Synthesis and Characterization of a Nano-Polyplex system of GNRs-PDMAEA-pDNA: An Inert Self-Catalyzed Degradable Carrier for Facile Gene Delivery

Engineering molecules at nano-scale is a promising approach in targeting and curing diseases. In this research, fabricated new hybrid system called nano-polyplex represents an example of the molecular engineering at nano-scale. Polymer of PDMAEAs with four different molecular weights were synthesized using the RAFT method, attached onto the gold nano-rod surface, which modified and produced a safe novel system with an average size less than 100 nm. The hybrid system was characterized by ultra violet-visible spectrophotometer (UV-Vis), dynamic light scattering (DLS), 1H NMR, gel permeation chromatography (GPC), Fourier transform-infrared (FT-IR) spectroscopy, Zeta potential analyzer and transmission electron microscopy (TEM). Features of higher transfection and lower toxicity compared to the previously reported polyplex of PDMAEA, as well as the gold standard PEI, have been shown in all molecular weights and defined N/P ratios (10–200). The ideal physicochemical properties for escaping from the cell barriers, covering the large volume of genetic material (pDNA) and high efficiency of loading polyplexes on GNRs’ surface make it an ideal carrier. The results of this effort pave way in designing a new generation of nanoparticle-based delivery systems for nucleic acid therapy and gene editing.


Results and Discussion
Characterization of GNRs. Surface plasmon resonance bands of gold nanorods were monitored in the visible and near infrared region, representing oscillation of the conduction band electrons along the short and long axis of GNRs (Fig. 1a). Appearance of a strong longitudinal surface plasmon resonance band (LSPR) at 720 nm, along with a transverse SPR band of weaker intensity at 520 nm is characteristic of formation of nanostructure with rod morphology. Figure 1b depicts the transmission electron microscopy image of the nanostructures. The mean aspect ratio of GNRs was estimated to be 3.2 ± 0.6, with average dimensions of 38.38 nm in length and 12.00 nm in width.
Characterization of PDMAEA. RAFT polymerization is known to offer many advantages for biomedical applications. For instance, controlled molecular weight and length as well as polydispersity, ability to design a wide range of compounds with a variety of reactive end-groups for modification and functionalization are considered to be some benefits of this method. According to the vast majority of studies, versatility of RAFT agents can be proof of the superiority of this technique 11,12 . In this study, PDMAEA was synthesized with four different molecular weights using the RAFT polymerization technique, as shown in Table 1. For the sake of convenience, polymers with different molecular weights have been marked as I, II, III and IV for all data representations. Analysis of the polymers by 1 H NMR and GPC showed controlled behavior of RAFT polymerization (Table 1). Functionalization of GNRs with PDMAEA. Previous studies have shown that the presence of an excess amount of cationic surfactant provides notable positive surface charge for GNRs, which in turn can result in nonspecific cell uptake and protein adsorption upon interaction with cells, disruption of their membranes and eventually cell death 13 . This is an important issue that limits the utilization of GNRs for biocompatible medical purposes. Therefore, displacement of CTAB with a more biocompatible molecule is necessary for further fruitful biomedical applications. So far, a number of strategies have been proposed to reduce the toxic effect of CTAB molecules, which include modification of GNRs with different ligands or polymers such as PEG, PSS, etc. 14 .
In order to modify the surface of GNRs with polymer, PDMAEA was attached to the GNRs based on a modified protocol 15,16 . Figure 2 displays a summary of the synthesis reactions of PDMAEA (a), GNR (b) and attaching of PDMAEA to GNR surface (c).
Typically, in this study, a "grafting to" approach was employed for functionalization of the nanostructures with PDMAEA, based on the high affinity of gold atoms for sulfur 16 . As a result, gold can form stable bonds with sulfur compounds such as thiols, dithioesters and trithiocarbonates. Figure 3 depicts the FT-IR spectra of bare GNRs and PDMAEA as well as their complex forms. The characteristic peaks in the GNRs-PDMAEA complex appeared as C=O (1734.00 cm −1 ), stretching bonding of C-S (1473.72 cm −1 ), and C-H bonding (2850-2950 cm −1 ) at the same position of the corresponding peaks observed for bare PDMAEA. The C-H bonding refers to C-H of monomers in the backbone of the polymer. Resonance binding of Au-S (1386.61 cm −1 ) indicated valuable evidence for the process of binding. Hence, the comparison of the FT-IR spectrum of the bare PDMAEA with its complex form with GNRs confirmed conjugation of the polymer to the matrix of nanostructures.
