Accepted Manuscript Gold-Nanorods-siRNA Nanoplex for Improved Photothermal Therapy by Gene Silencing Bei-Ke Wang, Xue-Feng Yu, Jia-Hong Wang, Zhi-Bin Li, Peng-Hui Li, Huaiyu Wang, Li Song, Paul K. Chu, Chengzhang Li PII:
S0142-9612(15)00921-7
DOI:
10.1016/j.biomaterials.2015.11.025
Reference:
JBMT 17202
To appear in:
Biomaterials
Received Date: 1 August 2015 Revised Date:
26 October 2015
Accepted Date: 12 November 2015
Please cite this article as: Wang B-K, Yu X-F, Wang J-H, Li Z-B, Li P-H, Wang H, Song L, Chu PK, Li C, Gold-Nanorods-siRNA Nanoplex for Improved Photothermal Therapy by Gene Silencing, Biomaterials (2015), doi: 10.1016/j.biomaterials.2015.11.025. 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.
ACCEPTED MANUSCRIPT
Gold-Nanorods-siRNA Nanoplex for Improved Photothermal Therapy by Gene Silencing
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Bei-Ke Wanga,1, Xue-Feng Yub*, Jia-Hong Wangc, Zhi-Bin Lib, Peng-Hui Lib,e, Huaiyu Wangb, Li Songd, Paul K. Chue, Chengzhang Lia*
The State Key Laboratory Breeding Base of Basic Science of Stomatology,
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a
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Hubei-MOST & Key, Laboratory of Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan 430079, P. R. China b
Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, P. R. China
d
School of Physics and Technology, Wuhan University, Wuhan 430072, P. R. China
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c
Department of Stomatology, The Second Affiliated Hospital to Nanchang University, Nanchang 330006, P. R. China
Department of Physics and Materials Science, City University of Hong Kong, Tat
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e
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Chee Avenue, Kowloon, Hong Kong, China
*Corresponding authors: Tel: +86-87686212; Fax: +86-87873260 (C. Li) E-mails:
[email protected] (C. Li);
[email protected] (X. F. Yu)
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ACCEPTED MANUSCRIPT Abstract
Nanomaterials-mediated photothermal therapy (PTT) often suffers from the
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fundamental cellular defense mechanism of heat shock response which leads to therapeutic resistance of cancer cells and reduces the therapeutic efficacy.
Herein, a
gold nanorods (GNRs)-siRNA platform with gene silencing capability is produced to After surface modification, the GNRs show the ability
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improve the PTT efficiency.
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to deliver siRNA oligos targeting BAG3 which is an efficient gene to block the heat-shock response.
The synthesized GNRs-siRNA nanoplex exhibits excellent
ability in the delivery of siRNA into cancer cells with high silencing efficiency which is even better than that of commercial Lipofectamine 2000.
The in vitro and in vivo
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studies demonstrate the ability of the GNRs-siRNA nanoplex to sensitize the cancer cells to PTT under moderate laser irradiation by down-regulating the increased BAG3 expression and enhancing apoptosis.
The GNRs-siRNA mediated PTT has large
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potential in clinical cancer therapy due to the elimination of therapeutic resistance and It also
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enhanced photothermal therapeutic efficacy by means of gene silencing. suggests an efficient platform for gene delivery and controllable gene therapy.
Keywords: gold nanorods; photothermal therapy; RNA interference; oral cancer; biomedical applications 2
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TOC Graphic
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ACCEPTED MANUSCRIPT 1. Introduction
Nanomaterials-mediated photothermal therapy (PTT) by means of near-infrared Owing to small light
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(NIR) illumination is an emerging tool in cancer therapy.
scattering and absorption from intrinsic chromophores in tissues, NIR light can penetrate tissues with sufficient intensity and high spatial precision [1-3]. Therefore,
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PTT treatment provides an efficient approach to convert photon energy into cytotoxic
surgery is difficult [1,4,5].
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heat to destroy cancer cells with high selectivity, especially in vital regions where Compared with conventional cancer treatment
approaches, PTT with highly localized heat delivery is minimally invasive and rapid and can be combined with chemotherapy and drug/gene delivery.
