Biocompatible Phenylboronic-Acid-Capped ZnS Nanocrystals

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Biocompatible Phenylboronic-Acid-Capped ZnS Nanocrystals Designed As Caps in Mesoporous Silica Hybrid Materials for onDemand pH-Triggered Release In Cancer Cells Yolanda Salinas,*,† Carolin Hoerhager,† Alba García-Fernández,‡,§ Marina Resmini,⊥ Félix Sancenón,‡,§,|| Ramón Matínez-Máñez,‡,§,|| and Oliver Brueggemann† †

Institute of Polymer Chemistry (ICP), Johannes Kepler University Linz, Altenberger Strasse 69, Linz 4040, Austria Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politécnica de València, Universitat de València, Valencia, Spain § CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain || Departamento de Química, Universidad Politécnica de Valencia, Camino de Vera s/n, Valencia E-46022, Spain ⊥ Department of Chemistry and Biochemistry, SBCS, Queen Mary University of London, Mile End Road, London E1 4NS, United Kingdom

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‡

S Supporting Information *

ABSTRACT: Biocompatible ZnS-based nanocrystals capped with 4-mercaptophenylboronic acid (ZnS@B) have been size-designed as excellent pHresponsive gatekeepers on mesoporous silica nanoparticles (MSNs), which encapsulate fluorophore safranin O (S2-Saf) or anticancer drug epirubicin hydrochloride (S2-Epi) for delivery applications in cancer cells. In this novel hybrid system, the gate mechanism consists of reversible pH-sensitive boronate ester moieties linking the nanocrystals directly to the alcohol groups from silica surface scaffold, avoiding tedious intermediate functionalization steps. The ∼3 nm size of the ZnS@B nanocrystals was tailored to allow efficient sealing of the pore voids and achieve a “zero premature cargo release” at neutral pH (7.4). The system selectively released the cargo in acidic conditions (pH 5.4 and 3.0) because of the hydrolysis of the boronate esters, which unblocked the pore voids. Delivery of the cargo by off−on cycles was demonstrated by changes in pH from 7.4 to 3.0, showing its potential pHswitching behavior. Cellular uptake of these nanocarriers within human cervix adenocarcinoma (HeLa) cells was achieved and the controlled release of the chemotherapeutic drug epirubicin was shown to occur within the endogenous endosomal/ lysosomal acidified cancer cell microenvironment and further diffused into the cytosol. Cytotoxicity tests done on the mesoporous support without cargo and covalently linked with ZnS@B nanocrystals as caps were negative, suggesting that the proposed system is biocompatible and can be considered as a very promising drug nanocarrier. KEYWORDS: pH-triggered release, mesoporous silica nanoparticles, molecular gates, biocompatible ZnS nanocrystals, HeLa cancer cells

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INTRODUCTION The possibility of delivering drugs in response to physiological changes has boosted the development of “smart” systems that aim to replace, when required, inefficient traditional drug formulations.1−4 An ideal delivery system should avoid premature release at unspecific sites and be able to tailor delivery by differentiating between healthy and non-healthy cells or tissues. In this context, change in pH is one of the most-studied endogenous stimuli,5,6 along with temperature,7,8 for drug delivery using smart nanodevices, because of the pH gradients present in tumors or in internal parts of the cell such as, for instance, in early endosomes (pH 6−6.2) and lysosomes (pH 5.0−4.5).9 Among carriers suitable for pH-controlled drug delivery, those based on mesoporous silica nanoparticles (MSNs) are ideal because of the remarkable features of the © XXXX American Chemical Society

mesoporous silica scaffold, such as chemical inertness, presence of a regular porous network (suited to load drugs or other molecules), high specific surface areas, non-toxicity, and ease of functionalization using the well-known alkoxysilane chemistry.10 In fact, in recent years, a number of nanodevices based in MSNs have been reported containing different (bio)molecules or supramolecular assemblies acting as “molecular gates”,11 that can deliver the cargo following the trigger of a specific stimuli. A significant number of chemical species have been used as capping agents for MSNs when change in pH is the external trigger, including organic polymers,12 polyReceived: August 10, 2018 Accepted: September 13, 2018

