BSA Protein-Mediated Synthesis of Hollow Mesoporous Silica

Letter www.acsami.org

BSA Protein-Mediated Synthesis of Hollow Mesoporous Silica Nanotubes, and Their Carbohydrate Conjugates for Targeting Cancer Cells and Detecting Mycobacteria Nanjing Hao,*,†,‡ Laifeng Li,† and Fangqiong Tang*,† †

Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire 03755, United States

‡

S Supporting Information *

ABSTRACT: A straightforward method was developed to synthesize hollow mesoporous silica nanotubes (HMSNTs) using bovine serum protein (BSA) as the protective coating and phosphate buffered saline (PBS) as the etching agent at room temperature. Galactose-grafted HMSNTs significantly reduced phagocytosis by macrophages, and enhanced cellular uptake by A549 cells via caveolae-mediated uptake pathway. Trehaloseconjugated HMSNTs interacted strongly with mycobacteria, showing the linear detection range from 1 × 104 to 1 × 108 bacteria/mL and the detection limit of 1 × 103 bacteria/mL. In all cases, the hollow nanotube structure showed higher cellular uptake, bacterial binding, and detection efficiency than their spherical counterpart. KEYWORDS: carbohydrate, hollow mesoporous silica, targeting, detection, mycobacteria

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templates. Therefore, straightforward and scalable synthesis of well-defined HMSNTs is still a great challenge. Herein, we report a simple and general protocol to synthesize HMSNTs by selective dissolution of mesoporous silica nanorods (MSNRs) using bovine serum protein (BSA) as protective layer and phosphate buffered saline (PBS, pH 7.4) as mild etching agent at room temperature. Carbohydrate functionalization was conveniently realized using the photocoupling chemistry by employing PFPA-silane (N-(3-trimethoxysilylpropyl)-4-azido-2,3,5,6-tetrafluorobenzamide) as the coupling agent (Figure S1).14−16 Galactose- and trehalosemodified HMSNTs (HMSNTs-Gal and HMSNTs-Tre) were further examined for targeting cellular uptake and specific bacteria detection (Scheme 1). The results were compared with hollow mesoporous silica nanospheres (HMSNSs). Well-defined MSNRs having long-range pore channels with an average diameter of 100 nm and length of 500 nm were first synthesized by co-condensation of cetyltrimethylammonium bromide and tetraethyl orthosilicate using aqueous ammonia as the catalyst (Figure 1A, Figure S2, and other details in the Supporting Information). Nitrogen adsorption−desorption analysis showed that MSNRs exhibited a type IV isotherm (Figure S3A), typical for mesoporous materials.17 The average pore size was determined to be 2.76 nm by the Barrett−

ver the past decade, theranostics have gradually shifted toward the more specific and targeted approaches. Of the targeting ligands, carbohydrates are especially unique as glycans are ubiquitously found on cells and pathogens, and are involved in many biological processes. Glyconanomaterials, nanomaterials functionalized with carbohydrates, combine the unique properties of nanoscale objects with the ability to present multiple copies of carbohydrate ligands, greatly enhancing the weak affinity of individual ligands to their binding receptors. Selected carbohydrate ligands, when conjugated to nanomaterials, resulted in multivalent glyconanomaterials that could target cells and bacteria with high specificity.1−3 Nonspherical mesoporous silica nanomaterials are shown to have superior performance, especially in cellular binding, cellular uptake, and in vivo circulation and stability, over their spherical counterparts.4−6 Among them, hollow mesoporous silica nanotubes (HMSNTs) with typical long large inner void have shown unique advantages in catalysis,7 biosensing,8,9 drug release,10 and cellular uptake.11,12 To date, HMSNTs are synthesized using template-based strategies in which either a hard template7,8,10,11 or a soft template9,12,13 is employed. However, template-based strategies have inherent drawbacks. For example, hard templating methods require time-consuming multistep procedures, including core synthesis, surface modification of core, silica deposition, and core etching using corrosive/toxic etching agents. Soft templating approaches require less synthetic steps, but the resulting products often have irregular size and shape because of deformation of soft © XXXX American Chemical Society

Received: August 10, 2016 Accepted: October 18, 2016

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DOI: 10.1021/acsami.6b10051 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Letter

