 200 μl of PBS.
Results and Discussion
- Characterization of Nanoparticles and Nanocomplexes
- Chemical analysis
- UV-Vis optical spectrophotometry analysis and dialysis
- Qualitative Ninhydrin test for amine groups
- Transmission electron microscopy
- Nanoparticle tracking analysis (NTA)
- Assessment of CS and CS-AuNP binding to Fluc-mRNA
- Gel retardation assay
- Dye Displacement/Intercalation assays…
- Nuclease Protection assay
- In Vitro cell culture studies
- Reconstitution and cell culture maintenance
- MTT cytotoxicity assay
- Luciferase reporter gene assay
- Acridine Orange/Ethidium Bromide (AO/EB) apoptosis assay…
Upon CS-AuNP and Fluc-mRNA complexation (Figure 3.9 C), the formation of aggregates can be seen due to the condensation of Fluc-mRNA and the overall interactions of the citrate-capped AuNPs, CS, and Fluc-mRNA. This can be attributed to the electrostatic condensation of the negatively charged FlucmRNA by the positively charged CS-AuNP particles. However, if Fluc mRNA is completely bound to the Cs-AuNP, this will not be possible.
For transfection purposes, it is imperative to determine the concentration of CS-AuNP and CS required to completely bind the Fluc mRNA. For the HEK293 cell line (Figure 3.15 A), a general trend was observed whereby cell viability (%) increased as the ratio of CS and CS-AuNP to Fluc mRNA increased. In the MCF-7 cell line (Figure 3.15 C), cell viability (%) decreased as the ratio of CS to Fluc mRNA increased, but increased as the ratio of CS-AuNP to Fluc mRNA increased.
Conclusion
Chitosan functionalization of gold nanoparticles encourages particle uptake and induces cytotoxicity and proinflammatory conditions in phagocytic cells, as well as enhancing particle interactions with serum components. Gold nanoparticles: composition, supramolecular chemistry, quantum size-related properties and applications for biology, catalysis and nanotechnology. UV-Vis and NMR study of the formation of gold nanoparticles by citrate reduction: observation of gold-citrate aggregates.
Synthesis and ultraviolet-visible spectroscopy studies of chitosan-encapsulated gold nanoparticles and their reactions with analytes. Preparation and characterization of gold nanoparticles in chitosan suspension by one-pot chemical reduction method. The effect of conjugation to gold nanoparticles on the ability of low molecular weight chitosan to transfer DNA vaccine.
Introduction
The high surface-to-volume ratio of AuNPs enables easy functionalization with biomolecules to anchor therapeutic modalities and control reactivity, stability, charge, cellular uptake, tissue biocompatibility, and toxicity (Sivaramakrishnan et al., 2014). In addition, their inert nature, low toxicity, favorable interaction with cells and ability to stabilize and protect nucleic acids have encouraged the use of AuNPs as gene delivery vectors (De Long et al., 2010). Nanoparticles that have been functionalized with cationic polymers greatly favor solubility, biodegradability, bioavailability, and circulation half-life ( Pedrosa et al., 2015 ).
Poly-l-lysine (PLL) has been one of the first cationic polymer vectors used in gene therapy (Jin et al., 2014). Poly-l-lysine is derived from polymerization of the N-carboxy anhydride component of lysine and is therefore biodegradable, a property suitable for gene delivery (Gascón et al., 2013). In addition, PLL has been reported to cause stress-induced apoptosis and therefore used as an antitumor agent (Symonds et al., 2005).
Materials and methods
- Materials
Synthesis, Characterisation and Nanocomplex Evaluation
- Synthesis of citrate reduced colloidal AuNP solution
- Functionalisation of AuNPs with poly-l-lysine (PLL)
- Inductively Coupled Plasma (ICP) analysis
- Fourier Transformed Infrared Spectroscopy (FTIR) analysis
- Qualitative ninhydrin test for amino groups
- Formation of mRNA:PLL-AuNP complexes
- UV-Visible spectrophotometry analysis
- Transmission electron microscopy (TEM)
- Nanoparticle Tracking Analysis (NTA)
- Gel retardation assay
- Dye displacement/Intercalation assays
- Ethidium bromide dye displacement
- SYBR Green dye displacement
- Nuclease Protection assay
The ninhydrin reaction was used as a simple qualitative test to confirm the successful functionality and the presence of PLL via its amine groups in the PLL-AuNP solution. The nanocomplexes were prepared with different PLL-AuNP/PLL:Fluc-mRNA (w/w) mass ratios by adding Fluc-mRNA (0.25 μg/μl in RNase-free water) to increasing amounts of the PLL-AuNPs and free PLL for comparative purposes. The nanocomplexes were supplemented to a constant volume of 10 µl with HEPES-buffered saline [HBS, (20 mM HEPES; 150 mM NaCl), pH 7.4] and incubated for 30 min at room temperature to induce the formation of PLL-AuNP/PLL to ease. :Fluc mRNA nanocomplexes.
