Plasmid DNA Delivery: Nanotopography Matters - Journal of the

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Plasmid DNA Delivery: Nanotopography Matters Hao Song, Meihua Yu, Yao Lu, Zhengying Gu, Yannan Yang, Min Zhang, Jianye Fu, and Chengzhong Yu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08974 • Publication Date (Web): 20 Nov 2017 Downloaded from http://pubs.acs.org on November 20, 2017

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Journal of the American Chemical Society

Plasmid DNA Delivery: Nanotopography Matters Hao Song, Meihua Yu, Yao Lu, Zhengying Gu, Yannan Yang, Min Zhang, Jianye Fu, Chengzhong Yu* Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia KEYWORDS: silica nanoparticles ∙ nanotopography ∙ plasmid DNA ∙ gene delivery ∙ transfection.

ABSTRACT: Plasmid DNA molecules with unique loop structures have widespread bio-applications, in many cases relying heavily on delivery vehicles to introduce them into cells and achieve their functions. Herein, we demonstrate that control over delicate nanotopography of silica nanoparticles as plasmid DNA vectors has significant impact on the transfection efficacy. For silica nanoparticles with rambutan-, raspberry- and flower-like morphologies comprised of spike, hemisphere and bowl type subunit nanotopographies, respectively, the rambutan-like nanoparticles with spiky surfaces demonstrate the highest plasmid DNA binding capability and transfection efficacy of 88%, higher than reported silica based nano-vectors. Moreover, it is shown that the surface spikes of rambutan nanoparticles provide a continuous open space to bind DNA chains via multivalent interactions and protect the gene molecules sheltered in the spiky layer against nuclease degradation, exhibiting no significant transfection decay. This unique protection feature is in great contrast to a commercial transfection agent with similar transfection performance but poor protection capability against enzymatic cleavage. Our study provides new understandings in the rational design of non-viral vectors for efficient gene delivery.

Introduction Cellular delivery of genetic molecules has gained significant attention during the past two decades with diverse applications.1-3 Naked nucleic acid, especially deoxyribonucleic acids (DNA) molecules are large and fragile, which cannot cross the cell membrane and suffer from rapid degradation in the presence of serum nuclease.4 Viral systems have been employed for gene delivery with high efficacy, but the inherent immunogenicity and toxicity limit their wide use.5-6 Therefore, the development of safe and effective non-viral vectors is decisive for gene delivery applications.7-8 Thanks to the remarkable progress in nanotechnology enabled drug delivery systems, various types of delivery vehicles have been fabricated, such as cationic liposomal and polymeric formulations7-9 as well as inorganic nanoparticles.10 Among them, silica based nano-vectors allow delicate nanostructure tailoring with versatile surface chemistry and good biocompatibility.11-12 Existing knowledge on the contribution of various physiochemical parameters, such as particle size,13 shape,14-16 porosity17-18 and surface functionalities,19-21 has been applied in the design of silica nanoparticles (SNPs) for plasmid DNA (pDNA) delivery. It is noteworthy that most reported nano-vectors only offer a moderate improvement in pDNA delivery efficacy,13-15, 17, 22-23 possibly due to the limited understanding of the interaction between cargo molecules and nanoparticle vectors at the nanoscale interface. Notably, distinct from small molecular drugs, therapeutic

proteins and siRNA, whose sizes are much smaller than nano-vectors, pDNA possesses unique rope-like loop structures with several micrometers in length (Scheme 1a), an average loop size around 50 nm,24 and a chain width of 2-3 nm.25 To our knowledge, taking this specific feature into consideration in custom-design of nextgeneration nano-vectors with high pDNA delivery efficacy is rarely reported. In the present study, we report that the precise control over nanotopography of SNPs plays a significant role in successful pDNA delivery. SNPs with controllable nanoscale surface topographies, including rambutan-26, raspberry-27 and flower-like28 morphologies (Scheme 1b-d) constructed by spike, hemisphere and bowl type subunits (Scheme 1e-g), have been fabricated. Although their applications in protein and siRNA delivery have been reported separately,26-29 there is no report on the impact of nanotopography on pDNA delivery. Inspired by the multivalent interaction model from Velcro,30 we demonstrate that the polyethylenimine (PEI) modified spike type subunits (Scheme 1e) enable stronger binding affinity toward pDNA molecules and allow effective protection against nuclease degradation (Scheme 1h left) compared to the other structures (Scheme 1f, g) and a commercial transfection agent. Rambutan nanoparticles facilitate efficient cellular uptake, endosomal escape and nuclei delivery of pDNA, leading to successful intracellular gene expression (Scheme 1h) and reaching a high transfection rate among silica based nano-vectors of 88%.

