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Preparation of Self-activated Fluorescence Mesoporous Silica Hollow Nanoellipsoids for Theranostics Rajendra Kumar Singh, Tae-Hyun Kim, Chinmaya Mahapatra, Kapil Dev Patel, and Hae-Won Kim Langmuir, Just Accepted Manuscript • Publication Date (Web): 22 Sep 2015 Downloaded from http://pubs.acs.org on September 25, 2015

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Preparation of Self-activated Fluorescence Mesoporous Silica Hollow Nanoellipsoids for Theranostics

Rajendra Kumar Singha,b, Tae-Hyun Kima,b, Chinmaya Mahapatrab, Kapil Dev Patelb, and Hae-Won Kima,b,c,*

a

Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 330-714, South Korea

b

Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine,

Dankook University, Cheonan 330-714, South Korea c

Department of Biomaterials Science, School of Dentistry, Dankook University, Cheonan 330-714, South Korea

-----------

*

Corresponding author: Tel) +82 41 550 3081; Fax) +82 41 550 3085; E-mail) [email protected]

For: Langmuir

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Abstract The newly-developed multifunctional (self-activated fluorescent, mesoporous and biocompatible) hollow mesoporous silica nanoellipsoids (f-hMS) are potentially useful as a delivery system of drugs for therapeutics and imaging purposes. For the synthesis of f-hMS, self-activated fluorescence hydroxyapatite (fHA) was used as a core template. Mesoporous silica shell was obtained by a silica formation and a subsequent removal of fHA core, which resulted in a hollow-cored f-hMS. While the silica shell provided a highly mesoporous structure, enabling an effective loading of drug molecules, the fluorescent property of fHA was also well preserved in the f-hMS. Cytochrome C and doxorubicin, used as model protein and anticancer drug, respectively, were shown to be effectively loaded onto f-hMS, and then were released in a sustainable and controllable manner. The f-hMS was effectively taken up by the cells and exhibited fluorescent labeling, while preserving excellent cell viability. Overall, the f-hMS nanoreservoir may be useful as a multifunctional (self-activated fluorescent, mesoporous and biocompatible) carrier system for drug delivery and cell imaging.

Keywords: Inorganic nanocarrier; Fluorescent; Delivery system; Hydroxyapatite; In situ imaging;

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Introduction The integration of two or multiple functions into one nanoparticulate system is currently under significant investigation to find their widespread biomedical applications 1, 2. For example, when nanoparticles are effective in loading and delivering of therapeutic molecules while enabling imaging and diagnostics by optical, fluorescence or magnetism sources, their potential for medical uses significantly increases 2-4. Mesoporous silica nanoparticles (MS) have attracted a lot of interest due to their combination of exceptional porosity with versatile surface functionality and high biocompatibility. They hold great promise for a diversity of applications, and have emerged as vehicles or reservoirs in a wide range of fields such as drug delivery 5-7. Efforts are ongoing to optimize their porous parameters, such as pore volume, size, and surface area 8, 9

, with a view to improving the storage capacity of cargo. The introduction of a hollow cage into the core area

of the MSs is an effective way to significantly increase the pore volume relative to weight or surface area, which is also tunable by the change in hollow cavity size and mesoporous shell thickness. This structure is of critical importance for targeting delivery systems, because more drug molecules can be secured inside of a large hollow space, rather than loosely adsorb on the pore walls, which leading to sustained releases 10. Hollow nanoparticles can be synthesized with various materials such as organic polymers, silicates, carbon, titania, and phosphates 11-16. Moreover, to produce the cavity in the nanoparticles, various removable templates, such as polymers, micelles, surfactants, silicates, magnetite, gold nanoparticles, semiconductor quantum dots (QDs), and emulsion droplets have been used 17-31. Compared to soft core templates, like surfactants, emulsions, and micelles, the hard core templates have been shown to generate hollow structures with more defined, reproducible and controllable sizes. Here we use a novel core template material - hydroxyapatite (HA) with a fluorescent property, namely fHA. HA nanoparticles with different sizes and shapes are easily produced by many synthesis methods and are relatively cheap to be properly used as a core template material. Moreover, HA has an excellent biocompatibility that has allowed widespread medical uses as bone substitutes. Herein, well defined HA rod-type nanocrystals are produced by a hydrothermal process, and the use of citrate in the HA process followed by a thermal treatment generates fluorescent property in the structure. In terms of the fluorescent materials used in biomedicine, fluorescent organic molecules and semiconductor QDs are currently most widely investigated for biological staining and diagnostics 32, 33. However, major serious concerns are raised on the photo-bleaching and quenching of fluorescent organic molecules and the toxicity of 3