Zeta potential is another useful technique for monitoring PDMAEA interaction with GNRs. According to the DLS results shown in Table 2, there was notable decrease in the zeta potential value for all the nano-polyplexes (GNR-PDMAEA-pDNA) compared to that of the untreated GNRs (without PDMAEA), which had a much higher value due to the presence of cationic surfactant (CTAB), i.e. +37.8 mV. Such changes in the zeta potential values can represent interaction of the GNRs with PDMAEA and formation of the nano-polyplexes.
In addition to the mentioned analytical techniques, TEM image which is shown in Fig. 2(c) approved the attachments of PDMAEA on GNRs surfaces. The arrows in the image clearly define the attachment process.
DNA-binding and Protection Assay of PDMAEA. The prominent feature of an ideal gene delivery carrier system is the capability to transport the foreign gene to its target without any damage. In this regard, capability is defined as the ability of the carrier to escape the processes that affect the natural state of exogeneous DNA 7 . Covering and condensing the DNA molecule with polymers is considered as a useful protection strategy against nuclease digestion. Recently, PDMAEA is found to have shown excellent properties as well as degradation in an independent manner in the presence of external or environmental triggers. The self-catalyzed hydrolysis mechanism of this polymer in a defined time interval is useful in the delivery of genes and drugs. To study the role of PDMAEA in condensing pDNA molecules and also measurement of the size of polyplexe, pDNA (GFP and luciferase separately) was mixed with polymers of four molecular weights (as shown in Table 1) at different N/P ratios (10 to 200 as mentioned). Figure 4 depicts the capability of PDMAEA for DNA binding/condensing by agarose gel retardation assay. The gel images have been obtained with time intervals of 30 min, 24, 72 and 120 hours, respectively, after formation of the polyplexes. From Fig. 4, it can be seen that the migration of the polyplexes in the agarose gel is retarded. This indicates the strong binding of pDNA with four different molecular weights with all N/P ratios (10 to 200). As shown in Fig. 4(a1-a4), the binding capability is gradually reduced until completion of pDNA release. Based on the running pattern in the gel, it is presumable that increasing the molecular weight and N/P ratios leads to formation of durable complexes that provides a better coverage as well as a better release of pDNA.
Previous reports on the effect of molecular weights and charge ratios of another polymer, i.e., chitosan, have shown similar results 17 . A study on PEI as a popular cationic gene carrier system has provided valuable information about the role of molecular weights and N/P ratios on polyplex formation, durability and dissociation (release of DNA from polymer) 18 . The main release mechanism for DNA molecules is based on a self-catalyzed degradation pattern of polymer in water, leading to the formation of the negatively charged poly acrylic acid and N, N-dimethylamino ethyl ethanol. The average size of the nano-polyplex system (pDNA-PDMAEA-GNR) and its distribution was measured using the dynamic light scattering technique (see Table 2). According to DLS data, the size of the nano-polyplex was estimated to be 100 nm without a noticeable difference at different N/P ratios. This could be due to the presence of GNRs which act as matrix-making dense polyplex structures (Fig. 1a). However, larger complexes were also observed, independent of molecular weight and N/P ratio. It seems that the larger size of such complexes resulted from the misconnection of polyplexes on GNRs surface and assemblies of the polyplexes by themselves. Comparison of the bare polyplexes (PDMAEA-pDNA) with nano-polyplexes  revealed that the latter has a smaller size ( Table 2). It should be noted that sizes of all samples at an N/P ratio of 10 and 50 were smaller, especially at the higher molecular weights. The remarkable difference between the average size of nano-polyplexes and polyplexes implies the positive impact of GNRs in condensing the overall nano-polyplex system. Assessment of the potential of PDMAEA to protect pDNA was investigated and compared with the PEI (25 kDa-branch) as gene transfers agents. In this regard, experiments showed an excellent potential of PDMAEA for protecting pDNA from nuclease digestion (Fig. 4b). Treatment of polyplexes (PDMAEA-pDNA) with DNase I was carried out in tow separate parts as following: (1) the first part comprised the samples treated with DNase I immediately after half an hour of complex formation. The obtained results showed an excellent protection of polymer for covering pDNA samples as well as an enhanced parallel protective effect upon increase in the molecular weight ( Fig. 4(b1)). (2) The second part comprised the samples stored at room temperature for one week and subsequently treated with DNase I. The results showed no pDNA band upon exposure to DNase I, and full digestion of pDNA occurred reflecting the gradual changing nature of the PDMAEA (Fig. 4(b2 and b3)). Different molecular weights of PDMAEA with N/P ratios of 10 and 100 were evaluated. The protective effect was found to increase with increase in the molecular weight of the polymer. Furthermore, in all the molecular weights, there was no perceptible difference between N/P ratios of 10 and 100. On the other hand, comparison of PDMAEA with PEI (N/P ratios of 5, 10) showed that the PDMAEA ability for gene protection was higher than that of PEI. All obtained results were similar to the results of previous researchers 9 .
