Tobacco Mosaic Virus-Functionalized Mesoporous Silica

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Tobacco Mosaic Virus-Functionalized Mesoporous Silica Nanoparticles, a Wool-Ball-like Nanostructure for Drug Delivery Laura Marín-Caba,† Paul L. Chariou,‡,§ Carmen Pesquera,∥ Miguel A. Correa-Duarte,*,† and Nicole F. Steinmetz*,‡,§

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Department of Physical Chemistry, Biomedical Research Center (CINBIO), Southern Galicia Institute of Health Research (IISGS), and Biomedical Research Networking Center for Mental Health (CIBERSAM), Universidade de Vigo, 36310 Vigo, Spain ‡ Department of Biomedical Engineering, Case Western Reserve University Schools of Medicine and Engineering, Cleveland, Ohio 44106, United States § Department of NanoEngineering, Moores Cancer Center, Department of Radiology, Department of Bioengineering, University of California-San Diego, La Jolla, California 92039, United States ∥ Department of Chemistry and Processes and Resources Engineering, Superior Technical School of Industrial and Telecommunications, University of Cantabria (UC), Sanitary Research Insitute, (IDIVAL, Valdecilla), Santander 39005, Cantabria, Spain S Supporting Information *

ABSTRACT: The design of versatile tools to improve cell targeting and drug delivery in medicine has become increasingly pertinent to nanobiotechnology. Biological and inorganic nanocarrier drug delivery systems are being explored, showing advantages and disadvantages in terms of cell targeting and specificity, cell internalization, efficient payload delivery, and safety profiles. Combining the properties of a biological coating on top of an inorganic nanocarrier, we hypothesize that this hybrid system would improve nanoparticle−cell interactions, resulting in enhanced cell targeting and uptake properties compared to the bare inorganic nanocarrier. Toward this goal, we engineered a hierarchical assembly featuring the functionalization of cargo-loaded mesoporous silica nanoparticles (MSNPs) with tobacco mosaic virus (TMV) as a biological coating. The MSNP functions as a delivery system because the porous structure enables high therapeutic payload capacity, and TMV serves as a biocompatible coating to enhance cell interactions. The resulting MSNP@TMV nanohybrids have a wool-ball-like appearance and demonstrate enhanced cell uptake, hence cargo delivery properties. The MSNP@TMV have potential for medical applications such as drug delivery, contrast agent imaging, and immunotherapy.

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INTRODUCTION A new era for diagnosis and therapy has emerged from the confluence of multidisciplinary fields such as biology, materials science, chemistry, and medicine. In the same way, the structures studied for biomedical applications are no longer either monofunctional or monomaterial.1 Over the past few decades, bionanotechnologies have been subjected to intense research efforts toward the development of new nano-sized materials.2,3 In particular, hybrid nanomaterials made of bioinorganic components have attracted the attention of the scientific community due to their huge potential in the nanomedicine field (e.g., nanotheranostics and advanced drug delivery systems).1,4−6 A fashionable case is nanohybrids composed of a polymeric or inorganic core functionalized with soft-matter bionanoparticles as a shell.6 Plant viruses working as biocoating deserve special attention because they can be regarded as naturally © XXXX American Chemical Society

occurring nanomaterials, which have been widely studied in the field of nanomedicine in the context of drug delivery, immunotherapy, and molecular imaging.7 In this regard, hybrid core−shell nanomaterials using plant viral nanoparticle (NP) as one of their components have been described in the literature; for example, polymer−virus hybrid core−shell nanoparticles have been reported using cowpea mosaic virus (CPMV) and tobacco mosaic virus (TMV).8−10 Other examples of virus−inorganic nanohybrids include polyelectrolyte-functionalized CPMV covered with gold NPs,11 selfassembled plant viruses by Pickering emulsion interfaces,12,13 and SiO2 nanospheres covered by the filamentous potato virus X (PVX).14 Received: October 2, 2018 Revised: November 30, 2018

