Synthesis of hollow mesoporous ruthenium nanoparticles: evaluation of physico-chemical properties and toxicity

Abstract

Hybrid nanoparticles (NPs) with a mesoporous hollow structure have attracted great interest for biomolecule delivery, due to the easy fabrication process, the multi-functionalization capability for navigating to specific hosts and the high surface area for encapsulating therapeutic moieties. In the present study, hybrid ruthenium NPs were prepared with a dual template method by using colloidal amine functionalized silica particles and poloxamer 407. The results indicate that the size of the NPs can be controlled; smooth, spherical, monodispersed, negative surface charge potential, polycrystalline, and hollow interior architecture particles can be prepared. Also, the Brunauer–Emmett–Teller (BET) analysis shows a specific high surface area of 75 m² g⁻¹. The hybrid NP system also exhibits fluorescent properties. Thus this system could be a great advantage for cell trafficking. Then, cell cytotoxicity was evaluated in a real-time manner for two different cell lines using the electric cell-substrate impedance sensing (ECIS) system, and the results show that a higher than 100 μg ml⁻¹ concentration is cytotoxic. The negatively charged surface NPs show higher cellular uptake through electrostatic interactions and binding at the cationic sites of the cell membrane, and also show high drug loading and potential for pH sensitive drug release. This hybrid NP system represents a promising nanocarrier for effective and favorable antitumor treatment and theranostic systems.

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Introduction

In recent years, hollow and mesoporous nanomaterials have received great attention in diverse fields due to their synergistic physico-chemical and biological functionality suitable for various applications, including as a drug carrier.^1–3 In drug delivery, porous nanoparticles (NPs) brought a new insight with their tunable porous channels, and their distinctive properties such as the ability to act as a carrier, absorption, release, and biocompatibility.⁴ Due to their nanometric size, they are often easily taken up by cells, and also the porosity and high surface area to volume ratio of NPs can encapsulate more guest species for targeted actions. From the existing reports, the mesoporous NPs have gained increasing interest for building materials with unique properties for use in drug delivery and imaging, including various metals⁵ and metal oxide NPs.^6,7 Compared to other metal oxide NPs, mesoporous NPs have an advantage of a high surface area for enhanced payloads. The selection of ruthenium (Ru) was due to its valence states and multiple oxidation states and it has efficient action against cancer cells. Ru based complexes are an improved replacement for the existing anticancer drugs.⁸ In order to attain a mesoporous structure in metals and related nanomaterials, several methods have been utilized. Of the various preparation methods, the template assisted method has been employed mostly.⁹ The hard template assisted synthesis has several advantages, such as controllable and tunable shapes and sizes.¹⁰ The soft template assisted synthesis method, as a facile one pot synthesis, enables easy self-assembly without an external template, and it is also easy to remove the templates.¹¹

Inspired by the physico-chemical characteristics of the aforementioned materials and methods, herein we designed a synthesis of hollow mesoporous Ru NPs by a dual template method for cell study. A simple and versatile dual templating approach by utilizing amine functionalized colloidal silica (AFSN) NPs as a hard template¹² and poloxamer 407 (P407) non-ionic surfactant as a soft template¹³ was used to prepare the hollow mesoporous Ru NPs. The AFSN (100 nm) were used to obtain a hollow sphere (core) with the assistance of P407 to form a Ru hollow core shell, following a consecutive reduction reaction and coordination chemistry. Here, we report the facile synthesis of porous Ru NPs, which will accelerate the diffusion of guest species better. In this synthesis, the metal species are accumulated as a layer of silica particles, and it is guided by simply adjusting the surfactant concentration to form hollow mesoporous nanostructured Ru particles. Notably, this optimization method is able to achieve uniform, monodispersed, spherical, hollow interior architectured particles for enhancing payloads.

To understand the physico-chemical characteristics and functionalities of the final hollow mesoporous nanostructured Ru particles, various characterizations were carried out to clarify the structural integrity and material surface properties of the nanoparticulate system, which is useful for biological application. The nanostructured Ru particles have gained much attention for their cytotoxic properties against cancer cells.¹⁴ However, a detailed study examining the cytotoxic properties of hollow mesoporous Ru particles towards different types of cells and the mode of toxicity is not yet reported, and no literature was available until now for real-time in vitro measurement. In general, conventional techniques were applied to evaluate the cytotoxicity, but these techniques are time point assays which are time consuming, need several reagents and are also complex. Here we used a label-free, unconventional, high sensitive, real-time measurement system. One of various real-time measurement systems, the electric cell substrate impedance sensing (ECIS) system is unique and ideal for monitoring cellular response against external analytes in a real-time manner as a biophysical approach.^15–18 From the experimental analysis, we observed that the nanoparticulate system will have a promising potential application in various fields, especially in the field of drug delivery and therapeutics.