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semiconductor QDs, which limit their potential biomedical applications33, 34. Recently, a new class of materials has thus been reported. The materials have fluorescence properties in the structure without the additional use of earth metal ions, quantum dots, and biomolecules, and this characteristic can be named ‘self-activated fluorescent’ or ‘self fluorescent’.35-38 These novel self-activated inorganic materials may be promising fluorescent platforms for bio-imaging due to their good optical properties and substantially reduced toxicity. As introduced, here we focus on fHA as the base inorganic material for this fluorescence property as well as the core template for hollow structure. HA is popularly known as an inorganic component of hard tissues and thus highly compatible to cells and tissues, finding extensive biomedical applications as bone grafts, implant coatings, and tissue engineering scaffolds39. Compared to the extensive researches on the HA as biomaterials for those purposes, there is a handful of works on the fluorescence properties of HA 35,40. Here, we design MS using this fHA as the core template (fHA@MS) for the creation of hollow cavity, consequently to produce hollow fluorescent MS (f-hMS). This novel class of self-activated fluorescent inorganic materials is considered to be a promising candidate for biomedical applications due to their decent optical properties and good biocompatibility.

Materials and Methods

Fluorescent Hydroxyapatite Nanocrystals (fHA) Supersaturated solutions were prepared by mixing 0.1 g of hexadecyltrimethylammonium bromide (CTAB), and calcium nitrate [Ca(NO3)2.4H2O ] as follows: NH4OH (28%) was dissolved in deionized water to adjust pH 9.0 to form solution A. Trisodium citrate and (NH4)2HPO4 were dissolved into deionized water to form solution B. Solution B was mixed with solution A to provide molar ratio of Cit/Ca 1:1. After stirring for 60 min, the mixture solution was transferred to a bottle held in a stainless steel autoclave, sealed, and maintained at 190 °C for 24 h. After cooling, the precipitates were separated by centrifugation, washed with deionized water and ethanol in sequence, and then dried in air at 80 °C for 24 h to obtain the final fHA nanocrystals.

Synthesis of fHA@MS Mesoporous silica coatings on the fHA template were obtained by mixing silica precursor, TEOS with fHA. Before adding TEOS, 30 mg of freshly synthesized fHA in 200 mL of ethanol was sonicated for 30 min in an ultrasonic bath and then stirred at 500 rpm to make sure all fHA particles were well-dispersed before silica 4


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coating. 5 g of CTAB in 8 mL of ammonium hydroxide and 22 mL of deionised water was added to the fHA solution and mixed under a high-power ultra-sound for 30 min. Afterwards, 200 µL of TEOS was added to the mixture and sonicated for 2 h to prevent agglomeration. The mixture was then stirred overnight at 1200 rpm to obtain well-dispersed silica-coated fHA. The solution was centrifuged and thoroughly washed with absolute ethanol and deionised water three times each to ensure CTAB removal. The wetted solution was then dried overnight under vacuum to obtain precipitate of fHA@MS.

Preparation of Fluorescent Hollow-Core Structure of Mesoporous Silica Shell (f-hMS) To obtain a fluorescent hollow-core structure of MS, the template of fHA was dissolved through the etching process. 0.15 g of fHA@MS precipitate in 30 mL of deionised water was mixed with 20 mL of 1N hydrochloric acid solution. The mixture was then sonicated for 2 h and stirred further at 1200 rpm to prevent agglomeration and ensure thorough mixing. An intensive washing procedure was conducted for f-hMS to remove CTAB molecules embedded in the silica shell wall inner-core. In brief, the mixture was dispersed in the solution containing 20 mg of ammonium nitrate dissolved in 40 mL of deionised water and kept at 60 °C overnight. This mixture was centrifuged and washed with absolute ethanol and deionised water for four times sequentially. The wetted precipitate was dried overnight under vacuum to obtain dried fluorescence f-hMS precipitate.

Loading and Delivery of Cytocrome C (cyt C) – A Model Protein For the loading test of cyt C, different amounts (from 25 to 400 µg) were pooled in 1 ml phosphate buffered saline (PBS). Within each solution, fHA@MS and f-hMS were added at 1 mg and left for 24 h at 37 °C. The absorbance of the upper solution was assessed at 409 nm using a Libra S22 ultraviolet-visible (UV-vis) spectrophotometer (Biochrom) to determine the remaining quantity of drug. The amount of drug loaded onto the fHA@MS and f-hMS was calculated referring to a cyt C standard curve. The adsorption quantity of drug was plotted as a function of the drug quantity initially pooled in PBS. To examine the release profile of cyt C from the fHA@MS and f-hMS samples, the cyt C-loaded nanocarriers, which were prepared in 300 µg/mL of initial cyt C concentration, were used. To test cyt C release kinetics, cyt C-loaded samples was dispersed in 1 mL of PBS and were then incubated at 37 °C. The amount of drug released into the medium was measured at different time points (up to ~two weeks), and the medium was refreshed at each testing. 5