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Although synthetic strategies for the preparation of such hybrid materials have been reported as outlined above, their potential in the medical sector has not yet been demonstrated. A plant-viral-protein-coated nanoparticle hybrid would be appealing because its protein coating is expected to enhance colloidal stability and promote nanoparticle−cell interactions through protein−protein and protein−lipid interactions.14 Additionally, this plant viral coating provides a threedimensional scaffold for further functionalization. Thus, through this approach, the plant viral nanoparticle could carry a therapeutic effect, whereas the core particle could function as a contrast agent for theranostics. Also, by means of this hybrid system, different drug combinations could be delivered with spatiotemporal control, which means that different drugs could be delivered at different times and different locations in the cells. Mesoporous silica nanoparticles (MSNPs) were chosen as a core because these materials have been extensively explored in drug delivery due to their ability to carry large payloads of drugs, their tunable particle size, and ease of functionalization.15−18 The MSNPs possess several unique features including a porous structure with tunable pore diameter (2− 30 nm) allowing for high drug loading capacity and temporally controlled drug release. In addition, MSNPs exhibit colloidal stability, chemical versatility, and high biocompatibility with low immunogenicity.1,19 Although MSNPs have been extensively explored for drug delivery in cancer treatment,18 in many cases, their efficacy is still limited.20 Thus, the development of functionalization strategies of the MSNP nanocarriers is required to enhance their cell targeting and uptake properties. TMV was chosen as the biological coating unit and has been studied in plant pathology and structural biology since the early twentieth century and more recently in biotechnology, nanomedicine, and energy research.21−23 Native TMV has a cylindrical structure (300 × 18 nm) formed by 2130 identical copies of the virus coat protein (CP). These CPs self-assemble around a single stranded RNA, forming hollow nanorods with a 4 nm wide interior channel.24 Because TMV is a plant virus, it cannot infect or replicate in mammalian cells. Furthermore, TMV is not only biocompatible and biodegradable but can also be chemically or genetically modified to impart new functionalities (e.g., tissue specificity or cargo for imaging or drug delivery).25 Additionally, from a manufacturing and quality control point of view, the monodispersity and low cost of production of TMV offer advantages over its synthetic counterparts. We report the synthesis of a novel nanohybrid consisting of a MSNP core functionalized with a TMV shell, MSNP@TMV. As a proof-of-concept of their potential for drug delivery applications, the MSNPs were loaded with rhodamine B isothiocyanate (RBITC) as a model drug. The pores were sealed with a gatekeeper system16 made of a high-molecularweight poly(diallyldimethylammonium chloride) (PDADMAC) polyelectrolyte. Moreover, the functionalization of the MSNPs with this positively charged polyelectrolyte enables the electrostatic assembly of TMV (TMV has an overall negative surface charge) onto the MSNP surface. Additionally, TMV were labeled with cyanine5 (Cy5) fluorophore for particle tracking in tissue culture experiments; the cell uptake properties of the nanohybrid system was assessed using a cervical cancer cell line (HeLa).

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EXPERIMENTAL METHODS

Chemicals. p-Toluenesulfonic acid (p-TSA) was supplied by Fisher Scientific and sulfo-cyanine5-azide (Cy5) by Lumiprobe. Aminoguanidine (AMG, 98%), L-ascorbic acid sodium salt (Sod Asc, 99%), copper(II) sulfate (CuSO4, 98%), hexadecyltrimethylammonium bromide (CTAB, 99%), and glycerol (99%) were obtained from ACROS Organics. Sodium nitrite (NaNO3) and 3-ethynylaniline (3E), poly(diallyldimethylammonium chloride) (PDADMAC, Mw = 400 000−500 000 Da), tetraethyl orthosilicate (TEOS, 98%), rhodamine B isothiocyanate (RBITC, 70% labelling efficiency), and (3aminopropyl)triethoxysilane (APTS) were purchased from SigmaAldrich. AlexaFluor 488 antibodies were obtained from Life Technologies and AlexaFluor 488 antihuman CD107a (Lamp-1) antibodies were supplied by Biolegend. Milli-Q water was prepared in a three-stage Millipore Milli-Q plus 185 purification system with a resistivity higher than 18.2 MΩ cm. TMV−Cy5 Preparation. Virus Propagation and Purification. T158K mutant of TMV was propagated in Nicotiana benthamiana and purified through established protocols, yielding approximately 5 mg of virus per gram of infected leaf material.25−28 TMV Sulfo-Cy5 Azide Labeling. The surface-exposed tyrosine (Tyr) residues of TMV were labeled with the sulfo-cyanine5-azide (Cy5) dye using a two-step reaction. First, a diazonium salt (DS) was formed by reacting 0.3 M p-TSA with 3 M NaNO3 and 0.68 M 3ethynylaniline on ice for 1 h. The resulting diazonium salt contains a reactive alkyne for the subsequent click chemistry. Then, 15 equiv of the in situ formed diazonium salt was added to TMV (2 mg mL−1) in 0.1 M borate buffer of pH 8.8 for 30 min on ice.26 The resulting TMV−DS was purified over a 40% (w/v) sucrose cushion using an Optima MAX-TL ultracentrifuge (Beckman) and a TL-55 rotor at 50 000 rpm for 1 h. Subsequently, click chemistry was performed through a Cu(I)-catalyzed alkyne−azide cycloaddition reaction. Two equivalents of Cy5 per coat protein were added to 2 mg mL−1 TMV− DS in the presence of 2 mM AMG, 2 mM Sod Asc, and 1 mM CuSO4 in 10 mM KP buffer of pH 7.4 on ice for 30 min. The final TMV− Cy5 product was purified by ultracentrifugation as described above. MSNP Preparation. MSNP Synthesis. MSNPs were synthesized in an aqueous solution at pH 7. First, 10 mM CTAB and 12% (v/v) glycerol were dissolved in 0.686% (w/v) of KH2PO4 and 1.16% (w/v) NaOH in deionized water under vigorous magnetic stirring at 95 °C. After the solution became homogeneous, 161 mM TEOS was added every 30 min for 8 h. The resulting MSNPs were centrifuged and washed three times with ethanol before they were dried at 70 °C for 2 h. Surfactant removal was carried out by calcination at 550 °C during 6 h using a heating rate of 1 °C min−1. The resulting product was centrifuged and washed with ethanol several more times.29,30 MSNP Dye Loading and Taping with Polyelectrolyte. Two hundred forty-two micromole RBITC was mixed with 10 mg of MSNP in ethanol solution for 18 h under magnetic stirring. RBITC− MSNP were collected by centrifugation at 13 000 g for 10 min and washed with Milli-Q water. The dye concentration of the supernatant was calculated to quantify the mass of dye loaded in the MSNP carrier using UV−vis spectroscopy (see below). The entrapment efficiency (EE) was calculated as shown in eq 131 EE (%) = (M t /M i) × 100

