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Capacity of mesoporous bioactive glass nanoparticles to deliver therapeuticmolecules†

Ahmed El-Fiqi,abc Tae-Hyun Kim,ab Meeju Kim,ab Mohamed Eltohamy,abc Jong-Eun Won,ab Eun-Jung Leeab

and Hae-Won Kim*abcd

Received 8th July 2012, Accepted 6th October 2012

DOI: 10.1039/c2nr31775c

Inorganic bioactive nanomaterials are attractive for hard tissue regeneration, including

nanocomponents for bone replacement composites and nanovehicles for delivering therapeutics.

Bioactive glass nanoparticles (BGn) have recently gained potential usefulness as bone and tooth

regeneratives. Here we demonstrate the capacity of the BGn with mesopores to load and deliver

therapeutic molecules (drugs and particularly genes). Spherical BGn with sizes of 80–90 nm were

produced to obtain 3–5 nm sized mesopores through a sono-reacted sol–gel process. A simulated body

fluid test of the mesoporous BGn confirmed their excellent apatite forming ability and the cellular

toxicity study demonstrated their good cell viability up to 100 mg ml�1. Small molecules like chemical

drug (Na-ampicillin) and gene (small interfering RNA; siRNA) were introduced as model drugs

considering the mesopore size of the nanoparticles. Moreover, amine-functionalization allowed

switchable surface charge property of the BGn (from �20–30 mV to +20–30 mV). Loading of

ampicillin or siRNA saturated within a few hours (�2 h) and reflected the mesopore structure. While

the ampicillin released relatively rapidly (�12 h), the siRNA continued to release up to 3 days with

almost zero-order kinetics. The siRNA–nanoparticles were easily taken up by the cells, with a

transfection efficiency as high as �80%. The silencing effect of siRNA delivered from the BGn, as

examined by using bcl-2 model gene, showed dramatic down-regulation (�15% of control), suggesting

the potential use of BGn as a new class of nanovehicles for genes. This, in conjunction with other

attractive properties, including size- and mesopore-related high surface area and pore volume, tunable

surface chemistry, apatite-forming ability, good cell viability and the possible ion-related stimulatory

effects, will potentiate the usefulness of the BGn in hard tissue regeneration.

1. Introduction

Regeneration of hard tissues including bone has been signifi-

cantly facilitated by the use of materials developed and engi-

neered to react actively with biological environments where the

bone-associated cells are favored to anchor and differentiate to

secrete bone extracellular matrices (ECMs).1–3 Among this class

of bone-bioactive materials, the group of bioactive glasses (BGs)

has been the most actively and popularly studied. Initially

developed to specific compositions by the melt-quenching, the

BGs take the form of blocks, microfibers and crushed granules in

aDepartment of Nanobiomedical Science and WCU Research Center,Dankook University, South KoreabInstitute of Tissue Regeneration Engineering (ITREN), DankookUniversity, South Korea. E-mail: kimhw@dku.edu; Fax: +82 41 5503085; Tel: +82 41 550 3081cGlass Research Department, National Research Center, EgyptdDepartment of Biomaterials Science, School of Dentistry, DankookUniversity, South Korea

† Electronic supplementary information (ESI) available. See DOI:10.1039/c2nr31775c

This journal is ª The Royal Society of Chemistry 2012

micron-size.4–6 A sol–gel technique has spurred the development

of BGs with compositions in a broader range compared to melt-

quenching, enabling the incorporation of bioactive molecules

and allowing nanoscale formulations such as nanofibers and

nanoparticles.7–12

In particular, the BGs developed at the nanoscale (possibly

tens to hundreds of nanometers in tuned dimensions) possess

substantially increased surface properties, accelerating the

possible interactions with other materials/molecules and bio-

logical reactions, which compel their use for either nano-

composites in concert with biopolymers13–15 or delivery systems

incorporating candidate therapeutic molecules.16,17 A nano-

fibrous form of the sol–gel BGs, previously developed by elec-

trospinning and studied as the nanocomponent of the inorganic

phase within biopolymeric matrices, including collagen, poly-

(lactic acid) (PLA) and poly(caprolactone) (PCL), has been

demonstrated to play a significant role in accelerating acellular

mineral formation as well as the in vitro cellular proliferation and

osteogenic differentiation.18–20 Some recent studies have shown

the development of nanoparticulates of BG, with sizes ranging

from tens to hundreds of nanometers.21,22 This class of BG

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nanoparticles has also shown high surface bioreactivity inducing

ionic release and mineral precipitation, which is highly attractive

for fabrication of nanocomposites with the biopolymers.23,24

Furthermore, BG nanoparticles are of potential interest in

dentistry because of their antibacterial properties25,26 and the

ability to remineralize dentine.27

One attractive application of the BG nanoparticles is as a

delivery system for therapeutic molecules. The size of BG

nanoparticles can be modulated to allow intracellular uptake by

endocytosis, while not harming the tissue cells. In tandem with

excellent bone-bioactivity, BG nanoparticle-mediated delivery of

therapeutic molecules will potentiate the capacity of these

nanomaterials for hard tissue regeneration.

To this end, the present study developed spherical BG nano-

particles destined for utilization as delivery vehicles for thera-

peutic molecules. For this we provided a level of mesoporosity to

the nanoparticles using surfactant templates during an ultra-

sound assisted base-catalyzed sol–gel process applied newly in

this study for the nanoparticle preparation. As the model mole-

cules, we applied either chemical drug (antibiotic) or nucleic acid

(small interfering RNA, siRNA) and examined the capacity to

load and release those biomolecules.

Although previous studies have reported the potential of

mesoporous BG in bulk, scaffold or granular form for loading

and delivery of therapeutic molecules, such as drugs and

proteins,16,28 there have been few works using the nanoparticle

form of BG. This nanoparticle form is considered much more

intriguing than other forms as it is possibly taken up by the cells

to carry therapeutic molecules and temporarily release them

inside of cells, directly regulating genetic functions. This allows

more potential use of the BGn in gene delivery systems, which

was demonstrated for the first time in this study. This report on

the utilization of a novel inorganic mesoporous BG nanodelivery

system should provide valuable information for the development

of bioactive delivery systems, particularly for the hard tissue

regeneration.

2. Materials and methods

2.1. Preparation of mesoporous BG nanoparticles

2.1.1. Mesoporous structuring. Tetraethyl orthosilicate

(TEOS, C8H20O4Si, 98%), calcium nitrate tetrahydrate (Ca(N-

O3)2$4H2O, 99%), poly(ethylene glycol) (PEG; (C2H4)nH2O,Mn:

10 000), hexadecetyltrimethyl ammonium bromide (CTAB,

C19H42BrN, $98%), ammonium hydroxide (NH4OH, 28.0%

NH3 in water, $99.99% metal basis), methanol anhydrous

(CH4O, 99.8%), toluene anhydrous (C7H8, 99.8%), 3-amino-

propyl triethoxysilane (APTES, C9H23NO3Si, $98%), and

ampicillin sodium salt (C16H18N3NaO4S) were all purchased

from Sigma-Aldrich and were used as-received without any

further purification.

