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Antibody fragment-armed mesoporous silica nanoparticles for the targeted delivery of bevacizumab in ovarian cancer cells
Accepted Manuscript Title: Antibody Fragment-armed Mesoporous Silica Nanoparticles for the Targeted Delivery of Bevacizumab in Ovarian Cancer Cells Author: Ying Zhang Jing Guo Xiao-Ling Zhang Da-Peng Li Ting-Ting Zhang Fu-Feng Gao Nai-Fu Liu Xiu-Gui Sheng PII: DOI: Reference:
S0378-5173(15)30347-1 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.10.080 IJP 15335
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
17-8-2015 16-10-2015 30-10-2015
Please cite this article as: Zhang, Ying, Guo, Jing, Zhang, Xiao-Ling, Li, DaPeng, Zhang, Ting-Ting, Gao, Fu-Feng, Liu, Nai-Fu, Sheng, Xiu-Gui, Antibody Fragment-armed Mesoporous Silica Nanoparticles for the Targeted Delivery of Bevacizumab in Ovarian Cancer Cells.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.10.080 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Antibody Fragment-armed Mesoporous Silica Nanoparticles for the Targeted Delivery of
2
Bevacizumab in Ovarian Cancer Cells
3 4 5 6 7 8 9 10 11 12 13
Ying Zhang1,2, Jing Guo 1,2, Xiao-Ling Zhang1, Da-Peng Li1, Ting-Ting Zhang1, Fu-Feng Gao 1, Nai-Fu Liu1, Xiu-Gui Sheng1* 1
Department of Gynecologic Oncology, Shandong Cancer Hospital and Institute， Jinan, Shandong 250117 P.R.China 2
School of Medicine and Life Sciences, University of Jinan - Shandong Academy of Medical Sciences, Jinan, Shandong 250022 P.R.China
14 15 16 17 18 19 20 21 22 23 24 25
Corresponding author: Xiu-Gui Sheng, Department of Gynecologic Oncology, Shandong Cancer Hospital and Institute, 440 Jiyan Road, Jinan, Shandong 250117 P.R.China Tel&Fax:0086-531-67626976 Email:[email protected]
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Graphical abstract
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Abstract
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In order to enhance the therapeutic efficacy and intracellular concentration of bevacizumab
30
(BVC), we have designed a novel tumor endothelial marker 1 (TEM1)/endosialin (Ab-/scFv)-
31
conjugated mesoporous silica nanoparticles (MSN) to target ovarian cancer cell. The Ab-/scFv1
32
conjugated MSN were prepared by the conjugation of amine functional group of antibody of the
33
carboxyl group of MSN. The resultant MSN was nanosized, spherical shaped, and exhibited a
34
controlled release phenomenon in pH 7.4 conditions. Furthermore, BMSN/Ab was found to
35
increase the cellular uptake and intracellular distribution of BVC in OVCAR-5 cancer cells. The
36
Ab- conjugated MSN exhibited a superior anticancer effect with profound apoptosis in cancer
37
cells in a time- and concentration dependent manner. Consistently, BMSN/Ab effectively
38
inhibited the colony formation in transwell plate. Finally, BMSN/Ab showed a notable increase
39
in the proportion of cells in G2/M phase of cell cycle indicating promising anticancer efficacy
40
profile. Overall, Ab-/scFv-conjugated MSN might provide an effective strategy for the
41
therapeutic management of ovarian cancers.
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Keywords
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Antibody, bevacizumab, mesoporous silica nanoparticles, apoptosis, ovarian cancer
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Introduction
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Ovarian cancer is one of the most lethal cancers with an annual death rate of 15000 patients in
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United States (Jelovac et al., 2011). Approximately, 90% of ovarian cancers are of epithelial
2
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origin and therefore most lethal in nature. The 5-year survival rate of ovarian cancer remained at
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<45% despite the widespread advancement in cancer diagnostics and treatment methods
54
(Vaughan et al., 2011; Chobanian et al., 2008). The high mortality rate of this cancer was due to
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the recurrence and subsequent resistance to chemotherapeutic treatments. Moreover, ovarian
56
cancers progress without any specific symptoms and spread throughout the peritoneal cavity
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(Siegel et al., 2013; Johnson et al., 2013). At present, chemotherapy is the main treatment option;
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however it suffers from profound toxicity to normal cells and often drug resistance.
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In this perspective, bevacizumab (BVC) is a humanized recombinant monoclonal antibody
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against vascular endothelial growth factor (VEGF) (Shih et al., 2006). This blood circulation
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growth factor is responsible for the growth of blood vessels and up-regulated in multiple cancers
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(solid tumors). Therefore, BVC is used to inhibit the VEGF function in vascular endothelial cells
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and thereby inhibit the tumor angiogenesis which is important for the cancer cell proliferation
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and metastasis (Bashshur et al., 2008; Avery et al., 2006). BVC has been shown to exhibit
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antitumor properties in many tumors either as a single agent or in combination with other
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anticancer drugs. Despite its potential advantage, BVC suffers from severe systemic side effects
67
including hypertension, proteinuria, gastrointestinal perforation and increased thromboembolic
68
events when administered intravenously (Arevalo et al., 2008). Therefore, nanocarrier delivery
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systems that can specifically deliver the drug to the cancer at high concentrations and least toxic
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to the normal cells have to be developed. These includes the enhanced stability of drug in the
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systemic circulation, easy modification of particle surface (active targeting), and preferable
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accumulation in the tumor tissues.
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Many drug delivery systems constructed from different materials including polymers, peptides,
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lipids have been developed to increase the stability of anticancer drugs. In this regard, 3
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mesoporous silica nanoparticles (MSN) is one of the most efficient carrier due to its unique
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properties such as large loading capacity, microporous structure, controlled release property, and
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excellent biocompatibility (Lai et al., 2003; Yang et al., 2012). It has been previously reported
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that MSN could efficiently endocytosed and could escape the endolysosomal entrapment (Miele
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et al., 2012; Tsai et al., 2009). Despite the great progress in use of MSN as a drug delivery
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system, distribution of MSN in the in vivo conditions is unpredictable and challenging.
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Although, nanoparticles might passively accumulate in the tumor tissues utilizing the enhanced
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permeation and retention (EPR) effect, it would be insignificant. Therefore, efficient ways have
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to be found to precisely increase the therapeutic payload in the tumor tissues (Lu et al., 2009;
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Danhier et al., 2010).
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In this work, MSN was functionalized with carboxyl groups. The advantage associated with
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functionalization of carboxyl group on the MSN surface includes conjugation of targeting moiety
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to the nanoparticle surface. In the current work, we aimed to engineer antitumor endothelial
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marker 1 [TEM1] antibody [Ab]/single-chain variable fragment [scFv]-armed MSN.