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DOI: 10.1021/acsami.8b13698 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces amines,13−15 inclusion assemblies,16−18 metal complexes,19−21 biomolecules22 and inorganic nanoparticles (e.g., Fe3O4 or Au).23,24 Among them, the use of quantum dots (QD) or nanocrystals as inorganic ensembles for capping is of particular interest because these materials could be a useful alternative to organically-based caps, given their stable photophysical properties and the possibility to prepare them in a wide size range.25 However, despite their interesting features, to date their use as gatekeepers in capped MSNs has not been investigated extensively. In a pioneering work, Lin and coworkers26 prepared redox-triggered QDs-capped MSNs. These CdS QDs were covalently linked to the outer surface of MSNs using an organic stalk prepared to contain disulfide bonds, which were detached from the surface in the presence of reducing agents (dithiothreitol or mercaptoethanol) with subsequent cargo delivery. Guo, Zhu and co-workers27 used aminopropyl-functionalized ZnO QDs linked onto the external surface of loaded and previously carboxylic acid-functionalized MSNs using a linker containing amide bonds, which were hydrolyzed in acidic media allowing cargo release. More recently, in 2014, Fu and co-workers28 capped the pores of loaded acetal-modified MSNs with graphene QDs. In that case, acidic hydrolysis of the acetal moieties detached the QDs from the surface, allowing cargo release. In most of those examples, complex and lengthy synthetic protocols and functionalization steps are often required to decorate the inorganic caps as well as the silica surface, to allow the linking to different pHsensitive sites. In addition, despite the potential applications in gating protocols, there are still significant issues regarding toxicity, solubility, and biocompatibility that limit their clinical applications.29 In the last years, nanocrystals made of different materials have been synthetically improved in order to obtain the highest quality colloidal semiconductors.30,31 These nanocrystals are single fragments from the bulk crystals, which present well-controlled particle size distribution in the nanometer range, and their potential lies on their versatile preparation and their general interesting in tailoring sizedependent properties.32,33 Hence, given our interests in the preparation of straightforward capped MSNs for different biotechnological applications, we began to look into the use of non-toxic materials such as nanocrystals based on Zn and S to act as gatekeepers in the design of pH-controlled gated MSNs for drug delivery into cells. Zinc sulfide nanocrystals (ZnS) were among the first semiconductors to be discovered,34 and their physicochemical characteristics led to increased popularity, resulting in multiple applications.35 The surface modification of ZnS with molecules such as trioctylphosphine oxide, thioacetamide, and thioglycerol amides or aniline derivatives35−37 is known to prevent aggregation, control size, and make them suitable for further functionalization. Among potential molecules for their functionalization, boronic acid has the ability to form reversible covalent complexes with 1,2 or 1,3-diols.38 We postulated that the presence of hydroxyl moieties onto the surface of silica materials (i.e., silanol groups) would allow the simple and direct attachment of ZnSbased nanocrystals coated with phenylboronic acid moieties on the surface of mesoporous silica via formation of boronate ester bonds. Herein, we report the preparation of novel pH-responsive drug nanocarriers based on as-made MSNs loaded with the dye safranin O (S2-Saf) or the anticancer drug epirubicin (S2Epi). These nanoparticles were capped directly with phenylboronic acid capped ZnS nanocrystals of specific size (ca. 3

nm), allowing the perfect closure of the pore voids through pH-sensitive boronate ester linkages (see Scheme 1). These Scheme 1

(a) MSNs loaded with safranin O (S2-Saf) or epirubicin (S2-Epi) and capped with boronic-acid-coated ZnS nanocrystals (ZnS@B) (closed gate, no release of the cargo); (b) cargo release achieved by hydrolysis of boronate esters, formed between the boronic acid moieties of nanocrystals and silanols (−OH) from the silica surface at acidic pH (open gate, release of the cargo)

pH-responsive capped nanoparticles have been designed to achieve a more precise and selective controlled delivery of drugs in cancer cells. Our proposed biocompatible material could be injected directly into the tumor or transported within the bloodstream, protecting and diminishing the cargo side effects and responding by unblocking the pores only under acidic conditions, such as the one typically found in cancer cell surroundings.

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EXPERIMENTAL SECTION

Chemicals. Zinc acetate dihydrate (Zn(OAc)2·2H2O, 99%), sodium sulfide nonahydrate (Na2S·9H2O, ≥ 98%), tetraethylorthosilicate (TEOS), N-cetyltrimethylammonium bromide (CTAB), Safranin O (≥85%) and epirubicin hydrochloride (European Pharmacopoeia Reference Standard) were purchased from SigmaAldrich. 4-mercaptophenylboronic-acid (95%) was provided by Fluorochem. Sodium hydroxide (NaOH), hydrochloric acid (HCl) and sodium chloride (NaCl) were provided by J. T. Baker Chemicals. Diethyl ether and dimethylformamide were provided by VWR. Ethanol was purchased from Merck. Dulbecco’s Phosphate Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM) - high glucose, Foetal Bovine Serum (FBS) and Hoechst 33342 were purchased from Sigma-Aldrich. Lysotracker Green DND-26 was purchased from ThermoFisher Scientific. Cell proliferation reagent WST-1 was obtained from Roche Applied Science. All the solvents and reagents were used as received, without further purification. General Methods. Powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), N2 adsorption−desorption, Fourier transform infrared (FTIR), UV−visible (UV−vis) and fluorescence spectroscopy were used to characterize the materials. X-ray measurements were performed on a Bruker AXS D8 Advance using CuKα radiation. Thermogravimetric analyses were carried out on a TAQ5000, using nitrogen (25 mL min−1) with a heating program consisting of a dynamic segment (10 °C per min) from 313 to 1173 K. TEM images were obtained with a Jeol JEM-2200 FS microscope. SEM images were captured with a Jeol 6400 microscope. DLS was used to determine the size of the particles by a Malvern Zetasizer nano ZSP. Milli-Q water was used as dispersant at 25 °C and disposable cuvettes (DTS 0012) were used. B