Mesoporous binary 85SiO2/15CaO (mol%) BG nanoparticles

with well-developed spherical morphology were synthesized by a

novel ultra-sound assisted base-catalyzed sol–gel method using

PEG and CTAB as templates (coded as ‘BGn1’ and ‘BGn2’,

respectively). In a typical synthesis, 5 g PEG for BGn1 or 5 g

CTAB for BGn2 was completely dissolved by stirring in 120 ml

absolute methanol. The solution pH was then adjusted to 12.5 by

7476 | Nanoscale, 2012, 4, 7475–7488

adding about 30 ml NH4OH. To this clear solution, 0.179 g

Ca(NO3)2$4H2O was dissolved while gently stirring. In a sepa-

rate vessel, 0.895 g TEOS was diluted with 30 ml absolute

methanol and then added drop-wise to the vigorously stirred pH

12.5 solution with the simultaneous application of a high-power

ultra-sound using a Sonoreactor, LH700S ultra-sonic generator

(Ulsso Hitech, South Korea) operating at 20 kHz and 700 W.

The output power was 220 W in a 10 s on/10 s off cycle for

20 min. After 24 h of vigorous stirring, the white precipitate was

separated by centrifugation at 5000 rpm for 5 min in aMega17 R

centrifuge (Hanil Science, South Korea), and washed, and re-

dispersed three times with de-ionized water and twice with

absolute ethanol. The final white precipitate was dried at 70 �Cfor 12 h. Finally, organics and nitrates were removed from the

dried powder by calcination at 600 �C under air for 5 h at a

heating rate of 1 �C min�1.

2.1.2. Surface functionalization. The calcined BGn1 and

BGn2 were surface-functionalized with aminopropyl groups

through a post-synthesis procedure. Briefly, 100 mg nanopowder

was dispersed in 50 ml anhydrous toluene and refluxed with 1 ml

APTES at 60 �C by stirring for 6 h. The solution was then

allowed to cool to room temperature and the nanoparticles were

collected by centrifugation at 5000 rpm for 5 min, washed,

re-dispersed three times with toluene, and dried in an oven at

80 �C overnight. The amine-functionalized BGn1 and BGn2

samples were designated as BGn1(A) and BGn2(A), respectively.

2.2. Characterizations of BG nanoparticles

2.2.1. Physicochemical properties. The morphology of the

mesoporous BG nanoparticles was observed by field-emission

scanning electron microscopy (TESCAN,MIRA II LMH, Czech

Republic). The particle size and mesoporous structure of the

nanoparticles were also observed by a high-resolution trans-

mission electron microscope using a JEM-3010 apparatus

(JEOL, Japan).

The phase of the samples was analyzed by X-ray diffraction

(XRD) using an Ultima IV apparatus (Rigaku, Japan) with

CuKa radiation (l ¼ 1.5418 �A). X-ray was generated at 40 mA

and 40 kV, and data were obtained at diffraction angles (2q) from

4–70� with a step size of 0.02� and a scanning speed of 2� min�1.

Small angle X-ray diffraction was also performed using a Rigaku

D/Max-2500 diffractometer in the 2q range of 0.5–5�, to examine

the characteristic of mesopores. Infrared spectra of the samples

were obtained with a resolution of 4 cm�1 on a Varian 640-IR

Fourier transform infrared (FT-IR) spectrometer (Varian, Aus-

tralia) in the range 4000–400 cm�1 using the KBr method.29 The

particle size was also analyzed by a Zetasizer Nano ZS, dynamic

light scattering (DLS) instrument (Malvern Instruments, UK).

Thermogravimetric analysis of the samples was carried out on a

TGA N-1500 instrument (Scinco, South Korea) at a heating rate

of 10 �C min�1 and a nitrogen flux of 40 ml min�1.

The specific surface area, pore volume, and pore size distri-

bution were determined based on N2 adsorption–desorption

measurements. The N2 adsorption–desorption isotherms were

obtained at 77 K using a Quadrasorb SI automated surface area

and pore size analyzer (Quantachrom Instruments, UK).

Samples were degassed under vacuum at 300 �C for 12 h prior to

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analysis. The specific surface area was determined according to

the Brunauer–Emmett–Teller (BET) method.30 The pore size

distribution was determined from the N2 desorption branch of

the obtained N2 adsorption–desorption isotherms on the basis of

the density functional theory (DFT) method.31 The total pore

volume was calculated from the amount adsorbed at a maximum

relative pressure (P/P0).

The surface electrical potential and dispersion stability of the

nanoparticles were investigated by zeta (z) potential measure-

ments. The z-potential of the nanoparticles was measured with a

Zetasizer Nano ZS laser Doppler electrophoresis (LDE) instru-

ment (Malvern Instruments, UK). The nanoparticles were

dispersed in deionized water at different pH values. The

z-potential was measured at 25 �C with an applied field strength

of 20 V cm�1 five times (each measurement being the average of

40 runs) and the mean � standard deviation (n ¼ 5) was calcu-

lated. The instrument automatically calculates electrophoretic

mobility (U), and z-potential according to the Helmholtz–

Smoluchowsky equation: z ¼ Uh/3, where z is the zeta potential,

U is the electrophoretic mobility, h is the dispersing medium

viscosity, and 3 is the dielectric constant.

2.2.2. In vitro apatite forming ability. The in vitro hydroxy-

apatite forming ability of the prepared BG nanoparticles was

tested in a simulated body fluid (SBF).32 SBF has an ionic

composition and concentration similar to those of the human

body plasma. Briefly, SBF was prepared by dissolving NaCl,

NaHCO3, KCl, K2HPO4$3H2O, MgCl2$6H2O, CaCl2,

and Na2SO4 in deionized water and buffering at pH 7.4 with

tris(hydroxymethyl) aminomethane (HOCH2)CNH2 and HCl.

The BG nanoparticles were well-dispersed in SBF at a concen-

tration of 1 mg ml�1 in clean, sterile (sterilized with 70% ethanol

and washed with deionized water), tightly stopped 100 ml poly-

ethylene bottles. The bottles were placed inside an incubator at a

controlled temperature of 37 �C for different times up to 28 days,

and the SBF medium was refreshed regularly. At each selected

time point, a bottle was removed from the incubator and the

powder was separated by centrifugation at 5000 rpm for 5 min,

washed, redispersed gently with deionized water and pure

ethanol, and dried at 50 �C overnight. For the next run, the

medium was refreshed. The samples were examined by scanning

electron microscopy (SEM) to detect crystallite formation. The

phase and chemical bond structure of the SBF-treated nano-

particles were examined using XRD and FT-IR, respectively.

2.3. In vitro cytotoxicity assays

The in vitro cytotoxicity of the BGn1(A) and BGn2(A) was

assessed by using different types of cells: HeLa cell line, pre-

osteoblastic MC3T3-E1 cells, and rat bone marrow mesen-

chymal stem cells (rMSCs). The procedures of isolation and

maintenance of rMSCs were described in detail elsewhere.33 Cells

were maintained in Dulbecco’s modified Eagle’s medium

(DMEM; Welgene, Korea) containing 10% fetal bovine serum

(FBS; Gibco, USA) and 1% penicillin–streptomycin under an

atmosphere of 5% CO2 at 37�C. One hundred microliter aliquots

of the cells prepared at a density of 1 � 105 ml�1 were plated in

each well of 96-well plates. After culture for 24 h, the culture

medium was refreshed with that containing the nanoparticles

This journal is ª The Royal Society of Chemistry 2012

prepared at specific concentrations (0, 5, 10, 20, 40, 60, 80 and

100 mg ml�1). After incubation for further 24 h, the cells were

collected and analyzed for the viability using a CCK-8 cell

counting kit (Dojindo, Japan). According to the manufacturer’s

instructions, the reaction medium was added to each well and the

cells were incubated for 4 h at 37 �C, after which 100 ml of each

cell culture supernatant was collected and the optical density was

measured at 450 nm using an iMark microplate reader (BioRad,

USA). Absorbance values were normalized to those of control

free of BG nanoparticles for each test group, and data were

averaged from triplicate samples (n ¼ 3).