Experimental

Materials

Ruthenium chloride (RuCl₃), sodium borohydride (NaBH₄), poloxamer 407 (P407), amine functionalized silica NPs (AFSN) (100 nm) and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), Alexa Fluor 488 were purchased from Sigma Chemical Co., St Louis, MO, USA. Hydrofluoric acid (HFL) was purchased from J. T. Baker, PA, U.S.A. Human epidermal melanocyte-adult (HEMa) cell line with respective growth media and supplements were purchased from CEFO Ltd, Seoul, South Korea. Mouse murine melanoma (B16-F10) cell line was purchased from ATCC, VA, U.S.A. Fetal bovine serum (FBS), trypsin–EDTA, and penicillin–streptomycin was purchased from Gibco Laboratories (Grand Island, NY, U.S.A). Doxorubicin (DOX) hydrochloride, Alexa Fluor 488, phalloidin, 4′,6-diamidino-2-phenylindole (DAPI) and phosphate buffer saline (PBS) were purchased from Sigma-Aldrich. All other chemical reagents were supplied by Sigma-Aldrich.

Synthesis of hollow mesoporous Ru NPs

The mesoporous hollow Ru NPs were prepared by the double template method¹⁹ with a slight modification. The RuCl₃ was dissolved in 7.0 ml of water containing different concentrations of perchloric acid, and then P407 was added into the solution and dissolved by vortexing for 1–3 min. Subsequently, 3.0 mg of AFSN particles were added through ultrasonication. After complete dissolution, 7.0 ml of NaBH₄ was added and then the mixture was sonicated for 4 h in an ultrasonic cleaner (MUJIGAE-SD-120H, Korea) at 50 Hz and equilibrated at 37 °C throughout the experiment by circulating water. The obtained particles were washed three times and redispersed in 20% hydrofluoric acid for 12 h to remove the silica template. Then the final product was washed several times with water and dried for 2 days at room temperature. The PEO groups of P407 were easily removed by dissolving the final product in water. The synthesis of Ru NPs with various concentrations of P407 and perchloric acid is shown in ESI Table S1.†

Characterization methods

NP reduction was confirmed by using UV-Visible spectroscopy (Optizen 3220; Mecasys, Korea). Morphological characterization of the NPs was done by using field emission scanning electron microscopy (FE-SEM) (JEOL-ISM-7500F; JEOL, Japan) and the interior nature of the NPs was characterized by transmission electron microscope (TEM-FEI TECHNAI G² F30, USA) equipped with EDX spectroscopy (EDAX Analyzer (DPP-II)), with an acceleration voltage of 300 kV. The morphology and roughness of the NPs were analyzed by bio-atomic force microscopy (Bio-AFM, Nanowizard II; JPK Instruments, Germany) in contact mode, and the root mean square values were obtained by using JPK software in an offline process. The average particle size distribution was measured by using dynamic light scattering (DLS) spectrophotometer (Otsuka electronics, Photal ELSZ-1000). The crystalline nature of the NPs was identified by using an X-ray diffractometer (XRD) (Model D/Max-2200; Rigaku, Woodlands, TX), with copper K(alpha) radiation at a wavelength 1.5406 Å and a scan range from 10° to 90° run at 0.02 second intervals. Thermogravimetric analysis and differential scanning analysis (Universal V4.4A TA Instruments, Korea) were used to evaluate the thermal behavior of the obtained particles and the heat rate was 10 °C min⁻¹ with the temperature range between 35–1000 °C under a nitrogen atmosphere. The functional groups of the sample were identified by Fourier-transform infrared spectroscopy (FT-IR) (JASCO FT/IR 4100, Japan) in the range of 500–4000 cm⁻¹. The fluorescence emission spectra of the hollow mesoporous Ru NPs suspension was measured by using a fluorescence spectrophotometer (Quanta Master, Photon Technology International, U.S.A). The fluorescence emission intensity of hollow mesoporous Ru NPs was calculated using the precursor solution. The surface area and pore size of hollow mesoporous Ru NP distribution were measured by the N₂ adsorption isotherm method (ASAP2020, MICROMERITICS, USA).