Based on previous studies, packaging and protecting of nucleic acids (DNA, siRNA) and the stability of their complexes are the main parameters to protect them against cellular compartments such as endosomal/lysosomal and nuclease degradation 19,20 . Cationic polymers such as PEI, chitosan and PDMAEMA are good candidates for masking the anionic charges of nucleic acids. Complexes generated by these polymers have an appropriate protection against degradation 21 . The point that should not be forgotten is that the strength of complexes and balance of electrostatic interactions between polymer and DNA are important issues that affect the release pattern of DNA inside the cells 22 . However, defective release of DNA has common problems associated with the above-mentioned polymers. Meanwhile, strong electrostatic interactions (which are used to make durable polyplexes), conversion of the ionic composition, and full dissociation and release of DNA inside the cell without limitation, are superior features of PDMAEA.
MTT-assay. Conversion of tetrazolium salt to formazan in MTT assay represents mitochondria activity and cell survival in abnormal conditions such as drugs treatments. Therefore, this assay is useful in investigating the safety or toxicity of pharmaceutical agents 23 . In addition, cytotoxicity of vector-mediated gene transfer can be evaluated by MTT assay. The toxic effect of a cationic gene carrier refers to their positive charge, and influences their capability and efficiency in gene therapy attempts 4 . Previous research on cytotoxicity of PDMAEA has shown that polymers with a molecular weight of 5600 or lower do not induce toxic effect. The cell cytotoxicity increases with increase in the N/P ratio as well as the molecular weight of the polymer 9 . To monitor the safety of the nano-polyplex system, all four different molecular weights (I, II, III, and IV) with the specified N/P ratios were treated with the HEK293 cell line. As shown in Fig. 5, toxicity and cellular damage increased with increase   in the molecular weight and N/P ratio. Comparison of the nano-polyplex system (pDNA-PDMAEA-GNRs) with ordinary complexes (PDMAEA-pDNA) and PEI-pDNA (the control) indicated that the nano-polyplex system affected the cells in a safer fashion. The bar chart clearly shows that the role of the N/P ratio is more prominent than the molecular weight. This is consistent with a previous study by Truong et al., who reported a negligible effect of PDMAEA on cytotoxicity in all molecular weights and N/P ratios, except that of the N/P ratio of 200 9,24 . Furthermore, a lower toxic effect of PDMAEA exists with respect to PEI. This occurs due to the fact that the amine groups of the polymer chain in PEI act as a positive charge source, thus producing a high density of charge and cell membrane disruption 25 .