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Loading and Delivery of Doxorubicin (DOX) – A Model Anticancer Drug DOX was dissolved in PBS at pH 7.4 to produce a stock solution at a concentration of 120 µg/mL. A standard curve was obtained by measuring optical intensities of serial dilutions of the stock solution (20, 30, 40, 60, 80, 100, 120 µg/mL) using a UV-vis spectrophotometer at a maximum absorption intensity of 483 nm. For the DOX loading test, 1 mg of nanoparticles samples (LHA@MS and L-hMS) was ultrasonically dispersed in each DOX solution for 5 min and were then kept in a 37 °C water bath for 4 h. To quantify the drug loading amount, the nanoparticles were centrifuged at 10000 rpm for 5 min and the supernatant was gathered for the assay using a UV-vis spectrophotometer (482 nm). To examine the release profile of DOX from the nanocarrier samples, the DOX-loaded nanocarriers (LHA@MS and L-hMS), which were prepared in 120 µg/mL of initial DOX concentration, were used. To test DOX release kinetics, 2 mg of each sample was dispersed in 2 mL of PBS prepared with different pH values (5.5 and 7.4), and were then incubated at 37 °C. The amount of drug released into the medium was measured at different time points (up to ~two weeks), and the medium was refreshed at each testing.

In Vitro Cell Viability Test of f-hMS Human cervical cancer cell line HeLa cells were purchased from the American Type Culture Collection (Rockville, USA) and maintained at 37°C in an atmosphere of 5% CO2 in alpha minimum essential medium (aMEM; Gibco, USA) with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin. The medium was replaced three times per week and cells were passaged at sub-confluency. Cells were seeded in 96 well plates with a density of 1 x 104 cells per well and allowed to attach for 24 h. The attached cells were then treated with f-hMS (0, 5, 10, 20, 40, 80, 160, 320 and 640 µg/ml) in culture medium for 24 h. Cell viability was measure using CCK-8 assay (Dojindo, Japan) by incubating samples with the CCK-8 reagent for 3 h in dark condition at 37°C. Optical density values at 450 nm were measured using the iMark microplate reader (BioRad, USA) and converted into cell viability.

f-hMS Delivery to Cells Cells was seeded (1 X 105 cells) in each well of 6-well plates and the 20 µg f-hMS was added to each well and incubated for 4 h. After the incubation, the cells were harvested and fixed with 4 % paraformaldehyde solution 6


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for 30 min on a coating slide glass. The fixed cells were then washed with cold PBS and stained with propidium iodide (PI; Invitrogen, USA) for nucleus stain. As a negative control, the same cells without f-hMS incubation were used.

Characterizations The crystalline phase of the nanomaterials was determined by X-ray diffraction (XRD; Ragaku). The samples were scanned in the range of diffraction angle 2θ = 5-60º at a rate of 2º min-1 with a step width of 0.02 º 2θ using Cu Kα1 radiation at 40 kV and 40 mA current strength. Fourier transform infrared spectroscopy (FT-IR; Varian 640-IR) was used to determine chemical bond status of the samples. For each spectrum, 20 scans in the wave number of 400-4000 cm−1 were recorded in the transmission mode by potassium bromide (KBr) pellet method. Surface pore levels of the nanomaterials including porosity, specific surface area and pore volume were determined by N2 gas adsorption/desorption using Brunauer–Emmett–Teller (BET) method. Pore size distribution was obtained by the Barret-Joner-Halenda (BJH) method. The morphology of the samples was characterized by transmission electron microscopy (TEM, JEOL-7100). The photoluminescence (PL) excitation and emission spectra were recorded with a Hitachi F-4500 spectrophotometer equipped with a 150W xenon lamp as the excitation source. Hydrodynamic particle size was measured by using a Malvern Zetasizer (ZEN3600; Mlavern) particle analyzer. Particles were suspended in distilled water (200 µg/mL) at pH 7 for the measurement. The electron paramagnetic resonance (EPR) property of the samples was assessed by using a JEOL Spectrometer (model JES-FA200) operating at X-band frequency (ν = 9.4 GHz) with 100 kHz magnetic field modulation at room temperature. The fluorescent images of the cells were observed by confocal laser scanning microscopy (CLSM; LSM 510, Carl Zeiss, Germany).