In vitro evaluation

The in vitro evaluations, including cell cytotoxicity behavior by ECIS (Applied biophysics, USA) method and cellular uptake study by fluorescence microscope (NIKON ECLIPSE 80I, USA), loading of doxorubicin (DOX) onto hollow mesoporous Ru NPs and drug release behavior were studied, and the detailed experimental procedures are given in the ESI under Section S1.†

Results and discussion

Mechanism of hollow mesoporous nano structure formation

We employed a facile one pot synthesis method to obtain the mesoporous structure of Ru NPs, schematically represented in Fig. 1. In this approach, AFSN was dispersed into Ru which contains a surfactant (P407) using ultrasonication at 37 °C with circulating water. The temperature 37 °C plays an important role in micellar formation of P407. Unimer to micelle transition occurs at high temperature according to the critical micelle concentration (CMC) which results in aggregation or entanglement between polyethylene oxide (PEO) chains of P407. Previous studies reported the reduction of P407 at CMC from 1.75 weight% at 10 °C to 0.008 weight% at 30 °C.²⁰ P407 plays a crucial role and acts as a structure directing agent,²¹ wherein the poloxamer chains assist in the deposition of Ru. The interaction between the hydrophobic polypropylene oxide (PPO) chains and Ru surface leads to the growth of Ru shells over the silica surface.¹³ This reveals that the self-assembly of surfactant micelles and the reduction of metal species are the main key factors in the formation of mesostructured NPs. NaBH₄ acts as a reducing agent, which leads to the accumulation of Ru over silica, followed by reduction to form Ru⁰. The reducing potential of Ru was controlled by the addition of perchloric acid, with the increase of chloride and hydrogen ions decreasing the reduction rate as a result of acid catalyzed enolisation.²² The deposition of Ru over the silica particles was achieved with the aid of a surfactant, and the coupling interaction between the template and surfactant was promoted by the formation of hydrogen bonds between the terminal hydroxyl group of P407 and the surface amine group of AFSN. This kind of mechanism was proved in the synthesis of Pt spheres by using AFSN, and gave a reported high surface area with a hollow interior.²³ During the synthesis, the amine group plays a vital part in the attachment site for Ru deposition. During the formulation procedure, we utilized different concentrations (ESI Table S1†) of perchloric acid and P407 for optimization named as F1, F2, and F3 to achieve the uniform monodispersed hollow mesoporous Ru NPs.

Fig. 1 Schematic representation showing the formulation of hollow mesoporous Ru NPs based on the dual template approach.

Particle characterization

The FE-SEM and DLS images (Fig. 2) show the morphology and size distribution of hollow mesoporous Ru NPs of various formulations (F1, F2 and F3). Fig. 2a shows the low magnification and Fig. 2b shows the high magnification SEM micrographs revealing that the particles are mesoporous, uniformly distributed and without significant aggregation. Ru NPs obtained from F1 formulation had a size range of 162 nm with a surface charge of −22.06 mV. The presence of OH– on the particle surface contributed to the negative zeta potential and also making a relatively stable formation of a particle.²⁴ Increasing the concentration of P407, the formulation F2 shows the incomplete and random deposition of metal over the silica, also the formulation F2 shows major accumulation of surfactant and the formation of large size polymeric NPs. In higher concentrations of perchloric acid, the reducing rate was controlled for nucleation,²¹ which leads to tuned pore formation (F1 formulation). Simultaneously, by increasing the concentrations of P407, particles tended to aggregate (F2 and F3 formulation). The physico-chemical characterizations of all the preparations and different concentrations of the excipients are shown in ESI Table S1†. All the preparations show a relatively uniform homogeneous size distribution with a polydispersity index (PDI) between 0.17 to 0.41 and a zeta potential between −23 to −17 mV. Among the 3 formulations (F1, F2 and F3), F1 shows the smallest (162 nm) sized, uniform, and most pores for a negatively surface charged particle. However, the negatively surface charged particle minimizes the nonspecific binding of the particle to the cells, and this kind of nanocarrier can enhance the circulatory lifetime.²⁵ On the other hand, this type of NP can have a lower internalization into the cells than positively charged particles. For example, positively charged Au nanoparticles show a more rapid internalisation than negative surface charged particles, due to affinity to the negatively charged cell membrane.²⁶ So, our prepared negatively charged surface NPs can have the ability to increase the circulation lifetime, which leads to high accumulation in the tumor tissue compared to normal tissues.²⁷