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ACS Applied Materials & Interfaces Silica based solids were measured in a concentration of 1 mg mL−1, sonicated for 15 min and filtered through 0.2 μm nylon filters before performing measurements to avoid aggregations. N2 adsorption− desorption isotherms were recorded on a Micromeritics TriStar 3000 automated sorption analyzer. The samples were degassed at 60 °C under vacuum overnight. The measurements were performed at 77.30 K. Specific surface areas were calculated from the adsorption data at low pressure range using Brunauer, Emmett and Teller (BET) model. Pore size was determined following the Barret, Joyner and Halenda (BJH) method. FT-IR spectra were recorded on a PerkinElmer Spectrum 100. UV−visible spectroscopy was carried out with a Lambda 35 UV/vis spectrometer (PerkinElmer Instruments). Fluorescence spectroscopy measurements were taken on a Fluorolog Horiba Jobin Yvon. Nanocrystals absorbance, fluorescence emission and hydrodynamic diameter were measured from aqueous concentration of 0.1 mg mL−1. HeLa cells slides were visualized using a confocal microscope Leica TCS SP8 HyVolution II. Synthesis of ZnS Nanocrystals Capped with 4-Mercaptophenylboronic Acid (ZnS@B). The synthetic procedure followed was a modification of the method reported by Sarma et al.40 Under inert conditions and in a 2-neck round-bottom flask, zinc acetate dihydrate (240.5 mg, 1.10 mmol) and 4-mercaptophenylboronic-acid (401.3 mg, 2.61 mmol) were stirred in 25 mL of dimethylformamide (DMF). Sodium sulfide nonahydrate (33.4 mg, 0.14 mmol) dissolved in 4 mL of water was added dropwise, and the pH was adjusted with 1 mL of 0.1 M sodium hydroxide. The reaction was heated up slowly up to 150 °C and refluxed for 12 h. After this time, the reaction was cooled down to room temperature and the solvents were removed under vacuum. The resulting particles were washed three times by centrifugation with ethanol (3 × 30 mL) and diethyl ether (1 × 10 mL). Finally, the white nanocrystals were dried under air. The obtained ZnS@B nanocrystals were characterized by standard techniques. Similar nanocrystals were prepared without adding the boronic acid capping agent (so-called ZnS nanocrystals). Synthesis of Silica Mesoporous Nanoparticles (MSNs). Surfactant CTAB (1 g, 2.74 mmol) was dissolved in 480 mL of Milli-Q water, followed by the addition of 3.5 mL of NaOH (2 mol L−1). The temperature was then adjusted to 80 °C and TEOS (5 mL, 2.57 × 10−2 mol) was added dropwise to the surfactant mixture. The mixture was stirred for 2 h and a white precipitate was obtained. The precipitated solid was isolated by centrifugation and washed with deionized H2O until neutral pH. The sample was dried at 60 °C for 12 h. The surfactant was removed by calcination at 550 °C for 5 h and the final porous material (S0) was obtained. Synthesis of S1-Saf and S2-Saf. Calcined MSNs (201.4 mg) and safranin O (57.97 mg, 0.165 mmol) were suspended in CH3CN (40 mL) in a round-bottom flask. To remove water from the pores of the calcined MSNs, 10 mL of the solvent were removed via azeotropic distillation. Then, the suspension was stirred at room temperature for 24 h with the aim of loading the pores of MSNs scaffolds. The resulting solid (S1-Saf) was isolated by centrifugation, rinsed with 15 mL of CH3CN and finally dried at 38 °C for 12 h. For the preparation of the material S2-Saf, the solid S1-Saf (76.91 mg) was suspended in ethanol (25 mL) and NaOH (40 μL, 2M) as well as ZnS@B nanocrystals (2.3 mg) were added. The mixture was stirred for 16 h. Then, the material was centrifuged and washed once with 10 mL of ethanol, in order to eliminate the residual dye and the unreacted nanocrystals. The obtained product S2-Saf was dried at 37 °C for 12 h. Synthesis of S1-Epi and S2-Epi. Calcined MSNs (150 mg) and epirubicin hydrochloride (45 mg, 0.078 mmol) were suspended in distilled water (50 mL) in a round-bottom flask. Then, the suspension was stirred at room temperature for 24 h with the aim of loading the pores of MSNs scaffolds. The resulting solid (S1-Epi) was isolated by centrifugation, rinsed with 15 mL of ethanol and finally dried at 38 °C for 12 h. For the preparation of the material S2-Epi, the previous material S1-Epi (150 mg) was suspended in a mixture of slightly basic distilled water (pH 8) and ethanol 2:1 (50 mL:25 mL). ZnS@B nanocrystals (5 mg) were added and the mixture was stirred for 16 h at room temperature. The material was then centrifuged and washed