2.4. Drug loading and release studies

2.4.1. Sodium ampicillin loading and release. Na-ampicillin

was used as a model drug for the loading and release tests of

antibiotics. Trials were first made to load Na-ampicillin into BG

nanoparticles, lacking surface functionalization. No significant

loading was detected. This may reflect charge effects, with the

negatively charged Na-ampicillin and BG nanoparticles repelling

each other. Therefore, the loading of Na-ampicillin was con-

ducted on aminopropyl surface-functionalized BG nanoparticles,

BGn1(A) and BGn2(A). The amount of loaded Na-ampicillin

was measured by a depletion method, in which the difference in

Na-ampicillin concentration in the loading solution before and

after the loading process was ascertained. Na-ampicillin was

assayed by UV-VIS spectroscopy using a Libra S22 apparatus

(Biochrom, UK) by monitoring the changes in the absorbance at

a characteristic wavelength, 230 nm. A series of standard Na-

ampicillin solutions in deionized water (10–100 mg ml�1) were

prepared to obtain a linear calibration curve (r2 ¼ 0.99) that

obeys the Beer–Lambert law A¼ abc, where A is the absorbance,

a is a constant known as absorptivity coefficient, c is the

concentration, and b is the cell bath length, which is constant.

The loading procedure was as follows. Five milligrams of amine-

functionalized nanoparticles, BGn1(A) or BGn2(A), were

completely dispersed in Na-ampicillin solutions (1–20 mg). These

concentrations were selected to investigate the effect of the initial

Na-ampicillin concentration on the adsorption kinetics. The

solutions were incubated at 37 �C for up to 4 h. At each selected

time point, 1 ml of each solution was withdrawn and centrifuged

at 10 000 rpm for 3 min using a model 5415D centrifuge

(Eppendorf, Germany). The separated clear solution was

analyzed for the Na-ampicillin concentration by UV-VIS spec-

trophotometry. A blank solution of the same pH as the sample

was used as a reference. After determining the optimal time point

for the loading of Na-ampicillin, the same procedure was

repeated for all other solutions. For further release tests, the Na-

ampicillin loaded nanoparticles were washed with distilled water

and dried at 37 �C overnight.

The Na-ampicillin release profile was estimated in vitro using

phosphate buffered saline (PBS, pH 7.4). For the in vitro release

test, 5 mg of each Na-ampicillin loaded sample was dispersed in

10 ml PBS and the solution was incubated at 37 �C for up to 24 h.

At each selected time point, 1 ml of each solution was withdrawn

and centrifuged at 10 000 rpm for 3 min. The clear solution was

analyzed for the Na-ampicillin concentration by UV-VIS spec-

trophotometry using PBS as a blank. The same procedure was

repeated for all other samples.

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2.4.2. siRNA loading and release. The siRNA loading test

onto the nanoparticles of either BGn1(A) or BGn2(A) was per-

formed using fluorescein isothiocyanate (FITC)-conjugated

siRNA for the nanoparticle detection by fluorescence. 10 mg of

FITC-conjugated siRNA was mixed with varying quantities (10,

20, 50 and 100 mg) of BG nanoparticles (resultant

RNA : nanoparticles ¼ 1 : 1, 1 : 2, 1 : 5 and 1 : 10) within 1 ml

release medium (RNase-free TE buffer) to find out an optimal

mixing ratio for high loading efficiency of siRNA. Tests gave a

ratio of 1 : 5 with the highest loading. Next, 10 mg of siRNA was

mixed with 50 mg of BG nanoparticles for different times (30 min,

1 h, and 2 h) at room temperature. The highest loading quantity

was attained at 2 h. For the loading quantity assay, the siRNA–

nanoparticle mixture was centrifuged at 10 000 rpm at 4 �C, andthe supernatant was collected and the siRNA quantity was

assessed by using a SpectraMax M2e multi-detection microplate

reader (Molecular Devices, USA) at an absorbance of 210 nm.

Subtraction of the quantity from the initial amount of siRNA

resulted in the loaded amount of siRNA onto the nanoparticles.

For the siRNA release study, the siRNA-loaded nanoparticles

were pooled into 10 ml of release medium (RNase-free TE buffer)

and stored at 37 �C for various time points (up to a saturation

point). The supernatant was collected by centrifugation at 10 000

rpm and then assessed using a multi-detection microplate reader.

2.4.3. siRNA delivery to cells. Intracellular uptake experi-

ments of the siRNA–nanoparticles as well as the functional

activity of gene silencing were performed using BGn2(A) as the

representative sample as this type presented higher loading

capacity of siRNA. FITC-conjugated AccuTarget Validated

siRNA specific to human bcl-2 mRNA was designed (Bioneer,

South Korea). As a negative control, the same nucleotides were

scrambled to form a non-genomic combination and conjugated

to FITC (scRNA). As the siRNA transfection mixture, 10 mg of

BGn2(A) loaded with siRNA (either bcl-2–siRNA or scrambled

RNA; scRNA) in Opti-MEM (Invitrogen, USA) was used. HeLa

cells were seeded (1 � 105 cells) in wells of 6-well plates and the

transfection mixture was added to each well and incubated for

4 h. As the negative control, 1 mg ml�1 of free siRNA (i.e.,

without loading onto the nanoparticles) was also used for the

transfection. After the siRNA transfection, the cells were har-

vested and used for the following experiments. For the quanti-

fication of bcl-2 mRNA expression, the transfected HeLa cells

were cultured in a-MEM and harvested 24 h later.

The harvested cells were fixed with 4% paraformaldehyde

solution for 30 min on a coating slide glass, which was followed

by washing with cold (4 �C) PBS and mounting on a cover glass.

The fluorescent images were observed and analyzed by confocal

laser scanning microscopy (CLSM; model LSM 510, Carl Zeiss,

Germany). Cells were counterstained with propidium iodide (PI;

Invitrogen, USA) to observe the nucleus of the cells. For the

quantification of the transfection efficiency, cells were analyzed

using a fluorescence activated cell sorting (FACS) Calibur flow

cytometer (BD Biosciences, USA). The data acquired for 10 000

cells in each sample were analyzed using the CellQuest Pro

software (BD Biosciences, USA).

For examination of the ultrastructure of cells, the transfected

cells with bcl-2 siRNA loaded BGn2(A) were harvested and fixed

in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde

7478 | Nanoscale, 2012, 4, 7475–7488

with 0.2 M PBS for 8 h at pH 7.2. Post-fixation was conducted

using 1% osmium tetroxide in PBS for 2 h. Subsequently, the

fixed cells were dehydrated in ethanol of ascending concentra-

tions (70%, 80%, 90%, 95%, and 100%) and embedded in EMbed

812 resin (EMS, USA) via propylene oxide. Ultrathin sections

were obtained using an ultramicrotome (Leica, USA) and were

double-stained with uranyl acetate and lead citrate. The stained

sections on the grids were then examined with a H7000 TEM

(Hitachi, Japan) operating at an acceleration voltage of 80 kV.

The expression of bcl-2 in the cells treated with bcl-2 siRNA

loaded BGn2(A) was confirmed by quantitative real-time RT-

PCR. The first strand cDNA was synthesized from the total

RNA (2 mg) using a SuperScript first strand synthesis system for

real-time PCR (Invitrogen, USA) according to the manufactur-

er’s instructions. The reaction mixture was made up to 50 ml.