Fig. 2 Surface morphology and particle size distribution of Ru NPs with different formulations (F1, F2 and F3). (a and b) SEM image of the surface morphology with different magnification of mesoporous Ru NPs. (c) Particle size (DLS) distribution histogram of Ru NPs with different formulations (top to bottom; F1 to F3 respectively).

The morphological structure of the TEM image correlates well with the FE-SEM images. The overall spherical shape of the final product is maintained upon AFSNPs reaction with Ru and P407. At a higher magnification, the image shows a well-defined surface modification with a porous structure on the template revealing the nucleation by P407. Fig. 3a shows F1 NPs aligned well with a hollow interior without destruction of the Ru shell. The NPs have a smoother surface and a decrease in porous nature with an increase in concentration of P407, also the reduction in perchloric acid concentration results in a fast reduction rate and uncontrolled nucleation (Fig. 3b). At the highest concentration of P407, hydrophobic parts of P407 were concealed within the micelles, which led to the loss in ability of metal seed capping.²⁸ It results in large polymeric sized particles (Fig. 3c) without fine porosity in the F3 formulation. EDX (Fig. 3d) elemental analysis data confirms the presence of Ru. From the examination of SEM and TEM, F1 was found to be less aggregated and have a uniform size distribution. The desired preliminary physicochemical properties of F1 are more suitable for biological studies, and were also selected for further studies.

Fig. 3 TEM images of different formulations of synthesized Ru NPs for interior structure analysis. Left to right TEM images show low to high magnification. (a) Formulation 1 (F1); (b) formulation 2 (F2); (c) formulation 3 (F3), and (a-inset) the selected area electron diffraction (SAED) pattern.

Microtopography and surface roughness were investigated by bio-atomic force microscopy (Fig. 4). The height and vertical deflection image helps to visualize the particles assumed to be spherical, and shows that the size as well as the homogeneous distribution with the desired surface texture is well consistent with other morphological evaluations. Also the crest in the line profile (Fig. 4b) indicates the availability of pores in the NPs.²⁹ The standard deviation of the root mean square (RMS) roughness was found to be 11.28 nm, which indicates that the obtained particles with a high surface roughness is due to the high porosity and particle size. Petr et al. reported that an RMS value of 10 nm represents the surface morphology of a particle with minimum to maximum range of 100 nm.³⁰

Fig. 4 Bio-AFM images show topographical characterization of hollow mesoporous Ru NPs. (a) AFM images show the vertical deflection, height, 3D height and a corresponding line profile of hollow mesoporous Ru NPs with a scan range of 2.5 × 2.5 μm size. (b) Magnified AFM images of ‘a’ with a scan range of 500 × 500 nm size.

Based on N₂ adsorption isotherm curves (Fig. 5a and b) for the F1 formulation, the Brunauer–Emmett–Teller (BET) surface area was found to be 75 m² g⁻¹ and the bimodal pore distribution of maximum and minimum size is 19 nm and 6 nm respectively, measured by the Barrett–Joyner–Halenda (BJH) method. The BET measurement indicates a high surface area of 75 m² g⁻¹ and this value is comparatively higher than previously reported hollow mesoporous NP values,³¹ and smaller than Ru nanocomposites which were reported as 741.2 m² g⁻¹.³² The pore size distribution (Fig. 5 (inset)) shows that pores from 17–19 nm were more common. Thus, the adsorption results were in good agreement with SEM and TEM profiles (Fig. 2(F1) and 3a). The abundant internal areas are attractive for more drug payloads and the guest species can easily permeate into the host species. Zhou et al. reported that more drug payloads due to a large surface area and accessible pores also provide a better drug release profile for core–shell NPs.³³

Fig. 5 N₂ adsorption isotherm measurement of hollow mesoporous Ru NPs. The image shows the BET surface area profile, and the inset image shows the BJH pore size distribution of the particles.