once with 25 mL of water, in order to eliminate the residual dye and the unreacted nanocrystals. The obtained product S2-Epi was dried at 37 °C for 12 h. Moreover, an extra material was prepared following the same procedure above but without cargo and grafted with ZnS@B nanocrystals (S2-control). Cargo Release Studies (S2-Saf and S2-Epi). In a typical release experiment, 5 mg of S2-Saf or S2-Epi were suspended in 12.5 mL of PBS solutions at pH 7.4, 5.2, and water solution at pH 3.0. Then, aliquots of 1.0 mL were collected at fixed times and filtered (to remove the nanoparticles) and the absorbance of safranin O at 520 nm (S2-Saf) or epirubicin (S2-Epi) at 480 nm was measured. Partial dye release studies were followed only with S2-Saf nanoparticles, as a function of pH variations between pH 7.4 and pH 3.0. For this experiment, 5 mg of S2-Saf was suspended in 12.5 mL of PBS at pH 7.4 and stirred for 30 min, while aliquots of 1.0 mL were collected every 5 min and centrifuged and the absorbance of safranin O at 520 nm was measured. After 30 min, the pH was decreased to pH 3.0 by adding 100 μL of 1 M of HCl triggering to a rapid release of the entrapped dye and same samples collecting and measuring procedure was followed. A second pH change was completed and the pH was set back to 7.4 by adding 100 μL of 1 M of NaOH. The whole experiment was performed over 150 min. Cell Culture Conditions. HeLa human cervix adenocarcinoma cells were purchased from the German Resource Centre for Biological Materials (DSMZ) and were growing in DMEM supplemented with 10% FBS. Cells were incubated at 37 °C in an atmosphere of 5% carbon dioxide and 95% air and underwent passage twice a week. Cellular Uptake of Solid S2-Saf in HeLa Cell Line. Internalization and cargo delivery studies using S2-Saf were performed in HeLa cells. For this purpose, Hela cells were seeded over glass coverslips at 250.000 cells mL−1 in 6-well plate and incubated at 37 °C for 24h. Then, S2-Saf at 50 μg mL−1 and lysotracker at 0.1 μM were added to HeLa cells and cells were incubated at 37 °C for 2 h. After 2 h, cells were washed several times with PBS and DNA marker Hoechst 33342 was added at 2 μg mL−1. Controlled Release of Solid S2-Epi in HeLa Cells. Internalization and cargo delivery studies using S2-Epi were performed in HeLa cells. For this purpose, HeLa cells were seeded over glass coverslips at 250 000 cells mL−1 in 6-well plate and incubated at 37 °C for 24 h. Then, S2-Epi was added to HeLa cells at 100 μg mL−1 and cells were incubated at 37 °C for 4 h. After 4 h, cells were washed several times with PBS and DNA marker Hoechst 33342 was added at 2 μg mL−1. Activity Assays with S2-Epi in HeLa Cell Line. To evaluate the efficacy of the S2-Epi system, a solid with epirubicin was tested in HeLa cells. For this purpose, Hela cells were seeded in a 24-well plate at 50 000 cells well−1 and incubated at 37 °C for 24 h. Then, solid S2control as control and solid S2-Epi were added to the cells at 50 and 100 μg mL−1 and incubated for 24 h. Finally, viability and thus epirubicin activity, was evaluated by adding the cell proliferation WST-1 reagent for 1 h, and absorbance was measured at 480 nm (emission at 595 nm).

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RESULTS AND DISCUSSION Gated Material. ZnS-based nanocrystals functionalized with phenylboronic acid moieties (ZnS@B) as capping agent were prepared by reacting zinc acetate, sodium sulfide and 4mercaptophenylboronic acid in DMF under basic and inert conditions at 150 °C for 12 h (see the Experimental Section for further details). DMF was chosen instead of water because of the low solubility of the capping agent 4-mercaptophenylboronic-acid. This capping agent was covalently bound to the surface atoms of the nanocrystals to prevent agglomeration and to increase the monodispersity between particles enhancing the individual silica pores blocking. MSNs were prepared by polymerization of tetraethylorthosilicate (TEOS) under alkaline conditions in the presence of the structure-directing agent N-cetyltrimethylammonium broC