Results and Discussion

As schematically shown in Figure 1, the fHA used as the core template was shelled with mesoporous silica (fHA@MS) by a sol-gel reaction using silica precursor tetraethyl orthosilicate (TEOS). The inner fHA was subsequently leached out in HCl to form fluorescent hollowed mesoporous silica nanoellipsoid (f-hMS). This nanoellipsoidal particle is considered to load and release drugs effectively while enabling in situ imaging system. First, the fHA nanoparticles were synthesized by an autoclave hydrothermal process at a temperature 190 ºC in 7


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the presence of trisodium citrate and CTAB. The fHA was then covered by MS through the sol-gel reaction. Colloidal HA solution was added with TEOS, where the nucleation reaction occurs via adsorption of partially hydrolyzed TEOS molecules into the fHA nanocrystal surface. Under basic conditions (pH 10-11), neighboring silanol groups react to form siloxane bonds via condensation reaction 7 . The TEM image of the fHA template showed the development of fHA nanorods with a size of approximately 20 nm x 50 nm (Figure 2a), and the XRD pattern showed the typical HA crystal phase (Figure 2b). The mesoporous silica layer formed was further evidenced by TEM images (Figure 3a). The thickness of the silica layer formed on the fHA nanocrystal surface was measured 15 ± 3.4 nm and the resultant fHA@MS nanoellipsoid shape are thus sized ~62 nm length x 35 nm width. We subsequently removed out the fHA core part by the acidification treatment and the typical hollow-cored nanoellipsoid shape of the f-hMS was revealed (Figure 3b). The mesoporous structure of the silica shell was shown to be intact after the removal out process. The particle size distribution of f-hMS was measured by DLS (Figure S1). The hydrodynamic size was 132 nm, a value larger than that observed in TEM images. In general, the hydrodynamic size of nanoparticles is measured to be larger than the TEM-measured size due to the particle scattering effect; and for the case of fhMS, a certain level of agglomeration is also considered to contribute to this increase. The hollow-cored mesoporous shell nanocontainer will be used to contain drug molecules within the hollow space at large quantity, not only in the mesopore channels of the shell. The presence of MS formed on the surface enables mesoporous characteristics of the nanoparticles, which are important parameters in determining the capacity to loading drug molecules. We measured the mesoporosity by BET method. The fHA@MS showed a typical type IV isotherm, representing narrow hysteresis loop area (Figure 3c). On the other hand, the hollowed f-hMS presented a type IV isotherm with a type H3 hysteresis loop, and this particular large loop found in the P/P0 range of 0.45 < P/P0 < 0.9 should be attributed to the hollow part of the nanoparticles, which is a characteristic of large-pored or cavity-structured nanoparticles, as similarly observed elsewhere (Figure 3d)41, 42. The pore size distribution, presented in the inset of N2 hysteresis loop, showed a narrow peak at ~3 nm for fHA@MS whilst a dual pore size distribution in f-hMS with a peak ~ 4 nm and another peak at ~15 nm, which is ascribed to the hollow cavity. The BET results, including specific pore volume and surface area, and pore size are summarized in Table 1. The hollowed nanoparticles showed significantly higher surface area and pore volume, suggesting the hollow cavity part will play effective roles in taking up a large quantity of drug molecules. The mesopore sizes of the nanocarries (fHA@MS and f-hMS) 8


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were also obtained by BJH method. For the fHA@MS samples the mesopores were comparable in size of 3.12 nm, and for the f-hMS, the average mesopore size was increased to 4.32 nm. It might be due to that the mesopores were enlarged during the removal process of the fHA core structure. The cavity size was 15.1 nm for the f-hMS. The phase of the fHA@MS and f-hMS nanomaterials was examined from XRD (Figure 4a). When mesoporous silica was shelled, there appeared one distinct broad peak at 2θ = 23º, which corresponds to a typical amorphous phase of silica. There were no other peaks except fHA and silica for the fHA@MS samples. When the fHA core was removed (f-hMS sample), only silica broad amorphous peak was noticed, confirming the complete removal of the fHA core phase. Small-angle XRD was employed to further characterize the mesoporous structure of the synthesized nanoparticles (Figure 4b). The XRD pattern of fHA@MS has a narrow and strong peak at 2θ = 2.51 (with a d spacing of 3.41 nm), whilst that of f-hMS shows a much broader peak at lower 2θ = 2.1 (with a d spacing of 4.3 nm), which suggests that fHA@MS has relatively uniform-sized and smaller mesopores than f-hMS. The EDS atomic analyses also proved the chemical composition of each nanocarrier system (Figure 4c). This atomic compositional result also supports the complete removal of the cored HA crystalline phase which consisted of Ca and P ions. The IR spectrum of the nanomaterials was also investigated (Figure 4d). In the fHA@MS, along with the fHA core bands including phosphate (1096 cm−1, 1020 cm−1, 960 cm−1, 600 cm−1, and 565 cm−1) and hydroxyl (OH− stretching vibration at 3430 cm−1), silica-related bands also appeared such as Si–O–Si (symmetric stretching at 1095 and 1220 cm−1 and asymmetric stretching at 801 cm−1), Si–OH (symmetric stretching at 954 cm−1), and Si–O (bending at 465 cm−1) are the major bands