(1)

where Mt is the molarity of the total amount of dye loaded in nanoparticles and Mi is the molarity of dye added initially in the suspension, taking into account that RBITC has λmax = 555 nm and ε = 9.6 × 10−3 M−1 cm−1 in 70% ethanol. After the last washing step, RBITC−MSNP were resuspended in a solution of PDADMAC (1 μL mL−1, NaCl 0.5 M), for 3 h with magnetic stirring. Subsequently, RBITC−MSNP were washed three times at 13 000 g, 15 min, in deionized water. MSNPs were also functionalized with APTS. First, 100 μL of APTS was added to 10 mg of MSNP in an ethanol solution for 3 h and then washed several times by centrifugation. The procedure followed was same as that described above. The resulting sample was RBITC− MSNP−APTS. B

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Figure 1. (a) Sequential synthetic steps of wool ball MSNP@TMV. (I) Loading of RBITC uncoated MSNP. (II) Functionalization of MSNP with PDADMAC to make the surface electropositive (RBITC−MSNP). (III) Coverage of RBITC−MSNP with TMV−Cy5, forming a wool-ball-like nanoparticle (MSNP@TMV). (b) TMV particle showing both exterior Lys in pink dots and exterior Tyr in green dots. Coupling of exterior Tyr (green ball in the single CP and green dots on the TMV particle) to a diazonium salt followed by conjugation of an azide-terminated Cy5 dye (yellow ball) to the alkyne-modified Tyr using copper-catalyzed click chemistry. Assembly of TMV−Cy5 onto RBITC−MSNP (MSNP@TMV). Once the MSNPs were loaded with RBITC, TMV−Cy5 was deposited on the surface of RBITC−MSNP through electrostatic interaction using PDADMAC. Briefly, 0.12 mg RBITC−MSNP was resuspended in 0.4−1 mg of TMV−Cy5 and Milli-Q water up to 2 mL overnight with mild rotation. Then, four washing steps were conducted at 16 000 g for 10 min at 4 °C in deionized water (Milli-Q water) to remove excess TMV−Cy5 particles and collect the resulting MSNP@TMV assemblies. We estimated the TMV/MSNP ratio by subtracting the amount of free TMV recovered after washing the MSNP@TMV assemblies from the total amount of TMV that was incubated with the MSNPs. Characterization of Nanoparticles. MV Particles. To assess particle integrity, TMV particles (0.1 mg mL−1, in Milli-Q water) were placed on copper grids and negatively stained with 0.2% (w/v) uranyl acetate. Copper grids were imaged using Zeiss Libra 200FE transmission electron microscope (TEM) operated at 300 kV. Denatured protein samples (30 μg) of TMV were separated on 4− 12% bis-Tris Nu−polyacrylamide gel (PAGE, Life Technologies) in 1× MOPS running buffer (Life Technologies) at 200 V for 45 min. Protein bands were observed under white light before and after staining with 0.25% (v/v) Coomassie blue. UV−vis spectroscopy was used to determine the number of dyes per TMV, the concentration of TMV, and the number TMV coated on the surface of MSNP@TMV particles using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). The number of dyes per TMV and concentration of TMV were quantified as previously described.26,27 MSNP. To assess particle integrity, MSNPs were deposited on copper grids, which were observed by TEM using a JEOL JEM 1010 microscope operated at 80 kV. Dynamic light scattering (DLS) and ζ-potential (ZP) measurements were performed using a disposable cuvette for a Zetasizer Nano ZS instrument (Malvern Instrument). The resulting hydrodynamic radius, polydispersity indices (PDI), and ZP were calculated using Zetasizer Nano software. Reported hydrodynamic radius and PDI values are the average of 5 measurements, each consisting of 10 runs.