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ACS Applied Materials & Interfaces mide (CTAB),39 following previously reported procedures. The mesoporous inorganic scaffold was obtained after calcination of the as-made material at 550 °C using an oxidizing atmosphere to remove the template (solid S0). The calcined material was then loaded with the dye safranin O (solid S1-Saf) or with the anticancer drug epirubicin hydrochloride (solid S1-Epi). Finally, the pores were capped upon addition of ZnS@B through the formation of reversible covalent boronate esters bonds between boronic acids from the nanocrystals and genuine silanols groups from the external surface of the loaded MSNs (solids S2-Saf and S2-Epi). The final material was purified by extensive washing with ethanol to ensure the removal of free unreacted caps. Characterization of Nanocrystals and Hybrid Solids. The grafting of 4-mercaptopropyl boronic acid on the external surface of the nanocrystals was assessed by FT-IR spectroscopy (Figure S3). In particular, the IR spectrum of ZnS@B did not show any band at around 2560 cm−1 (S−H stretching) which suggested the successful attachment of capping agent to the nanocrystals. Moreover, bands for O−H (3250−3400 cm−1), B−O (1370 cm−1), and aromatic C−H (1580−1620 cm−1) stretching vibrations were observed, confirming the presence of the boronic acid on the nanocrystals. Besides, ZnS@B nanocrystals showed a characteristic absorption peak at 300 nm and emission at 435 nm (Figure S1). The absorption was used to estimate an average size of ZnS@B nanocrystals of ca. 3 nm, results confirmed by DLS and TEM (Figures S2 and S4), and in agreement with previously reported data by Sarma and co-workers.40 Low-angle PXRD patterns of MSNs as synthesized, calcined MSNs (S0), S1-Saf, and S2-Saf solids are shown in Figure 1a. PXRD of MSNs as-synthesized showed four low-angle peaks, typical of a hexagonal-ordered pore array that can be indexed as (100), (110), (200), and (210) Bragg reflections. A shift of the (100) peak and a remarkable broadening of the (110) and (200) in S0 (calcined MSNs) was observed and ascribed to further condensation of silanols during the calcination step. The presence of the characteristic (100) reflection in the diffraction spectra obtained for solids S1-Saf and S2-Saf indicated that the mesoporous structure was preserved throughout the filling with safranin O and capping with ZnS@B nanocrystals processes. The same features were observed in the low angle PXRD patterns of S1-Epi and S2Epi nanoparticles. Moreover, Figure 1b shows the PXRD patterns of ZnS@B nanocrystals compared with S2-Saf nanoparticles. The peaks found in the 20−60 2θ range for ZnS@B were indexed as (111), (220) and (311) reflexions and were attributed to lattice planes of nanocrystals. The same peaks were detected in the PXRD of S2-Saf nanoparticles confirming the immobilization of the nanocrystals onto the silica surface. Additionally, FT-IR spectrum of S2-Saf was also analyzed and compared with that of ZnS@B nanocrystals (Figure S5). In the spectrum, it is shown the characteristic bands corresponding to the nanocrystals in the solid S2-Saf, which proves their presence in the mesoporous silica support. The morphology of the MSNs was studied by TEM and SEM techniques. The TEM image of S0 nanoparticles in Figure 2a shows the typical repeated patterns of well-organized hexagonal and longitudinal array of mesopores of the silica matrix, visualized as alternate white and black stripes. TEM images also showed that the final solid was obtained as nanoparticles with diameters in the 100−130 nm range, presenting a smooth external surface due to the absence of

Figure 1. (a) Low-angle PXRD patterns of as-synthesized MSNs, calcined MSNs (S0), loaded MSNs (S1-Saf), and ZnS@B-capped MSNs (S2-Saf). (b) High-angle PXRD patterns of ZnS@B nanocrystals and ZnS@B-capped MSNs (S2-Saf).

further surface functionalization. A SEM image of S0 was additionally captured (Figure 2b), confirming the expected spherical shape of the nanoparticles. Dynamic light scattering (DLS) measurements of S0, S1-Saf, S2-Saf, S1-Epi, and S2Epi nanoparticles were in agreement with TEM images and average hydrodynamic diameters (HD) between 125 and 132 nm were estimated for all prepared solids (data collected in Table 1). Interestingly, the diameter of the silica nanoparticles increased by approximately 6 nm following the nanocrystals grafting step (from 124 nm of solid S1-Saf to 130 nm of solid S2-Saf), which may be attributed to their ca. 3 nm size. The size distribution of the nanoparticles was found to be narrow. A very important result was the clear presence of nanocrystals in the final material (S2-Epi) by TEM microscope. Figure 2c shown the contrast between the mesopores channels from the initial solid S0, which were well observed by white and black stripes in Figure 2a, and lighter areas covering the whole silica-based nanoparticle (final solid S2-Epi). That lumpy external surface of the silica nanoparticles was attributed to the presence of nanocrystals. Figure 2d showed the narrow nanocrystal size−shape distribution and high crystallinity (see inset FFT). Specific surface area, pore volume, and pore size of the solids were determined by applying the BET41 and BJH42 models to D


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Figure 2. (a) TEM image of S0 showing the typical mesoporous structure of the silica matrix, the spherical morphology and smooth external surface of the nanoparticles; (b) SEM image of S0; (c) TEM image of the final material S2-Epi,showing the presence of ZnS@B nanocrystals on the surface of S2-Epi and a lumpier external surface; and (d) ZnS@B nanocrystals image showing their monodisperse size distribution. Inset: nanocrystal-FFT.

Table 1. Structural Parameters of the Prepared Solids Calculated from DLS and N2 Adsorption−Desorption Isotherms, and Content (α) of Anchored Nanocrystals and Cargo (dye or drug) in g/(g of SiO2)−1 for S0, S1-Saf, S1-Epi, S2-Saf, and S2-Epi solid S0 S1-Saf S1-Epi S2-Saf S2-Epi

HDa(nm) 121.2 124.8 129.3 130.1 136.8

± ± ± ± ±

0.8 0.5 0.1 0.7 0.1

SBET(m2 g−1)

pore vol.b(cm3 g−1)

pore sizeb(nm)

971.9 511.7 439.2 276.9 164.2

0.79 0.50 0.51 0.31 0.26

3.06

αnanocrystals(g/(g of SiO2−1))

αcargo (g/(g of SiO2−1))

0.039 0.043

0.303 0.293 0.181 0.211

a Hydrodynamic diameters by DLS measurements (average values from 6 independent measurements). bPore volumes and sizes associated with intraparticle mesopores. Pore sizes estimated by using BJH model.