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surface nano-textures. The 3D nanotopographies were reconstructed from a series of tomograms and presented in Figure 1j-l. Ram-SNPs exhibit slightly curved spike type subunits on the shell (Figure 1j). The silica spikes have an average diameter of 8 nm, length of 42 nm and distance of 18 nm between adjacent spikes. Ras-SNPs (Figure 1k) and Flw-SNPs (Figure 1l) present hemisphere and bowl type subunits on the surface, respectively. As measured in the 3D models, the radius of irregular hemispheres on RamSNPs surface ranges from 15 to 40 nm, and the bowl type subunits of Flw-SNPs give an average pore opening of around 20 nm with wall thickness of 2-3 nm. Apart from the above SNPs engineered with different nanotopographies, S-SNPs, i.e., silica hollow spheres with smooth surfaces, were also fabricated with a uniform particle size around 350 nm for comparison (Figure S2).

Scheme 1. An illustration of nanotopography design for plasmid DNA delivery. (a) A 3D model (top) and an AFM image (bottom) of pDNA-EGFP on a mica surface. (b-g) 3D model images displaying silica nanoparticles featured with rambutan- (b), raspberry(c) and flower-like (d) morphologies and spike (e), hemisphere (f) and bowl (g) type subunit nanotopographies conjugated with plasmid DNA at the interface. (h) Schematic representation of pDNA-EGFP DNase I protection, cellular delivery and transfection process by Rambutan SNPs.

Results and Discussion Rambutan-like silica nanoparticles (Ram-SNPs) were fabricated via the assembly of resorcinol-formaldehyde (RF) resin and silica primary particles under Stöber synthesis condition according to our recent report (Figure S1a I).26 To justify the advantage of spiky nanotopography design for pDNA delivery, raspberry-like SNPs (Ras-SNPs) were prepared using a similar approach as illustrated in Figure S1a II. Variation in this approach produced smoothsurfaced SNPs (S-SNPs) for comparison (Figure S1a III). Flower-like SNPs (Flw-SNPs) were synthesized according to a literature report (Figure S1 b) 28. In this study, all particles were designed to have a hollow structure and comparable sizes. Scanning electron microscopy (SEM) images (Figure 1a-c) and transmission electron microscopy (TEM) images (Figure 1d-f) reveal that well dispersed Ram-SNPs, RasSNPs and Flw-SNPs in similar particle sizes of around 300-350 nm are obtained, which exhibit an inner hollow structure but distinct outer surface nanotopographies. To characterize the detailed nanostructures of these SNPs, electron tomography (ET) technique was employed.31-32 The tomograms sliced from the center of each single particle (Figure 1g-i) clearly present the hollow interior and