The reduction of the trivalent Ru compound occurred upon the addition of NaBH₄ to the zerovalent state of Ru. The UV-Vis spectral data shows (ESI Fig. S1a†) the disappearance of characteristic Ru³⁺ transitions peaks at 405 nm in Ru⁰ confirming the complete reduction of Ru.³⁴ Also, the appearance of blue shifts in the UV absorption band confirms the π → π* transition of intermolecular hydrogen bonds. Also the color change from brownish red to black confirms the complete reduction of RuCl₃.

UV-Vis results were in good agreement with photoluminescence (PL) spectral data (ESI Fig. S1b†), confirming the existence of fluorescent properties. The emission is at the wavelength of 478 nm for synthesized hollow mesoporous Ru NPs. From the normalized PL data, the peak intensity of synthesized Ru NPs was comparatively much lower than RuCl₃.

The obtained particles show fluorescent properties with low energy, due to metal to ligand and ligand to metal charge transfer states.³⁵ These fluorescent properties confirmed that the prepared NPs have efficient and high photochemical stability. Also they can be used for fluorescent tracing and may be a promising candidate for theranostics, optical and bio-imaging studies.

Fig. 6 shows the XRD profile of the synthesized particles, the obtained peaks confirming the presence of Ru; the broadening and sharp major peak indicates that the particles are hollow and crystalline in nature. The peaks obtained at 38.50°, 44.0°, 82.0° for Ru NPs were assigned to the rings of (100, 101, 200). It represents the hexagonal closely packed (hcp) structure of Ru (JCPDS card #06-0663), and the diffraction rings of the SAED pattern (Fig. 2a, inset) from one particle reveals the polycrystalline nature of hollow mesoporous NPs. The minor peak at 64° may be reflections of Ru.³⁶

Fig. 6 XRD profile of the obtained NPs shows the hcp structure of Ru.

From the FT-IR functional group analysis results (ESI Fig. S2†), the broad –OH stretch at the region of 3350 cm⁻¹ confirms the formation of core shell NPs by strong hydrogen bonding by amine (silica)-hydroxyl group (surfactant), and the peak in the region of 1630 cm⁻¹ assigned to the C=C stretch of the alkene indicates the PPO group of P407. A stretch at the region of 2051 cm⁻¹ confirms the existence of Ru.³⁴ This strong hydrogen bonding result is consistent with the UV results.

From the TGA curve, a continuous weight loss has been observed in the first two stages (ESI Fig. S3†). The initial weight loss was attributed to the loss of free and associated water molecules in the sample, and further changes in the curve is due to loss of P407. FTIR data supports the weight loss, which is indicated by the broad OH peak obtained for the prepared sample. The major weight loss of 33.32% has been observed at the second stage from 310 °C to 690 °C respectively. After 700 °C, the TGA signal is related to the residue, which is attributed to traces of Ru. A total of 47.30% relative weight loss has been observed in 700 °C. The TGA curve represents the slow rate of the hollow structured NPs deformation while raising the temperature, due to the degradation of P407. Also the DSC indicates that the weight loss occurs from 55 °C indicating the glass transition of P407. The melting (T_m) transition temperature of the particles was measured using DSC, which is characterized by the peak corresponding to the latent heat of melting. The initial endothermic peak below 100 °C is due to the loss of water molecules; T_m curve of Ru occurring at 672.98 °C reveals the endothermic transition corresponding to its melting point with heat of fusion about −62.73 mW. The result confirms the thermal stability of the synthesized NPs.

In vitro cytotoxic evaluation of Ru NPs by real-time bioimpedance method

ECIS is the ideal technique which provides the complete dynamic onset cytotoxic biophysical information in a real-time manner. In brief, the system consists of 250 μm circular gold electrodes and it is submerged into the bottom of the tissue culture wells. After the cell seeding, cells drift downwards to the substrate then attach, proliferate and spread over the surface of electrodes. The proliferated cells resist the normal current flow, then the current flows through the spaces between cellular gaps. The changes in impedance were monitored during attachment and spreading, after the addition of various concentrations of NPs, which leads to alterations in cellular behaviors, and the resulting biophysical information can be measured by changes in impedance.¹⁵