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The main objective was to increase the therapeutic efficacy and cancer specificity of BVC to
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ovarian cancers. For this purpose, we have constructed a scFv antibody surface conjugated
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mesoporous silica nanoparticles in which BVC is loaded in the microporous structures. Particle
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size distribution and release kinetics were evaluated to observe the physicochemical properties.
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Preferential cellular uptake of antibody conjugated MSN was evaluated in SK-OV3 cancer cells.
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Furthermore, intracellular distribution of targeted and non-targeted NP was tested. The
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therapeutic potency of antibody-MSN was tested in SK-OV3 ovarian cancer cells. Various
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biological parameters including cytotoxicity assay, cell apoptosis analysis, western blot analysis,
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colony formation assay, and cell cycle analysis have been investigated. 4
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Materials and Methods
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Materials
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Cetyltrimethyl ammonium bromide (CTAB, 98%), tetraethyl orthosilicate (TEOS, 99%), 2-
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cyanopropyltriethoxysilane (CPTES, 99%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
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hydrochloride (EDAC), N-Hydroxysulfosuccinimide sodium salt (sulfoNHS), and 2-N-
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morpholino-ethanesulfonic acid (MES) were purchased from Sigma–Aldrich. All other
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chemicals were of reagent grade and used without further purifications.
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Preparation of carboxylated mesoporous silica nanoparticles
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cetyltrimethyl ammonium bromide (CTAB, 1200 mg) was dissolved in a aqueous ammonia
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solution (~200 ml). Bevacizumab (BVC) in ethanolic solution was added. The solution was
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stirred for 30 min at 60°C, followed by the addition of 2 ml of TEOS and 0.4 ml of 2-
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cyanopropyltriethoxysilane to the CTAB solution. The resulting mixture was stirred for 120 min
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at 60°C. The solution was maintained at the same temperature for 24h. The MSN was collected
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after centrifuging at 20000 rpm for 15 min. The MSN were washed, re-dispersed several times,
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and freeze dried. The dried MSN was treated with sulphuric acid at 100°C to obtain a carboxyl-
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functionalized MSN. The nanoparticles were stored in cold conditions until further use.
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Preparation of antibody-conjugated MSN
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100 mg of acid-terminated MSN was suspended in a MES buffer (pH 6) and activate with
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EDC/NHS reaction for 2h. The activated MSN was washed and re-dispersed in a PBS and
5
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incubated with 100 µL of antibody (at a molar ratio of 10:1) for 6h at room temperature. The
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antibody-conjugated MSN was separated by centrifugation.
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Particle size and zeta potential analysis
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The particle size distribution and zeta potential analysis was determined using Zetasizer Nano-
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ZS (Malvern Instruments, Malvern, UK). The samples were suitably diluted such that optimized
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concentration of particles would be present in the formulations. The experiments were performed
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in triplicate.
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Morphological analysis
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Tecnai 12 microscope (FEI, Hillsboro, OR, USA) equipped with a Gatan, Inc (Pleasanton, CA,
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USA) 896 2.2.1 US1000 camera was used to observe the morphology of nanoparticles. For this
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purpose, dried samples were dispersed in water and a drop of suspension should be placed over a
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carbon-coated copper grid. The samples were allowed to dry and the image was observed at 120
130
KeV.
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Release kinetics
132
The release study was performed in phosphate buffered saline (pH 7.4) at 37°C. The drug-loaded
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nanoparticle dispersion was loaded in a dialysis bag (molecular weight cut-off: 3000 Da), and the
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dialysis tube was sealed and immersed in 20 mL of the release buffer in a 50 mL centrifuge tube.
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The centrifugal tube was placed in a rotary shaker which is set at 100 rpm and 37°C. At specific
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time points, samples were withdrawn and replaced with equal amount of fresh release medium.
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The amount of drug released in release media was evaluated by means of HPLC method.
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Cellular uptake analysis 6
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The quantitative cellular uptake of targeted and non-targeted MSN was evaluated by means of
140
flow cytometry (FACS; BD Biosciences. Rhodamine B was used as a fluorescent probe. For this
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purpose, 1×105 cells were seeded in a 12-well plate and incubated overnight. The cells were
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treated with antibody-targeted and non-targeted MSN containing rhodamine B and incubated for
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2h and 4h, respectively. The cells were washed twice with PBS buffer and redispersed to 1ml
144
again. The amount of cellular uptake of individual nanoparticle was evaluated by flow
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cytometry.
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Cellular internalization with confocal microscopy
147
The cellular internalization process was confirmed by CLSM (Carl Zeiss LSM510, Dresden,
148
Germany). For this purpose, 2×105 cells were seeded in a 6-well plate and incubated overnight.
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The cells were treated with antibody-targeted and non-targeted MSN containing rhodamine B
150
(instead of drug). The cells were incubated for 2h and then washed twice with PBS buffer. The
151
nuclei were stained with DAPI (blue color) and then lysosome was stained with lysotracker
152
green (green color). The cells were washed and fixed with 4% paraformaldehyde for 10 min. The
153
intracellular distribution of nanoparticles were observed using CLSM.
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Cell culture and cytotoxicity assay
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The OVCAR-5 ovarian cancer cell was grown in RPMI 1640 media supplemented with 10%
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fetal bovine serum and 1% penicillin/streptomycin mixture. The cells were maintained at
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ambient conditions of 37°C at 5% CO2 atmosphere.
7
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The cell viability was determined using MTT assay protocols. Briefly, 1×104 cells were seeded
160
in a 96-well plate and incubated for 24h. The culture medium was removed next day and
161
replaced with fresh medium containing the formulations at different concentrations. The cells
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were further incubated for 24h. The medium was removed and treated with 20 µl of MTT
163
solutions (5 mg/ml) into each well and incubated for 24h. The cells were further treated with 100
164
µl of DMSO solution and kept aside for 15 min to dissolve formazan crystal. Finally, absorbance
165
of cells was read at 570 nm using Victor 3 microplate reader (PerkinElmer, Boston, MA, USA).
166
Apoptosis analysis
167
Annexin V/FITC/PI apoptosis assay kit (Molecular Probes, OR) was used to analyse that
168
apoptosis rate. Annexin V possess the high affinity towards PS and whereas PI could be taken up
169
by necrotic cells. The phosphatidyl serine (PS) which resides on the cell surface was used to
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quantify the cell apoptosis. To determine the apoptosis rate, cells were seeded in a 12-well plate,
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incubated overnight, and treated with 1 µg of respective formulations (20h). The cells were
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washed, trypsinized, centrifuged, and re-dispersed in a binding buffer. Cells were treated with 1
173
µl of Annexin and 1 µl of PI and incubated for 15 min at room temperature. Cell apoptosis was
174
analyzed using flow cytometer FACS Aria II (Beckton and Dickinson, Sanjose, CA).
175 176
Western Blot analysis
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Western blot analysis was performed to observe the apoptotic activity of OVCAR-5 cancer cells.