The dynamic light scattering (DLS) analysis was conducted to determine the size and dispersity of silica nanoparticles with different surface nanotopographies in both Milli-Q water and phosphonate buffer solution (PBS, 10 mM). As shown in Figure S3, these silica nanoparticles show a similar particle size distribution in both water and PBS, and also good dispersity in the aqueous solutions with polydispersity index (PDI) less than 0.3. The particle sizes of silica nanoparticles measured from DLS are relatively larger than those determined by TEM analysis due to the hydration of silica surface by surrounding water molecules.33 The particle size of Ram-SNPs can be easily tuned by varying the RF precursor amount. As shown in Figure S4, nanoparticles show a relatively uniform particle size of 270 and 180 nm, where the surface silica spikes are well maintained. To further identify the particle stability in physiological solutions, the suspension of Ram-SNPs in PBS kept for 1, 2, 3 and 5 days were then measured by DLS. Their particle size distributions and average particle sizes were presented in Figure S5, which shows a narrow particle size distribution ranging from 445 to 521 nm. No obvious aggregation and sedimentation were observed, which demonstrates the good stability of Ram-SNPs. To characterize the pore structures and texture properties of these SNPs, nitrogen sorption analysis was employed. The nitrogen sorption-desorption isotherms of S-SNPs, Ras-SNPs, Ram-SNPs and Flw-SNPs are plotted in Figure S6a. Both Ram-SNPs and Flw-SNPs show a characteristic type IV isotherm which indicates the abundance of mesopores. S-SNPs and Ras-SNPs show a typical type I isotherm, where no obvious mesopores can be identified. The Barrett-Joyner-Halenda (BJH) pore size distribution curves derived from adsorption branch (Figure S6b) further reveal that Ram-SNPs and Flw-SNPs have similar pore sizes around 18 nm, in accordance with the measurement from 3D reconstruction models (Figure 1j, l). Flw-SNPs show the highest Brunauer-Emmett-Teller (BET) surface area and pore volume of 639 m2∙g-1 and 2.3 cm3∙g-1, respectively. Ram-SNPs give a relatively low BET surface area and pore volume of 166 m2∙g-1 and 0.68 cm3∙g1 , respectively. S-SNPs and Ras-SNPs exhibit the smallest surface area and pore volume (Table S1) compared to the


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Journal of the American Chemical Society other two types of SNPs due to the limited porous struc-

tures.

Figure 1. (a-c) SEM images, (d-f) TEM images, (g-i) electron tomogram slices and (j-l) reconstructed subunits of Ram-SNPs (a, d, g, j), Ras-SNPs (b, e, h, k) and Flw-SNPs (c, e, I, l), respectively. Ram-, Ras- and Flw-SNPs have spike (j), hemisphere (k) and bowl (l) type nanotopographies, respectively.

To facilitate successful binding with plasmid DNA, negatively charged bare SNPs were modified with branched PEI with a molecular weight around 10 kDa. The nanostructures of these particles were maintained well after PEI conjugation as shown in the TEM images (Figure S7). The PEI amounts modified on each type of SNPs were quantified via elemental analysis according to their nitrogen content (Table S1). Flw-SNPs with a large surface area exhibit a PEI weight content of 21.9 wt%, much higher than Ram-SNPs (9.6 wt%), Ras-SNPs (7.2 wt%) and SSNPs (2.4 wt%). Plasmid DNA expressing enhanced green fluorescent protein (pDNA-EGFP) was loaded on the PEI modified SNPs (SNPs-PEI). Ram-SNPs-PEI achieve a high pDNA loading capacity up to 133 ng∙μg-1, much higher than the typical loading amount of reported silica based pDNA vectors. 2223 Even with a large surface area/pore volume and high PEI content (Table S1), Flw-SNPs-PEI show a loading capacity of 114 ng∙μg-1, slightly lower than Ram-SNPs-PEI (Figure 2a). In comparison, Ras-SNPs-PEI and S-SNPsPEI have significantly lower loading capability (89 and 38 ng∙μg-1, respectively) due to their limited surface area and PEI content to accommodate DNA molecules. To characterize the binding of SNPs and pDNA molecules, nanoparticle surface zeta potential change was recorded in 10 mM PBS. As shown in Figure 2b, SNPs with different surface nanotopographies are all negatively charged with zeta potential ranging from -16 to -28 mV. After PEI modification, Ram-SNPs-PEI, Ras-SNPs-PEI and S-SNPs-PEI exhibit a similar positive surface charge