Fig. 7 shows the cytotoxic effects of hollow mesoporous Ru NPs on HEMa and B16-F10 cells. Initially, the electrodes were stabilized with 200 μl of the medium and the measurements were conducted for nearly 30 minutes without the cells to nullify the background impedance. During the initial measurement, the impedance value is low due to cell free conditions with a result of unblocked current flow. Fig. 7a and a1 shows the cytotoxic effects of NPs on HEMa cells. An increase in HEMa cells impedance value illustrates the attachment and spreading until 16 h. It also shows that the current flow was resisted by the insulating plasma membrane of the cells and the flow of current through the gaps between cells. After cell attachment, five different concentrations of Ru NPs (50–250 μg ml⁻¹) were added into the HEMa cells. 50 μg ml⁻¹ shows no cytotoxicity and no changes in normal cellular growth. But the 100 μg ml⁻¹ concentration shows minimal cytotoxic effect and increasing concentration shows increased toxicity. The magnified image of normalized impedance data (Fig. 7a1) clearly shows the concentration dependent toxicity. Hollow mesoporous Ru NPs with ≥100 μg ml⁻¹ concentration started to inhibit the cellular proliferation. The inhibitory effects of the concentration range of 150 μg ml⁻¹ and above lead to more cell death from the initial time of Ru NP addition, suggesting they are an acute toxicant. Additionally, the cytotoxic effects of NPs on B16-F10 cells have been evaluated, as shown in Fig. 7b and b1. The cell impedance showed a quick growth rate in contrast to HEMa and within 4 h it shows complete attachment and spreading. From the time of 8 h, the cells were exposed to various (50–500 μg ml⁻¹) concentrations of hollow mesoporous Ru NPs and the cells were monitored over 24 h. The impedance curve shows no obvious changes in cell growth up to 7.5 h (Fig. 7b), which means the cell has been tolerating the Ru NPs, and after an increase in the time of exposure, the cell impedance shows a decline in a concentration dependent manner. The magnified image of normalized impedance data (Fig. 7b1) clearly shows the concentration dependent toxicity. The impedance values for 100 μg ml⁻¹ concentration show minimal inhibitory effects with maximum toleration capacity, followed by 200 μg ml⁻¹ concentration showing a decrease in impedance value. However, both 250 and 500 μg ml⁻¹ concentrations show no viability, which means cell death and reveals that a higher concentration serves as a more potent toxicant to cells than lower concentrations. Overall, the result depicts the variation in the cytotoxic level of Ru NPs for both cell lines, and 100 μg ml⁻¹ of Ru NP found to be non-toxic for both cell lines in the initial time and tolerated. Also, it reveals that higher than 100 μg ml⁻¹ concentrations lead to more toxicity in HEMa cells than B16-F10 cell lines. For the comparison of Ru NPs with other noble metals including Au NPs, they have less cytotoxic potential. For instance, Keith et al. reported that the synthesized Au NPs showed a highly cytotoxic effect³⁷ and it is comparatively higher than synthesized Ru NPs. These real-time bioimpedance cytotoxicity results reveal the synthesized hollow mesoporous Ru NPs found to be less cytotoxic.

Fig. 7 ECIS data shows the normalized impedance value of cytotoxic effects of hollow mesoporous Ru NPs. (a) Bioimpedance profile of HEMa cells with different concentrations. (a1) Normalized impedance value of magnified image of ‘a’ showing the concentration dependent impedance decline for 24 h upon adding the NPs on HEMa cells. (b) Bioimpedance profile of B16-F10 cells with different concentrations. (b1) Normalized impedance value of magnified image of ‘b’ showing the concentration dependent impedance decline for 24 h upon adding the NPs on B16-F10 cells.

