Triggered RNAi Therapy Using Metal Inorganic Nanovectors

Article Cite This: Mol. Pharmaceutics 2019, 16, 3374−3385

pubs.acs.org/molecularpharmaceutics

Triggered RNAi Therapy Using Metal Inorganic Nanovectors Eva Villar-Alvarez,*,†,‡,∥ Baltazar H. Leal,†,∥ Adriana Cambón,† Alberto Pardo,†,‡ Raquel Martínez-Gonzalez,† Javier Fernández-Vega,† Sonia Al-Qadi,† Víctor X. Mosquera,§ Alberto Bouzas,§ Silvia Barbosa,†,‡ and Pablo Taboada*,†,‡ Grupo de Física de Coloides y Polímeros, Departamento de Física de Partículas, Facultad de Física, and ‡Instituto de Investigaciones Sanitarias (IDIS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain § Departamento de Cirugía Cardíaca, Complexo Hospitalario Universitario A Coruña, Instituto de Investigación Biomédica de A Coruña (INIBIC), 15006 A Coruña, Spain Downloaded via RUTGERS UNIV on August 6, 2019 at 03:50:51 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

S Supporting Information *

ABSTRACT: The administration of small interfering RNA (siRNA) is a very interesting therapeutic option to treat genetic diseases such as Alzheimer’s or some types of cancer, but its effective delivery still remains a challenge. Herein, Au nanorod (GNR)-based platforms functionalized with polyelectrolyte layers were developed and analyzed as potential siRNA nanocarriers. The polymeric layers were successfully assembled on the particle surfaces by means of the layer-bylayer assembly technique through the alternating deposition of oppositely charged poly(styrene)sulfonate, PSS, poly(lysine), PLL, and siRNA biopolymers, with a final hyaluronic acid layer in order to provide the nanoconstructs with a potential targeting ability as well as colloidal stability in physiological medium. Once the hybrid nanocarriers were obtained, the cargo release, their colloidal stability in physiological-relevant media, cytotoxicity, cellular internalization and uptake, and knockdown activity were studied. The present hybrid particles release the genetic material inside cells by means of a protease-assisted and/ or a light-triggered release mechanism in order to control the delivery of the oligonucleotides on demand. In addition, the hybrid nanovectors were observed to be nontoxic to cells and could efficiently deliver the genetic material in the cell cytoplasms. The GNR-based nanocarriers proposed here can provide a suitable environment to load and protect a sufficient amount of the genetic material to allow an efficient and sustained knockdown gene expression for long (up to 93% for 72 h), thanks to the slow degradation of PLL, without the observation of adverse side toxic effects. It was also found that the silencing activity was enhanced with the number of siRNA layers assembled in the nanoplatforms. KEYWORDS: hybrid nanoplatform, theranostics, enzymatic release, siRNA, phototherapy

1. INTRODUCTION

explored to target the delivery and sustain the release of siRNA in vitro and in vivo.2−6 In this regard, nanoparticles (NPs) may be considered as potential efficient nonviral vectors to encapsulate polynucleotides such as plasmid DNA and siRNA for their in vitro and in vivo delivery.3,4,7,8 Their bioactivity and bioperformance seems to be better than current viral vectors as a consequence of negligible associated carrier toxicity and immune-mediated reactions, potential carcinogenicity, and development of viral pathogenic forms.9−12 Various kinds of nanocarriers as cationic polymers,13,14 lipids,15,16 and organic and inorganic NPs17−20 have been used to overcome the aforementioned limitations and, particularly, silica,7,20,21 iron oxide,19,22 and gold NPs (Au

Small interfering RNA (siRNA) is configured as a doublestranded RNA molecule usually composed of 21−23 base pairs able to degrade complementary messenger RNA sequences by the knockdown of the disease-related gene expression.1 In this manner, gene silencing using siRNAs rapidly rises as a powerful therapeutic option to achieve gene knockdown,2 which can be of key importance in numerous disease consequences of an abnormal gene expression such as cancer or neurodegenerative diseases (Alzheimer’s or Parkinson’s diseases). Consequently, a decrease or complete suppression of these genes in specific cells would then be therapeutically beneficial. However, to achieve such a goal in clinical trials some challenges must be first overcome. For example, the use of free siRNA-based therapies is hindered by its poor biodistribution, circulation stability, intracellular trafficking, fast degradation by ubiquitous nucleases, and associated nonspecific immune reactions. To address such issues, different methodologies have been © 2019 American Chemical Society