around +13 mV, indicating successful PEI conjugation on the particle surface. To be noted, the zeta potential value of Flw-SNPs-PEI reaches as high as +29 mV, contributed by the high content of PEI conjugation. After loading negatively charged pDNA molecules, the zeta potential of S-SNPs-PEI and Ras-SNPs-PEI reverses to around -16 mV, indicating that the pDNA molecules mostly cover the surface of nanoparticles.13, 17 The zeta potential of pDNA/Flw-SNPs-PEI complex also shows a negative charge around -7 mV, which suggests that at least a part of DNA molecules are located on the outer surface of the nanoparticles. In contrast, pDNA/Ram-SNPs-PEI complex gives an almost neutral surface charge, implying that the pDNA molecules are mostly entrapped inside the spiky silica network, rather than exposed on the particle outer surface. These results support our hypothesis: the unique spike type nanotopography favors the multivalent interaction through the entanglement with pDNA molecules with a high loading capacity (Scheme 1e). To further study the binding affinity of pDNA toward nanoparticles with different surface nanotopographies, gel retardation assay was performed. A constant amount of pDNA (0.5 μg) was mixed with various amounts (2.5 to 20 μg) of SNPs-PEI. As shown in Figure 2c, a complete electrophoretic shift is observed for naked pDNA. A high content of compact supercoiled form of pDNA molecules appears as the main band, while the slower-running open circular form and the relaxed closed circular form appear as the minor faint band.34



Figure 2. (a) Loading capacity of pDNA-EGFP on Ram-SNPs-PEI, Flw-SNPs-PEI, Ras-SNPs-PEI and S-SNPs-PEI. (b) Zeta potential change of nanoparticles before and after PEI modification and further conjugation with pDNA-EGFP. (c) Gel retardation assay of PEI modified nanoparticles/pDNA-EGFP at different pDNA/SiO2-PEI weight ratios. (d-i) pDNA localization on nanoparticles with different nanotopographies by immunogold labelling process. (d) A schematic illustration of immunogold labelling process for pDNA identification. (e) TEM image of gold labelled pDNA-EGFP where 10 nm gold nanoparticles can be identified conjugated on pDNA (inset). (f-i) TEM images of gold labelled pDNA-EGFP on Ram-SNPs-PEI (f), Flw-SNPs-PEI (g), Ras-SNPsPEI (h) and S-SNPs-PEI (i), respectively. Red arrows identify gold nanoparticles conjugated on silica particle surface, while blue arrows indicate gold nanoparticles outside silica particles.

When pDNA and Ram-SNPs-PEI were mixed at different weight ratios, the release of pDNA molecules was observed at a ratio of 1:10 as evidenced by a very weak band of supercoiled pDNA during the electrophoresis (Figure 2c). A complete binding was achieved at a ratio of 1:20. In comparison, the complete binding of pDNA was observed at a lower pDNA/particle ratio of 1:30 for Flw-SNPs-PEI and 1:40 for Ras-SNPs-PEI, suggesting weaker interaction compared to pDNA/Ram-SNPs-PEI. At the lowest pDNA/particle ratio of 1:40 under study, the release of pDNA was still observed for S-SNPs-PEI. The pDNA release behavior varies among these four types of SNPs, which provides further support to our proposed mechanism (Scheme 1): the designed spiky nanotopography is favorable to binding rope-like pDNA chains via multivalent interactions (Scheme 1a, e), achieving the highest binding affinity between Ram-SNPs-PEI and pDNA as evidenced by both high loading capacity and minimum release under electrophoresis. Flw-SNPs-PEI with the largest surface area and PEI content exhibit even weaker binding capability compared to Ram-SNPs-PEI, further highlighting the significant contribution of nanotopographies in the design of nanoparticles as DNA vectors. The unfavorable nanotopography, low surface area and limited PEI conjugation amount in Ras-SNPs-PEI and S-SNPs-PEI result in the weakest binding toward pDNA molecules. The pDNA molecules after loading on SNPs-PEI were monitored by TEM using immunogold labelling technique.35 As shown in Figure 2d, pDNA molecules were conjugated specifically by anti-double-strained DNA (anti-ds DNA) antibody on TEM grid. Then the anti-ds DNA antibody was conjugated by the secondary antibody,