The adsorption isotherm of N2 at 77 K was determined in a Micromeritics ASAP 2010 with a micropore system, after outgassing the samples at 140 °C for at least 16 h. Mesopore size distribution was calculated using the adsorption branch of the nitrogen adsorption isotherm and the Barrett−Joyner−Halenda (BJH) approach.32 UV−vis spectroscopy was used to determine the RBITC loading capacity of MSNP by a NanoDrop 2000 spectrophotometer. MSNP@TMV Assembly. Copper grids were imaged using FEI Helios 650 NanoLab microscope using TLD-SE mode 2 at 1 kV. RBITC and Cy5 Release. RBITC−MSNP, RBITC−MSNP−APTS, and MSNP@TMV were incubated either with phosphate-buffered saline (PBS) pH 7.4 or acetate buffer pH 5.0 at room temperature in the dark with mild rotation. At specific time points (t = 1, 3, 6, 24, 48, and 72 h), the samples were centrifuged and the supernatant was analyzed by UV−vis spectroscopy with a Hewlett Packard 8453 spectrophotometer to quantify the concentration of RBITC and Cy5 fluorescent dye released from each nanoparticle formulation. Cellular Uptake and Cytotoxicity. Cell Culture. HeLa cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies), supplemented with 10% (v/v) fetal bovine serum (FBS, Atlanta Biologicals) and 1% (v/v) penicillin streptomycin (Life Technologies). Confluent cells were washed with PBS and collected in enzyme-free Hank’s-based cell dissociation buffer (Fisher). Cellular Uptake and Intracellular Trafficking. HeLa cells, 2.5 × 104, were seeded overnight on coverslips in an untreated 24-well plate (500 μL per well). The cells were then incubated with either 8 × 105 TMV nanoparticles/cell or 2 × 102 MSNP/cell (the number of MSNP was normalized to the number of TMV) for 8 h at 37 °C, 5% CO2. Following incubation, the cells were washed three times with Dulbecco’s phosphate-buffered saline (DPBS) and fixed with both 5% (v/v) paraformaldehyde and 0.3% (v/v) glutaraldehyde in DPBS for 10 min at room temperature. After fixing, the cell membrane was stained using wheat germ agglutinin conjugated to AlexaFluor 488 (1:1000 in 5% (v/v) goat serum (GS) in DPBS) for 45 min at room temperature. Additionally, intracellular trafficking was studied by staining endolysosomes with AlexaFluor 488 antihuman Lamp-1 CD107a (Biolegend). First, the cells were permeabilized with 0.2% (v/v) Triton X-100 in DPBS for 2 min at room temperature to allow C