specific volumes and specific surface areas, suggesting the right blocking of the pores due to the presence of nanocrystals. In fact S2-Saf and S2-Epi show flat isotherm curves when compared with the starting support (S0) or with the intermediate loaded solids (S1-Saf and S1-Epi). These facts successfully confirmed the correct cargo loading and nanocrystals blocking steps. The content of nanocrystals and cargo (dye or drug) in S0, S1-Saf, S1-Epi, S2-Saf, and S2-Epi were determined by thermogravimetric analysis and are also shown in Table 1. Solids S2-Saf and S2-Epi functionalized with the nanocrystals showed very similar final contents of 0.181 and 0.211 g of the cargo per g of SiO2 and 0.039 and 0.043 g of nanocrystals per g of SiO2, respectively. These contents were similar to other capped silica mesoporous nanoparticles described in literature for on-command delivery applications.45 Functional pH-Driven Controlled Release. Studies of pH-driven drug release using S2-Saf and S2-Epi were carried out. Safranin O released from solid S2-Saf at pH 7.4 was

N2 adsorption−desorption isotherms (see Table 1). Typical type IV isotherms were obtained for all prepared silica-based nanoparticles (Figure S6) presenting an adsorption step at an intermediate P/P0 value between 0.1 and 0.3. In this range, an absence of hysteresis loop and a narrow pore size distribution indicated the presence of cylindrical and uniform mesopores (pore diameter and pore volume calculated for initial solid S0 were 3.06 nm and 0.79 cm3 g−1, respectively). A large total specific surface area (971.9 m2 g−1) for solid S0 (close to 1000 m2 g−1, typical of calcined MSNs) was calculated using the BET model. In the case of S1-Saf and S1-Epi, the N2 adsorption−desorption isotherms (Figure S6) were consistent with those solids with partially filled mesopores previously reported.43,44 Lower BJH mesopore specific volumes and surface areas were obtained for S1-Saf and for S1-Epi, in comparison with the original S0 (see values at Table 1), indicative of a high loading of the pores with the selected cargo. Both final nanocrystals-capped materials (S2-Saf and S2-Epi) showed similar features and the lowest BJH mesopore E


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Figure 3. (a) Cumulative release profiles of safranin O from PBS suspensions of S2-Saf nanoparticles at different pH (dye absorbance measured at 520 nm). Inset: release observed after 60 min at pH 7.4, 5.2, and 3.0. (b) Cumulative release profile of safranin O suspensions from S1-Saf at pH 7.4, 5.2, and 3.0. Inset: cumulative safranin O release vs the square root of the time for S2-Saf at pH 3.0. (c) Cumulative partial dye release (absorbance of safranin O measured at 520 nm) from S2-Saf as a function of pH modulations. Inset: UV−vis spectra of solutions of solid S2-Saf at pH 7.4 and at pH 3.0 after 30 min. (d) Cumulative release profiles of the drug epirubicin hydrochloride from PBS suspensions of S2-Epi nanoparticles at different pH. Inset: release observed after 60 min at pH 7.4, 5.2, and 3.0. Error bars expressed as σ from three independent experiments.

for encapsulating safranin O or epirubicin molecules and their tailored pH triggered release. Additionally, it was found that the emission of detached ZnS@B nanocrystals was quenched by the cargo molecules also released from the pores (see quenching studies in Supporting Information, Figure S8). This behavior was consistent with the capability of ZnS nanocrystals to exchange electrons with a diverse range of molecules upon excitation, followed by their subsequent photoluminescence quenching, already well reported in the literature.46,47 Therefore, in this work, the emission of the nanocrystals for tracking the whole system inside cells was not meaningful, and only the emission from the respective dye/drug was used. To test the role played by the nanocrystals in the gating mechanism, we also tested cargo release from S1-Saf (without nanocrystals as gatekeepers) at pH 7.4, 5.2, and 3.0. As can be seen in Figure 3b, a fast delivery of safranin O from S1-Saf nanoparticles was observed at all selected pHs, showing nonpH responsiveness. In addition, data from the release profile of S1-Saf at pH 3.0 were fitted to the Higuchi model.48,49 This model has been widely applied to describe drug release kinetics from porous carrier matrices, and it is based on Fickian diffusion processes taking into account the hypothesis that initial cargo concentration in the matrix is much higher than its solubility, that dye diffusion takes place only in one dimension and that dye diffusivity is constant. Figure 3b (inset) shows the good fitting of the model, suggesting that under these