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The treated cells were lysed by adding SDS sample buffer and scraped. The cell lysates were
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prepared and protein was quantified. Proteins in the buffer was loaded on a 10% SDS
180
polyacrylamide gels and transferred to membranes (Millipore, Bedford, MA, USA). The 8
181
membranes were incubated with primary antibodies of cleaved caspase-3 and cleaved PARP at
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4°C overnight. The membranes were treated with secondary antibodies at room temperature. The
183
blots (bands) were developed using chemiluminescence (LumiGLO, KPL Europe, Guildford,
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UK) according to the manufacturer’s protocol.
185
Colony formation assay
186
Colony formation assay was performed in order to further confirm the cytotoxic effect of
187
formulations on the cancer cells. Cells were seeded in a 6-well plate and treated with free drug
188
and drug-loaded formulations (1 µg/ml) and the medium was changed every 3 days once. The
189
cells were incubated for 2 weeks to form colonies. The number of colonies was counted using an
190
optical microscope after crystal violet staining procedure.
191
Cell cycle analysis
192
To determine the apoptosis rate, cells were seeded in a 12-well plate, incubated overnight, and
193
treated with 1 µg of respective formulations (20h). The cells were then fixed in 70% ethanol at
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4°C for 24h. The cells were then stained with PI (200 µg/ml) for 10 min. The cell cycle
195
distribution was then evaluated by means of flow cytometry.
196 197
Statistical analysis
198
Data were statistically analyzed using t-test or one-way analysis of variance. A P-value less than
199
0.05 were used to show statistical significance. All of the experiments were performed two or
200
more times for reproducibility.
201 9
202
Results and Discussion
203
In this study, we aimed to harness both passive and active targeting mechanisms by engineering
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the mesoporous silica nanoparticle conjugated with TEM1-targeting Ab/scFv (Figure 1). The
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presence of targeting ligand on the surface is expected to increase the chemotherapeutic potency
206
of anticancer agents.
207
Particle size and zeta potential analysis
208
The particle size and zeta potential measurement were carried out by dynamic light scattering
209
technique. The average particle size of drug-loaded MSN was ~135 nm while the particle size of
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antibody-conjugated MSN was ~166 nm (Figure 2a). The slight increase in particle size was
211
owing to the presence of antibody on the surface of preformed nanoparticles. Nevertheless, the
212
size was within the limit of EPR targeting suggesting its suitability to cancer drug delivery.
213
These nanoparticles were small enough to evade detection and destruction by the
214
reticuloendothelial system (RES) which would prolong their time in the systemic circulation
215
(Gong et al., 2012). The zeta potential of MSN was 22 mV while the surface charge slightly
216
decreased to 16 mV upon antibody conjugation.
217
Morphological analysis
218
The morphological analysis of optimized antibody-conjugated MSN was investigated by TEM
219
imaging. The particles were spherical with clear cut boundary (Figure 2b). The particles were
220
uniformly spread on the copper grid indicating the homogeneity of nanoformulations. A shallow
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greyish outer surface might be attributed to the presence of antibody on the surface of the
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nanoparticles.
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In vitro drug release
224
The in vitro release of BVC from MSN was investigated under a simulated phosphate buffered
225
saline (pH 7.4) (Figure 3). The study clearly showed that both the nanoparticle system effectively
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controlled the release of drug in the PBS medium. It can be seen that presence of antibody on the
227
surface significantly inhibited the release of drug in the medium. Approximately ~25% of drug
228
released at the end of 24h from the carrier system. Whereas at the end of 96h, nearly 70% of
229
drug released from BMSN/Ab comparing to 95% of drug released in the BMSN carrier system.
230
From the release data, it is evident that both the nanoparticle controlled the release of drug in the
231
pH 7.4 conditions and much of the drug was encapsulated in the nanoparticles. Therefore,
232
limited release of drug in pH 7.4 conditions would effectively reduce the side effects.
233
Furthermore, it can be safely expected that when the nanoparticles reach the tumor site, it will
234
release the payload in the cancer cell and thereby will increase the therapeutic efficacy (Wang et
235
al., 2012).
236
Cellular uptake analysis
237
Flow cytometer was used to quantitatively asses the cellular uptake potential of targeted and non-
238
targeted nanoparticles in OVCAR-5 cancer cells (Figure 4). Flow cytometric analysis showed
239
that BMSN/Ab significantly increased the cellular uptake by comparison to BMSN. It can be
240
seen that BMSN/Ab exhibited a 3-fold increase in cellular uptake at the end of 2 and 4h,
241
respectively. In another observation, nanoparticle showed a typical time-dependent cellular
242
uptake in cancer cells. It can be expected that the superior uptake of BMSN/Ab was mainly
243
attributed to the specific affinity of antibody conjugated NP towards the receptor over expressed
244
in OVCAR-5 cancer cells (Conejo-Garcia et al., 2005).
11
245
Intracellular internalization of nanoparticles
246
The cellular internalization of BMSN and BMSN/Ab was studied by means of confocal laser-
247
scanning microscopy (CLSM). The CLSM study will reveal the difference in uptake between
248
targeted and non-targeted nanoparticles. For this purpose, nanoparticles were loaded with
249
rhodamine B as a fluorescent probe. The green fluorescence on the cell indicates the staining of
250
lysosome and blue fluorescence corresponds to the nucleus. As shown in Figure 5, BMSN/Ab
251
exhibited a strong red fluorescence on the cell cytoplasm compared to that of non-targeted
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BMSN which exhibited a weak fluorescence on the cell surface. The non-targeted BMSN was
253
weakly localized on the outer surface, whereas, BMSN/Ab was abundantly localized in lysosome
254
and nucleus region as well. Fluorescence in the nucleus region was mainly attributed to the
255
accumulation of rhodamine B in the nucleus as intracellular drug molecules in the cytosol could
256
transport rapidly to the nucleus and avidly bound to the nuclear components. The superior
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cellular internalization of BMSN/Ab was attributed to the receptor which is over expressed on
258
the OVCAR-5 cancer cells. The specific receptor-mediated cellular uptake resulted in the high
259
colocalization of nanoparticles. This observation clearly inferred that antibody conjugated
260
nanoparticle was very much effective as a delivery system for targeted anticancer drugs.
261
Cytotoxicity assay
262
The cytotoxicity effect of free drug, BMSN, and BMSN/Ab were investigated by MTT assay.