Real-time PCR was conducted using SYBR GreenER qPCR

SuperMix reagents (Invitrogen, USA) and a Bio-Rad iCycler.

The relative transcript quantities were calculated using the DDCt

method with glyceraldehyde 3-phosphate dehydrogenase

(GAPDH) as the endogenous reference gene amplified from the

samples.

2.4.4. Statistical analysis.Datawere expressed as themeans�one standard deviations. Statistical analysis was carried out by

one-way analysis of variance (ANOVA) and p-values <0.05 were

considered significant.

3. Results

3.1. Properties of mesoporous BG nanoparticles

The morphology of the mesoporous BG nanoparticles (BGn1

and BGn2) prepared via a sol–gel route using different templates

(PEG and CTAB) was observed by high resolution SEM and

TEM. Spherical nanoparticles mono-dispersed with sizes

<100 nm were abundant in SEM examination (Fig. 1, panels a1

and b1). TEM images show that the nanoparticles contained

mesopores throughout their inner structure (Fig. 1, panels a2, a3,

b2, and b3). The size of the nanoparticles was measured from

arbitrarily selected TEM images (Fig. 1c); the average size of the

BGn1 and BGn2 nanoparticles was approximately 85 � 15 nm

and 95 � 15 nm, respectively. DLS instrumental analysis showed

that the particle sizes in ranges slightly increased, 105 nm for

BGn1 and 115 nm for BGn2 (Fig. 1d), possibly due to the

aggregation effect of nanoparticles.34 The XRD patterns

confirmed the amorphous phase for both BG nanoparticles

(Fig. 1e). Small angle XRD patterns (inset in Fig. 1e) revealed a

single peak (2q ¼ 2.959�) for BGn2 whilst no peaks for BGn1.

Results demonstrate that the mesopores in BGn2 are disordered

but uniform-sized; on the other hand, those in BGn1 are not

uniform-sized.16,28,35,36 The chemical compositions of BG nano-

particles were confirmed by EDS analyses, which showed Si and

Ca peaks at similar atomic ratios to the glass stoichiometry

(Fig. 1f).

The pore characteristics of the nanoparticles were evaluated

from the N2 adsorption–desorption isotherms by the BET

method. Fig. 2 shows the N2 adsorption–desorption isotherms

and the corresponding pore size distributions (inset graphs) of

BGn1 and BGn2. The N2 isotherms exhibited characteristics of

This journal is ª The Royal Society of Chemistry 2012

Fig. 1 (a and b) Nano-structural morphologies of the mesoporous BG nanoparticles (BGn1 and BGn2) developed by a sol–gel method; (a1–a3) BGn1

and (b1–b3) BGn2; (a1 and b1) SEM and (a2, a3, b2 and b3) TEM images. Spherical nanoparticles were mono-dispersed (SEM images) and contained a

large amount of mesopores inside the structure (TEM images). (c and d) Particle size distributions of the BGn1 and BGn2, as measured from the TEM

images arbitrarily chosen (c); average sizes were 85 nm (�15 nm) and 95 nm (�15 nm), respectively, for BGn1 and BGn2, as well as from the DLS

measurement (d), which gave slightly increased sizes (105 nm for BGn1 and 115 for BGn2). (e) XRD patterns showing the amorphous state of the

mesoporous BG nanoparticles, moreover, small angle XRD patterns in the inset revealed one peak at 2q ¼ 2.959� for BGn2, a characteristic of

disordered but uniform-sized mesopores, whilst no peaks were noticed for BGn1. (f) EDX analysis confirming the glass composition (BGn1 repre-

sentatively shown).

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the type IV isotherm associated with mesoporous materials

according to the IUPAC (international union of pure and

applied chemistry) classification. BGn1 exhibited a small

hysteresis loop of the desorption branch, indicative of the exis-

tence of large pores. BGn2 showed a little hysteresis loop,

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indicating that the adsorption process was completely reversible.

The pore size distribution in BGn1 showed multi-sized pores

with broad distribution and relatively low pore volume, whilst

BGn2 showed mono-sized pores with narrow distribution and

large pore volume.

Nanoscale, 2012, 4, 7475–7488 | 7479

Fig. 2 Nitrogen adsorption–desorption isotherms of the BG nanoparticles after the amine functionalization; (a) BGn1 and (b) BGn2. The pore size

distribution is shown in the insets of both graphs, as determined by the DFT method. Both isotherms exhibited a type IV isotherm characteristic of

mesoporous materials. While the pore size distribution in BGn1 was relatively broad with diffused pore sizes towards large pores, that in BGn2 was

much narrower with a sharp peak at �3 nm. The average pore size was 4.9 and 3.2 nm, respectively, for BGn1 and BGn2.

Table 1 Summary of the mesopore structures of BG nanoparticles,including pore size, specific surface area and pore volume, as determinedfrom N2 adsorption–desorption isotherms

Parameter BGn1 BGn2

Pore size (nm) 4.9 3.2Specific surface area (m2 g�1) 54 830Specific pore volume (cm3 g�1) 0.133 0.415

Fig. 3 Characteristics of the mesoporous BG nanoparticles. (a) FT-IR spect

functionalization. FT-IR results showed that the aminated nanoparticles, B

TGA curves showed weight losses at 250–580 �C, which is ascribed to the a

potentials of the nanoparticles at pH 7.0 changed from highly negative (�29

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The average pore size of BGn1 and BGn2 was 4.9 nm and 3.2

nm, respectively, as determined from the DFT method (Table 1).

The pore volume of BGn1 and BGn2 was 0.133 cm3 g�1 and

0.415 cm3 g�1, respectively, and the corresponding surface area

of BGn1 and BGn2 was 54 m2 g�1 and 830 m2 g�1, respectively,

as determined on the basis of the BET approximation. Conclu-

sively, BGn2 showed a greater level of pores with more uniform

pore size and higher surface area than BGn1. These mesopore

ra, (b) TGA and (c) zeta-potential of the samples before and after amine

Gn1(A) and BGn2(B), revealed characteristic N–H vibration bands (a).

mine groups present in the nanoparticles (b). After amination, the zeta-

mV and �19.5 mV) to highly positive (+28.5 mV and +22.5 mV) (c).

This journal is ª The Royal Society of Chemistry 2012

Fig. 4 Cytotoxicity tests of the BGn1(A) and BGn2(A) using different

types of cells including (a) bone marrow MSCs, (b) pre-osteoblast

MC3T3-E1 cells, and (c) HeLa cells. Varying concentrations (0, 5, 10, 20,

40, 60, 80 and 100 mg ml�1) of either BGn1(A) or BGn2(A) pooled in cell

growth medium were applied to the cells for 24 h and the cell viability was

assessed by an MTS assay. Data were normalized to those control media

without the addition of nanoparticles. Data are shown as means and

standard deviations from triplicate samples.

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configurations of the BG nanoparticles are of special importance

in loading and releasing therapeutic molecules.