ACS Applied Materials & Interfaces

To explore the formation mechanism of HMSNTs by this simple treatment protocol, we investigated the specific roles of BSA, PBS, and temperature, separately. In the absence of BSA, the dissolution exhibited no selectivity, both the particle surface and the inner core were slightly etched (Figure S7). This confirmed that BSA acted as a protetive layer for the particle surface. When replaced PBS with water, the nanostructures were intact (Figure S8), indicating that the dissolution process in water is quite slower than that in a highly salted PBS environment. If the reaction temperature was raised to 80 °C, the dissolution process accelerated, and HMSNTs could be formed within only 3 h (Figure S9). Because of their well-ordered morphology, long large inner void, and robust shell, the as-synthesized HMSNTs could be favored in many application fields, especially the current emerging needs of targeting cells and bacteria. Toward this end, we functionalized HMSNTs with galactose as targeting moeity for A549 cells, which are known to overexpress galectin (a galactose-binding protein) and have enrichment of galactose receprors on cell surface,19−21 and trehalose as specific ligand for mycobacteria (M. smegmatis mc2 651), which have abundant free form trehalose in the cytosol and glycolipids form in the cell wall.22,23 Galactose or trehalose was conjugated on HMSNTs using the photocoupling chemistry by treating HMSNTs with PFPA-silane followed by photolysis in the presence of galactose or trehalose (see details in the Supporting Information). The functionalization process was monitored by FTIR (Figure S10). Modification of mesoporous silica nanomaterials with PFPA-silane resulted in a typical azide signal at around 2125 cm−1. Upon photoinitiation, this azide signal disappeared, indicating that carbohydrates were successfully functionalized on the material surface. The conjugation density of galactose and trehalose on HMSNTs was determined to be about 2.1 and 1.3 molecules per nm2, respectively, obtained using the anthrone/sulfuric acid colorimetric assay.2,24 Thermogravimetric analysis (TGA) results showed that the weight ratios of galactose and trehalose to corresponding glyconanomaterials are about 4.9% and 5.8%, respectively (Figure S11). To visualize the interactions of nanoparticles with cells or bacteria, we further grafted HMSNTs-Gal and HMSNTs-Tre with fluorescein isothiocyanate (FITC) to give HMSNTs-Gal-FITC and HMSNTs-Tre-FITC, respectively. An extra weight loss of about 9.5% was found in both fluorescent glyconanomaterials (Figure S11). Because phagocytosis by macrophages is an undesired process for most diagnostic and therapeutic applications involving nanomaterials,25 the impact of the glycosyl coating on phagocytosis was investigated. We incubated RAW264.7 macrophage cells with HMSNTs-Gal-FITC and Gal-free HMSNTs-FITC, and then analyzed by confocal microscopy and flow cytometer. As is shown in Figure 2, there was almost no green fluorescent signal for HMSNTs-Gal-FITC-treated macrophage cells, whereas HMSNTs-FITC-treated cells showed strong FITC signal inside cells. Quantitative results showed that the mean fluorescent intensity of HMSNTs-FITCtreated cells is ca. 18 times higher than that of HMSNTs-GalFITC-treated cells (Figure 2C). These results indicate that glycosyl functionalization could effectively prevent macrophage cell uptake and thus enable nanoparticles to circulate to the target disease loci. The targeted cellular uptake of HMSNTsGal-FITC by A549 cells was next tested (Figure 3). It can be clearly seen that HMSNTs-Gal-FITC efficiently entered into A549 cells (Figure 3A, control). When trehalose was

Scheme 1. Fabrication of Carbohydrate-Functionalized HMSNTs for Cell Targeting and Bacteria Detection

Figure 1. TEM images showing the evolution of HMSNTs at different reaction time: (A) 0, (B) 1, (C) 3, (D) 6, and (E) 12 h. (F) TEM image of HMSNTs after heating sample in E at 450 °C for 1 h. The inset in B shows protein coating on the particle surface.