263
Results showed that all the formulations were effective in controlling the proliferation of
264
OVCAR-5 cancer cells (Figure 6a,b). Typically, all the formulations showed a time- and
265
concentration dependent cytotoxicity in cancer cells. The IC50 value was calculated to quantify
266
the effect of individual formulations. The IC50 value of BVC, BMSN, and BMSN/Ab after 24h
12
267
incubation was 10.56, 5.98, 1.86 µg/ml, respectively. Upon 48h incubation, IC50 value were
268
4.53, 2.39, 0.89 µg/ml, respectively for these formulations. The superior cytotoxic effect of
269
BMSN/Ab was mainly attributed to the high intracellular concentration of drug in the cancer
270
cells which was in turn due to the specific interaction between antibody on the MSN surface and
271
the receptor in cancer cells. We expected that Ab-/scFv-conjugated nanoparticle could
272
accumulate in the cancer cells via both passive as well as active targeting mechanisms. TEM1
273
has been reported to be expressed by the endothelial cells of ovarian tumor vasculature (Nanda et
274
al., 2004; Christian et al., 2008). Ab-/scFv-conjugated MSN showed stronger effect on killing
275
tumor cells, which was consistent with the intracellular distribution study. Therefore, it can be
276
safely expected that BMSN/Ab could be a promising therapeutic vehicle for the delivery of BVC
277
in cancer therapy.
278
Apoptosis analysis
279
The cell apoptosis was evaluated by means of Annexin-V/PI staining protocol. As shown in
280
Figure 6c, BMSN/Ab exhibited a remarkable apoptosis of cancer cells. BMSN/Ab induced ~60%
281
of early as well as late apoptosis of cancer cells whereas free drug induced only ~15% of
282
apoptosis. The enhanced cell apoptosis was due to the preferential internalization of BMSN/Ab
283
in the cancer cells. The enhanced anticancer effect of Ab-/scFv-conjugated nanoparticle on
284
TEM1-positive MS1 cells than was due to specific interaction and high intracellular
285
concentrations.
286
Western blot analysis
13
287
Apoptosis effects of formulations were further confirmed by western blot analysis. As shown in
288
Figure 6d, BMSN/Ab exhibited a strong band for cleaved PARP and cleaved caspase-3 which
289
are typical indicators of cellular apoptosis.
290
Colony formation assay
291
The cytotoxicity potential of individual formulation was further confirmed by colony formation
292
assay. As shown in Figure 7, BMSN/Ab significantly controlled the colony formation of cancer
293
cells. The untreated control did not have any effect on the cell proliferation and resulted in
294
maximum colony formation. Exposure of free BVC as well as BMSN did control the formation
295
of colony however could not inhibit completely. In contrast, BMSN/Ab inhibited the colony
296
formation by nearly 10 folds when compared with control and 6-folds by comparison with the
297
free drug. Overall, results were consistent with the cytotoxicity assay.
298 299
Cell cycle analysis
300
Cell cycle analysis of OVCAR-5 cancer cell was evaluated using flow cytometer. As shown in
301
Figure 8, free BVC as well as BVC-loaded formulations induced a strong G2/M phase cell cycle
302
arrest. In particular, G2/M phase arrest was more prominent upon BMSN/Ab treatment. It can be
303
clearly seen that free BVC induced around 25% of G2/M phase arrest while BMSN showed
304
around 30% of G2/M phase arrest. Among all, BMSN/Ab showed a significantly higher G2/M
305
phase arrest of ~50% indicating its superior anticancer efficacy. Consistently, proportion of G1
306
cells was gradually decreased with the formulations. The G1 cells were ~70% in untreated
307
control where as it decreased to less than 30% for BMSN/Ab treatment. The notable increase in
308
G2/M phase cell for BMSN/Ab exposure indicating its promising anticancer efficacy profile. It 14
309
can be expected that MSN played an important role in drug encapsulation and cellular
310
internalization whereas Ab-/scFv increased the targeting efficiency of delivery vehicle. Overall,
311
results were consistent with cellular uptake, cell cytotoxicity and apoptosis analysis.
312 313
Conclusions
314
In summary, we have demonstrated that Ab-/scFv-conjugated MSN could increase the
315
intracellular concentration of BVC and enhance the anticancer effect of active therapeutic agent
316
in ovarian cancer cell. The Ab-/scFv-conjugated MSN were prepared by the conjugation of
317
amine functional group of antibody of the carboxyl group of MSN. The resultant MSN was
318
nanosized, spherical shaped, and exhibited a controlled release phenomenon in pH 7.4
319
conditions. Furthermore, BMSN/Ab was found to increase the cellular uptake and intracellular
320
distribution of BVC in OVCAR-5 cancer cells. The Ab- conjugated MSN exhibited a superior
321
anticancer effect with profound apoptosis in cancer cells in a time- and concentration dependent
322
manner. Consistently, BMSN/Ab effectively inhibited the colony formation in transwell plate.
323
Finally, BMSN/Ab showed a notable increase in the proportion of cells in G2/M phase of cell
324
cycle indicating promising anticancer efficacy profile. Overall, Ab-/scFv-conjugated MSN might
325
provide an effective strategy for the delivery of Bevacizumab in ovarian cancers.
326 327
Competing interests
328
The authors report no conflict of interest in this work.
329 330
Acknowledgement 15
331 332 333
This research was supported by the National Natural Science Foundation of China (No: 81372778)
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and disposition of nanomedicine using biodegradable PEG/PCL polymers as drug carriers, Curr.
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Drug Metab. 13, 338–353.
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Yang, P., Gai, S., Lin, J., 2012. Functionalized mesoporous silica materials for controlled drug
385
delivery. Chem. Soc. Rev. 41, 3679–3698.
386 387 388 389
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390 391 392 393 394 395 396 397 398 399
Figure captions
400
Figure 1: Illustration of preparation of antibody-conjugated mesoporous silica nanoparticles
401
(MSN). The amine group of antibody reacted with the carboxyl functional group of MSN.
402
Figure 2: (a) Dynamic light scattering measurements of size distribution for BMSN/Ab (b)
403
Transmission electron microscopic image of BMSN/Ab.
404
Figure 3: In vitro drug release profile of BMSN and BMSN/Ab in phosphate buffer saline (pH
405
7.4) at 37°C. Results are expressed as means ± the standard error from three independent
406
experiments.
407
Figure 4: Cellular uptake of BMSN and BMSN/Ab in human ovarian cancer cell. The cellular
408
uptake was determined using flow cytometer. Rhodamine B was used as a fluorescent probe. 19
409
Figure 5: Intracellular distribution of BMSN and BMSN/Ab in human ovarian cancer cell. Cells
410
were stained with lysotracker green to stain lysosome and DAPI to stain nucleus. The cells were
411
treated with respective formulation for 2h and observed using a confocal microscope.
412
Figure 6: (a,b) Cytotoxicity of free BVC, BMSN, BMSN/Ab in OVCAR-5 cancer cells. Cells
413
were treated with respective formulations and incubated for 24 and 48h, respectively. The
414
cytotoxicity was determined using MTT assay. Results are expressed as means ± the standard
415
deviation from three independent experiments. (c) Annexin-V/PI based apoptosis analysis of
416
cancer cells. (d) Western blot analysis of cancer cells.
417
Figure 7: Colony formation capacity of OVCAR-5 cancer cells. The cells were treated with free
418
BVC, BMSN, BMSN/Ab and colonies were then stained in crystal violet (0.5%).