The surface of the mesoporous BG nanoparticles was modified

with amine groups, which can impart a positive charge to the

surface. The prepared nanoparticles had abundant OH groups

on their surface, conferring a net negative charge. We aminated

BGn1 and BGn2 nanoparticles with APTES to allow the meso-

porous surface to contain NH2 functional groups, which are

considered highly useful for the loading of negatively charged

molecules including nucleic acids. FT-IR spectra of the aminated

nanoparticles, BGn1(A) and BGn2(A), revealed N–H vibration

bands characteristic of NH2 functional groups (Fig. 3a). Fig. 3b

shows the TGA curves of the aminated and non-aminated BG

nanoparticles. For BG1, there was only a 4% weight loss before

approximately 250 �C, being mainly associated with the loss of

water molecules (from about 30–120 �C) adsorbed onto the

surface of the nanoparticles and the condensation of surface

silanol groups (from 120–250 �C). In the range of 250–550 �C,only 1% weight loss was recorded, which may be due to the

residual PEG template, after which the weight became stable

above 550 �C. As a result of its large surface area, BG2 showed a

15% weight loss before 250 �C, and in the range of 250–550 �Conly 2% weight loss was recorded, which might also be from the

residual CTAB template, after which the weight stabilized above

550 �C. For aminated BG nanoparticles, the TGA curves showed

different weight loss behavior compared to non-aminated ones.

BG1(A) showed 10% weight loss between 250 �C and 550 �C and

BG2(A) showed a similar weight loss step with 13% weight loss

between the region, which was attributed to the decomposition of

NH2 functional groups.37

After the amination, the surface charge of the nanoparticles

was totally changed. Fig. 3c shows the z-potential of the BG

nanoparticles with variation in pH. At pH 7.0 the z-potentials for

BGn1 and BGn2 before amination were highly negative, being

�29.0 mV and �19.5 mV, respectively. However, after amina-

tion, the z-potentials of the nanoparticles became positive within

the pH range measured (from pH 3.0–9.0). In particular, at

pH 7.0, the z-potentials were highly positive, +28.5 mV and

+22.5 mV, for BGn1(A) and BGn2(A), respectively. The results

obtained by FT-IR and z-potential analyses demonstrate that the

amination process was properly complemented to provide posi-

tively charged amine groups to the surface of BG nanoparticles.

The mesoporous BG nanoparticles, with their ultra-high

specific surface area resulting from the small particle size and

inner mesopores, should have excellent bone bioactivity. As a

first index, we investigated the hydroxyapatite forming ability in

vitro. Samples were incubated in SBF for periods up to 28 days,

and the development of crystalline phases, changes in nano-

morphology, and chemical structures were observed (ESI 1†).

The XRD phase change of the BGn2(A) samples after immersion

in SBF showed the evolution of typical hydroxyapatite peaks,

and the intensity at the main peak 2q ¼ 32� increased with

increasing immersion time (ESI 1a†). FT-IR spectra also showed

the development of bands related to carbonated hydroxyapatite

(PO4 bands at 565, 605 and 964 cm�1 and CO3 bands at 1420 and

1458 cm�1) (ESI 1b†). The high resolution field emission-SEM

images show the changes on the surface of the BGn2(A). During

SBF incubation as short as 1 day, some crystallites started to

form (ESI 1†, panel c1). With prolonged immersion for 7 and

This journal is ª The Royal Society of Chemistry 2012

14 days (ESI 1†, panels c2 and c3, respectively), the formation of

hydroxyapatite crystallites was pronounced, to the point where

the spherical shape of the nanoparticles completely disintegrated.

High resolution TEM images taken at 1 and 7 days also sup-

ported the development of hydroxyapatite crystallites on the

surface of the BG nanoparticles (ESI 1†, panels c4 and c5).

Comparing the two types of BG nanoparticles, no significant

differences in XRD and FT-IR could be found.

To utilize the BG nanoparticles as a delivery system for

biomolecules, their cellular toxicity needed to be first clarified.

We assessed the cytotoxicity of the nanoparticles using several

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types of cells including rat bone marrow mesenchymal stem cells

(rMSCs), pre-osteoblastic MC3T3-E1 cells and HeLa cells. Fig. 4

shows the relative cell viability in response to varying concen-

trations (0, 5, 10, 20, 40, 60, 80 and 100 mg ml�1) of the BGn1(A)

and BGn2(A) during culture for 24 h. In rMSCs, the cell viability

appeared to be dose-dependent for both types of nanoparticles;

the cell viability up-regulated at low doses (<20 mg ml�1) was

reduced at higher doses. The up-regulation at low doses

(particularly in BGn2(A)) was more apparent in MC3T3-E1

cells. In the HeLa cell line, the viability was largely maintained at

all the doses used for both types of nanoparticles. In particular,

the cell viability at a dose of 10 mg ml�1, which was chosen for

further drug and gene delivery tests, was preserved well (equal to

or even higher than the control).

3.2. Capacity to load and deliver therapeutic molecules

As a first model therapeutic molecule, we selected the hydrophilic

antibiotic, Na-ampicillin. First, drug loading tests were per-

formed. Ampicillin loading was not possible with the BGn1 or

BGn2 nanoparticles without amine-functionalization. Conversely,

Fig. 5 (a and b) Na-ampicillin loading tests onto the aminated-BG nanoparti

presented as the cumulative total amount or the relative amount to the initial

different times to observe the optimal loading time (a); based on this, the load

by varying the initial concentration of ampicillin, resulting in about 180 and 3

the higher pore volume and surface area of BGn2 allowed better drug loadin

recorded in cumulative after testing in PBS for different time points; drug relea

d). Three replicate samples were tested for each condition.

7482 | Nanoscale, 2012, 4, 7475–7488

when loaded onto the amine-functionalized BGn1(A) or

BGn2(A), we could detect the loading quantities with increasing

time of loading. The ampicillin loading was shown to saturate at

approximately 120 min for both types of nanoparticles, which was

chosen as the optimal loading time (Fig. 5a). After the loading, the

mesopore structure of the BG nanoparticles was observed to

change (shown in ESI 2†). Surface areas and pore volumes were

reduced significantly, and the pore sizes were also decreased. In

particular, the decrease in the pore volume of BGn1(A) and

BGn2(A) caused by the ampicillin loading was as high as �30 to

40%, demonstrating the incorporation of ampicillin molecules

within the mesopore structure (summarized in ESI 3†). Further-

more, we sought to find the loading capacity by obtaining an

adsorption isotherm. The initial concentrations of ampicillin were

varied and the corresponding loaded amounts were measured

(Fig. 5b); the maximal loading was obtained at approximately 180

and 300 mg of ampicillin per gram of BGn1(A) and BGn2(A),

respectively, both of which were reached at the maximal concen-

tration of ampicillin that has been tested (500 mg ml�1). It can be

deduced that the high pore volume and surface area of BGn2(A)

allowed enhanced loading capacity of ampicillin. The release

cles, BGn1(A) and BGn2(A) and (c and d) the release profiles for the drug

loading. Na-ampicillin was loaded onto either BGn1(A) or BGn2(A) for

ing time was determined at 120 min. The adsorption isotherm was plotted

00 mg of ampicillin per gram of BGn1(A) and BGn2(A), respectively (b);

g capacity. The release profiles of ampicillin from the nanoparticles were

se was almost completed at�12 h for both BGn1(A) and BGn2(A) (c and

This journal is ª The Royal Society of Chemistry 2012

Fig. 6 Loading quantity of siRNA onto BGn1(A) and BGn2(A); 10 mg

of siRNA, initially used onto 50 mg of nanoparticles with varying loading

time, provided a loading saturation of 1.93 mg (�19% of initial loading)

for BGn1(A) and 2.59 mg (�26% of initial loading) for BGn2(A) attained

at 2 h. The loading efficiency (maximal quantity of siRNAwith respect to

nanoparticles) was thus 3.86% for BGn1(A) and 5.18% for BGn2(A),

demonstrating significantly higher loading efficiency in BGn2(A) than in

BGn1(A).