which was labelled with 10-nm gold nanoparticles for easy TEM observation. TEM image of gold labelled bare pDNA molecule is shown in Figure 2e, indicating a coiled ropelike structure (Figure 2e, inset). The width of the “rope” is less than 20 nm which includes the size of conjugated antibodies. This observation is consistent with the AFM result shown in Scheme 1a. Gold labelled pDNA/Ram-SNPs-PEI complex showed many gold nano-dots embedded in the spiky silica shell (Figure 2f, red arrows), although the silica spikes cannot be clearly identified in the Ram-SNPs-PEI after conjugation with pDNA and antibody staining. Unlike the spike type nanotopography, TEM image of gold labelled pDNA/Flw-SNPs-PEI showed a smaller number of gold nanoparticles attached to the bowl at the external surface (red arrow), or even detached from the SNPs (indicated by blue arrows in Figure 2g). The number of gold nanoparticles detached from the pDNA/Ras-SNPs-PEI and pDNA/S-SNPs-PEI significantly increased (Figures 2h, i), in accordance with the weak binding capability observed in the loading and gel retardation tests. To further characterize the entanglement of pDNA molecules to the PEI modified silica spikes, ET analysis under a low dose mode was conducted for both Ram-SNPs-PEI and pDNA/Ram-SNPs-PEI. As shown in the tomogram slice, compared to bare silica nanoparticles (Figure S8a), Ram-SNPs-PEI (Figure S8b) maintained the spiky nanostructure, although the edges of these silica spikes were relatively blurred which may result from the PEI conjugation. Notably, after pDNA loading, the void space between silica spikes in pDNA/Ram-SNPs-PEI was filled up (Figure S8c), suggesting that the negatively charged pDNA molecules are entangled to the positively charged


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Journal of the American Chemical Society silica spikes of Ram-SNPs-PEI. The gold nanoparticles were also observed in the silica layer, which further demonstrates the existence of pDNA molecules between the silica spikes. All the above results consistently support our hypothesis: the pDNA molecules can be entangled and hidden inside spiky shell of Ram-SNPs-PEI thanks to the spike type nanotopography. Indeed, large pore mesoporous silica nanoparticles reported in literature17 as well as Flw-SNPs-PEI show similar pore sizes to Ram-SNPs. However, there is an intrinsic difference in the structure of porous space, i.e., whether the porous space is laterally continuous at the nanoparticle surface to accommodate the pDNA chains with an average diameter of 2-4 nm and a larger loop size (> 50 nm).24 For the spiky architecture, one "pore" is formed by adjacent spikes and interconnected to adjacent pores, forming a laterally continuous porous space. This unique structure favors the binding between positively charged silica spikes (due to PEI modification) and the negatively charged pDNA chains, with the long pDNA chain easily penetrating into the continuous space. This configuration is evidenced by the immunogold labelling observation (Figure 2f), and the nearly neutral zeta potential (Figure 2b). On the other hand, for Flw-SNPs-PEI and large pore mesoporous silica nanoparticles,17 laterally the adjacent mesopores are mostly separated by silica walls. Even though a small portion of pDNA could be embedded in one mesopore, the chance for the entire DNA loop to fit into adjacent pores over a large area is hindered. This non-continuous nature in porous space is not favorable to the entrapment of pDNA molecules, leading to a weak binding affinity as demonstrated in the electrophoresis (Figure 2c) and immunogold labeling analysis (Figure 2g). pDNA/Ram-SNPs-PEI with the strongest pDNA binding affinity was monitored in simulated body fluids (SBF) by TEM. After soaking for 1 day, the wall thickness of pDNA/Ram-SNPs-PEI decreases from 90 to 60 nm and the number of surface silica spikes reduces significantly (Figure S9 a, b). After 2 days, no silica spikes can be found on the particle surface, leaving a silica wall as thin as 25 nm with many holes (Figure S9 c). The above results indicate that pDNA/Ram-SNPs-PEI can gradually release pDNA molecules in SBF due to silica dissolution and the breaking down of surface spikes (Figure S9 d), in accordance with the excellent biodegradability of silica nanoparticles reported in literature.36, 37 The ultimate goal of an ideal gene vector is to deliver pDNA intracellularly and accomplish superior transfection efficacy, which relies on the pDNA binding, cellular uptake, endosomal escape and delivering gene molecules into the nuclei without significant degradation to trigger the target protein expression.38 Here, pDNA/SNPs-PEI formulations with different types of nanotopographies were compared for their cellular uptake ability in human embryonic kidney cells 293T (HEK-293T). SNPs-PEI were firstly labelled with rhodamine B isothiocyanate (RITC) and then loaded with pDNA. Cells were incubated with the formulations for 4 h. The nuclei staining was conducted using 4',6-diamidino-2-phenylindole (DAPI). The