419
Figure 8: Cell cycle analysis of OVCAR-5 cancer cells following the treatment of free BVC,
420
BMSN, BMSN/Ab. The cells were treated with 1 µg/ml equivalent of drug and incubated for 24h
421
before the analysis.
422 423
Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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Fig. 7
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Fig. 8
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S0378-5173(15)30347-1 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.10.080 IJP 15335
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
17-8-2015 16-10-2015 30-10-2015
Please cite this article as: Zhang, Ying, Guo, Jing, Zhang, Xiao-Ling, Li, DaPeng, Zhang, Ting-Ting, Gao, Fu-Feng, Liu, Nai-Fu, Sheng, Xiu-Gui, Antibody Fragment-armed Mesoporous Silica Nanoparticles for the Targeted Delivery of Bevacizumab in Ovarian Cancer Cells.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.10.080 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Antibody Fragment-armed Mesoporous Silica Nanoparticles for the Targeted Delivery of
2
Bevacizumab in Ovarian Cancer Cells
3 4 5 6 7 8 9 10 11 12 13
Ying Zhang1,2, Jing Guo 1,2, Xiao-Ling Zhang1, Da-Peng Li1, Ting-Ting Zhang1, Fu-Feng Gao 1, Nai-Fu Liu1, Xiu-Gui Sheng1* 1
Department of Gynecologic Oncology, Shandong Cancer Hospital and Institute， Jinan, Shandong 250117 P.R.China 2
School of Medicine and Life Sciences, University of Jinan - Shandong Academy of Medical Sciences, Jinan, Shandong 250022 P.R.China
14 15 16 17 18 19 20 21 22 23 24 25
Corresponding author: Xiu-Gui Sheng, Department of Gynecologic Oncology, Shandong Cancer Hospital and Institute, 440 Jiyan Road, Jinan, Shandong 250117 P.R.China Tel&Fax:0086-531-67626976 Email:[email protected]
26
Graphical abstract
27 28
Abstract
29
In order to enhance the therapeutic efficacy and intracellular concentration of bevacizumab
30
(BVC), we have designed a novel tumor endothelial marker 1 (TEM1)/endosialin (Ab-/scFv)-
31
conjugated mesoporous silica nanoparticles (MSN) to target ovarian cancer cell. The Ab-/scFv1
32
conjugated MSN were prepared by the conjugation of amine functional group of antibody of the
33
carboxyl group of MSN. The resultant MSN was nanosized, spherical shaped, and exhibited a
34
controlled release phenomenon in pH 7.4 conditions. Furthermore, BMSN/Ab was found to
35
increase the cellular uptake and intracellular distribution of BVC in OVCAR-5 cancer cells. The
36
Ab- conjugated MSN exhibited a superior anticancer effect with profound apoptosis in cancer
37
cells in a time- and concentration dependent manner. Consistently, BMSN/Ab effectively
38
inhibited the colony formation in transwell plate. Finally, BMSN/Ab showed a notable increase
39
in the proportion of cells in G2/M phase of cell cycle indicating promising anticancer efficacy
40
profile. Overall, Ab-/scFv-conjugated MSN might provide an effective strategy for the
41
therapeutic management of ovarian cancers.
42 43
Keywords
44
Antibody, bevacizumab, mesoporous silica nanoparticles, apoptosis, ovarian cancer
45 46 47 48 49
Introduction
50
Ovarian cancer is one of the most lethal cancers with an annual death rate of 15000 patients in
51
United States (Jelovac et al., 2011). Approximately, 90% of ovarian cancers are of epithelial
2
52
origin and therefore most lethal in nature. The 5-year survival rate of ovarian cancer remained at
53
<45% despite the widespread advancement in cancer diagnostics and treatment methods
54
(Vaughan et al., 2011; Chobanian et al., 2008). The high mortality rate of this cancer was due to
55
the recurrence and subsequent resistance to chemotherapeutic treatments. Moreover, ovarian
56
cancers progress without any specific symptoms and spread throughout the peritoneal cavity
57
(Siegel et al., 2013; Johnson et al., 2013). At present, chemotherapy is the main treatment option;
58
however it suffers from profound toxicity to normal cells and often drug resistance.
59
In this perspective, bevacizumab (BVC) is a humanized recombinant monoclonal antibody
60
against vascular endothelial growth factor (VEGF) (Shih et al., 2006). This blood circulation
61
growth factor is responsible for the growth of blood vessels and up-regulated in multiple cancers
62
(solid tumors). Therefore, BVC is used to inhibit the VEGF function in vascular endothelial cells
63
and thereby inhibit the tumor angiogenesis which is important for the cancer cell proliferation
64
and metastasis (Bashshur et al., 2008; Avery et al., 2006). BVC has been shown to exhibit
65
antitumor properties in many tumors either as a single agent or in combination with other
66
anticancer drugs. Despite its potential advantage, BVC suffers from severe systemic side effects
67
including hypertension, proteinuria, gastrointestinal perforation and increased thromboembolic
68
events when administered intravenously (Arevalo et al., 2008). Therefore, nanocarrier delivery
69
systems that can specifically deliver the drug to the cancer at high concentrations and least toxic
70
to the normal cells have to be developed. These includes the enhanced stability of drug in the
71
systemic circulation, easy modification of particle surface (active targeting), and preferable
72
accumulation in the tumor tissues.
73
Many drug delivery systems constructed from different materials including polymers, peptides,
74
lipids have been developed to increase the stability of anticancer drugs. In this regard, 3
75
mesoporous silica nanoparticles (MSN) is one of the most efficient carrier due to its unique
76
properties such as large loading capacity, microporous structure, controlled release property, and
77
excellent biocompatibility (Lai et al., 2003; Yang et al., 2012). It has been previously reported
78
that MSN could efficiently endocytosed and could escape the endolysosomal entrapment (Miele
79
et al., 2012; Tsai et al., 2009). Despite the great progress in use of MSN as a drug delivery
80
system, distribution of MSN in the in vivo conditions is unpredictable and challenging.
81
Although, nanoparticles might passively accumulate in the tumor tissues utilizing the enhanced
82
permeation and retention (EPR) effect, it would be insignificant. Therefore, efficient ways have
83
to be found to precisely increase the therapeutic payload in the tumor tissues (Lu et al., 2009;
84
Danhier et al., 2010).
85
In this work, MSN was functionalized with carboxyl groups. The advantage associated with
86
functionalization of carboxyl group on the MSN surface includes conjugation of targeting moiety
87
to the nanoparticle surface. In the current work, we aimed to engineer antitumor endothelial
88
marker 1 [TEM1] antibody [Ab]/single-chain variable fragment [scFv]-armed MSN.
89
The main objective was to increase the therapeutic efficacy and cancer specificity of BVC to
90
ovarian cancers. For this purpose, we have constructed a scFv antibody surface conjugated
91
mesoporous silica nanoparticles in which BVC is loaded in the microporous structures. Particle
92
size distribution and release kinetics were evaluated to observe the physicochemical properties.