Received: Revised: Accepted: Published: 3374

January 7, 2019 June 6, 2019 June 12, 2019 June 12, 2019 DOI: 10.1021/acs.molpharmaceut.9b00021 Mol. Pharmaceutics 2019, 16, 3374−3385

Article

Molecular Pharmaceutics NPs)23,24 have been largely investigated as new siRNA delivery carriers. Au NPs possess several interesting characteristics such as their attainment by simple wet chemical methodologies, easily tunable sizes and shapes, a wide range of easily affordable surface functionalizations, and conjugation with biomolecules, as well as biocompatibility.25 Amongst the different types of Au NPs, GNRs have gained a special interest as a consequence of their outstanding optical properties. GNRs exhibit two surface plasmon resonance (SPR) bands: the longitudinal surface plasmon resonance (LSPR), tunable from the visible to the near-infrared (NIR), where the light is transparent to tissues (the so-called biological window at ca. 700−1200 nm), and the transversal one which lies in the visible range of the electromagnetic spectrum. Moreover, GNRs have been widely studied because of their exceptional NIR absorption crosssections and photothermal conversion efficiencies26 being a potential new tool for cancer diagnosis and therapeutics.27 Additionally, GNRs are easily surface-functionalized, which make them attractive nanocarriers for chemodrugs,28,29 photosensitizers,30 small biomolecules,31 and genetic materials.32,33 Very recently, several different examples of multifunctional GNR-siRNA nanocomplexes have been developed to fight against diverse types of cancers such as breast,32 pancreas,34 colorectal,35 and head and neck tumors,36 being highlighted as potential nanovectors for simultaneous siRNA, drug delivery, and/or photothermal and imaging agents for cancer theranostic purposes.37−39 Nevertheless, to use GNRs as gene delivery platforms several common limitations must be overcome: (i) their positively charged surfaces and inherent cytotoxicity of the CTAB surfactant layer resulting from the synthesis process;40,41 (ii) nonspecific cargo delivery, and (iii) unspecific targeting ability. The positively charged GNRs may also induce the nonspecific binding of blood serum components onto their surfaces favoring their aggregation in the physiological medium, consequently, increasing the risk of embolism within the body. 42 Besides, the nonspecific interactions between positively charged GNRs and negatively charged cell membranes may also induce nonspecific siRNA delivery, thereby, largely decreasing the therapeutic efficacy.42 To overcome such drawbacks, different surface coatings and functionalizations have been developed and tested with the aim of reducing GNRs cytotoxicity as “hiding” the CTABcoating layer with polyelectrolytes (PEs)40 and phospholipids,43 or by exchanging this surfactant with other molecules such as poly(ethylene glycol).44 Here, we developed a layer-by-layer (LbL) polymeric assembly strategy to coat GNRs with poly(styrene sulfonate) (PSS, as the anionic layer) and poly(lysine) (PLL, as the cationic one) in order to (i) efficiently mask the remaining CTAB layer after the cleaning process;40,45 (ii) to ensure a suitable colloidal stability of the nanoplatform; (iii) to overcome some toxicity issues related to other commonly used synthetic cationic electrolytes in LbL self-assembly procedures such as poly(allylamine) chloride, polyethylenimine, or polydiallyldymethylammonium chloride, among others; and (iii) to provide a stimuli-responsive release mechanism of the cargo molecules making use of the enzymatic degradation ability of PLL by endogenous proteases. To achieve the latter goal, siRNA is assembled in the polymeric coating surrounding the metallic core in one or two layers through electrostatic interactions with the underlying positively