7, 35

. In particular, a large band appeared at 1577

cm−1 and 1375 cm−1 which are assigned to carbonate. In the hollow-cored nanocontainer, silica-related bands were revealed in majority, however, a close look at the region of 1300−1600 cm−1 clearly revealed carbonaterelated bands at similar positions but with much lowered intensity when compared with fHA@MS, demonstrating there is a common chemical bond related with all the nanocarriers. This is acknowledged to the presence of carbon-related impurities. In fact, pure fHA, the key fluorescent factor is the CO2· − radical ions, and the bands were also ascribed to this. Importantly, this radical group also exists in the fHA-removed hollow MS sample, which suggesting the radical ions still remained within the hollow space without being washed out even after the fHA removal process. More illustrations on the fluorescent behaviors will be discussed in the following section. 9


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We next sought to examine the fluorescent property of the nanocarriers. It has been reported that when citric acid is used as a chelating agent for the sol-gel preparation of HA, the R-C-COO− (Cit3−) group is readily adsorbed onto HA surface to form Ca-citrate chelating complex. During further processing, Cit3− group may cleave to R-C· and CO2· −, and CO2· − radicals are trapped in the HA lattice or interstitial positions, which consequently resulting in the formation of fluorescent centers. The PL emission spectra of the fHA, fHA@MS and f-hMS are shown in Figure 5a. First, an excitation spectrum had a broad (300-700 nm) band with a maximum at 341 nm. The corresponding emission spectrum of fHA, fHA@MS and, f-hMS was shown to consist of two bands centered at 427 nm and 466 nm. The emission spectra of fHA@MS samples are understandable as this originates from the cored fHA, and the lowered intensity in the emission band reflects the shielding effects of the existing MS shell. However, the comparable emission spectrum noticed in the hollowed MS sample is somewhat surprising as the fHA core was removed out. It is thus reasoned that the CO2· − radical which is believed to play a key role in fluorescent behavior of fHA should be trapped in the hollow core even after the removal process. While some part of the CO2· − radical may be lost during the removal process, most part is considered to be remained, as we could notice only slight decrease in the fluorescence intensity of f-hMS when compared to that of fHA@MS (sample before hollowing process). Inset images exhibit the photograph of the fHA, fHA@MS, and f-hMS nanorods dispersed in water under UV lamp (70%) in the dark, illustrating a strong blue emission of the samples. We further analyzed the fluorescent mechanism of the nanocarries samples through the electron paramagnetic resonance (EPR) spectrum (Figure 5b). The fHA nanocrystals showed characteristic EPR bands at g = 2.1177 and 1.9805 (where g is Lande g- factor), which resulted from the existence of paramagnetic defects in the interstitials of apatite crystal lattice. Since the EPR signal cannot be caused by P5+, Ca2+, and O2− (no single electron in these ions), it must arise from some radical-related defect, such as peroxyl radicals or carbon dioxide radical anions (CO2•−) 35, 37, 43, 44. Similarly in other nanocarrier samples (fHA@MS and f-hMS), the EPR signals were noticed while the signal intensity was decreased when the MS shell and also when the nanocarrier was hollowed (f-hMS). The EPR results in coincidence with the PL measurement demonstrate all the nanocarriers have effective paramagnetic defects which are most likely CO2•− radical ions. Together with the self-fluorescent properties, the high mesoporosity of the fHA@MS and f-hMS nanocarries signifies their biomedical uses in the detection and drug delivery systems. Therefore, we next sought to address the efficacy of the fHA@mSi and f-hMS nanocarriers in loading biomolecules and delivering within 10