93
Preferential cellular uptake of antibody conjugated MSN was evaluated in SK-OV3 cancer cells.
94
Furthermore, intracellular distribution of targeted and non-targeted NP was tested. The
95
therapeutic potency of antibody-MSN was tested in SK-OV3 ovarian cancer cells. Various
96
biological parameters including cytotoxicity assay, cell apoptosis analysis, western blot analysis,
97
colony formation assay, and cell cycle analysis have been investigated. 4
98 99
Materials and Methods
100
Materials
101
Cetyltrimethyl ammonium bromide (CTAB, 98%), tetraethyl orthosilicate (TEOS, 99%), 2-
102
cyanopropyltriethoxysilane (CPTES, 99%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
103
hydrochloride (EDAC), N-Hydroxysulfosuccinimide sodium salt (sulfoNHS), and 2-N-
104
morpholino-ethanesulfonic acid (MES) were purchased from Sigma–Aldrich. All other
105
chemicals were of reagent grade and used without further purifications.
106
Preparation of carboxylated mesoporous silica nanoparticles
107
cetyltrimethyl ammonium bromide (CTAB, 1200 mg) was dissolved in a aqueous ammonia
108
solution (~200 ml). Bevacizumab (BVC) in ethanolic solution was added. The solution was
109
stirred for 30 min at 60°C, followed by the addition of 2 ml of TEOS and 0.4 ml of 2-
110
cyanopropyltriethoxysilane to the CTAB solution. The resulting mixture was stirred for 120 min
111
at 60°C. The solution was maintained at the same temperature for 24h. The MSN was collected
112
after centrifuging at 20000 rpm for 15 min. The MSN were washed, re-dispersed several times,
113
and freeze dried. The dried MSN was treated with sulphuric acid at 100°C to obtain a carboxyl-
114
functionalized MSN. The nanoparticles were stored in cold conditions until further use.
115
Preparation of antibody-conjugated MSN
116
100 mg of acid-terminated MSN was suspended in a MES buffer (pH 6) and activate with
117
EDC/NHS reaction for 2h. The activated MSN was washed and re-dispersed in a PBS and
5
118
incubated with 100 µL of antibody (at a molar ratio of 10:1) for 6h at room temperature. The
119
antibody-conjugated MSN was separated by centrifugation.
120
Particle size and zeta potential analysis
121
The particle size distribution and zeta potential analysis was determined using Zetasizer Nano-
122
ZS (Malvern Instruments, Malvern, UK). The samples were suitably diluted such that optimized
123
concentration of particles would be present in the formulations. The experiments were performed
124
in triplicate.
125
Morphological analysis
126
Tecnai 12 microscope (FEI, Hillsboro, OR, USA) equipped with a Gatan, Inc (Pleasanton, CA,
127
USA) 896 2.2.1 US1000 camera was used to observe the morphology of nanoparticles. For this
128
purpose, dried samples were dispersed in water and a drop of suspension should be placed over a
129
carbon-coated copper grid. The samples were allowed to dry and the image was observed at 120
130
KeV.
131
Release kinetics
132
The release study was performed in phosphate buffered saline (pH 7.4) at 37°C. The drug-loaded
133
nanoparticle dispersion was loaded in a dialysis bag (molecular weight cut-off: 3000 Da), and the
134
dialysis tube was sealed and immersed in 20 mL of the release buffer in a 50 mL centrifuge tube.
135
The centrifugal tube was placed in a rotary shaker which is set at 100 rpm and 37°C. At specific
136
time points, samples were withdrawn and replaced with equal amount of fresh release medium.
137
The amount of drug released in release media was evaluated by means of HPLC method.
138
Cellular uptake analysis 6
139
The quantitative cellular uptake of targeted and non-targeted MSN was evaluated by means of
140
flow cytometry (FACS; BD Biosciences. Rhodamine B was used as a fluorescent probe. For this
141
purpose, 1×105 cells were seeded in a 12-well plate and incubated overnight. The cells were
142
treated with antibody-targeted and non-targeted MSN containing rhodamine B and incubated for
143
2h and 4h, respectively. The cells were washed twice with PBS buffer and redispersed to 1ml
144
again. The amount of cellular uptake of individual nanoparticle was evaluated by flow
145
cytometry.
146
Cellular internalization with confocal microscopy
147
The cellular internalization process was confirmed by CLSM (Carl Zeiss LSM510, Dresden,
148
Germany). For this purpose, 2×105 cells were seeded in a 6-well plate and incubated overnight.
149
The cells were treated with antibody-targeted and non-targeted MSN containing rhodamine B
150
(instead of drug). The cells were incubated for 2h and then washed twice with PBS buffer. The
151
nuclei were stained with DAPI (blue color) and then lysosome was stained with lysotracker
152
green (green color). The cells were washed and fixed with 4% paraformaldehyde for 10 min. The
153
intracellular distribution of nanoparticles were observed using CLSM.
154 155
Cell culture and cytotoxicity assay
156
The OVCAR-5 ovarian cancer cell was grown in RPMI 1640 media supplemented with 10%
157
fetal bovine serum and 1% penicillin/streptomycin mixture. The cells were maintained at
158
ambient conditions of 37°C at 5% CO2 atmosphere.
7
159
The cell viability was determined using MTT assay protocols. Briefly, 1×104 cells were seeded
160
in a 96-well plate and incubated for 24h. The culture medium was removed next day and
161
replaced with fresh medium containing the formulations at different concentrations. The cells
162
were further incubated for 24h. The medium was removed and treated with 20 µl of MTT
163
solutions (5 mg/ml) into each well and incubated for 24h. The cells were further treated with 100
164
µl of DMSO solution and kept aside for 15 min to dissolve formazan crystal. Finally, absorbance
165
of cells was read at 570 nm using Victor 3 microplate reader (PerkinElmer, Boston, MA, USA).
166
Apoptosis analysis
167
Annexin V/FITC/PI apoptosis assay kit (Molecular Probes, OR) was used to analyse that
168
apoptosis rate. Annexin V possess the high affinity towards PS and whereas PI could be taken up
169
by necrotic cells. The phosphatidyl serine (PS) which resides on the cell surface was used to
170
quantify the cell apoptosis. To determine the apoptosis rate, cells were seeded in a 12-well plate,
171
incubated overnight, and treated with 1 µg of respective formulations (20h). The cells were
172
washed, trypsinized, centrifuged, and re-dispersed in a binding buffer. Cells were treated with 1
173
µl of Annexin and 1 µl of PI and incubated for 15 min at room temperature. Cell apoptosis was
174
analyzed using flow cytometer FACS Aria II (Beckton and Dickinson, Sanjose, CA).
175 176
Western Blot analysis
177
Western blot analysis was performed to observe the apoptotic activity of OVCAR-5 cancer cells.