charged PLL chains. Finally, an external outer layer composed of hyaluronic acid (HA) was assembled to avoid nonspecific interactions with serum components,46 to reduce the free diffusion of the underlying assembled siRNA, and to actively target CD44 receptors typically overexpressed in, for example, tumoral cervical and breast cancerous cells,47,48 consequently, ensuring a high localization of the hybrid particles and subsequent cargo release in the diseased cells/tissues. In this manner, we developed an active targeting GNR-based nanoplatform, which provides a dual-triggered mechanism to control siRNA cargo release kinetics on demand by making use of the protease-based biodegradability of the PLL polymer and the temperature enhancements facilitating cargo diffusion on NPs surfaces, thanks to the light responsiveness of the hybrid particles under NIR light irradiation of suitable wavelengths. In addition, an enhanced protection to cargo biomacromolecules and control on release kinetics is provided by the controlled configuration of the polymeric coating. In this manner, a complementary targeted dual mechanism, silencing + photothermal is achieved in order to induce toxicity in malignant cancerous cells. The successful formation of the different coating layers onto the metallic NPs was confirmed by the shifts in the LPSR bands of GNRs as well as the observation of the typical zig-zag patterns in their ζ-potential values. It was observed that the construction of the hybrid platform has also an important influence on both their release and photothermal properties, that is, the presence of a two-layered siRNA coating onto the NPs provides slower cargo release rates and lower photothermal enhancements in vitro than the one-layered coated counterparts. The present hybrid PE/siRNA-coated GNRs were shown to be biocompatible and were effectively uptaken and internalized by tumoral cells. In addition, they were able to release their content inside the cells as observed by fluorescence microscopy, giving rise to the expression knockdown of the green fluorescent protein (GFP) overexpressed on a modified GFP-HeLa cell line. This knockdown was more efficient when the nanoconstructs bear a two-layered siRNA coating under NIR-light irradiation of low intensity (0.5 W/ cm2), being better than that of the commercially available Lipofectamine RNAiMAX transfection reagent used as a control).

2. EXPERIMENTAL SECTION 2.1. Materials. Tetrachloroauric acid (HAuCl4·3H2O), silver nitrate (AgNO3), hexadecyltrimethyl ammonium bromide (CTAB), sodium borohydride (NaBH4), poly(sodium-4styrenesulfonate) (PSS) of molecular weight (Mw) ≈ 70 000 g/ mol, poly-L-lysine hydrobromide (PLL) of Mw ≈ 22 000 g/ mol, HA of Mw ≈ 15 000 g/mol were from Sigma-Aldrich. BLOCK-iT control fluorescent oligo (1 mM) was from Invitrogen. siRNA against GFP was purchased from Ambion [silencer GFP (eGFP) siRNA]. Heat-inactivated fetal bovine serum and trypsin 0.25% with ethylenediaminetetraacetic acid were from Hyclone (Thermo Scientific, USA). All other reagents were of analytical grade and suitable for cell culture. All organic solvents were of HPLC grade. DNa−RNase-free water was used in all experiments. 2.2. Synthesis of GNRs. GNRs were obtained using a seed-mediated growth synthetic procedure with some modifications, as previously described.28 The GNRs concentration was calculated by the combination of inductively coupled plasma mass spectroscopy (ICP-MS) and transmission 3375

DOI: 10.1021/acs.molpharmaceut.9b00021 Mol. Pharmaceutics 2019, 16, 3374−3385

Article

Molecular Pharmaceutics

Scheme 1. Schematic Representation of Single-Layer siRNA-Coated (Left) and Two-Layered siRNA-Coated GNRs (Right)

residues at 280 nm using a Cary Bio 100 UV−vis spectrophotometer (Agilent Technologies, USA). Fluorescence spectra were obtained with a Cary Eclipse spectrophotometer (Agilent Technologies, USA). The encapsulation efficiency (EE) and loading capacity (LC) of the hybrid particles were calculated as