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Figure 2. Characterization of nanoparticles. (a) TMV−Cy5: (i) on the left: TEM image of a group of TMV−Cy5; on the right: TEM image of a single TMV−Cy5; (ii) sodium dodecyl sulfate (SDS)−PAGE gel, under white light, before and after Coomassie staining: 1 = TMV wild-type (TMV-wt) and 2 = TMV−Cy5. Yellow dot lines highlight the coat protein (CP). The staining at the bottom of the gel is the loading dye that runs at the front of the sample; (iii) UV−vis spectra of TMV−Cy5 (red line) vs TMV-wt (black line). (b) RBITC−MSNP: (i) TEM image of RBITC− MSNP; (ii) on the left: RBITC−MSNP DLS distribution; on the right: MSNP@TMV DLS distribution; (iii) distribution pore volume using the BJH method. (c) Scanning electron microscopy (SEM) characterization: (i, ii) MSNP@TMV and (iii) RBITC−MSNP. Lamp-1 to internalize in the cells. Following permeabilization, the cells were blocked with 10% (v/v) GS in DPBS for 45 min at room temperature and conjugated to Lamp-1 (1:200 in 5% (v/v) GS in DPBS) for 1.5 h at room temperature. In between each step, cells were washed three times with DPBS. Coverslips were mounted with Fluoroshield (4′,6-diamidino-2-phenylindole (DAPI), Sigma-Aldrich). Slides were imaged using Zeiss Axio Imager Z1 fluorescent inverted high-resolution microscope with motorized stage. Two replicates per sample were evaluated and five images per replicate were taken using a Leica TCS SPE microscope and the LASAF software. The images were analyzed by ImageJ, using JACoP (Just Another Co-localization Plugin)32 to calculate the Manders’ coefficient (M).33 Flow Cytometry. To quantify cellular uptake, 2.5 × 105 HeLa cells per mL in 200 μL per well were seeded in a 96-well v-bottom plate. Then, either 8 × 104 TMV particles/cell or 2 × 10 MSNP/cell (the number of MSNP was normalized to the number of TMV) were added in triplicate and incubated for 8 h at 37 °C, 5% CO2. Following incubation, the cells were washed twice with 200 μL flow cytometry staining (FACS) buffer (0.2% (v/v) 0.5 M ethylenediaminetetraacetic acid, 1% (v/v) FBS, and 2.5% (v/v) 1 M (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) pH 7.0 in DPBS) and fixed with 2% (v/v) paraformaldehyde in FACS buffer for 10 min at room temperature. Following fixation and washings, the cells were resuspended in 200 μL FACS buffer and stored at 4 °C. The cells were analyzed using a BD LSR II Flow Cytometer and 1 × 104 events were recorded. Data were analyzed using FlowJo v8.6.3 software.

hybrid structure by coating them with the proteinaceous softmatter nanorods formed by tobacco mosaic virus (TMV), as outlined in Figure 1. The MSNPs were synthesized based on previously described methods.29 By adjusting the glycerol percentage and tetraethyl orthosilicate (TEOS) concentration, we synthesized MSNPs with a diameter of ∼550, 2.4, and 3.5 nm pores (Figure 2b). TMV was purified from Australian tobacco plants as previously described;25 yields were 5 mg g−1 of infected leaf material. The synthesis of the hybrid nanostructure MSNP@TMV was carried out as follows (Figure 1): first, MSNPs and TMV were labeled with distinct fluorophores enabling tracking in cell studies. The MSNPs were loaded with RBITC (λem/ex = 540/ 566 nm) through a diffusion mechanism of the fluorophore into the silica pores (Figure 1a-I).34 The entrapment efficiency of RBITC into the mesoporous silica structure was evaluated by UV−vis spectroscopy and was equal to 54.5 wt %. The TMV was labeled at solvent-exposed Tyr 13935 with sulfocyanine5-azide (Cy5, λem/ex = 647/670 nm) using diazonium coupling and click chemistry (Figure 1b) as previously described.25 The TEM images of TMV−Cy5 confirmed that the particles retained their structural integrity after chemical labeling (Figure 2a-i). Furthermore, denaturing SDS−PAGE gel electrophoresis confirmed the covalent conjugation of Cy5 dye to TMV (Figure 2a-ii). Before staining with Coomassie, a ∼17.5 kDa band was visible under white light (lane 3) corresponding to the colorful Cy5. After Coomassie staining, the Cy5-band colocalized with the TMV CP band (17.5 kDa, lane 1, TMV-wt), thus confirming the covalent attachment of

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RESULTS AND DISCUSSION Synthesis and Characterization of the Wool Ball MSNP@TMV Assembly. This work is based on the engineering of MSNPs into a wool-ball-like MSNP−biological D

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Figure 3. HeLa cell interactions with RBITC−MSNP (red), RBITC−MSNP + TMV−Cy5 (green) and MSNP@TMV (blue) after 8 h of incubation. (a, b) Flow cytometry histograms for both Cy5 and RBITC channels, respectively. (c, d) Quantitative flow cytometry analysis by mean fluorescence intensity (MFI, error bars represent standard deviation; *statistical analysis by Student’s t-test) for both Cy5 and RBITC channels, respectively. Values represent two independent experiments, each sample by triplicate.