Despite these In
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advantages, the full potential of PTT is still hindered by some challenges.
particular, to achieve sufficient heating for complete killing of cancer cells, a high-power laser or large agent concentration has been suggested [6-8]. However, a
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high laser power or agent concentration produces safety risks including uncertain
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cytotoxicity, unnecessary morbidity due to collateral damage, and lack of patient tolerance at a high temperature [9-11].
It is especially true for some special cancer
such as head and neck cancer due to their special anatomic sites, vital biological functions, and cosmetic requirements [12].
Therefore, it is imperative to develop a
general strategy to improve the PTT efficiency without a high laser power or large agent amount. Heat shock response is a mechanism to prevent cancer cells from hyperthermia 4
ACCEPTED MANUSCRIPT and has been shown to undermine the therapeutic efficacy of thermal therapy due to their cytoprotective and antiapoptotic effects, termed as thermoresistance [13-15]. Recent studies indicate that PTT can trigger the heat shock response in cancer cells
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consequently decreasing the therapeutic efficacy by suppressing apoptosis [16-19]. Several families of heat shock proteins (such as HSP70) and BAG3 (Bcl-2 associated athanogene domain 3, also known as Bis and CAIR) induced by the heat shock
The application of RNA interference to cancer
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role in the thermoresistance [20-23].
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response exert central cytoprotective effects preventing cell death and play a major
therapy has recently attracted attention as a promising therapeutic modality [24,25]. By interfering with the expression of specific genes, the small-interfering RNA (siRNA) acts as an effective vehicle in RNA interference thereby suggesting a
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possible strategy to inhibit the heat shock response and renders the cancer cells more susceptible to PTT by silencing the expression of HSPs or BAG3. Among the various nano-agents for PTT, gold nanorods (GNRs) with the unique
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surface plasmon resonance (SPR) bands have been studied extensively on account of
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their strong NIR absorption and high photothermal conversion efficiency [26], and PTT mediated by GNRs has been suggested to be a promising treatment for oral cancer [27].
Moreover, possessing a large surface area to volume ratio and being
easily and controllably surface functionalized, GNRs are attractive nanocarriers for different types of drugs to cells [28-31], photosensitizers [27,32-35], and small biomolecules [36,37]. The effectiveness of GNRs in a gene delivery system has been demonstrated in vitro and also preliminarily in vivo [38-45]. 5
It has further been
ACCEPTED MANUSCRIPT shown that GNRs can penetrate the blood brain barrier and silence expression of DARPP-32 in the delivery system for specific siRNAs into the neuron cells [46]. Owing to the versatility, GNRs can play an important role in a single multifunctional With regard to
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nanotherapeutic platform for combined PTT and gene delivery.
cancer therapy, it has shown that different kinds of synthesized multifunctional GNRs-siRNA nanoplex can be applied to treat different types of cancer such as breast
In these studies, GNRs-siRNA complexes not only function as nanovectors
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[51].
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cancer [38,47-49], pancreatic adenocarcinoma [50], as well as head and neck cancer
[52-54] for siRNA and chemotherapy agents delivery [55-57], but also act as photothermal or imaging agents for theranostic purposes (summarized in Table S1) [55-57].
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In this work, the GNRs-siRNA nanoplex is utilized in a therapeutic platform to introduce gene silencing technology to improve the PTT efficacy (see Scheme 1). The CTAB-GNRs are prepared by a seed-mediated growth method followed by surface
modification
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sequential
with
negatively-charged
poly(sodium
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4-styrenesulfonate) (PSS) and positively-charged poly(diallyldimethylammonium chloride) (PDDAC) to form the GNRs/PSS/PDDAC with cationic charges.
The
GNRs/PSS/PDDAC can complex with anionic charged siRNA oligos targeting BAG3 via electrostatic interaction to form the GNRs-siRNA nanoplex.
The nanoplex
shows the ability to silence the increased BAG3 expression induced by a high temperature both on the mRNA and protein levels and produces good NIR-activated photothermal properties.