electron microscopy (TEM) data. For GNRs with a LSPR band at 780 nm, 1 mL of three different GNRs batches at an OD ≈ 1 has a Au concentration of ca. 49.0 ± 0.5 μg/mL, considering the particle length and width obtained by TEM. The final particle concentration obtained under these conditions was of ca. 5 × 1014 rods/L. 2.3. LbL Polymeric Coating of GNRs. 2.3.1. PSS/PLL/ siRNA/HA-Coated GNRs. Multilayers of PSS and PLL were successfully deposited onto the GNR surfaces as described in a previous publication (see Scheme 1).28 For the siRNA assembly, within the polymeric coating, the desired amount of siRNA (typically 0.5 nmol) was diluted in 1 mL of RNasefree water and stirred at 500 rpm for 30 min in a water bath at 37 °C. Then, 1 mL of PSS/PLL-coated GNRs was added dropwise. After 2 h of adsorption at 37 °C, this mixture was centrifuged once at 15 000 rpm for 10 min and redispersed in 1 mL of RNase-free water. The final HA layer was assembled onto the BP surfaces by previously mixing 60 μL of an 1 mg/ mL HA solution with 1 mL of RNase-free water under stirring at 500 rpm for 10 min. Then, PSS/PLL/siRNA-coated GNRs were added dropwise to the mixed solution. After 1 h, the obtained hybrid NPs were centrifuged at 15 000 rpm for 10 min and resuspended in 1 mL of RNase-free water. 2.3.2. (PSS/PLL/siRNA)2/HA-Coated GNRs. A process similar to that for the deposition of a single monolayer was followed, but repeated exactly twice. Finally, the outer HA layer was added as described above. 2.4. Dynamic Light Scattering. Dynamic light scattering (DLS) data were obtained by means of an ALV-5000 digital correlator system (ALV 5000/E, ALV GmbH, Germany) equipped with a 488 nm solid-state laser (2 W), as previously described.28 For stability measurements, the temporal evolution of the hydrodynamic radii of (PSS/PLL/siRNA)2/HA-coated GNRs were evaluated in water, water + 10% (v/v) fetal bovine serum (FBS), phosphate-buffered saline (PBS) buffer pH 7.4, and PBS buffer pH 7.4 + 10% (v/v) FBS. 2.5. Electrophoretic Mobilities. ζ-potential values of bare and PE-coated GNRs were obtained from electrophoretic mobilities measured by means of a Nano ZS instrument (Nanoseries, Malvern Instruments, UK) at 25 or 37 °C. Each NP sample in 10 mM phosphate buffer pH 7.4 was fed into a folded capillary cell and measured in triplicate. 2.6. Quantitative Analysis of siRNA. To quantify the amount of siRNA loaded in the hybrid nanoplatforms, PSS/ PLL/siRNA/HA coated-GNRs and (PSS/PLL/siRNA)2/HAcoated GNRs were centrifuged at 15 000 rpm at 20 °C for 20 min, and the siRNA concentration in the supernatant was determined by UV−vis and fluorescence spectroscopies in triplicate for three different batches and averaged. Previously, calibration curves for free FITC-labeled siRNA in RNase-free water at 37 °C were obtained. UV−vis data were obtained at 260 nm after subtraction of the contribution of protein

EE (%) =

total amount of SiRNA feeded − siRNA in supernatant × 100 total amount of SiRNA feeded

(1a) LC (%) =

total amount of SiRNA feeded − siRNA in supernatant × 100 total weight of nanoparticles

(1b)

2.7. In Vitro Release. The temporal evolution of the siRNA release from PSS/PLL/siRNA/HA-coated GNRs and (PSS/PLL/siRNA)2/HA-coated GNRs was monitored at different solution conditions, in particular, at pH 7.4 and 5.5 at 37 °C under 300 rpm stirring in the presence and absence of proteases and/or NIR light irradiation by dialysis. For details, please see the Supporting Information. 2.8. Tumor Cells. HeLa cervical cancer cells, GFPmodified HeLa, and breast MDA-MB-231 cells were purchased from Cell Biolabs (San Diego, CA). Cells were cultured under standard conditions (5% CO2 at 37 °C) in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 1% (v/v) penicillin/streptomycin, 1 mM sodium pyruvate, and 0.1 mM MEM nonessential amino acids. 2.9. In Vitro Cell Cytotoxicity. The cytotoxicity of siRNA-loaded GNRs was analyzed in vitro using the CCK-8 proliferation assay. For further details, please see the Supporting Information. 2.10. Cellular Uptake by TEM. HeLa cells were seeded in 6-well plates (5 × 104 cells per well) and grown for 24 h at standard conditions. Each type of hybrid particle (200 μL) (2.5 × 1010 NP/mL) was added to the wells. After 6 h, cells were washed three times with ice cold PBS, trypsinized, and centrifuged at 1500 rpm for 4 min. Cell pellets were fixed with 500 μL of a 2.5% (v/v) glutaraldehyde solution, included in an agar pellet, postfixed with osmium tetraoxide in 0.1 M cacodylate buffer at 1% (w/v), and, finally, pelletized with Eponate (Ted Pella Inc, Redding, CA, USA). Ultrathin cuts were obtained with an ultramicrotome (UltraCut S, Leica Microsystems GmbH) and analyzed with a transmission electron microscope (JEOL JEM 1011, Japan). 2.11. Cellular Uptake by ICP-MS. The gold concentration and, thus, the number of GNRs inside the cells were quantified by ICP-MS. PSS- (200 μL), and PSS/PLL/HAcoated GNRs (2.5 × 1010 NP/mL) were added to HeLa and MDA-MB-231 cells (1 × 105 cells/well) and incubated for 6 h. Cells were washed three times with ice-cold PBS, harvested, and measured by ICP-MS, as previously explained.28 To test whether the hybrid nanoplatforms are internalized inside cells 3376