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cells while allowing their detection in the cells. For this, cyt C was selected as the first candidate molecule for the loading and release study. The cyt C loading study was carried out using representative nanocarriers (mesopored; fHA@MS and hollow spaced; f-hMS). The loading amount of cyt C was recorded at a given nanocarrier content (1 mg) while varying the cyt C concentrations from 0.020 to 0.4 mg. On fHA@MS sample, cyt C loading amount increased gradually with increasing the concentration of cyt C used and recorded a maximum value (0.037 mg) at 0.1 mg/ml (Figure 6a). For f-hMS case, the cyt C loading amount also increased linearly with increasing the cyt C content used up to 0.2 mg/ml where the maximum loading amount was as high as 0.18 mg. Results clearly showed the effects of the hollowed space in loading cytc C molecules since the fhMS was produced from the fHA@MS sample by eluting out the core fHA part and thus the silica shell parts were almost same between the two nanocarriers. As the cyt C is oppositely charged to the MS surface and smallsized the incorporation into the mesopore channels are considered to easily occur and even the penetration through the channels into the hollow space should also be enabled effectively. In this manner, the cyt C drug molecule is considered properly chosen to demonstrate the loading capability of the currently developed hollowed nanocarriers. Based on the cyt C loading and release study, the hollowed MS is considered as an effective delivery carrier. The cyt C release from fHA@MS was initially (~10h) rapid, with ~17% release, and then sustained release over a two weeks with completion (Figure 6b). The cyt C release pattern from f-hMS was initially (~10h) similar to the HA@MS, the release amount was ~28%, moreover, further sustained release continued over a two weeks (Figure 6b), showing a slightly faster release than the case in fHA@MS. It is impressing to note that loading of cyt C molecules at large quantity even within the hollow space as well as the long-term sustainable delivery with almost a constant diffusion-controlled mechanism over 14 days. In fact, biocompatible silica nanoparticles have been widely studied for the drug delivery purposes. However, a rapid drug release from the nanostructure is a common feature for them

7, 18, 45

. Therefore, many efforts have been made to enable sustained delivery of

drugs. For example, mesoporous silica nanoparticles were decorated by poly(ethylene glycol) on the surface to slow down drug release due to the pore narrowing effect 46. The hollow structuring of MS, implemented in this study, was shown to be highly helpful for slowing the release profile of drug molecules particularly in cyt C case. It is not thought however that this effectiveness of the hollowed MS incurred in cyt C can be universally applicable to other types of drugs as the mesopore structure (pore size and porosity) and surface functional groups should be properly adjusted to the type of drug molecules, and this remains as further application studies. 11


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We further examined the loading and delivery capacity of f-hMS using different therapeutic molecule, DOX, a representative anticancer drug (Figure 6c). The loading capacity of both nanocarriers was significantly different between the two nanocarriers, i.e., the amount of DOX loaded onto f-hMS (71 µg) was more than double that loaded onto fHA@MS (32 µg), demonstrating significant role of the hollow space played in loading DOX molecules. As DOX is oppositely charged to the MS surface, and is small in size, its incorporation into the hollow space through mesopore channels should be easily enabled. The release of DOX from the nanocarriers was subsequently monitored under different pH conditions (7.4 and 5.3) (Figure. 6d). While the pH 7.4 represents normal physiological conditions, the acidic pH 5.3 is representative of the extracellular tissues of tumor cells (ca. pH = 5-6)8. Initially, DOX was released more rapidly under acidic conditions than under neutral conditions from both nanocarriers, indicating a pH-dependent release pattern. A possible explanation for this pH-sensitive phenomenon is that the DOX hydrophilicity and solubility increases at acidic environment due to the stronger protonation of –NH2 groups present in DOX 8. Compared to fHA@MS, f-hMS showed higher release % for both pH values, and this was due to that the f-hMS contained initially higher loading amount of DOX. Based on the DOX loading and release study, the hollow MS is considered to be an effective delivery carrier of anti-cancer drug, particularly DOX, due to its high loading capacity and the release profile with high pH sensitivity. Next we examined the applicability of the f-hMS in biomedical uses, particularly for the imaging in anticancer treatment. As a first step, we observed the toxicity of nanoparticles against representative tumor cells (HeLa cell line). CCK-8 assay demonstrated that the f-hMS had fairly good cell viability; over a wide concentration range (up to 320 µg/ml), the viability level was almost equivalent to or even higher than the case for f-hMS free cell control (Figure 7a). After confirming the cellular compatibility of the f-hMS, we next examined the fluorescence labelling of cells. The self-fluorescent activity of f-hMS in the cells was analyzed by CLSM. In the f-hMS treated group, most of the cells showed positive for fluorescence signals (blue) due to fhMS, as well as positive for the PI (red) (Figure 7b). Compared to the f-hMS treated cells, no positive results were observed in the control cells (not shown here). The above results on the responses of f-hMS to cells in vitro support the possible application of the developed nanocarriers in biomedicine. Firstly, the nanocarriers are highly compatible to cells over a wide concentration range, which allows the treatment of nanocarriers at high concentrations and ultimately broadens the window of therapeutic dose levels of drug molecules to the target disease and injury sites. Secondly, the 12