Transfection Assays. Herein, PDMAEA has been used as a carrier to transport pDNAs (GFP and luciferase) into the nuclei of the KEK-293 cell line in order to investigate its potential in transportation of pDNA, consistent with previous research which reported transport of siRNA molecules 24 . Green Fluorescent Protein (GFP) was mixed with GNRs-PDMAEA, bare PDMAEA and PEI as a control, for HEK-293 cells transfection. Experiments were conducted according to the modified protocols of previous studies 26 . Figure 6 depicts the transfected cells imaged by fluorescence microscopy. The specified N/P ratios and four molecular weights were studied for both nano-polyplexes of GNRs-PDMAEA-pDNA and polyplexes of PDMAEA-pDNA and compared with PEI-pDNA. It should be noted that the optimized time interval for cell transfection is less than approximately 10 h after complex formation. This is due to the fact that dissociation of polymer and release of pDNA inside the cells usually occurs after 10 h 9 . As shown in Fig. 6, the maximum transfection was observed for an N/P ratio of 50. It has been noted that the rate of transfection with nano-polyplexes is higher than that of complexes with all molecular weights. The results suggest that an increase in the molecular weight and the N/P ratio leads to enhancement of the transfection efficiency. It is noteworthy that higher N/P ratios (100 and 200) with polymers of lower molecular weight induced better transfection efficiency, whereas polymers of higher molecular weights did not show the same efficiency. Findings of this effort are consistent with previous research that reported the use of PDMAEA as well as other polymers such as chitosan, PDMAEMA, and PEI as a gene carrier 3,9,27,28 .

Dark-Filed imaging and Confocal microscopy.
Dark-field microscopy can be utilized to monitor cellular uptake and transfection process by orange-red plasmonic scattering phenomenon that relies on the strong longitudinal surface plasmon resonance of GNRs in the near IR spectrum. Such a characteristic phenomenon enables potential application of GNRs as contrast agents in tracking nanopolyplex systems 29,30 . Figure 6(b) shows dark-field microscopy images of cellular uptake of nano-polyplex system (pDNA-PDMAEA-GNRs) by HEK-293. Cell internalization was also monitored by confocal microscopy as a powerful technique for high resolution optical sectioning of samples and three dimensional detection 31 . This technique provides useful information on location of nanoparticles within cell environment 32 . Figure 6(c) shows confocal microscopy images of HEK-293 cells treated with SYBR Green-labeled nano-polyplex and DAPI stained nuclei, confirming internalization and accumulation of the nano-polyplex around the nuclei. Another evaluation based on reported gene technology includes bioluminescence and luciferase assay which is widely used for gene expression analysis in mammalian cells. Rapid, sensitive and nonradioactive detection indicates that the firefly luciferase gene is a suitable candidate for monitoring promoter activity and observing the activity of living cells 33 . Plasmids of pGL3 were mixed with GNRs-PDMAEA with N/P ratios of 10-200, and incubated with HEK293 cells as described for the GFP gene. Transfection results indicated the effectiveness of the nano-polyplex system in delivery of pGL3 to the HEK293 cell nucleus, and subsequent expression of the luciferase gene (Fig. 7). According to these results, an inverse relationship between molecular weight and N/P ratio is observed, such that increasing the molecular weight is associated with decreasing the proper N/P ratio of transfection. Such behavior is considerable for molecular weights of II, III and IV polymers and N/P ratios of 100, 50 and 10, respectively. However, the overall data represents an important point, i.e. the average transfection of the nano-polyplex system is more than PEI and the bare-polyplex of PDMAEA.