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profiles of ampicillin from the nanoparticles were further recorded

in a cumulative manner after incubation in PBS for different time

points (Fig. 5c and d). The drug release pattern showed an initial

linear profile (up to a few hours), a further slow-down in the

release rate, and then final saturation at approximately 12 h.

Although the release patterns of BGn1(A) and BGn2(A) were

similar, the release amount of ampicillin with time was substan-

tially different because the loading amount was initially different.

Next, we sought to find the potential of the BG nanoparticles

in loading and delivery of genes. We selected siRNA as a model

Fig. 7 TEM ultrastructure of the delivered BG nanoparticles (BGn2(A) sh

particles taken up by the cells, distributed in cytoplasm around the cellular c

cells) are shown for comparison.

This journal is ª The Royal Society of Chemistry 2012

gene, as this has been one of the most fascinating genetic mole-

cules used in many diseases and injuries of target tissues.38,39 For

the loading tests, we used standard siRNAwithout endowing any

genetic functions to the sequence, but we conjugated FITC to

assess the fluorescence intensity and further to detect the intra-

cellular process of the siRNA–nanoparticle complex. First, we

determined the maximal loading quantity of siRNA onto the

nanoparticles and the proper loading time. A preliminary test

revealed that the mixing ratio of siRNA–nanoparticles affected

the loading quantity of siRNA (not shown here), and the optimal

loading was determined at the mixing ratio of 1 : 5 (siR-

NA : nanoparticles). We further changed the time of complexa-

tion at a ratio of 1 : 5. Amaximal loading quantity of 1.93 mg and

2.59 mg siRNA per 50 mg BGn1(A) and BGn2(A), respectively,

was found to be attained at approximately 2 h, after which a

loading saturation was observed, indicating that BGn1(A) and

BGn2(A) had a loading capacity of siRNA of approximately

3.86% and 5.18%, respectively (�1.3 times higher in BGn2(A))

(Fig. 6). The loading conditions were thus determined at a ratio

of siRNA : nanoparticles 1 : 5 and a loading time 2 h for further

gene delivery and cell studies.

The typical ultrastructure of the cells that received the deliv-

ered siRNA–BGn2(A) complex was observed by TEM (Fig. 7).

Many electron dense nanoparticles with sizes <100 nm were

readily apparent in the cytoplasm around intracellular compo-

nents, including the Golgi apparatus and the rough endoplasmic

reticulum. There was no appearance of cellular stressing associ-

ated with the intracellular uptake of the nanomaterials, such as

signs of apoptosis and necrosis.

The intracellular uptake of the siRNA–BGn2(A) complex was

analyzed by CLSM and FACS (Fig. 8). Both scramble RNA

(scRNA) and bcl-2 target gene siRNA complex with BGn2(A)

own representatively) within HeLa cells showing electron dense nano-

omponents. Cell images without the treatment of nanoparticles (control

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were used for the experimental groups. On the other hand,

control groups without any addition (w/o), siRNA only, and

BGn2(A) only were also tested for comparison purposes. For the

experimental groups, a large number of cells were stained posi-

tive (green) for fluorescence FITC dye, as well as co-stained

positive (red) for the nucleus-specific dye PI (Fig. 8a). This was

not readily observed in other control groups; w/o, siRNA only,

or BGn2(A) only. The fraction of the cells that were positive for

the FITC from the whole cell mass was examined by FACS

analysis. Compared to the cells in control groups, where no

positive fractions were noticed, those with the siRNA–BGn2(A)

or scRNA–BGn2(A) complex presented as high as approxi-

mately 80% of FITC-positive cells (Fig. 8b), confirming the high

intracellular delivery capacity of the complexes.

We further sought to find the functional genetic effects of the

delivered siRNA–BGn2(A). Prior to this, we investigated the

Fig. 8 (a) FITC-conjugated siRNA loaded within BGn2(A) was transfected

BGn2(A) complex in green as well as co-staining of nuclei with PI in red; in

siRNA only, or with BGn2(A) only, there was no green fluorescence (only nucl

significant transfection of siRNA–BGn2(A) or scRNA–BGn2(A) delivery int

7484 | Nanoscale, 2012, 4, 7475–7488

release profile of the siRNA from the nanoparticles. The siRNA

release in PBS exhibited a pattern of almost linear increase for up

to 3 days (Fig. 9a). When the release amount was converted to

the percentage of initial loading amount the total release corre-

sponded to approximately 45%. The release profile of siRNA for

up to 3 days indicated the potential value of the BGn2(A) system

in the delivery of siRNA and its possible genetic regulation at

least for up to those periods, i.e., the temporal expression of

target genes and further silencing effects for a certain period was

expected. To elucidate the functional activity of the siRNA, we

designed a simple well-known target gene siRNA that silences

bcl-2 gene; although not specified for bone cells, it is considered

proper as a pilot study to find out functional gene-silencing

efficacy of siRNA when delivered from the mesoporous BG

nano-vehicle. The silencing effect of the siRNA delivery by

the designed system is shown in Fig. 9b. A significant effect

into HeLa cells, which was visualized under CLSM to reveal the siRNA–

contrast, other control cells, including those without any additions, with

ei with PI in red). (b) FACS analysis of the cells positive for FITC showing

o the cells (approximately 80%), in direct contrast to other cases (<1%).

This journal is ª The Royal Society of Chemistry 2012

Fig. 9 (a) Release profile of siRNA from the BGn2(A) delivery system, showing a continual release (zero-order kinetics) for up to 3 days with a final

quantity of approximately 45% of the initial loading and then almost saturation. (b) Assay on biological activity of the siRNA delivered from the

BGn2(A) nanocarrier, designed to silence bcl-2 gene, demonstrated clearly the silencing effect down to approximately 15% maintained, in contrast to

other control cases (either siRNA only, approximately 89%; BGn2(A) only, approximately 79%; or scramble siRNA–BGn2(A), approximately 90%

maintained). Data are shown as means and standard deviations from triplicate samples.

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(down-regulation to 15%) was evident, when compared to other

cases used as controls (siRNA only; 89%, BGn2(A) only; 79%, or

scramble siRNA–BGn2(A); 90% remained).

4. Discussion

Development of proper delivery systems for the regeneration of

tissues including bone is of special importance to maximize the

therapeutic efficacy of bioactive factors by delivering them in a

timely and sustainable way.40,41 The currently introduced BG

nanoparticles, especially those retaining the large volume of

mesopores within the structure, satisfy some promising aspects

targeting bone tissue, as their composition has already been

proven as a suitable platform for novel inorganic bioactive

nanomaterials.42,43 Endowing a capacity to deliver therapeutic

biomolecules improves the bone regenerative potential of the

bioactive nanomaterials, which seeks to target and modulate

specific cellular behaviors.40,41

Here, we developed the BG nanoparticles into spherical mes-

oporous structures using two different templates: PEG as a non-

ionic surfactant and CTAB as a cationic surfactant. The char-

acteristics of mesopores, including pore size, volume, and surface

area, are very important in determining the capacity of the

nanoparticles to incorporate and release candidate therapeutic

molecules. The use of two different templates to generate mes-

opores within the BG nanoparticles resulted in mesopore volume

(PV) and surface area (SA) with different levels depending on the

template type. The use of CTAB produced significantly higher

levels of PV and SA than those of PEG. This difference originates

from the differences in chemical structure and properties of

CTAB and PEG in the micelle formation, such as chemical

structure and size, and the maximum number of surfactant

molecules per micelle. The large quantity of mesopores, espe-

cially in the case of BGn2(A), is considered highly beneficial for

providing enough space to home small molecules in large

amounts, including chemical drugs and genes.43 In fact, the

effectiveness of the BG nanoparticles with such a mesoporous

structure in the loading of biomolecules can be presumed when

This journal is ª The Royal Society of Chemistry 2012

referenced from the accumulating studies on pure mesoporous

silica nanoparticles (MSNs).44–46 MSNs generally developed by

means of CTAB as surfactants present typical values of large

PV in the range 0.5–1 cm3 g�1 and high SA of approximately

900 m2 g�1.45,47 A very close range (PV: 0.415 cm3 g�1 and SA:

830 m2 g�1) was obtained for our BGn2 templated with the same

surfactant. Along with the total space volume and area of pores,

the size of individual pores is also crucial, determining the size of

biomolecules that can be allowed to enter into the mesopores.