Their ability to silence the increased BAG3 expression 6
ACCEPTED MANUSCRIPT attenuates the heat shock response induced by PTT sensitizing the cancer cells to easier PTT-mediated cell death.
Our in vitro and in vivo studies demonstrate the
ability of the GNRs-siRNA nanoplex to overcome the thermoresistance during PTT
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and produce the maximal tumoricidal effects with minimal damage to normal tissues.
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platform.
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Scheme 1. Schematic illustration of the design of GNRs-siRNA in the improved PTT
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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Materials The Dulbecco's modified Eagle's medium (DMEM) was obtained from Hyclone
USA).
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(UT, USA) and fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide),
serum-free
medium
(opti-MEM),
(4 ′ ,6-diamidino-2-phenylindole),
DAPI
The BAG3 siRNA and fluorescently labeled siRNA
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Invitrogen (Carlsbad, CA).
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lipofectamine 2000 (lipofectamine), and Trizol reagent were purchased from
(BAG3 siRNAFAM) were synthesized by GenePharma (Shanghai, China). sequences
were
as
follows:
AAGGUUCAGACCAUCUUGGAA-3’.
BAG3
siRNA:
The 5’-
The primary antibodies against human
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BAG3 were obtained from proteintech (Wuhan, China), HSP27, HSP60, HSP70, and HSP90 were purchased from ABclonal (Wuhan, China), cleaved PARP was purchased from Cell Signaling Technology (Danvers, MA, USA), and GAPDH was
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purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Chloroauric acid
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(HAuCl4·4H2O, 99.99%), sodium chloride (NaCl, 96.0%), and hydrochloric acid (HCl, 36–38%) were bought from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).
Hexadecyltrimethylammonium
bromide
(CTAB,
99.0%),
sodium
borohydride (NaBH4, 96%), silver nitrate (AgNO3, 99.8%), L-ascorbic acid (99.7%), Poly(sodium
4-styrenesulfonate)
(PSS,
MW
~70,000
g/mol),
and
Poly(diallyldimethylammonium chloride) solution 20 wt% in H2O (PDDAC, MW 100,000~200,000 g/mol) were obtained from Aldrich (America). 8
All the chemicals
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Ultrapure water with a resistivity of
about 18.25 MΩ·cm was used as the solvent in the experiments.
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2.2 Synthesis of GNRs-siRNA The GNRs were synthesized in an aqueous solution using a seed-mediated growth method as previously described [58].
For preparation of the gold seed
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particles with a size of 3-4 nm, 5 ml of 0.5 mM HAuCl4 were mixed with 5 ml of 0.2
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M CTAB and 600 µl of freshly prepared ice-cold 10 mM NaBH4 were immediately injected into the solution under vigorously stirring. to bright brown.
The color changed from yellow
The seed solution was left at least 2 h before use.
In the GNR
synthesis, 1.2 ml of 5 mM HAuCl4 and 15 µl of 0.1 M AgNO3 were added to 6 ml of
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0.2 M CTAB and then 15 µl of 1.2 M HCl and 720 µl of 10 mM ascorbic acid were added and gently swirled until the color changed from dark orange to colorless. Afterwards, 12 µl of the seed solution were added.
Finally, the GNRs were collected by
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mixed and left undisturbed for 8 h.
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centrifugation at 12,000 rpm for 15 min.
The supernatant was removed and
precipitate was resuspended in ultrapure water. estimated to be about 0.65 nM.
The resulting solution was gently
The GNRs concentration was
In the synthesis of GNRs-siRNA, GNRs were first
coated with PSS by the method previously reported [59].
2 ml of 10 mg/mL PSS
dispersed in 1 mM NaCl and 1 ml of 10 mM NaCl were added to 10 ml of GNRs and stirred for 1 h at room temperature.
A centrifugation cycle of 10,000 rpm for 10 min
was performed to remove the excess PSS and NaCl, obtaining PSS-coated GNRs 9
ACCEPTED MANUSCRIPT (GNR/PSS).
The PDDAC was further coated using a similar method.
The
prepared cationic GNRs/PSS/PDDAC (GNRs) were mixed with different amounts of siRNAs by pipetting and incubated for 30 min at room temperature before the
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operation to form the nanoplex electrostatically.