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Figure 1. UV−vis spectra for (a) PSS/PLL/siRNA/HA-coated GNRs and (b) (PSS/PLL/siRNA)2/HA-coated GNRs after each surface-assembled PE layer on the NP surfaces.

Figure 2. Changes in the position of LPSR maxima (●), decreases in the longitudinal (green ■) and transversal absorption bands (green □), and ξ-potential values (■) after each polymeric assembled layer for (a,b) PSS/PLL/siRNA/HA-coated and (c,d) (PSS/PLL/siRNA)2/HA-coated GNRs. LSPR shifts of the hybrid GNRs with assembled coating layer i are defined as: Δλ = λ(i) − λ(CTAB). Decays of longitudinal and transversal absorption bands are determined as decay (SPR) = abs(i)/abs(CTAB), that is, the ratio between the optical absorbance in the layer i regarding that of the underlying CTAB layer. (e) Hydrodynamic radii for CTAB-coated (black), PSS-coated (red), PSS/PLL/siRNA/HA-coated (blue), and (PSS/PLL/siRNA)2/HA-coated (green) GNRs. (f) TEM images of (PSS/PLL/siRNA)2/HA-coated GNRs.

by a receptor-mediated endocytosis process, control experiments were performed by adding either free HA (200 μM) or

free anti-CD44 (Abcam Inc, USA) to cells 1 h before the incorporation of the nanoplatforms. 3377


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Molecular Pharmaceutics 2.12. Cellular Internalization and Gene Expression Silencing by Fluorescence Microscopy. Particle cell uptake and siRNA transfection were analyzed using fluorescence microscopy by seeding HeLa (for internalization experiments) and GFP-HeLa (for gene silencing) cells, respectively, in the presence of PSS/PLL/siRNA/HA-coated GNRs, (PSS/PLL/siRNA)2/HA-coated GNRs (2.5 × 1010 NP/mL), and siRNA-Lipofectamine RNAiMAX complexes used as controls. The silencing activity was monitored by the loss of fluorescence in the GFP-HeLa cell line, whereas cell internalization was followed by the increases of cell fluorescence signals in normal HeLa cells after the internalization of both type of hybrid NPs. For further details, please see the Supporting Information. 2.13. Cellular Uptake and Gene Expression Silencing by Flow Cytometry. siRNA uptake was quantified by measuring the fluorescence of a FITC-labeled siRNA oligo (1 mM, Invitrogen) assembled on PSS/PLL/siRNA/HAcoated and (PSS/PLL/siRNA)2/HA-coated GNRs (2.5 × 1010 NP/mL) by flow cytometry, as reported elsewhere (see Supporting Information for details).49 2.14. Statistical Analysis. All data were expressed as means ± standard deviations from at least three independent experiments. Statistical significance was assayed by a two-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test using GraphPad Prism 6 software.