monitored for 1 day (1440 min). The same procedure was repeated at more acidic pHs (5.2 and 3.0). The release profiles at different pHs are shown in Figure 3a, where it can be observed that at neutral pH the amount of dye released was minimal (ca. 10% after 180 min and steady during 24 h). This result highlights the capping efficiency of the nanocrystals linked on the outside pores, blocking the release of the cargo and minimizing unspecific release. This tight closing of the pores at neutral pH was the main drive for employing these ZnS-based blockers, given the current needs in nanomedicine applications for delivery systems able to give tailored and controlled releases. Furthermore, a very significant cargo delivery was observed after 180 min at pH 5.2 (ca. 55%). The highest amount of safranin O released was found at pH 3.0 (ca. 82%), which was detectable by naked eye after 1 h (Figure 3a, inset). The cargo release observed in acidic conditions (pH ≤ 5.4) was attributed to the hydrolysis of boronate esters that linked the ZnS-based nanocrystals to the external surface of the silica-based nanoparticles, unblocking the pores and releasing the entrapped cargo as required. As expected, very similar release profiles were observed for S2-Epi loaded with the chemotherapeutic drug epirubicin (Figure 3d and Figure S7). Again, in this case, a poor release was observed at neutral pH and only under acidic conditions a marked delivery occurred (at pH 5.4 and 3.0 cumulative dye release of 20 and 65%, respectively, after 180 min). These results provide evidence of the excellent capping efficiency of the nanocrystals F


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Figure 4. Internalization studies of S2-Saf in the HeLa cells. Cells were incubated for 1 h in the presence of solid (50 μg mL−1) and lysosomes were marked with lysotracker green DND-26 (0.1 μM). (a) The internalization of the nanoparticles was followed by safranin O fluorescence (red) in the presence of lysosomes marker Lysotracker green DND-26 (green) and DNA marker Hoechst 33342 (blue). Yellow signals in the merged image evidence a high colocalization, as we expected, associated with the green signal from lysosomes and the red fluorescence of the S2-Saf nanoparticles. (b) Bottom images are zoomed in from the white dashed square marked in the a images above.

(see Figure S4), which could even favor the proposed pHopening mechanism in acidic conditions. Cargo Delivery from the Gated Materials in Intracellular Media. Cargo delivery from S2-Saf and S2-Epi at the cellular level was also evaluated. For this purpose, human cervix adenocarcinoma (HeLa) cells were used and cellular uptake of solid S2-Saf in this cell line was studied. Confocal microscopy analyses were used to evaluate whether or not nanoparticles were internalized in cells by tracking safranin O associated fluorescence. Moreover, cell nuclei and lysosomes were stained with Hoechst 33342 and lysotracker green DND26, respectively. A dotted pattern of a safranin O fluorescent signal associated with intracellular vesicles was observed (Figure 4), which suggests the successful internalization of nanoparticles. Moreover, a partial colocalization of the lysosome-associated signal (green) and nanoparticle associated fluorescence (red) was observed (yellow) thus suggesting lysosomal localization of the hybrid nanoparticles. In Figure 4b it could be better appreciated the accumulation of nanoparticles inside the lysosome (yellow signal). In this compartment, with a pH around 5, nanoparticles released the cargo that also diffused to the cytosol (in red). In a further step, the controlled release of epirubicin from S2-Epi nanoparticles in HeLa cells was assessed by confocal microscopy studies. The cells were incubated with 50 μg mL−1 of solid for 4 h and drug-associated fluorescence was observed. The images of the HeLa cells without treatment, as control, were measured and autofluorescence was discarded (Figure 5a). In contrast, a significant drug release was observed (Figure 5b) when cells were treated with S2-Epi. Finally, cell viability studies were performed with S2-Epi (loaded with the anticancer drug epirubicin) and with S2control in HeLa cells. S2-control nanoparticles consisted of MSNs without cargo but capped with ZnS@B nanocrystals. Results showed in Figure 5c demonstrated that the presence of S2-control (without anticancer drug) nanoparticles was welltolerated by HeLa cells at concentrations up to 100 μg mL−1

conditions the delivery of the cargo from the pores is basically a diffusive process. In order to corroborate the effect of the phenyl boronic acid moieties in the pH-sensitive gating process, a new material was prepared that consisted of solid S1-Saf treated with uncoated ZnS nanocrystals. The controlled release profile of this solid at different pH resembles those obtained with solid S1-Saf (data not shown), confirming the crucial role of the boronic acid-functionalized nanocrystals in the pH-responsive mechanism. In a further step, the potential switching mechanism (closeopen-close) of the hybrid gated system S2-Saf was investigated due to the reversible boronate ester bonds formation. The partial dye release from S2-Saf as a function of pH variations between pH 7.4 and pH 3.0 was followed for 150 min (Figure 3c). After stirring S2-Saf suspended in aqueous solution at pH 7.4 for 30 min, an expected negligible release of safranin O was observed (closed gate), similarly to the results observed in Figure 3a. After the same solution was acidified to pH 3.0 (by adding hydrochloric acid) a marked dye release occurred (open gate), also in agreement with the previously confirmed pH-triggered behavior of S2-Saf. This experiment was followed by switching the pH for 2 cycles more and interestingly, the increase of the pH back to 7.4 (by adding sodium hydroxide) resulted in the blocking of the pores with the nanocrystals located in the solution, with the subsequent inhibition of dye release (closed gate). This switching behavior was attributed to the reversible well-known hydrolysis (at acidic conditions) or formation (at neutral conditions) of boronate esters bonds between the ZnS@B nanocrystals and the silanol moieties onto the external silica surface. Furthermore, in a different experiment the stability of the ZnS-based nanocrystals was checked by DLS measurements of 0.1 mg mL−1 in different solutions (at pH 7.4 for 24 h and at pH 3 for 96 h). The size differences were negligible between particles at pH 7.4 or pH 3.0 despite the longest time of contact with acidic environment, suggesting a slightly disaggregation between particles G