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imaging that a majority of cells are positive for nanocarrier-derived fluorescence signals supports the possibility of cell labeling and the in vivo application. In this case however, the surface of f-hMS should be tailored to target specific cells (like tumor cells), and the in vivo imaging capacity should also be confirmed with further studies. In fact, many fluorescent dye materials have been developed to label cells and to track and image in vivo tissues. Some examples are QDs

51

and upconversion nanoparticles

52, 53

, in which case the nanoparticles are

mostly encapsulated by a silica shell to enable loading of drug molecules. These fluorophore-doping methods are however time-consuming and relatively expensive, and the materials are commonly associated with issues like high toxicity, and dye-leaking51,54,55. Although the currently developed nanocarrier may not be superior in every aspect of the theranostic properties to those dye nanoparticles, the f-hMS can be a promising candidate or replacement to target similar theranostic applications, considering its straightforward and label-free method, high loading capacity of drug molecules, and excellent cell compatibility. To be more applicable for in vivo cell imaging and tracking, and eventually for tissue treatment and healing, the f-hMS needs further development for targeted action and more in vivo studies in the future.

Conclusions The newly developed f-hMS nanocarrier exhibited mesoporous and hollow structure, enabling loading of therapeutic molecules at high quantity and releasing them in a sustained and controlled manner. Furthermore, fhMS had a self-activated fluorescence property, allowing in situ imaging of cells, while possessing good cell viability and high cellular uptake level. These multifunctional aspects support the possible utility of the nanomaterials for therapeutic delivery and diagnostic purposes.

Acknowledgements: This work was supported by the Priority Research Centers Program (No. 2009-0093829) through the National Research Foundation, Republic of Korea, and the research fund of Dankook University (2013, BK21 Plus program).

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List of Figures

Figure 1. Schematic showing the procedure of producing fluorescent hollow mesoporous silica nanoellipsoid for use as self-luminescent drug delivery carrier. Fluorescent HA (fHA) was shelled with mesoporous silica via an reaction with TEOS in basic ethanol/water solution added with CTAB (fHA@MS), and the inner fHA was subsequently leached out in HCl to form hollowed mesoporous silica shell (f-hMS) which is to possess fluorescent property. Cyto C drug can be loaded onto the mesoporous of the fHA@MS or inner hollow space of the f-hMS. Figure 2. (a) TEM image of fHA used as a template and (b) its XRD pattern showing apatite peaks. Figure 3. TEM images of (a) fluorescent HA shelled with mesoporous silica (fHA@MS) and (b) hollowed fluorescent mesoporous silica nanoellopsoid (f-hMS). Analysis of mesopore structure and cavity size by BET method; N2 adsorption/desorption curve of (c) fHA@MS and (d) f-hMS samples, pore and cavity size distribution of fHA@MS and f-hMS samples, including insets. Figure 4. (a) XRD pattern, (b) small angle XRD pattern, (c) TEM-EDS analysis, and (d) IR analysis of the fhMS and fHA@MS. Figure 5. (a) Luminescent property of f-hMS showing a high emission peak at 427 nm with excitation at 341 nm, assigning blue range, a behavior similar to fHA@MS while with reduction in peak intensity and inset Optical image of samples showing fluorescence in blue color. (b) EPR spectroscopy showing the fluorescent defect property. Figure 6. Capacity of the hollow nanoellipsoids to load and deliver therapeutic biomolecules. (a,b) Cyt C was used as the model protein; (a) Loading amount measured at varying concentration of cyt C initially used. Significantly higher loading saturation was attained for f-hMS vs. fHA@MS (~0.18 mg at 0.2 mg cyt C used for f-hMS vs. ~0.037 mg at 0.1 mg cyt C used for fHA@MS). (b) Cyt C release from fHA@MS was initially (~ 10 h) rapid, with ~17% release, and then sustained release over a two weeks with completion. The cyt C release pattern from f-hMS was initially (~ 10 h) similar to the fHA@MS, the release amount was only ~28%, moreover, further sustained release continued over a two weeks, showing a big difference from the case in fHA@MS. (c,d) DOX used as the model anticancer drug; (c) DOX loading curve, obtained by measuring the amount of DOX loaded onto the nanocarrier while varying the initial DOX concentration (20 to 140 µg/ml). The maximal loading attained was significantly higher on the f-hMS (~71 mg) vs. fHA@MS (~32 µ g). (d) DOX release from 19


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the nanocarrier samples observed at two different pH values (5.3 and 7.4) at 37 °C. Figure 7. (a) Cell viability assay of f-hMS. Cells were treated with various concentrations of f-hMS (0, 5, 10, 20, 40, 80, 160, 320, 640 µg/ml) for 24 h and the cell viability was determined by CCK assay. (b) Cellular uptake detection of the hollow nanoellipsoids f-hMS under fluorescent microscopy.