Cy5 to TMV. Five hundred five Cy5 dyes per TMV particle were quantified by using UV−vis spectroscopy and Beer− Lambert’s law (Figure 2a-iii), which corresponds to 23.7% TMV CPs modified with Cy5. We have previously reported that a minimum conjugation of Cy5 to 8% of TMV CPs yields an optimal fluorescence intensity for imaging experiments.36 MSNPs were functionalized with PDADMAC to (1) avoid the diffusion of the RBITC out of the silica pores and (2) promote the electrostatic assembly of negatively charged TMV−Cy5 onto the MSNP surface (Figure 1a). Although TMV has a zwitterionic structure because of its proteinaceous nature, under physiological conditions, its overall surface charge is negative, as has been reported by Tiu et al.37 MSNPs and TMV were mixed at 0.3−0.12 ratio in Milli-Q water overnight at mild rotation. The MSNP@TMV particles were then characterized by scanning electron microscopy (SEM, Figure 2c). The TEM analysis confirmed the MSNP were spherical (Figure 2b-i), whereas the N2 isotherm analysis revealed their mesoporous structure with the presence of 2.4 and 3.5 nm pores (Figure 2b-iii). Further DLS studies were performed to analyze the polydispersity and hydrodynamic radius size distribution of the MSNP. The resulting particles showed a hydrodynamic diameter of 565.01*/0.47 nm with a PDI: 0.273 ± 0.022 (Figure 2b-ii, left panel), which are in accordance with the size observed from the TEM images (547 ± 2.82 nm, data not shown). Finally, the SEM characterization of the MSNP@ TMV nanoparticle assembly confirmed the presence of TMV particles on the MSNP surface (Figure 2c-i and c-ii, MSNP@

TMV), resulting in a wool-ball-like structure compared to the smooth uncoated RBITC−MSNP (Figure 2c-iii). The hydrodynamic diameter of MSNP@TMV increased compared to that of RBITC−MSNP, up to 606.6*/1.47 nm and PDI: 1, revealing a high level of polydispersity (Figure 2b-ii, right panel). The fluorescence activity of the sample and the different refractive indexes of MSNP and TMV could explain the observed fluctuations in polydispersity.38 Based on the diameter of MSNP@TMV (606 nm) and RBITC−MSNP (565 nm), the calculated thickness of the TMV layer was 40 nm. The corresponding TMV/MSNP ratio was approximately 108 and the estimated percentage of MSNP surface coverage by TMV was equal to 48%. Furthermore, we confirmed that both the PDADMAC polymer (first step) and TMV (second step) were bound to the MSNP surface based on changes of the ζ-potential of each formulation: −30.3 ± 13.8 mV for bare MSNP; 22.9 ± 3.86 for MSNP−PDADMAC, whose positive charge comes from the PDADMAC polyelectrolyte; 19.0 ± 1.29 mV for MSNP@TMV due to the overall negative charge exhibited by TMV (Figure S1b). RBITC and Cy5 Azide Release. We evaluated the release profiles of RBITC (loaded into the MSNPs) and Cy5 (covalently attached to TMV). RBITC−MSNP, RBITC− MSNP−APTS, and MSNP@TMV were rotated gently in PBS at pH 7.4 to mimic blood and physiological pH and in acetate buffer at pH 5.0 to mimic the lysosomal and tumor microenvironment pH. The RBITC−MSNP−APTS sample was included to determine if reversing the negative charge of the MSNP (−25 mV at pH 5) by amine functionalization (+30 E

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Figure 4. Confocal imaging of HeLa cells showing cellular uptake of NPs. (a) Composite image of MSNP@TMV sample and (b) composite image of RBITC−MSNP sample. For both composites, nuclei are shown in blue (stained with DAPI); membrane is shown in green (stained with wheat germ agglutinin); and both MSNP@TMV and RBITC−MSNP particles are shown in red (arrows point both types of nanoparticles). Furthermore, MSNP@TMV sample is depicted in Cy5 (yellow) and RBITC (orange) channels, in which colocalization of both is shown in white with associated Manders’ coefficient (M) is indicated. (c) Cellular trafficking of MSNP@TMV. Inset shows MSNP@TMV in red and endolysosomes in green (stained with Lamp-1), where colocalization of both is shown in white as before. Scale bar: 25 μm.