2.3 Characterizations
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Transmission electron microscopy (TEM) was performed on a JEOL 2010 (HT) The zeta
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transmission electron microscope at an accelerating voltage of 200 kV.
potentials of the samples were determined on a Zeta sizer (Nano ZS90, Malvern Instruments, UK) at 25 ○C and the absorption spectra were taken on a TU-1810 UV-Vis-NIR spectrophotometer (Purkinje General Instrument Co. Ltd. Beijing,
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China).
2.4 Agarose gel electrophoresis
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The GNRs-siRNA nanoplexes with different GNRs/siRNA ratios were prepared
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by addition of the 1.5 µg BAG3 siRNA to the assigned amounts of 0.65 nM GNRs solution (ranging from 0-27 µl) for 30 mins at room temperature.
After incubation,
the RNase free water was added to the GNR-siRNA nanoplexes with different binding ratios to form the sample solution (30 µl total).
The solutions were centrifuged and
the supernatants were loaded onto 1% agarose gels containing ethidium bromide (0.5 mg/ml) in the 1 × TAE buffer (Tris-acetate-EDTA buffer) and electrophoresed at 100
10
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The gel was imaged under UV light on a GelDoc 2000 imager system
(Bio-Rad, Munich, Germany).
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2.5 Photothermal conversion measurement The photothermal efficiency was measured on a homemade setup as described A 1 cm quartz cuvette containing 2 ml of the sample was covered
with a foam cap.
The cuvette was clamped on the top part above the sample surface
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previously [27].
stirrer.
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and the bottom of the cuvette was kept at approximately 0.5 cm above the magnetic A fiber-coupled continuous semiconductor diode laser (810 nm,
KS-810F-8000, Kai Site Electronic Technology Co., Ltd. Shaanxi, China) with a power density of 2.7 W·cm-2 and beam diameter of approximately 0.5 cm illuminated A digital thermometer (TX3001, Xintengxing, Wuhan, China) was used
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the cuvette.
to monitor the temperature change.
The head of the thermometer was completely
submerged in the solution and carefully prevented from direct illumination by the Each sample in the cuvette was irradiated for 20 min under rigorous stirring
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laser.
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and the temperature was recorded per 30 s.
2.6 Cell culture
The human oral squamous cell carcinoma cell line Cal-27 (CRL-2095, ATCC)
was obtained from the Shanghai Research Institute of Stomatology, Affiliated Ninth People’s Hospital, Shanghai Jiaotong University, Shanghai, China.
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The cells were
ACCEPTED MANUSCRIPT maintained in DMEM supplemented with 10% FBS in a humidified atmosphere of 5% CO2 at 37 °C.
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2.7 Selection of the moderate laser irradiation power density for PTT To select the optimal light irradiation conditions for PTT, the cell viability of Cal-27 cells in the presence of GNRs irradiated with different laser power density was The GNRs were diluted in the complete DEME 8 × 103 Cal-27 cells/well were
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medium to achieve the concentration of 97.5 pM.
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assessed by the MTT assay.
seeded onto 96-well plates and incubated overnight.
Afterwards, the cells were
treated with the prepared GNRs medium for 24 h and irradiated by the 810 nm laser at power densities of 0 J·cm-2, 200 J·cm-2, 400 J·cm-2, 600 J·cm-2, 800 J·cm-2, and 1,000 The untreated cells were irradiated with a power density of 1000 J·cm-2.
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J·cm-2.
Afterwards, the cells were incubated in a complete medium up to 24 h. Finally, the cell
viability
was
determined
using
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) The formula, (ODtreated/ODcontrol) × 100%, was
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assay as previously described [60].
used to calculate the cell viability and the experiments were conducted in triplicate. The cells without any treatment serve as the viability control.
2.8 Heat shock response detection To confirm the occurrence of heat shock response in PTT at a moderate laser power, real-time PCR and western blots were performed to determine the expression 12
ACCEPTED MANUSCRIPT of HSP27, HSP60, HSP70, HSP90, BAG3 at different time points after the photothermal treatment.