optimal conditions for siRNA coating were established, the LbL coating process was monitored by UV−vis spectrophotometry. UV−vis data (Figure 1) show that the LPSR band of GNRs progressively shifts after each PE adsorption, confirming the successful deposition of the PE layers. It is also noted that almost no broadening of the longitudinal GNRs’ plasmon peak takes place as a result of the coating process, suggesting the absence of particle aggregation. The intensity of the LPSR peak abruptly drops when the first PSS layer is attached to the Au surface. This light damping can be the result of a decrease in the electron oscillation amplitude related to: (i) light absorption by the PE layer, (ii) a slow down of the electronic oscillations on the metallic surface.59,60 However, for subsequent deposited PE layers, no additional decreases were noted (see Figure 2a). The transversal plasmon band remains barely constant during the PE complexation process except when siRNA layers are adsorbed, for which an enhancement is noted. The observed increase at ca. 490−495 nm may stem from light absorption by the FITC dye present in the fluorescence-modified oligo-siRNA. ζ-potential data showed a complete change in the sign of the GNRs surface charge after the alternating deposition of the PSS and PLL layers. This characteristic zig-zag pattern (Figure 2b,d) confirms the alternating deposition of the negatively and positively charged PEs, respectively. However, this pattern is not observed when wrapping siRNA and HA. For siRNA, the amount of oligonucleotides is not enough to reverse the NP surface charge, and some PLL patches remain exposed. This effect was also taken into account in order to assemble the outer HA layer, this means, slightly positive NPs should interact to larger extents with negatively charged cell membranes. Therefore, HA was added in order to get an effective decrease in the net NPs positive surface charge but without a complete reversal. Figure 2e shows the hydrodynamic radii of siRNA-coated GNRs measured by DLS. The sizes of the present hybrid NPs increase as the number of assembled layers does (ca. 60 and 90 nm for PSS/PLL/siRNA/HA-coated and (PSS/PLL/ siRNA)2/HA-coated GNRs, respectively, where the subscript denotes the number of layers). Here, it is worth reminding that hydrodynamic sizes are calculated assuming spherical geometries of the nano-object, so these data should be considered as a rough estimation. To disregard any possible contribution coming from GNR aggregation, TEM images of (PSS/PLL/ siRNA)2/HA-capped GNRs were also acquired (Figure 2f). No important signs of particle aggregation were observed. Unfortunately, the polymeric coating wrapping the metallic NPs could be hardly observed. Finally, the EE for an initial siRNA feed amount of 0.5 nmol in a 5 × 1011 NR/mL particle solution was estimated to be 77.6 ± 8.3 and 44.2 ± 9.7% for the first and second wrapped siRNA layers, respectively (total efficiency ca. 62 ± 15%), that is, ca. 380 and 600 pmol of siRNA were loaded, which correspond to LC values of ca. 14.2 ± 7.1 and 11.0 ± 3.0%, respectively. 3.2. siRNA Release from siRNA-Coated GNRs. The influence of both endogenous enzymatic and exogenous NIR light-triggered processes on the siRNA release profiles from the hybrid particles were analyzed at pH 7.4 and 5.5. The degradability of PLL by endogenous proteases may provide a certain control over the degradation of the PE coating inside the cells to get a triggered and sustained siRNA release from the particles, which results in a long gene-silencing effect. On

3. RESULTS AND DISCUSSION RNAi methodologies for gene knockdown make use of synthetic RNA consisting of a negatively charged doublestranded siRNA (ca. 21−23 oligonucleotides) with rodlike dimensions of 5.5 nm × 2.0 nm.50 Some previous studies have shown that by using the LbL coating technique siRNA can be attached to positively charged particle surfaces either at room or higher temperatures.23,51 Here, we used CTAB-capped GNRs as the positively charged NPs synthesized by a modified seed-mediated growth methodology using CTAB as the stabilizing surfactant agent.52 The excess of CTAB was extensively removed by centrifugation; subsequently, CTABcapped GNRs were alternatively coated with negatively and positively charged PEs (PSS and PLL, respectively) following the LbL self-assembly technique. Here, PSS was selected by its biocompatible nature, whereas PLL was used taking advantage of its biodegradation by proteases to achieve an endogenoustriggered cargo release nanoplatform.41,53 siRNA was loaded on the metallic nanocarrier surfaces as a single or two assembled coating layers exploiting the electrostatic adsorption interactions of the negatively charged cargo biomolecule with the underlying PLL layer (see Scheme 1). Finally, an outer HA layer was wrapped around the particles as a targeting ligand to provide the carrier with colloidal stability and targeting ability to cancerous cells overexpressing CD44 receptors such as colorectal, pancreatic, lung, breast, liver, gastric, renal, and cervical ones.54−56 3.1. Obtaining and Physico-Chemical Characterization of siRNA-Coated GNRs. siRNA is a stiff rodlike molecule57 which is not expected to easily wrap around Au NPs. Thus, it is necessary to find suitable conditions to optimize its attachment. In this sense, the influence of solution temperature in siRNA wrapping onto LbL-functionalized GNRs was analyzed because this variable possesses a great importance in the ability of siRNA to be adsorbed onto NP surfaces (see Supporting Information for details).58 Once the 3378