with either CPMV or TMV.10,39 The hybrid assemblies of potato virus X (PVX) coated on SiO2 spheres also presents similar structural features.40 Next, the fully characterized RBITC-loaded MSNPs coated with TMV−Cy5 were evaluated in vitro to observe cellular internalization and trafficking of the MSNP@TMV assembly. Enhanced Cellular Uptake of the Wool-Ball-like MSNP@TMV Hybrid Assembly. To address whether the TMV coating layer of the MSNP@TMV would enhance cellular uptake, flow cytometry and confocal microscopy experiments were performed using HeLa cells (a cervical cancer cell line). Cells were incubated with either RBITC− MSNP, RBITC−MSNP + TMV−Cy5, or MSNP@TMV (made of RBITC−MSNP coated with TMV−Cy5) for 8 h at 37 °C, 5% CO2. Because TMV was conjugated to Cy5, whereas MSNPs were loaded with RBITC, its distinct fluorescent profiles were monitored. Whereas flow cytometry (Figure 3) provides quantitative data, confocal microscopy (Figure 4) gives insights into the cellular fates and trafficking of the assembly and its components. As compared to MSNP@TMV, the “naked” MSNP showed negligible cell interactions. The MSNP@TMV assembly exhibited strong cell interactions resulting in 15× greater mean fluorescence intensity (MFI) compared to MSNP alone. These data indicate that the biological TMV coating indeed enhanced the cell interactions of the MSNP. As a control, we

mV at pH 5) would enhance the interaction between RBITC and MSNP. Increased RBITC release rates were observed at low pH and cargo association with the MSNP was enhanced for the MSNP−APTS formulation (Figure S2b). At pH 5.5 and t = 24 h, less than 20% of RBITC was released from RBITC−MSNP−APTS; in contrast, more than 50% of the cargo was released from RBITC−MSNP. Complete release from RBITC−MSNP was observed after 72 h. At physiological pH, the RBITC release rate was slower for both samples: no more than 30% of the cargo was released from RBITC− MSNP−APTS after 72 h, whereas up to 80% of the cargo was released from both RBITC−MSNP and MSNP@TMV. Furthermore, RBITC−MSNP and MSNP@TMV showed similar RBITC release profiles because neither had an amino functionalization (Figure S2c). Overall, the amine-functionalized MSNP−APTS may be more suitable for future translational studies, as this formulation confers enhanced stability during storage and physiological conditions, thus avoiding premature drug release. Lastly, we confirmed that Cy5 was not released from the nanoparticles, which is consistent with its covalent attachment to TMV (Figure S3d). Our data demonstrate the successful synthesis of virus− inorganic nanohybrids MSNP@TMV. The resulting structures with their wool-ball-like morphology have some similarities to virus−inorganic nanohybrids reported previously. This includes poly(4-vinyl-pyrindine) polymer nanostructures coated F

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Langmuir also studied the cell uptake of TMV and MSNP mixtures; data showed that free TMV interacts with cells, resulting in a 20fold increase in Cy5 MFI compared to cells only controls. In contrast, free MSNPs show moderate cell interactions, resulting in a 3-fold increase in RBITC MFI vs cells only. Interestingly, cell interactions seemed to be reduced when TMV and MSNPs were mixed compared to when MSNPs were added to HeLa cells alone: the RBITC MFI was reduced by 0.5 compared with MSNP + TMV vs free MSNP, most likely because the enhanced TMV−cell interactions interfere with the MSNP interaction when the mixtures were added (Figure 3d). The analysis of the Cy5 (TMV) and RBITC (MSNP) channels indicates correlation (Figure 3a + c vs 3b + d, blue histogram, MSNP@TMV), i.e., data indicate that intact assemblies interact with the cells and that dissociation of the complex does not occur in biological media. Confocal imaging further confirmed the interaction between the assembly and HeLa cells (see Figure 4). Next, we investigated the intracellular trafficking of the MSNP@TMV assembly compared to bare RBITC−MSNPs. Confocal imaging revealed that MSNP@TMV particles (Figure 4a) accumulate in the cell to a greater degree than uncoated RBITC−MSNP (Figure 4b), which is consistent with the flow cytometry study described above. Furthermore, data indicate colocalization between TMV−Cy5 and RBITC− MSNP (Figure 4a, colocalization, M = 0.9), again indicating that TMV remains attached to the MSNP surface in biological media and upon cell internalization (Figure 4a). Further staining and imaging studies indicate that the MSNP@TMV complex enters cells via endocytosis and traffics to the endolysosomal compartment stained with Lamp-1 (Figure 4c, colocalization, M = 0.98 between MSNP@TMV and Lamp-1 channels). The Manders’ coefficients of MSNP + Lamp-1 (M = 0.98) and TMV + Lamp-1 (M = 0.97) confirmed both MSNPs and TMV colocalize with endolysosomes. Similar Manders’ coefficients were expected because it was determined that both RBITC−MSNP and TMV−Cy5 particles colocalize in Figure 4a. Following 8 h of incubation, the wool ball MSNP@TMV remained in the late endolysosome, within the perinuclear region of the cell, as can be observed from the overlay of the Lamp-1 endosomal staining and the nanoparticles. This corroborates with the study by He et al., who previously reported that MSNP with 420 nm diameter were colocalized with lysosomes.30 Together, our data show that nanohybrids composed of ∼550 nm-sized MSNP coated with the proteinaceous softmatter nanorods formed by TMV were successfully developed. The MSNP@TMV assembly demonstrated enhanced cellular uptake compared to the bare MSNP. Previous research indicates that the cell internalization rate of MSNPs is greatly affected by their size and surface modifications.16,41 For example, MSNPs with a diameter greater than 500 nm showed decreased cell internalization rates compared to smaller ones.30,42 Both positively charged polyelectrolyte-functionalized fluorescent-SiO2 spheres and MSNPs coated with carbon nanotubes (CNTs) have been previously used in our group for surface modification and cell internalization studies.43 In accordance with the findings reported here, we previously demonstrated MSNP@CNTs displayed a significantly increased cell uptake compared to naked MSNPs. However, in contrast to the present findings using MSNP@TMV, we found that MSNP@CNTs accumulate in the cell cytoplasm without apparent cell toxicities.15,43