cellular uptake was evaluated by confocal microscopy and flow cytometry. As shown in Figure 3a, the nuclei exhibit blue fluorescence while SNPs-PEI internalized by the cells show red color. Judged by the intensity of red fluorescence, RamSNPs-PEI exhibit the highest cellular uptake followed by Flw-SNPs-PEI, Ras-SNPs-PEI and S-SNPs-PEI formulations. Quantitative analysis by flow cytometry revealed the same trend (Figure 3b) judged from the median fluorescent intensity (MFI). As shown in Figure 3c, RamSNPs-PEI, Flw-SNPs-PEI and Ras-SNPs-PEI all achieve cellular uptake into HEK-293T cells by 100%. In comparison, S-SNPs-PEI only show cellular uptake of 80%. Our results are in accordance with literature findings that silica nanoparticles engineered with rough surfaces have enhanced cellular uptake capability,27, 39 and provide a direct comparison of the contribution of various nanotopographies (e.g., spike vs hemisphere) to cellular uptake performance of nanoparticles.

Figure 3. Cellular uptake analysis of pDNA/SNPs-PEI formulations in HEK-293T cells at a nanoparticle concentration of -1 40 μg∙mL . (a) Confocal images of cells incubated with pDNA loaded RITC-labelled SNPs-PEI (red fluorescent) for 4h. (b) Flow cytometry analysis of positive cell percentage and corresponding normalized median fluorescence intensity for four formulations.

The intracellular pDNA trafficking was monitored by confocal microscopy, using Ram-SNPs-PEI as one example and fluorescein-labelled pDNA with a green fluorescence. HEK-293T cells were incubated with the pDNA/RamSNPs-PEI formulation for 4 and 12 h with endo/lysosome stained with lysotracker to give a red color. As shown in Figure 4a, yellow dots (indicated by yellow arrows) come from the overlap of green and red fluorescence, indicating the entrapment of pDNA molecules in endo/lysosome within 4 h. At 12 h, more pDNA molecules (indicated by green arrows) can be found outside the endo/lysosome region, suggesting successful endosomal escape of pDNA/Ram-SNPs-PEI formulation. The introduction of PEI on silica particle surface not only endows nanoparti-



cles with positive surface charge for DNA binding, but also contributes to the endosomal escape of silica nanoparticles based on the proton sponge mechanism.19, 20

Lipofectamine-2000 which show a cell viability of only 59 ± 6 %, the Ram-SNPs-based delivery platform exhibits improved biocompatibility.

The intracellular transportation of pDNA/Ram-SNPs-PEI was also tracked by labelling pDNA with fluorescein (green) and Ram-SNPs-PEI with RITC (red). Most pDNA and Ram-SNPs-PEI were conjugated together at 4 and 12 h (Figure S10), indicating limited pDNA can be released from the pDNA/Ram-SNPs-PEI complex. After 24 h, the orthogonal side-view section of z-stack confocal image (Figure 4b) show that the green fluorescence (from pDNA) and blue color (from the nuclei) are overlapped (indicated by white arrows), suggesting the released pDNA molecules are delivered into the nuclei. Meanwhile, successful green fluorescent protein expression can be observed in some cells after 24 h (Figure S10).

The gene delivery efficacy was evaluated by transfecting pDNA-EGFP into HEK-293T cells mediated by SNPs-PEI with different nanotopographies. A certain amount of pDNA-EGFP was mixed with varied dosage of nanoparticles and then administered toward pre-cultured cells in serum containing culture medium for transfection of 48 h. The transfection efficacy was quantitatively analyzed by flow cytometry, and the successfully transfected cell percentages were compared in Figure 5a. A dose dependent transfection behavior is observed for all the formulations, and Ram-SNPs-PEI demonstrate a significantly higher transfection efficacy compared to the other three counterparts at all dosages. The transfection efficacy of Ram-SNPs-PEI can reach as high as 88% at a nanoparticle concentration of 80 μg∙mL-1, which is higher than the transfection rate reported in literature using silica based nano-vectors for pDNA-EGFP delivery in the same cell line.13, 15, 21