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Figure 3. siRNA release profiles for PSS/PLL/siRNA/HA-coated (■, □) and (PSS/PLL/siRNA)2/HA-coated GNRs (green ●, ○) when trypsin is within (closed symbols) or not (open symbols) the sampling solution of (a) pH 7.4 and (b) 5.5. The NIR light-induced siRNA release from (c) PSS/PLL/siRNA/HA-coated and (d) (PSS/PLL/siRNA)2/HA-coated GNRs. Laser light fluencies used were 3.0 (red ▼) and 0.5 (blue ▲) W/ cm2. In the latter plots, simple cargo diffusion (■, ●) and enzymatic-assisted release (□, ○) profiles were also included for comparison. The uncertainties were below 10.0% and not plotted for clarity.

siRNA)2/HA-coated ones, especially when the enzyme is not present in the surrounding medium. The burst phase is followed by a more sustained release, as occurring at pH 7.4. The release rates are larger for PSS/PLL/siRNA/HA-coated GNRs, with a faster siRNA release when the enzyme is present in solution by ca. 15−25%. Moreover, the amount of siRNA released is lower at acidic pH as a result of: (i) PLL can adopt a random coil conformation at this pH in contrast to a more expanded configuration at more basic conditions;63 and (ii) this PE is more densely charged at pH 5.5, thus, facilitating interactions and entanglements with siRNA strands. 3.2.2. NIR Light-Induced Release. Optical laser excitation at the LSPR frequency of GNRs results in strong light absorption by this type of NPs. This absorbed energy can help modulate the attractive interaction between the metallic particles and the surrounding molecules allowing the controlled release of the therapeutic cargo molecule (siRNA) on demand by two possible mechanisms, as previously stated:64 (i) a nonequilibrium thermal mechanism based on the localized heating around each NP surfaces which allows siRNA releases before bulk solution temperature increases; (ii) a nonthermal one, where the laser excitation generates hot electrons which modify the PE-siRNA interactions. PSS/PLL/siRNA/HA-coated and (PSS/PLL/siRNA)2/HAcoated GNRs were incubated at 37 °C (1 × 1011 NP/mL) and pH 5.5. After 6 and 24 h of incubation the hybrid nanoplatforms were exposed to a continuous wave (CW) NIR laser light (808 nm) for 5 min at a fluency of 0.5 and 3.0 W/cm2 (see Figures 3c,d, and S2 for temperature heating profiles in Supporting Information).

the other hand, the optical absorption properties and the subsequent ability to act as photothermal agents make GNRs suitable platforms to use NIR light as an external trigger in order to get a controlled release of the cargo molecules upon subtle temperature changes in the NP surroundings under light stimulation. 3.2.1. Enzymatic-Assisted Release. PLL has been previously shown as a promising material to develop gene-delivery vectors and drug nanocarriers, thanks to its protease biodegradability.61 PLL is positively charged at physiological pH, so it can electrostatically interact with the negatively charged siRNA phosphate backbone. To study the siRNA release, PSS/PLL/ siRNA/HA-coated and (PSS/PLL/siRNA) 2 /HA-coated GNRs were incubated in both presence and absence of trypsin at pH 7.4 and 5.5 at 37 °C for 4 days (Figure 3a−b). At pH 7.4, siRNA release profiles are independent of the number of polymeric layers but the release rates are not.62 There exists an initial burst phase within the first ca. 12−15 h followed by a more sustained release pattern, with cargo releases of up to ca. 85 and 50−65% in the presence and absence of trypsin, respectively. In the absence of the enzyme, 50% of siRNA was released from PSS/PLL/siRNA/HA-coated GNRs within 58 h of incubation in comparison to 96 h needed for (PSS/PLL/siRNA)2/HA-coated ones. This fact confirms that the bilayered hybrid nanoconstruct is more resistant to siRNA diffusion out of the coating probably as a consequence of the thicker layered structure, which may offer an enhanced protection for the genetic material together with a more sustained release. At pH 5.5, a clear burst phase for PSS/PLL/siRNA/HAcoated GNRs is also present, being shorter for (PSS/PLL/ 3379