This accumulation indicates that the cell uptake rates of MSNPs can be enhanced through surface coatings and that their intracellular fates and trafficking can be fine-tuned using appropriate surface coatings. In the future, it would be interesting to study the process in more detail and also to address whether the MSNP@TMV hybrid can escape from endolysosomes after 8 h; we have previously shown that MSNP@CNT can be released into the cytoplasm after 72 h, mimicking the mammalian virus cycle.42,43 Although future cytotoxicity assays will be necessary, potential applications of the MSNP@TMV nanohybrids include drug delivery, immunotherapy, or diagnostic applications. MSNPs are chemically tunable and demonstrate a high loading capacity, which make them suitable for carrying large quantities of drugs. In addition, TMV particles have been previously employed as drug delivery and immunotherapy platform for medical and environmental applications.44−49 Therefore, because TMV is chemically and genetically tunable, it can be modified to introduce further functionalitieseither for cell targeting/tissue specificity or for additional cargo delivery (i.e., nucleic acid, chemodrugs, peptide, or proteins).

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CONCLUSIONS Herein, we have synthesized an organic−inorganic hybrid composed of a 550 nm-sized MSNP covered by a TMV biological coating. This MSNP@TMV hybrid resembles a wool-ball-like structure, which improves the cell internalization compared to naked MSNP. The individual components of the hybrid can be exploited not only in terms of their intrinsic properties but also due to their synergistic effects. All together, these unique nanostructures can be considered as excellent candidates for the development of biomedical applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.8b03337.

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UV−vis spectra and ζ-potential of MSNP and its hybrid formulations; cumulative release assay of RBITC and Cy5 from the MSNP formulations (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (M.A.C.-D.). *E-mail: [email protected] (N.F.S.). ORCID

Laura Marín-Caba: 0000-0002-9638-9289 Paul L. Chariou: 0000-0002-7115-3878 Author Contributions

N.F.S. and M.A.C.-D. conceived and designed the experiments. L.M.-C. and P.L.C. performed all the experiments. Carmen Pesquera performed BET experiment. L.M.-C., P.L.C., N.F.S., and M.A.C.-D. wrote the paper. All the authors read and approved the final manuscript. Funding

This research has been supported in part by the Spanish MINECO Project (refs BES-2015-075567, CTM2014-58481R, and CTM2017-84050-R) and a grant from the National Science Foundation, CAREER DMR 1452257. G

DOI: 10.1021/acs.langmuir.8b03337 Langmuir XXXX, XXX, XXX−XXX

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Langmuir Notes

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The authors declare no competing financial interest. All data generated or analyzed during this study are included in this published article.

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ACKNOWLEDGMENTS Nanthawan Avishai (Swagelok Center for Surface Analysis of Materials (SCSAM), Case Western Reserve University) is thanked for technical assistance with SEM imaging. MINECOSpain (CTM2014-58481-R, CTM2017-84050-R), Xunta de Galicia (Centro Singular de Investigación de Galicia Accreditation 2016−2019 and EM2014/035), and European Union (European Regional Development Fund-ERDF).

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DOI: 10.1021/acs.langmuir.8b03337 Langmuir XXXX, XXX, XXX−XXX