178
The treated cells were lysed by adding SDS sample buffer and scraped. The cell lysates were
179
prepared and protein was quantified. Proteins in the buffer was loaded on a 10% SDS
180
polyacrylamide gels and transferred to membranes (Millipore, Bedford, MA, USA). The 8
181
membranes were incubated with primary antibodies of cleaved caspase-3 and cleaved PARP at
182
4°C overnight. The membranes were treated with secondary antibodies at room temperature. The
183
blots (bands) were developed using chemiluminescence (LumiGLO, KPL Europe, Guildford,
184
UK) according to the manufacturer’s protocol.
185
Colony formation assay
186
Colony formation assay was performed in order to further confirm the cytotoxic effect of
187
formulations on the cancer cells. Cells were seeded in a 6-well plate and treated with free drug
188
and drug-loaded formulations (1 µg/ml) and the medium was changed every 3 days once. The
189
cells were incubated for 2 weeks to form colonies. The number of colonies was counted using an
190
optical microscope after crystal violet staining procedure.
191
Cell cycle analysis
192
To determine the apoptosis rate, cells were seeded in a 12-well plate, incubated overnight, and
193
treated with 1 µg of respective formulations (20h). The cells were then fixed in 70% ethanol at
194
4°C for 24h. The cells were then stained with PI (200 µg/ml) for 10 min. The cell cycle
195
distribution was then evaluated by means of flow cytometry.
196 197
Statistical analysis
198
Data were statistically analyzed using t-test or one-way analysis of variance. A P-value less than
199
0.05 were used to show statistical significance. All of the experiments were performed two or
200
more times for reproducibility.
201 9
202
Results and Discussion
203
In this study, we aimed to harness both passive and active targeting mechanisms by engineering
204
the mesoporous silica nanoparticle conjugated with TEM1-targeting Ab/scFv (Figure 1). The
205
presence of targeting ligand on the surface is expected to increase the chemotherapeutic potency
206
of anticancer agents.
207
Particle size and zeta potential analysis
208
The particle size and zeta potential measurement were carried out by dynamic light scattering
209
technique. The average particle size of drug-loaded MSN was ~135 nm while the particle size of
210
antibody-conjugated MSN was ~166 nm (Figure 2a). The slight increase in particle size was
211
owing to the presence of antibody on the surface of preformed nanoparticles. Nevertheless, the
212
size was within the limit of EPR targeting suggesting its suitability to cancer drug delivery.
213
These nanoparticles were small enough to evade detection and destruction by the
214
reticuloendothelial system (RES) which would prolong their time in the systemic circulation
215
(Gong et al., 2012). The zeta potential of MSN was 22 mV while the surface charge slightly
216
decreased to 16 mV upon antibody conjugation.
217
Morphological analysis
218
The morphological analysis of optimized antibody-conjugated MSN was investigated by TEM
219
imaging. The particles were spherical with clear cut boundary (Figure 2b). The particles were
220
uniformly spread on the copper grid indicating the homogeneity of nanoformulations. A shallow
221
greyish outer surface might be attributed to the presence of antibody on the surface of the
222
nanoparticles.
10
223
In vitro drug release
224
The in vitro release of BVC from MSN was investigated under a simulated phosphate buffered
225
saline (pH 7.4) (Figure 3). The study clearly showed that both the nanoparticle system effectively
226
controlled the release of drug in the PBS medium. It can be seen that presence of antibody on the
227
surface significantly inhibited the release of drug in the medium. Approximately ~25% of drug
228
released at the end of 24h from the carrier system. Whereas at the end of 96h, nearly 70% of
229
drug released from BMSN/Ab comparing to 95% of drug released in the BMSN carrier system.
230
From the release data, it is evident that both the nanoparticle controlled the release of drug in the
231
pH 7.4 conditions and much of the drug was encapsulated in the nanoparticles. Therefore,
232
limited release of drug in pH 7.4 conditions would effectively reduce the side effects.
233
Furthermore, it can be safely expected that when the nanoparticles reach the tumor site, it will
234
release the payload in the cancer cell and thereby will increase the therapeutic efficacy (Wang et
235
al., 2012).
236
Cellular uptake analysis
237
Flow cytometer was used to quantitatively asses the cellular uptake potential of targeted and non-
238
targeted nanoparticles in OVCAR-5 cancer cells (Figure 4). Flow cytometric analysis showed
239
that BMSN/Ab significantly increased the cellular uptake by comparison to BMSN. It can be
240
seen that BMSN/Ab exhibited a 3-fold increase in cellular uptake at the end of 2 and 4h,
241
respectively. In another observation, nanoparticle showed a typical time-dependent cellular
242
uptake in cancer cells. It can be expected that the superior uptake of BMSN/Ab was mainly
243
attributed to the specific affinity of antibody conjugated NP towards the receptor over expressed
244
in OVCAR-5 cancer cells (Conejo-Garcia et al., 2005).
11
245
Intracellular internalization of nanoparticles
246
The cellular internalization of BMSN and BMSN/Ab was studied by means of confocal laser-
247
scanning microscopy (CLSM). The CLSM study will reveal the difference in uptake between
248
targeted and non-targeted nanoparticles. For this purpose, nanoparticles were loaded with
249
rhodamine B as a fluorescent probe. The green fluorescence on the cell indicates the staining of
250
lysosome and blue fluorescence corresponds to the nucleus. As shown in Figure 5, BMSN/Ab
251
exhibited a strong red fluorescence on the cell cytoplasm compared to that of non-targeted
252
BMSN which exhibited a weak fluorescence on the cell surface. The non-targeted BMSN was
253
weakly localized on the outer surface, whereas, BMSN/Ab was abundantly localized in lysosome
254
and nucleus region as well. Fluorescence in the nucleus region was mainly attributed to the
255
accumulation of rhodamine B in the nucleus as intracellular drug molecules in the cytosol could
256
transport rapidly to the nucleus and avidly bound to the nuclear components. The superior
257
cellular internalization of BMSN/Ab was attributed to the receptor which is over expressed on
258
the OVCAR-5 cancer cells. The specific receptor-mediated cellular uptake resulted in the high
259
colocalization of nanoparticles. This observation clearly inferred that antibody conjugated
260
nanoparticle was very much effective as a delivery system for targeted anticancer drugs.
261
Cytotoxicity assay
262
The cytotoxicity effect of free drug, BMSN, and BMSN/Ab were investigated by MTT assay.