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Figure 4. Cell cytotoxicity induced under NIR light irradiation of PSS/PLL/siRNA/HA-coated and (PSS/PLL/siRNA)2/HA-coated GNRs in (a) HeLa and (b) MDA-MB-231 cancer cells. Cells were illuminated with a CW NIR laser at 808 nm at 0 (blue), 0.5 (light blue), 1.0 (light red), and 3.0 W/cm2 (red) for 5 min. Each treatment was referred to the survival rate of the negative control (HeLa cells) (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

PLL/siRNA/HA-coated and (PSS/PLL/siRNA)2/HA-coated GNRs in vitro, a CCK-8 proliferation assay was performed after particle administration (2.5 × 1010 NP/mL) and subsequent incubation for 24 and 48 h in both cervical HeLa and breast MDA-MB-231 cancerous cells. These cells were illuminated with the 88 nm laser light for 5 min at different fluencies (0, 0.5, 1.0, and 3.0 W/cm2). Some cells were also transfected with the siRNA oligo using Lipofectamine RNAiMAX as a positive control. When light irradiation is off, the nanoplatform-associated cytotoxicity is negligible in MDA-MB-231 cells, and within 10−20% in HeLa ones, respectively. An exception is observed for (PSS/PLL/siRNA)2/HA-coated GNRs after 48 h, for which their inherent cytotoxicity is ca. 35% (see Figure 4). This value might be associated to larger particle uptakes by CD44 receptor-mediated endocytosis to extents compromising cell viability (see Figure S4 in Supporting Information). Also, transfection of siRNA with Lipofectamine RNAiMAX induces similar cell toxicities (ca. 10−20%, particularly for HeLa cells which are more sensitive).65 On the other hand, NIR light irradiation does not seem to affect cell viability when hybrid platforms are not injected. When these are present in the cell culture medium and cells are NIR-irradiated, progressive higher cytotoxicities are noted as the light fluency increases. Specifically, HeLa cells are much more sensitive to laser light irradiation than MD-MB-231 ones, even at the lowest irradiation (0.5 W/cm2), with cell viabilities below 50%. In fact, for the latter class of cells survival rates are well above 50% in the whole range of fluencies analyzed except at the highest one (3.0 W/cm2). This behavior would stem from the higher particle uptake in HeLa cells as mentioned previously, which would enhance heat production inside cells, thus, improving photothermal cytotoxic effects. Irradiated (PSS/PLL/siRNA)2/HA-coated GNRs possess a lower photothermal cell killing activity than PSS/PLL/siRNA/HA-capped ones probably as a consequence of an attenuation of absorbed

The observed release profiles display again a burst phase within ca. 10−15 h followed by a sustained pattern, as already observed for the enzymatic-triggered release. Release rates and extents at 0.5 and 3.0 W/cm2 for PSS/PLL/siRNA/HA-coated GNRs were rather similar, with released siRNA percentages of up to ca. 85%. Conversely, (PSS/PLL/siRNA)2/HA-coated GNRs exhibit larger cargo releases at the largest fluency, which agrees with the need of a higher energy input to achieve enough light absorption and the subsequent required thermal increase to get the destabilization of the electrostatic interactions between the inner PE layers for successful siRNA release. Moreover, comparison of both enzyme and light triggered-profiles denotes that the latter provides much faster release rates, as observed in Figure 3c−d. At incubation times

Triggered RNAi Therapy Using Metal Inorganic Nanovectors

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