Joyner−Halenda (BJH) method (Figure S3B). When MSNRs were treated with BSA in PBS (pH 7.4) at room temperature, BSA was adsorbed and coated on particle surface (Figure 1B), which was also confirmed by FTIR measurement (Figure S4). The adsorption of BSA is likely due to electrostatic attraction since the isoelectronic point of BSA is ca. 4.918 and the Zeta potential of MSNRs in pH 7.4 PBS is about −21.76 mV. With increasing incubation time, the inner core began to dissolve, and many large pores were observed (Figure 1C). These pores became wider and interconnected with each other (Figure 1D). Finally, all pores were interconnected to form the hollow tube nanostructure, whereas the outer layer became as shell (Figure 1E). To the best of our investigations using TEM, the nanorod structure can be exclusively and completely evolved into nanotube structure. After thermal decomposition to remove coated proteins (Figure S4), HMSNTs with a shell thickness of 10 nm were obtained (Figure 1F). From nitrogen adsorption− desorption analysis (Figure S5), the resultant HMSNTs displayed a type IV character with H3 hysteresis loop, revealing the presence of macroporous structure and slit-shape pores that were formed by the dissolution process.17 This method of generating hollow macroporous in mesoporous silica nanomaterials is general. Using the same protocol, well-ordered hollow spherical nanostructures could also be formed (Figure S6). B


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Figure 2. (A) Confocal microscopy and (B) flow cytometer results of macrophage cells (RAW264.7) incubated for 3 h with 50 μg/mL of HMSNTs-Gal-FITC and HMSNTs-FITC. (C) Corresponding mean fluorescence intensity results from B. Cell nuclei in part A were stained with 5 μg/mL of Hoechst 33342 for 10 min. Scale bar = 50 μm.

conjugated instead of galactose, no obvious binding or internalization was seen in the cells (Figure S12). This suggests that the uptake of glycosyl nanomaterials by A549 cells is galactose-specific. The cellular uptake pathway of HMSNTsGal-FITC by A549 cells was further investigated using different chemical inhibitors (Figures 3A, B). Hypertonic challenge with sucrose is known to disrupt the formation of clathrin-coated vesicles, and has been employed as a specific inhibitor of clathrin-mediated endocytosis. 6 Methyl-β-cyclodextrin (MβCD), which is known to sequester and alter the cholesterol-rich domains within the plasma membrane, can be used to inhibit caveolae-mediated endocytosis.26 NaN3 treatment could disturb the production of ATP and thus block the energy-dependent endocytosis, including clathrin- and caveolae- mediated endocytosis.26 As is shown in Figure 3A, treating with MβCD could significantly reduce the uptake of HMSNTsGal-FITC by A549 cells, but the effect of sucrose treatment on cellular uptake was relatively minor. Quantitative analysis showed that the mean fluorescent intensity of MβCD- and sucrose- pretreated cells are ca. 21.8% and 88.4% of the control cells, respectively (Figure 3C). The obvious uptake inhibition was also observed for cells treated with NaN3 (46.6% of control). In addition, when free galactose was added as a competitive inhibitor, the uptake of HMSNTs-Gal-FITC by A549 cells almost completely diminished (6.5% of control). These results suggest that the uptake of HMSNTs-Gal-FITC by A549 cells is most likely via the energy-dependent caveolaemediated pathway. Comparatively, the uptake of spherical HMSNSs-Gal-FITC was significantly reduced by sucrose (29.1% of control) compared to MβCD-pretreated cells (84.3% of control), indicating that clathrin-mediated pathway was dominant for the uptake of HMSNSs-Gal-FITC by A549 cells (Figure S13). These findings are consistent with the previous studies that particles having high aspect ratios preferred to entry into cells via caveolae-mediated pathway

Figure 3. (A) Confocal microscopy and (B) flow cytometer results of A549 cells incubated for 3 h with 50 μg/mL of HMSNTs-Gal-FITC, or pretreated with sucrose (0.45 M), methyl-β-cyclodextrin (MβCD, 10 mM), NaN3 (5 mM), for 30 min. Free galactose (Gal, 10 mM) was added with HMSNTs-Gal-FITC for competitive assay. (C) Corresponding mean fluorescence intensity results from B. Cell nuclei in part A were stained with 5 μg/mL of Hoechst 33342 for 10 min. Scale bar = 50 μm.