Briefly, 1 µl of AuNP, PLL-AuNP, and PLL-AuNP:Fluc-mRNA solutions (0.7:1 m/m) were applied to separate carbon-coated Formvar grids and air-dried. AuNP, PLL-AuNP (1/500 dilution) and PLL-AuNP:Fluc-mRNA (1/100 dilution) solutions were prepared and screened according to the requirements of the NTA system. The extent of protection afforded by PLL-AuNPs and PLL to Fluc-mRNA in an environment mimicking that of the in vivo system was assessed by a nuclease protection assay.
In Vitro cell culture studies
- Cell culture materials and reagents
- Cell culture maintenance
- MTT cytotoxicity assay
- Luciferase reporter gene assay
- Acridine Orange/Ethidium Bromide (AO/EB) apoptosis assay
- Statistical analysis
The pelleted cells were suspended in 1 ml of fresh complete medium [EMEM, 10% (v/v) FBS and 1% (v/v) antibiotic mixture] and transferred to a cell culture flask containing 5 ml of complete medium. To monitor the action of trypsin and the effect of “rounding,” the cells were viewed under the inverted microscope. The cells were then incubated for 48 hours at 37°C, after which the spent medium was supplemented with 200 µl of fresh complete medium and 200 µl of MTT reagent (5 mg/ml in PBS) and the cells were incubated for 4 hours at 37°C . o'clock.
The cells were then incubated at 37 °C for 48 h, followed by removal of the spent medium and washing of the cells with 200 µl of PBS. Lysed cells were then harvested and cell lysates pelleted at 12000 x g using an Eppendorf centrifuge. After visual analyses, cells were harvested in 200 µl PBS and transferred to individual wells on a black FluorTrac flat-bottom microplate.
Results and Discussion
- Characterization of nanoparticles and complexes
- Chemical analysis
- UV-Vis optical spectrophotometry analysis
- Qualitative ninhydrin test for amine groups
- Transmission electron microscopy (TEM)
- Nanoparticle tracking analysis (NTA)…
- Assessment of PLL and PLL-AuNP binding to Fluc-mRNA
- Gel retardation assay
- Dye displacement/Intercalation assays
- Nuclease protection assay
- In Vitro cell culture studies
- Cell culture maintenance
- MTT cytotoxicity assay
- Luciferase reporter gene assay
- Acridine Orange/Ethidium Bromide (AO/EB) apoptosis assay
The PLL-AuNP:Fluc mRNA nanocomplexes (Figure 4.4 C) are believed to have “flower-like” branched structures due to the condensation of Fluc mRNA. The PLL-AuNP:Fluc-mRNA nanocomplexes produced a zeta () potential increase up to mV, indicative of excellent colloidal stability that could be attributed to the high condensation of the mRNA. The addition of SDS to the complexes after FBS treatment caused the release of Fluc mRNA from the nanocomplexes.
In the HEK293 cell line (Figure 4.10 A), a general trend was seen in which cell viability decreased as the ratio of PLL to PLL-AuNP to Fluc mRNA increased. For the Caco-2 cell line (Figure 4.10 B), cell viability increased as the ratio of PLL to PLL-AuNP to Fluc mRNA increased. The size of the PLL-AuNP:Fluc-mRNA complex was 89.4 nm, indicating their susceptibility to different surface particle activities where the cells contribute to the toxic effects seen in the HEK293 and Caco-2 cell lines at higher concentrations.
Conclusion
Furthermore, these nanocomplexes were able to induce apoptosis more in the cancer cell lines (Caco-2 and MCF-7) than in the normal HEK293 cell line, indicating their potential as antitumor agents. These results indicate that these PLL-AuNPs have the potential to be effective gene delivery vectors, especially for cancer therapy. Functionalized gold nanoparticles for the binding, stabilization and delivery of therapeutic DNA, RNA and other biological macromolecules.
Calculated absorption and scattering properties of gold nanoparticles of different size, shape and composition: applications in biological imaging and biomedicine. Gold nanoparticles (AuNPs) have been applied in the diagnostic and photothermal treatment of cancer and show promise as gene delivery vehicles as they provide multifunctional platforms for the delivery of a variety of therapeutic biomolecules. These functionalized nanocomplexes were able to induce greater apoptosis in the cancer cell lines (Caco-2 and MCF-7) than in the normal HEK293 cell line, confirming their cell specificity.
Future recommendations
Overall, the functionalized nanocomplexes were found to be more stable, less toxic and more efficient than the individual cationic polymer:mRNA complexes, indicating that the functionalization of AuNPs with CS and PLL improved the properties and biological functions of the nanocomplexes. Therefore, these CS/PLL-AuNPs have the potential to be effective gene delivery systems especially for cancer treatment, and the results obtained so far bode well for their use in future in vivo studies. However, further optimizations should be considered before these nanocomplexes can be translated into clinical applications.
There are several possible recommendations that could be addressed in future studies in this area.