Figure 4. Intracellular tracking of fluorescein labelled-pDNA (green) in HEK-293T cells delivered by Ram-SNPs-PEI at a -1 nanoparticle concentration of 80 μg∙mL . (a) Confocal images of cells incubated with pDNA/Ram-SNPs-PEI for 4 and 12 h (a) and 24 h (b). In (a), yellow arrows indicate pDNA entrapped in endo/lysosomes (stained by lysotracker, red), while green arrows indicate successful endo/lysosomal escape of pDNA. In (b), Ram-SNPs-PEI labelled with RITC (red). Orthogonal side-views from z-stack confocal images reveals the successful delivery of pDNA into nuclei as pointed by white arrows.

Ideal gene delivery nano-vectors should demonstrate good cellular biocompatibility. The cytotoxicity of these nanoparticle formulations was evaluated by 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. A dose dependent cytotoxicity behavior can be observed in Figure S11. Both S-SNPs-PEI and Ras-SNPsPEI DNA formulations show cell death less than 20% at nanoparticle dosage ranging from 5 to 100 ug∙mL-1. Complex of pDNA/Ram-SNPs-PEI exhibits a moderate toxicity, but can still maintain more than 70% of cell viability at nanoparticle dosage up to 100 ug∙mL-1. The high cytotoxicity of pDNA/Flw-SNPs-PEI complex may be explained by the large content of PEI conjugated on the particles. Compared to the commercial transfection agent of

Silica nanoparticles with different nanotopographies may result in variation in particle numbers administered for DNA transfection at the same dosage. Here, to compare the transfection efficacy of these samples under the same particle number, the geometries of Ram-SNPs, Flw-SNPs, Ras-SNPs and S-SNPs were simplified into 3D models as shown in Figure S12. The estimated particle number per milligram of each sample was calculated and shown in Table S3. The relative particle number ratio of Ram-SNPs: Flw-SNPs: Ras-SNPs: S-SNPs is 1:2.6:2.1:0.6 (Table S3). The transfection efficacy normalized by particle number follows the trend of Ram-SNPs > S-SNPs > Flw-SNPs > RasSNPs, where the rambutan-like silica nanoparticles still show the best pDNA delivery performance. The excellent pDNA-EGFP delivery performance mediated by Ram-SNPs-PEI was further demonstrated in Chinese hamster ovary K1 (CHO-K1) cell line. Both flow cytometry and confocal microscopy results (Figure S13) reveal that Ram-SNPs-PEI formulation achieves the highest transfection efficacy, which is 3-10 times higher than FlwSNPs-PEI, Ras-SNPs-PEI and S-SNPs-PEI formulations. In both cell lines, compared to the “gold standard” commercial product of Lipofecatmine-2000, Ram-SNPs-PEI provide comparable transfection performance (Figure 5b, Figure S13). However, the cationic lipoplex formulations have been flawed for their incapability of gene molecule protection upon nuclease degradation,40-42 resulting in quick inactivation under serum rich conditions.41 Silica nanoparticles are reported having good DNA protection property against DNase I degradation, the conclusion is based on electrophoresis results which demonstrate at most the DNA backbone is maintained,15, 17, 23 but not the function of DNA molecules which should be directly judged by transfection tests. It should be noted that whether the nanotopography of silica nanoparticles matters on pDNA protection has not been reported.


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Journal of the American Chemical Society

Figure 5. (a) pDNA-EGFP transfection efficacy assay determined by flow cytometry of SNPs-PEI with different nanotopographies -1 at nanoparticle concentrations of 40, 60 and 80 μg∙mL . (b, c) pDNA-EGFP expression determined by flow cytometry of SNPsPEI and a commercial product Lipofectamine-2000 without and with DNase I treatment. (d) Representative confocal images of GFP gene expression mediated by Lipofectamine (d1), Ram-SNPs-PEI (d2), Flw-SNPs-PEI (d3), Ras-SNPs-PEI (d4), S-SNPs-PEI -1 (d5), and corresponding formulations after DNase I treatment (d1* - d5*). In (d), the nanoparticle concentration was 80 μg∙mL and Lipofectamine-2000 was adapted according to the recommended optimized dosage. Transfection assay was performed in HEK-293T cell line with a constant pDNA-EGFP amount of 2 μg per well in 12 well plate.