and spherical particles favored to be internalized via clathrinmediated pathway.6,27 Knowing these could not only help us to fully understand the inner interactions between nanoparticles and cells but also help to rationally design more effective theranostic nanomedicines. The specific binding and detecting ability of HMSNTs-TreFITC was examined using mycobacteria (M. smegmatis mc2 651). We first investigated the binding interactions between nanoparticles and mycobacteria under confocal microscope. As is shown in Figure 4A, HMSNTs-Tre-FITC bound strongly to mycobacteria and induced severe aggregation of mycobacteria. In contrast, HMSNTs-Gal-FITC showed no specific binding or agglomeration with the bacteria. This suggests that the binding of the nanotubes with mycobacteria is trehalose-specific. This is further demonstrated by the lack of interactions between HMSNTs-Tre-FITC and E. coli ORN208 bacteria (Figure C


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strong agglomeration was used for the detection of mycobacteria, which exhibited a linear detection range from 1 × 104 to 1 × 108 bacteria/mL and a detection limit of 1 × 103 bacteria/mL. The cellular uptake and bacterial detection efficiency of HMSNTs were higher than that of their spherical counterparts. These hollow nanotube structures may find additional applications in drug delivery, catalysis, lithium-ion batteries, and separation.

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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.6b10051. Experimental details, materials characterization and additional data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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Figure 4. (A) Confocal microscopy images of mycobacteria incubated for 1 h with 50 μg/mL of HMSNTs-Tre-FITC and HMSNs-GalFITC. Scale bar = 50 μm. (B) Fluorescence spectra of HMSNTs-TreFITC (50 μg/mL) used as probes for the detection of mycobacteria (from 1 × 104 to 1 × 108). (C) Plot of relative fluorescence intensity [(IF − IF0)/IF0] at 517 nm versus bacteria number (R2 = 0.973).

ACKNOWLEDGMENTS The authors thank the National Science Foundation of China (81171454) for the financial support. Dr. Nanjing Hao thanks Prof. Mingdi Yan (University of Massachusetts Lowell, MA, U.S.A.) for her kind discussion and revision of this manuscript. Dr. Nanjing Hao also thanks Prof. John Xiaojing Zhang for his support at Dartmouth College, NH, U.S.A.

S14). In addition, obvious binding but with minor aggregation was investigated after treated mycobacteria with spherical HMSNSs-Tre-FITC (Figure S15), indicating that the long tube-like HMSNTs-Tre could act as a more efficient multivalent scaffold to present glycosyl moieties. The ability of HMSNTs-Tre-FITC to aggregate mycobacteria was used as a means for detecting mycobacteria. This was accomplished by incubating different concentrations of mycobacteria with HMSNTs-Tre-FITC, and then excess free trehalose was added to displace the particles from bacterial surface (see details in the Supporting Information).28,29 The fluorescence intensity from bacteria-binding particles increased with the increasing number of mycobacteria (Figure 4B). The correlation between the fluorescent intensity and the number of mycobacteria was linear from 1 × 104 to 1 × 108 bacteria/mL (R2 = 0.973) with a detection limit of 1 × 103 bacteria/mL. In comparison, the spherical HMSNSs-Tre-FITC displayed a linear detection range from 5 × 105 to 1 × 108 bacteria/mL (R2 = 0.957) with a detection limit of 1 × 104 bacteria/mL (Figure S16). These results again showed the superior advantages of nanorods over nanospheres in the detection of mycobacteria with an order of magnitude higher sensitivity. In summary, we developed a general and scalable selective dissolution strategy to synthesize hollow mesoporous silica nanotubes using BSA as a protective layer and PBS as mild etching agent at room temperature. Using the photocoupling chemistry, HMSNTs were functionalized with carbohydrates for cancer cell targeting and mycobacteria detection. HMSNTsGal showed efficient targeting of A549 cells while evading the undesired phagocytosis of macrophages. The uptake pathway of HMSNTs-Gal by A549 cells was determined as caveolaemediated. HMSNTs-Tre displayed specific interactions with mycobacteria, and caused the bacteria to agglomerate. This

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BSA Protein-Mediated Synthesis of Hollow Mesoporous Silica

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