Among nucleic acids, DNA-based therapy offers a better opportunity for gene delivery purposes because of its multivalent treatments 34 . Hence, stability of pDNA during interaction with cell membrane, uptake, intracellular movements and access to nucleus should be considered as a crucial issue 35 . In this regard, the design and fabrication of proper carriers would be the prerequisite for efficient transfection, i.e. the majority of pDNA can be delivered to the target area without any damage. Stability of the carriers during the long extra-or intra-cellular paths is a key factor that defines their fate and performance in biological environments 36 . Different release rates of nucleic acids from cationic nano-carriers are distinctive features of polymers in gene delivery. It has been shown that electrostatic interaction plays an important role in polyplex formation and dissociation in cellular conditions 1 . For instance, PEI with high positive charge can strongly interact with negatively charged nucleic acids with high transfection efficiency among other cationic counterparts. Nonetheless, PEI is found to be highly toxic, creating a great challenge to overcome for fruitful gene delivery purposes 37 . Formation of cavity structures in plasma membranes, membrane ablation and thinning of lipid bilayers are considered common phenomena when cationic carriers interact with anionic phospholipids of cell membranes 38,39 . Protonated polymers such as PEI, PDMAEMA and PAMAM, due to their buffering capacities, show easy endosomal escape. In fact, a phenomenon known as the proton sponge effect is related to the buffering effects of polymers in the acidic environment of endosomes. Subsequently, osmotic swelling and disruption of endosomes lead to release of polyplexes into the cytoplasm and resolve intracellular trafficking 40,41 . Nevertheless, the non-biodegradable nature and toxic effect can be considered as important limiting factors of such carriers systems with considerable proteins inactivation, strong linkage with cellular nucleic acids, and aggregation in cell organelles 42 . Meanwhile, PDMAEA can be regarded as a biocompatible and biodegradable polymer that is a promising carrier candidate in conjunction with GNRs that allows reduced intracellular trafficking and toxicity. Furthermore, the nano-polyplex of GNR-PDMAEA-pDNA simultaneously exploits gold atoms as an inert platform from the biological and chemical points of view 43 and PDMAEA with features of biocompatibility and self-catalyzed degradability. Another superior trait of the current nano-polyplex system with respect to other reported carriers is its size, which is estimated to be about 100 nm at all N/P ratios of PDMAEA. Previous studies have shown that an optimal size range exist for carriers to improve their cellular uptake. It has also been demonstrated that increasing size of the carrier leads to a decrease in the opportunity for cell entry. Moreover, nanostructures smaller than 100 nm are found to arrive in the cell cytoplasm faster and more easily 44 . Results of this effort are consistent with the above-mentioned studies. Hence, the present nano-polyplex can hopefully pave the way for further investigations on design and fabrication of nanoparticle-based gene delivery systems in the future.

Synthesis of Poly (2-(dimethylamino) ethyl acrylate) (PDMAEA).
All four PDMAEAs with different molecular weights (Table 1) were synthesized using the RAFT method, where methyl-2-(dodecylthiocarbonothioylthio)-2-methylpropionate was used as a RAFT agent. To obtain different molecular weights of PDMAEA, the variable parameters were reaction time and molar ratio of RAFT agent to the monomer. For a typical reaction, certain amounts of monomer, RAFT agent and AIBN were dissolved in dioxane as a solvent in a 10 mL The reaction mixture was then de-oxygenated by purging with nitrogen for 30 min followed by heating to 60 °C for 7 h. The reaction was stopped by incubation on ice and exposure to air. The precipitating and re-dissolving procedure was repeated three times in n-hexane and dioxane as nonsolvent and solvent, respectively. The polymer was dried under vacuum at 60 °C for 48 h and finally a yellow oily product was obtained. The polymer was characterized by nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC). NMR spectra were recorded by a Bruker Avarce DRX 500 MHz spectrometer, using deuterated chloroform (CDCl 3 ) and water (D 2 O) as the solvent for NMR analysis. To analyze the molecular weight distribution of the polymer GPC, tetrahydrofuran (THF) was applied as an eluent. GPC columns were calibrated with narrow molecular weight distribution polystyrene standards in the molecular weight range of polymers.

Conjugation of Polymer with GNRs.
Briefly, 3 mg of polymer were dissolved in 5 ml GNRs (1 OD) and stirred at 200 rpm for 6 hours. The solution was then centrifuged at 13000 rpm for 10 min at room temperature. The precipitate was re-dispersed in 5 ml deionized water. The purification procedure was repeated twice.
Binding Assay. Plasmids of GFP and luciferase (1 µg) were used to generate polyplexes with PDMAEAs at different N/P ratios (referring to the number of nitrogen (PDMAEA) per DNA phosphate (pDNA) in generated polyplexes). For this purpose, pDNA was complexed with polymers of 10 to 200 N/P ratios, followed by addition of water to the mixture up to 50 μL, and keeping at room temperature for 30 min (without stirring). All polyplexes were loaded onto a 2% agarose gel containing 1X TBE buffer. The gel was run for 1 hour at 80 V, stained with ethidium bromide and visualized using the UV Gel doc (Vilber Lourmat Gel doc, France).