Table 1. Summary of mesopore structure of the fHA@MS and f-hMS based on the BET analysis. There was significant improvement in pore volume and surface area in the hollow nanoellipsoids. Mesopores formed in silica were 3.1 nm in size and the cavity size in the hollow nanoellipsoids was 15.1 nm.

Supporting Information Figure S1. Hydrodynamic particle size distribution of f-hMS measured by a laser scattering method.

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Figure 1. Schematic showing the procedure of producing fluorescent hollow mesoporous silica nanoellipsoid for use as self-luminescent drug delivery carrier. Fluorescent HA (fHA) was shelled with mesoporous silica via an reaction with TEOS in basic ethanol/water solution added with CTAB (fHA@MS), and the inner fHA was subsequently leached out in HCl to form hollowed mesoporous silica shell (f-hMS) which is to possess fluorescent property. Cyto C drug can be loaded onto the mesoporous of the fHA@MS or inner hollow space of the f-hMS.

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Figure 2. (a) TEM image of fHA used as a template and (b) its XRD pattern showing apatite peaks.

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Figure 3. TEM images of (a) fluorescent HA shelled with mesoporous silica (fHA@MS) and (b) hollowed fluorescent mesoporous silica nanoellopsoid (f-hMS). Analysis of mesopore structure and cavity size by BET method; N2 adsorption/desorption curve of (c) fHA@MS and (d) f-hMS samples, pore and cavity size distribution of fHA@MS and f-hMS samples, including insets.

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Figure 4. (a) XRD pattern, (b) small angle XRD pattern, (c) TEM-EDS analysis, and (d) IR analysis of the fhMS and fHA@MS.

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Figure 5. (a) Luminescent property of f-hMS showing a high emission peak at 427 nm with excitation at 341 nm, assigning blue range, a behavior similar to fHA@MS while with reduction in peak intensity and inset Optical image of samples showing fluorescence in blue color. (b) EPR spectroscopy showing the fluorescent defect property.

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Figure 6. Capacity of the hollow nanoellipsoids to load and deliver therapeutic biomolecules. (a,b) Cyt C was used as the model protein; (a) Loading amount measured at varying concentration of cyt C initially used. Significantly higher loading saturation was attained for f-hMS vs. fHA@MS (~0.18 mg at 0.2 mg cyt C used for f-hMS vs. ~0.037 mg at 0.1 mg cyt C used for fHA@MS). (b) Cyt C release from fHA@MS was initially (~ 10 h) rapid, with ~17% release, and then sustained release over a two weeks with completion. The cyt C release pattern from f-hMS was initially (~ 10 h) similar to the fHA@MS, the release amount was only ~28%, moreover, further sustained release continued over a two weeks, showing a big difference from the case in fHA@MS. (c,d) DOX used as the model anticancer drug; (c) DOX loading curve, obtained by measuring the amount of DOX loaded onto the nanocarrier while varying the initial DOX concentration (20 to 140 µg/ml). The maximal loading attained was significantly higher on the f-hMS (~71 mg) vs. fHA@MS (~32 µ g). (d) DOX release from the nanocarrier samples observed at two different pH values (5.3 and 7.4) at 37 °C.

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Figure 7. (a) Cell viability assay of f-hMS. Cells were treated with various concentrations of f-hMS (0, 5, 10, 20, 40, 80, 160, 320, 640 µg/ml) for 24 h and the cell viability was determined by CCK assay. (b) Cellular uptake detection of the hollow nanoellipsoids f-hMS under fluorescent microscopy.

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Table 1. Summary of mesopore structure of the fHA@MS and f-hMS based on the BET analysis. There was significant improvement in pore volume and surface area in the hollow nanoellipsoids. Mesopores formed in silica were 3.1 nm in size and the cavity size in the hollow nanoellipsoids was 15.1 nm.

Materials

Pore Volume 3 (cm /g , BET)

Surface Area 2 (m /g , BET)

Pore size (nm, BJH)

Cavity size (nm, BJH)

fHA@MS

0.473

413.2

3.12

0

f-hMS

0.869

1287

4.32

15.1

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Graphic for the "Table of Contents”

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