To reveal the impact of nanotopography on protection of gene molecules against enzymatic degradation, pDNAEGFP loaded SNPs-PEI formulations and Lipofectamine2000 were treated with DNase I and then applied to transfection assay in HEK-293T cells. As shown in Figure 5b, the transfection efficacy of Lipofectamine-2000 decreases dramatically from 92% to 31% after DNase I treatment. In contrast, Ram-SNPs-PEI show no significant decay in transfection efficacy at three nanoparticle dosages (40 80 μg∙mL-1). For three other formulations (Ras-, Flw- and S-SNPs-PEI), the DNA transfection rate after adding DNase I experiences a significant decrease at a low dosage of 40 μg∙mL-1 (Figure 5c). At a higher dosage of 80 μg∙mL-1, the decrease is significant only for S-SNPs-PEI, not Rasand Flw-SNPs-PEI. To further quantify the DNA amount loaded on each nanoparticle as well as the DNA residual amount after DNase I treatment, pDNA/SNPs with and without nuclease degradation were analyzed by gel retardation. At the DNA: silica weight ratio of 0.5:40 (corresponding to silica dosage of 80 μg∙mL-1), all DNA molecules can be adsorbed on particle surface. As shown in Figure S14, all samples without DNase I treatment show similar band intensity. It is noted that more than 83% of pDNA molecules in pDNA/Ram-SNPs-PEI are well maintained after nuclease degradation. In contrast, S-SNPs-

PEI, Ras-SNPs-PEI and Flw-SNPs-PEI only show DNA retention of 23, 72, 76%, respectively, which is in accordance with their transfection performance. The specific DNA amount conjugated on single particle and residual amount after DNase I treatment were further calculated as shown in Table S3. Among the four samples under study, Ram-SNPs-PEI show the highest DNA loading capacity and DNA retention per single particle. The above results indicate that a rough surface (Ram-, Flw-, Ras-) enables pDNA protection against DNase I degradation compared to SNPs with a smooth surface, while the spike type nanotopography exhibits the highest extent of protection. The contributions of nanotopography to pDNA binding, transfection and protection against enzymatic degradation collectively support our proposed mechanism. The laterally continuous porous space of rambutan nanoparticles enables the entanglement of long DNA chains with the silica spikes. Upon DNase I approaching, the contact of enzyme toward DNA molecules “hidden” in the spiky layer is sterically hindered, providing the best protection efficacy among all the vectors under study. The GFP expression in HEK-293T cells incubated with different formulations before and after DNase I treatment



were visualized by confocal microscopy (Figure 5d). RamSNPs-PEI group (d2) exhibits a high GFP expression level similar compared to Lipofectamine-2000 (d1), but much better than Flw-SNPs-PEI (d3), Ras-SNPs-PEI (d4) and SSNPs-PEI (d5) groups. After DNase I treatment, RamSNPs-PEI group still maintains a high level of GFP expression. In contrast, a huge decay of GFP expression is observed for Lipofectamine-2000 treated with DNase I, similar to the flow cytometry results.

Conclusion In summary, the impact of nanotopography on pDNA delivery has been demonstrated. Compared to SNPs with other surface textures, rambutan nanoparticles engineered with a spiky nanotopography have enhanced pDNA loading capability and binding affinity, the highest pDNA-EGFP transfection efficacy compared to report silica based nano-vectors, and better protection of DNA molecules against nuclease degradation than a benchmark commercial product. The nanotopography design provides a new strategy in the development of non-viral vectors with improved performance and promising potential in applications such as gene therapy and DNA vaccines.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Experimental details on material synthesis and characterizations as well as supporting results.

AUTHOR INFORMATION Corresponding Author *[email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT The authors acknowledge the support from the Australian Research Council, Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis (CMM), University of Queensland, and the Queensland node of the Australian National Fabrication Facility. We acknowledge N4 Pharma PLC for providing fund for this research. We thank Mr Richard Webb (CMM) for great help in immunogold labelling experiment.

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Plasmid DNA Delivery: Nanotopography Matters - Journal of the

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