Cellular uptake study

The intracellular uptake of 100 μg concentrations of Ru NPs was monitored under fluorescence microscope for both HEMa and BI6-F10 (ESI, Fig. S4†). Both cell lines were incubated with the selected lowest concentration of 100 μg ml⁻¹ of Ru NPs and examined after 12 h. The cellular uptake mechanisms were varied according to the properties of the particle such as size, shapes and surface charges.³⁸ The NPs uptake is shown in the Fig. S4a and b† and is denoted by arrows in the merged images. Since the NPs are a larger size, negative surface charge will take more time for internalization and wrapping.³⁹ For these large sized NPs (162 nm) the internalization is possible via endocytosis through clathrin-coated pits,⁴⁰ and the figure shows that the NPs are present less in the cytoplasmic region and more accumulated on the cell surface of B16-F10 than HEMa cells. Furthermore, the NP internalization and attachment on the surface were confirmed by using confocal microscopy imaging with z-stacks (Fig. S5†). Usually, NPs with positive surface charge strongly bind to the cell and show a higher cellular uptake through pinocytosis, receptor-mediated endocytosis or phagocytosis.⁴¹ On the other hand, NPs with high negative surface charge showed a higher cellular uptake than low negative surface charge particles, because the NPs show a higher attraction to the cellular membrane mainly due to electrostatic interactions.⁴² So, our cellular uptake results reveal that the prepared negatively charged surface NPs show high electrostatic interactions with the cellular membrane, and show a higher cellular uptake through interactions, and also bind at the cationic sites of the cell membrane in the form of clusters.⁴³ This study confirms that the cellular uptake of Ru NPs was found low level in inside the cells and high level in cell membrane for both cell lines through electrostatic interactions and cationic sites binding. Furthermore, these hollow mesoporous NPs will be effective for intracellular drug release and targeted actions with selective ligands.

Evaluation of DOX-loading onto hollow mesoporous Ru NPs and drug-release properties

In order to confirm the possibility of hollow mesoporous Ru NPs acting as sustained drug release systems, DOX was loaded into the hollow mesoporous Ru NPs in PBS. The DOX-loading capacity of the hollow mesoporous Ru NPs was found to be 52 ± 1.8% of the total mass added. This suggests that, among various mesoporous NPs preparations, these functionalized hollow mesoporous Ru NPs show a high drug loading capacity. The amounts of DOX released from DOX-loaded hollow mesoporous Ru NPs in different PBS buffers (neutral or acid) were determined using UV-visible spectroscopy. Fig. 8 shows the results of the DOX release profile from DOX-loaded hollow mesoporous Ru NPs in different PBS buffers (Fig. 8a (neutral) and Fig. 8b (acidic)). Fig. 8a results reveal that in the acidic pH (pH 5.0) there was a fast initial release of around 40% within the first 12 hours, followed by slow continuous release of around 85% of the DOX released from DOX-loaded hollow mesoporous Ru NPs in the following 48 h. The DOX release rate is very high at pH 5.0 compared to pH 7.4. These results suggest that the initial burst release due to the solubility of doxorubicin in acidic pH aqueous medium causes the mesopores to open up to release DOX. The drug release profile reveals that the pH sensitive drug release is more favorable for antitumor treatment, because the tumor environments are mainly acidic in nature.

Fig. 8 Release of DOX from DOX-loaded hollow mesoporous Ru NPs in different pH PBS buffers. (a) Release of DOX in acid (pH 5.0) PBS buffer. (b) Release of DOX in neutral (pH 7.4) PBS buffer.

Conclusions

In summary, we successfully synthesized uniform mesoporous Ru NPs with a hollow interior architecture by the dual template method. The obtained particle shows a high surface area with a hollow interior, which reveals more loading sites for drug payloads and the pore size indicates easy diffusion of guest species. The precise tuning of material composition, especially P407 concentration, confirmed P407 plays a key role as a structural directing agent. From the overall evaluation, the synthesized hollow mesoporous Ru particle was found to have a high surface area, be stable, and be polycrystalline in nature. Moreover, this approach could be applied to various kinds of metals by using different templates with slight modification according to their material and synthetic conditions. From the in vitro cytotoxic evaluation for both cell lines, ≥100 μg ml⁻¹ of Ru NPs was found to be toxic. Cellular uptake confirms that the NPs show high electrostatic interactions with cationic sites binding to the cellular membrane, so the Ru NPs can be effective as drug carriers. And most importantly, the drug release profile revealed that the pH sensitive drug release, because of rapid diffusion and release by the acidic buffer, is more favorable for antitumor treatment. The overall report unveils that the synthesized Ru NPs will be suitable for biomedical application, especially for cancer drug carriers.

Acknowledgements

This research was supported by the R&D Program for the Society of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (2013M3C8A3078806 and 2013M3C1A8A01072922).

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Footnote

† Electronic supplementary information (ESI) available: Experimental, Section 1: ECIS cytotoxic analysis, cellular uptake evaluation and drug release study. Table S1: compositions of various formulations. Fig. S1–S4: absorption and emission spectral profile, FT-IR, TGA and DSC, cellular uptake. See DOI: 10.1039/c5ra10827f

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