Both BGn1 and BGn2 showed average pore sizes of 4.9 and

3.2 nm, respectively, values considered to facilitate loading of

relatively small molecules like chemical drugs and small sized

nucleic acids.48–52 Therefore, here we employed candidate (rela-

tively small) biomolecules of antibiotic and siRNA. In fact, the

mesopore size can be increased by using different types of

templates and with the help of some auxiliary organic molecules,

as have been well-studied in the case of MSNs.48,53 Thus, when

this will be exploited, large molecules such as growth factors can

also be considered as the candidate molecules to be delivered by

the engineered mesoporous BG nanoparticles, which remain as a

further interesting study.

Another factor to consider in tailoring the mesopores of the

BGn is the surface chemistry, particularly the surface charge, as

this can directly affect the nanoparticle–biomolecule interac-

tions.54,55 Biomolecules such as chemical drugs, proteins, and

genes can have a certain level of surface net charge under bio-

logical fluid conditions (generally at pH �7); drugs can be either

ionic or non-ionic, proteins are dependent on the sequence of

amino acids comprising the whole structure, and genes are highly

negatively charged due to the existence of a bunch of phosphate

groups.38 Here, the mesoporous BG nanoparticles initially

synthesized without surface treatment possessed a relatively high

negative charge; e.g., at pH 7, the z-potential of BGn1 and BGn2

before amination is in the range from �20 to �30 mV, which is

due to the presence of a high concentration of hydroxyl groups

on the surface of nanoparticles and, thus, it is considered

appropriate to afford chemically relevant sites for the ionic

interaction with positively charged drugs or biomolecules. For

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negatively charged drugs or biomolecules (e.g., Na-ampicillin

and siRNA), we aminated the surface with APTES. The func-

tionalization clearly showed the presence of amine groups on the

surface (from FT-IR spectra and TGA weight loss), and

dramatically changed the z-potential to high positive values (+20

to +30 mV).

We next sought to utilize the aminated BGn in the loading and

delivery of biomolecules. As the candidate molecules, we used

either chemical drug (anionic drug Na-ampicillin) or small

nucleic acid (siRNA). At first guess, the loading of siRNA was

presumed to be more effective than the ionic Na-ampicillin; while

the siRNA designed here with a base pair of �20 (20 bp) was

highly negatively charged, the Na-ampicillin is an amphoteric,

namely it forms dissociated ionic species in solution, either in

cationic, zwitterionic, and anionic form depending on the solu-

tion pH.56

Loading of Na-ampicillin within the nanoparticles was shown

to be attained completely at around 120 min. The loading

capacity of the nanoparticles showed a marked difference

between the two types: approximately 180 mg g�1 and 300 mg

g�1 of BGn1 and BGn2, respectively, with approximately

1.7 times higher in BGn2. This was primarily due to the differ-

ence in the total pore volume. Taken from the BET results, the

mesopores in BGn2 provided a large pore volume and high

surface area compared to those in BGn1 (0.415 cm3 g�1, 830 m2

g�1 vs. 0.131 cm3 g�1, 54 m2 g�1, respectively). The average pore

sizes of the BGn (4.9 nm and 3.2 nm) are much larger than the

molecular width of ampicillin (0.77 nm) reported.57 Thus we can

imagine the presence of ampicillin molecules inside the meso-

pores of both nanoparticles, presumably adsorbed onto the

mesopore channel surface. In fact, after the ampicillin loading,

the pore volumes of the BGn were significantly reduced (30–40%,

in ESI 3†), suggesting that a large number of ampicillin molecules

have filled the mesopore channel. Interestingly, although the

pore space of both nanoparticles shows big differences (3-times

in pore volume and 15-times in surface area), the difference in

ampicillin loading capacity was relatively small (1.7-times),

falling short of our expectation. A more detailed examination of

the pore size distribution demonstrated a broader pore size

distribution in BGn1 than in BGn2, particularly towards much

larger mesopores than the average value, which should be more

sufficient to incorporate the drug. The results explain well the

importance of the mesopore properties, including the size and

distribution of pores, and their volume and surface area, in

determining the loading capacity of drugs within the mesoporous

BGn. Although here we utilized the positive-charged mesopore

surface in capturing negatively charged drugs, cationic or

hydrophobic non-charged drugs can also be applied if the surface

of the nanoparticles is functionalized relevantly, namely using

hydroxylated nanoparticles (as-prepared without modification)

or after the carboxylation process for cationic drugs, and after

tailoring with alkyl groups for hydrophobic drugs.58

The loaded ampicillin was released over 12 h, presenting a

typical profile dominated by a diffusion process. This was simi-

larly observed for both types of nanoparticles, although the

BGn2 released more ampicillin, reflecting the higher loading

quantity. The release of ampicillin was not complete (80–90%

release). Possibilities for the retention of the drug are loss or

degradation of the drug during the test or the actual retention of

7486 | Nanoscale, 2012, 4, 7475–7488

10–20% of ampicillin within the mesopores. Loss/degradation is

more likely, but retention due to bonds between ampicillin and

the mesopore surface that are essentially irreversible is very

unlikely. In fact, although a somewhat sustainable release profile

was apparent over 12 h, the mesoporous BG nanoparticles are

not considered effective in retaining the ampicillin within the

pore structure in a manner that will prolong the release period for

days to weeks. It can be assumed that the Na-ampicillin would be

quite susceptible to ionic exchanges in saline solution, so the

positively charged amine groups will not allow strong bonds with

added ampicillin. However, such a delivery pattern (short but

diffusion-controlled and predictable) is beneficial for therapeutic

functions of certain drugs, like antibiotics.59 Strategies to prolong

the release period (days to weeks) would be needed to enhance

the potential of mesoporous BG nanoparticles as a delivery

system of small chemical drugs with weak ionic interactions. A

prolonged release will also allow better control of the drug

release kinetics.