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agent was selected to facilitate the covalent functionalization of the nanocrystals to the mesoporous silica scaffold through boronate ester bonds with the alcohol groups. This advantageous easy incorporation of the caps allowed minimizing any intermediate functionalization steps, often required in other reported similar materials. Furthermore, the pH-switching gate behavior of this hybrid system was demonstrated, which may bring interesting future applications. Silica mesoporous nanoparticles unloaded but capped with these novel ZnS-based nanocrystals were shown to be biocompatible in vitro when tested on HeLa cells in concentrations up to 100 μg mL−1 after 24 h of exposure. Successfully, the chemotherapeutic agent loaded material (S2-Epi) showed significant cancer cells death (ca. 40%) through internalization in the acidic lysosome, demonstrating the efficacy of this novel hybrid system. Our novel proposed biocompatible material was designed to be used by directly injection of the nanoparticles into the tumor or into the bloodstream. In an acidic environment, the caps will be removed and the cargo will be released to target viability of cancer cells. Future doping of these nanocrystals with transition metals, such as manganese, could add a beneficial effect on the nanocrystals for future tracking applications into cells by NIR, while avoiding photodamage.

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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/acsami.8b13698. Absorption and emission spectra, DLS and FT-IR spectra of ZnS@B nanocrystals, Bohr radius calculations, nanocrystals stability studies with pH, FT-IR spectra of solid S2-Saf, nitrogen adsorption−desorption isotherms of the solids S0, S1-Saf, S2-Saf, S1-Epi, and S2-Epi, fluorescence emission kinetic profiles of S2-Epi at different pHs, and quenching studies of the nanocrystals in the presence of safranin O (PDF)

Figure 5. Internalization and release of chemotherapeutic drug epirubicin from S2-Epi in HeLa cells. (a) HeLa cells without any treatment and (b) treated with 50 μg mL−1 of S2-Epi, where the internalization of the nanoparticles was followed by epirubicinassociated fluorescence (red) in the presence of DNA marker Hoechst 33342 (blue). (c) Cell viability studies in the presence of S2-control (black bars) and S2-Epi (grey bars) after 24 h of incubation. WST-1 reagent was added and cell viability was measured. Nanoparticles were added at 50 and 100 μg mL−1 and after 24 h viability were measured. Three independent experiments were completed, which gave similar results. Data are expressed as mean ± s.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Y.S.).

after 24 h of exposure (black bars showed 100% cells viability) and confirmed the biocompatibility features of our developed ZnS-based nanocrystals. In contrast, for HeLa cells treated with 50 and 100 μg mL−1 of S2-Epi (loaded with the chemotherapeutic agent Epirubicin hydrochloride) an expected significant decrease in cell viability (grey bars, Figure 5c) was in agreement with the internalization of S2-Epi material, the hydrolysis of the boronate esters in the acidic lysosomal environment and the subsequent release of the anticancer drug that resulted in cancer cell death (up to 40%).

ORCID

Yolanda Salinas: 0000-0002-1828-5839 Ramón Matínez-Máñez: 0000-0001-5873-9674 Author Contributions

The manuscript was written through contribution of all authors. Experiments were performed by Y.S., C.H., and A.G.-F. All authors have given approval to the manuscript. Notes

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The authors declare no competing financial interest.

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CONCLUSIONS In summary, we have demonstrated the possibility of directly attaching non-toxic ZnS-based nanocrystals as pore blockers on MSNs to efficiently control the release of uploaded molecules, such as fluorophore safranin O or anticancer drug epirubicin hydrochloride, at acidic pHs (≤5.2). The nanocrystals were intentionally designed to have similar size to the MSNs pores (ca. 3 nm), to tightly seal the voids at neutral pH (7.4) and achieve “zero premature cargo release”, which is an important target in nanomedicine to prevent uncontrolled released and side effects. The nanocrystals were synthesized in the presence of 4-mercaptophenyl boronic acid. This capping

ACKNOWLEDGMENTS The authors acknowledge P. Oberhumer from Centre for Nano- and Surface Analytics (ZONA) for the SEM and TEM measurements; Prof. A. Rastelli from Semiconductor Physics Division, Institute of Semiconductor and Solid State Physics, for helping in the Bohr radius calculations for excitons in ZnS; and the Prof. G. Knör from Institut für Anorganische Chemie, for the access to the fluorescence spectrometer, all located at Johannes Kepler University Linz. M. Kleindienst is acknowledged for her drawing contribution (Scheme 1,,). R.M.-M. and F.S. express their gratitude to the Spanish government H


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(MAT2015-64139-C4-1-R (MINECO/FEDER)) and the Generalitat Valencia (PROMETEOII/2014/047) for support. A.G-F. is grateful to the Spanish government for an FPU grant.

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Biocompatible Phenylboronic-Acid-Capped ZnS Nanocrystals

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