263
Results showed that all the formulations were effective in controlling the proliferation of
264
OVCAR-5 cancer cells (Figure 6a,b). Typically, all the formulations showed a time- and
265
concentration dependent cytotoxicity in cancer cells. The IC50 value was calculated to quantify
266
the effect of individual formulations. The IC50 value of BVC, BMSN, and BMSN/Ab after 24h
12
267
incubation was 10.56, 5.98, 1.86 µg/ml, respectively. Upon 48h incubation, IC50 value were
268
4.53, 2.39, 0.89 µg/ml, respectively for these formulations. The superior cytotoxic effect of
269
BMSN/Ab was mainly attributed to the high intracellular concentration of drug in the cancer
270
cells which was in turn due to the specific interaction between antibody on the MSN surface and
271
the receptor in cancer cells. We expected that Ab-/scFv-conjugated nanoparticle could
272
accumulate in the cancer cells via both passive as well as active targeting mechanisms. TEM1
273
has been reported to be expressed by the endothelial cells of ovarian tumor vasculature (Nanda et
274
al., 2004; Christian et al., 2008). Ab-/scFv-conjugated MSN showed stronger effect on killing
275
tumor cells, which was consistent with the intracellular distribution study. Therefore, it can be
276
safely expected that BMSN/Ab could be a promising therapeutic vehicle for the delivery of BVC
277
in cancer therapy.
278
Apoptosis analysis
279
The cell apoptosis was evaluated by means of Annexin-V/PI staining protocol. As shown in
280
Figure 6c, BMSN/Ab exhibited a remarkable apoptosis of cancer cells. BMSN/Ab induced ~60%
281
of early as well as late apoptosis of cancer cells whereas free drug induced only ~15% of
282
apoptosis. The enhanced cell apoptosis was due to the preferential internalization of BMSN/Ab
283
in the cancer cells. The enhanced anticancer effect of Ab-/scFv-conjugated nanoparticle on
284
TEM1-positive MS1 cells than was due to specific interaction and high intracellular
285
concentrations.
286
Western blot analysis
13
287
Apoptosis effects of formulations were further confirmed by western blot analysis. As shown in
288
Figure 6d, BMSN/Ab exhibited a strong band for cleaved PARP and cleaved caspase-3 which
289
are typical indicators of cellular apoptosis.
290
Colony formation assay
291
The cytotoxicity potential of individual formulation was further confirmed by colony formation
292
assay. As shown in Figure 7, BMSN/Ab significantly controlled the colony formation of cancer
293
cells. The untreated control did not have any effect on the cell proliferation and resulted in
294
maximum colony formation. Exposure of free BVC as well as BMSN did control the formation
295
of colony however could not inhibit completely. In contrast, BMSN/Ab inhibited the colony
296
formation by nearly 10 folds when compared with control and 6-folds by comparison with the
297
free drug. Overall, results were consistent with the cytotoxicity assay.
298 299
Cell cycle analysis
300
Cell cycle analysis of OVCAR-5 cancer cell was evaluated using flow cytometer. As shown in
301
Figure 8, free BVC as well as BVC-loaded formulations induced a strong G2/M phase cell cycle
302
arrest. In particular, G2/M phase arrest was more prominent upon BMSN/Ab treatment. It can be
303
clearly seen that free BVC induced around 25% of G2/M phase arrest while BMSN showed
304
around 30% of G2/M phase arrest. Among all, BMSN/Ab showed a significantly higher G2/M
305
phase arrest of ~50% indicating its superior anticancer efficacy. Consistently, proportion of G1
306
cells was gradually decreased with the formulations. The G1 cells were ~70% in untreated
307
control where as it decreased to less than 30% for BMSN/Ab treatment. The notable increase in
308
G2/M phase cell for BMSN/Ab exposure indicating its promising anticancer efficacy profile. It 14
309
can be expected that MSN played an important role in drug encapsulation and cellular
310
internalization whereas Ab-/scFv increased the targeting efficiency of delivery vehicle. Overall,
311
results were consistent with cellular uptake, cell cytotoxicity and apoptosis analysis.
312 313
Conclusions
314
In summary, we have demonstrated that Ab-/scFv-conjugated MSN could increase the
315
intracellular concentration of BVC and enhance the anticancer effect of active therapeutic agent
316
in ovarian cancer cell. The Ab-/scFv-conjugated MSN were prepared by the conjugation of
317
amine functional group of antibody of the carboxyl group of MSN. The resultant MSN was
318
nanosized, spherical shaped, and exhibited a controlled release phenomenon in pH 7.4
319
conditions. Furthermore, BMSN/Ab was found to increase the cellular uptake and intracellular
320
distribution of BVC in OVCAR-5 cancer cells. The Ab- conjugated MSN exhibited a superior
321
anticancer effect with profound apoptosis in cancer cells in a time- and concentration dependent
322
manner. Consistently, BMSN/Ab effectively inhibited the colony formation in transwell plate.
323
Finally, BMSN/Ab showed a notable increase in the proportion of cells in G2/M phase of cell
324
cycle indicating promising anticancer efficacy profile. Overall, Ab-/scFv-conjugated MSN might
325
provide an effective strategy for the delivery of Bevacizumab in ovarian cancers.
326 327
Competing interests
328
The authors report no conflict of interest in this work.
329 330
Acknowledgement 15
331 332 333
This research was supported by the National Natural Science Foundation of China (No: 81372778)
334
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Figure captions
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Figure 1: Illustration of preparation of antibody-conjugated mesoporous silica nanoparticles
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(MSN). The amine group of antibody reacted with the carboxyl functional group of MSN.
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Figure 2: (a) Dynamic light scattering measurements of size distribution for BMSN/Ab (b)
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Transmission electron microscopic image of BMSN/Ab.
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Figure 3: In vitro drug release profile of BMSN and BMSN/Ab in phosphate buffer saline (pH
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7.4) at 37°C. Results are expressed as means ± the standard error from three independent
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experiments.
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Figure 4: Cellular uptake of BMSN and BMSN/Ab in human ovarian cancer cell. The cellular
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uptake was determined using flow cytometer. Rhodamine B was used as a fluorescent probe. 19
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Figure 5: Intracellular distribution of BMSN and BMSN/Ab in human ovarian cancer cell. Cells
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were stained with lysotracker green to stain lysosome and DAPI to stain nucleus. The cells were
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treated with respective formulation for 2h and observed using a confocal microscope.
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Figure 6: (a,b) Cytotoxicity of free BVC, BMSN, BMSN/Ab in OVCAR-5 cancer cells. Cells
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were treated with respective formulations and incubated for 24 and 48h, respectively. The
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cytotoxicity was determined using MTT assay. Results are expressed as means ± the standard
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deviation from three independent experiments. (c) Annexin-V/PI based apoptosis analysis of
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cancer cells. (d) Western blot analysis of cancer cells.
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Figure 7: Colony formation capacity of OVCAR-5 cancer cells. The cells were treated with free
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BVC, BMSN, BMSN/Ab and colonies were then stained in crystal violet (0.5%).
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Figure 8: Cell cycle analysis of OVCAR-5 cancer cells following the treatment of free BVC,
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BMSN, BMSN/Ab. The cells were treated with 1 µg/ml equivalent of drug and incubated for 24h
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before the analysis.
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