The present study also assessed the potential of BGn as a gene

delivery vehicle. Genetic modification of cells is possible by the

proper delivery of genes (such as miRNA, siRNA and pDNA)

within cells, targeted to the cytoplasm or the nucleus. As the size

of the mesoporous BGn was presently in the range of 80–100 nm,

we consider that particle entry into the nucleus does not readily

occur, given the presence of the nuclear membrane.60 Therefore,

a strategy to deliver genes into the cytoplasm in a manner that

retains the functional activity of the delivered genes was

employed, utilizing siRNA. Once the designed siRNA is present

in the cytoplasm assisted by the synthetic nanocarrier after being

taken up by the cell, it is able to abrogate fundamental cellular

pathways through the well-established mechanism of RNA

interference (RNAi).60 To realize the therapeutic potential of

siRNA, safe and effective delivery systems are required. The

delivery of naked siRNA into target cells and tissues is not easily

implemented, mainly due to the degradation by endogeneous

enzymes, and the difficulties in penetrating cell membranes

imposed by large size (impermeable to ion channels) and highly

negative charge.59 For this reason, for the delivery of siRNA,

viral vectors were among the first vehicles introduced. However,

their tendency to produce unacceptable toxic effects associated

with immune rejection61 negated their routine use. Synthetic

nanoparticles from polymers and inorganics have thus gained

great attraction.44,62–64 Although many compositions and

formulations have been studied, there have been few reports on

the gene delivery with inorganic nanoparticles compared to

polymeric materials.44,64,65 One of the most fascinating non-viral

inorganic vectors, mesoporous silica nanoparticles (MSNs), have

been investigated in terms of their ability to incorporate genes

and deliver them into intracellular compartments.44,64 The mes-

oporous structure of the nanoparticles allows the uptake of a

large quantity of nucleic acids, and the particle size (<100 nm)

allows effective intracellular uptake through an endocytosis

mechanism. Furthermore, the entrapped genes within mesopores

can be secured and released sustainably.66 The presently devel-

oped mesoporous BGn share these basic concepts. Moreover,

calcium-containing BGn have beneficial effects on hard tissue

regeneration. Studies have suggested significant roles of such

nanoparticles in bone cell functions including stem cell differ-

entiation and hard tissue mineralization.1,3,22 Therefore, the

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presently developed mesoporous BGn are a promising candidate

non-viral vector for the delivery of genes targeting hard tissues

like bone and teeth.

Cytotoxicity tests confirmed that the mesoporous BGn were

very useful for cells in interest, including MSCs and osteoblasts.

Cellular toxicity is greatly influenced by factors of mesoporous

nanomaterials including size, pore structure, and surface chem-

istry.67 BGn1 and BGn2 with sizes of approximately 80–100 nm

containing mesoporous structures and having aminated

surface chemistry were not appreciably toxic (>90%, compared

to control) to a variety of cells, at doses ranging from 5 to 100 mg

ml�1. Compared to conventional nonbioactive silica nano-

particles, such as MSNs, the results on the BGn confirmed their

similar or even less toxic nature.67 Although more in-depth

investigations need to support, the observation of up-regulated

cell viability at very low doses particularly in the MC3T3-E1

osteoblastic cells makes it worthwhile to do further research on

the effective roles of the BGn in proliferation of bone-related

cells. The presently developed BGn are considered favorable for

preserving cell viability, at least to the level of MSNs which are

regarded as effective cell transfection nanocarriers.44 Specifically,

at the low dose of 10 mg ml�1 used for drug and gene delivery, the

cell viability level was almost equal to or even higher than that of

control.

If toxicity is not a concern, the efficacy of BGn for use as

siRNA delivery carriers relies on their ability to incorporate

genes and release them in a proper manner. Surface functional-

ization with amine groups is critical to utilize BGn in capturing

polyanionic nucleic acids. Presently, 2.59 mg of siRNA could be

loaded into 50 mg of BGn2, demonstrating a loading capacity of

�5.18%, and we used 10 mg of BGn2 for siRNA transfection,

which allowed a sufficient quantity of siRNA to be taken up into

cells if the transfection efficiency is high enough. Cells treated

with a FITC-conjugated siRNA–BGn2 complex demonstrated a

transfection efficiency as high as 80% in FACS analysis. This

cellular uptake phenomenon in BGn is considered to be

explained by the endocytosis mechanism similar to that reported

in MSNs. Although MSNs lack cell membrane-bound receptors,

such as low density lipoprotein or transferrin receptors, they are

known to have a great affinity for adsorbing onto cell membrane

surfaces, particularly the head groups of various phospholipids,

which thus leads to ‘adsorptive’ endocytosis of the nano-

particles.68–70 TEM also revealed pronounced intracellular

uptake of the siRNA-loaded nanoparticles, which were present

within the cytosol, localized in some intracellular compartments

such as mitochondria and rough endoplasmic reticulum. While

these sites are not exactly target-specific, it is presumed that the

internalized BG nanoparticles will further release siRNA in the

cytosol to allow the biological function of target-gene silencing.

The release profile of siRNA gene from the nanoparticles is

consistent with the view that very strong interactions are created

between the aminated-surface of BGn2 with siRNA than with

Na-ampicillin, facilitating effective complexation of the siRNA

within the mesopores, which is mainly due to the highly negative-

charged polyanion characteristic of nucleic acids. Thus, relatively

sustained and linear release of siRNA up to about 3 days could

be attained, and the total quantity released was almost 45% of

the initial loading quantity. Taken the result that an almost

saturation was attained after 3 days, parts of siRNA might be

This journal is ª The Royal Society of Chemistry 2012

degraded in the complexation process (but counted as the loaded

gene) or during the release period. However, a significant portion

of the siRNA was shown to release, profiling zero-order kinetics

up to 3 days, and this release time should be critically considered

in designing the siRNA delivery system, as we can presume the

possible intracellular action periods of gene silencing based on

the siRNA expression period.

We further investigated the possible biological interference of

siRNA within cells using a model silencing gene, bcl-2. The bcl-2

biological function in HeLa cells was almost completely

knocked-down when the target siRNA-loaded BGn2 nano-

particles were transfected, which was not readily recognizable in

other comparison groups including target siRNA alone, BGn2

alone, or scramble RNA-loaded BGn2, confirming that the

intracellular transfection of the siRNA–BGn2 complex was in

effect in silencing target gene. The bcl-2 gene is not specified for

hard tissue regeneration, but was utilized to confirm the parallel

action of transfection and the subsequent gene-silencing effect.

More relevant applications are possible and wait for further

studies.

Together with the excellent in vitro bone-bioactivity (namely

apatite forming ability) and the low cellular toxicity, the ability

to load therapeutic molecules and deliver them in a proper

manner as proved using model biomolecules demonstrates the

potential usefulness of the mesoporous BG nanoparticles for the

regeneration of bone. Specific diverse applications that are

immediately apparent include the use of nano-vehicles in the

osteogenesis of stem cells by the direct treatment of the gene-

loaded BG nanoparticles as well as in the bone regeneration as

implantable biomaterials and tissue engineering scaffolds when

incorporating drug-loaded BG nanoparticles.

5. Conclusions

Here we show for the first time the performance of mesoporous

BG nanoparticles as the delivery vehicles of biomolecules,

including drugs and genes. The particle sizes (<100 nm) and

extremely high surface area and large volume of mesopores

inside the nanoparticles, as well as the nontoxic and bioactive

traits are very suitable for such a purpose, by incorporating

candidate biomolecules and releasing them in a sustainable

manner. After the surface functionalization with amine groups,

specific utility of the mesoporous BG nanoparticles as a gene

delivery vector was demonstrated. As a model gene studied,

siRNA was shown to be effectively loaded within the nano-

particles, and then further released in vitro over a period of

3 days. The siRNA-loaded BG nanoparticles were shown to be

transfected efficiently into cells and to have a gene-silencing

function as well. The results support the possible promising uses

of the novel non-viral vector in delivering target genes such as

siRNA for the disease treatment or regeneration of hard tissues.

Acknowledgements

This study was supported by grants from the Priority Research

Centers Program (2009-0093829) and the WCU program (R31-

2008-000-100069-0), National Research Foundation, South

Korea.

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