Briefly, the Cal-27 cells were precultured on 24-well plates The cells were treated
with the 97.5 pM GNRs medium and incubated for 24 h.
After incubation, the
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at the confluence of 70% and allowed to adhere overnight.
treated cells were illuminated by the 810 nm laser with a power density of 600 J·cm-2. The irradiated cells were incubated in the complete medium and collected at 0 h, 2 h,
2.9 Cell uptake of GNRs-siRNA
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4 h, 8 h, and 12 h for real time PCR and western blots.
According to the fluorescence of BAG3 siRNAFAM (492 nm excitation and 518 nm emission), the cellular uptake of GNR-siRNA was analyzed using both
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fluorescence microscopy and flow cytometry.
In fluorescence microscopy, the
Cal-27 cells were seeded on glass coverslips at 40–50% confluency on 24-well plates and incubated overnight before transfection.
On the next day, the GNRs (97.5 pM),
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naked siRNAFAM (0.28 pM), and GNR-siRNAFAM nanoplexes (containing 97.5 pM
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GNRs and 0.28 pM siRNAFAM) were added to the complete DMEM medium and incubated with cells for 6 h, respectively.
The untreated cells served as the negative
control and cells transfected with BAG3 siRNAFAM by lipofectamine 2000 (invitrogen) served as the positive control following the manufacturer‘s instructions. Thereafter, the cells were rinsed twice with 1 × cold phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature.
After
washing with PBS buffer solution, the cells were mounted using an aqueous mounting 13
ACCEPTED MANUSCRIPT medium with DAPI (Invitrogen).
The cells were imaged under the the Leica
DM4000B fluorescence microscope (Leica, Nussloch, Germany).
In flow cytometry,
the Cal-27 cells were seeded on 6-well plates at a density of 5 × 105 cells/ml and
fluorescent microscopy.
After attachment, the cells were transfected for
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incubated under standard conditions.
Six hours after treatment, the cells were harvested and
analyzed on the FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes,
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NJ) at the excitation wavelength of 488 nm and emission wavelength range of
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515-545 nm and analyzed by the Cell Quest Software (Becton Dickinson).
2.10 Evaluation of gene silencing efficiency of BAG3 siRNA via different vectors: Before transfection, the Cal-27 cells were seeded on 24-well plates at the density 1.875 µg of the BAG3 siRNA
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of 5 × 104 cells/well and allowed to adhere overnight.
oligos were added to one aliquot of the 48.75 fmol GNRs in the eppendorf tubes and incubated for 30 min at room temperature to form the GNR-siRNA nanoplexes.
The
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GNRs,siRNA, and prepared GNR-siRNA were gently dipped into the complete
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DMEM medium making sure that the GNRs concentration and siRNA concentration in the final medium was 97.5 pM and 0.28 pM, respectively.
After aspirating the
media, the cells were treated with the prepared GNRs medium, siRNA medium, and GNRs-siRNA medium for 24 h according to the indicated treatment. cells were regarded as the control.
The untreated
Lipofectamine 2000 was used as the positive
control to transfect BAG3 siRNA and was performed according to the manufacturer’s instruction.
Briefly, 3 µl Lipofectamine diluted in 50 µl opti-MEM were mixed with 14
ACCEPTED MANUSCRIPT 5 µg of the siRNA diluted in 50 µl of the opti-MEM.
The mixture was incubated at
37 °C for 20 min and added to the wells producing a final siRNA concentration of 0.375 pM for the positive control.
Six hours after incubation, the medium was On the following
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exchanged by the 97.5 pM GNRs medium and incubated for 24 h.
day, all the groups except control were washed by PBS and irradiated by the 810 nm laser with a power density of 600 J·cm-2.
Eight hours after irradiation, all the cells
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were harvested for mRNA isolation and protein extraction and the expressions of
and western blotting, respectively.
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BAG3 and GAPDH on the mRNA and protein levels were analyzed by real time PCR
2.11 Analysis of cytotoxicity and apoptosis induced by combined treatment of
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GNRs-siRNA in vitro
The efficacy of the GNR-siRNA in PTT of cancer cells irradiated with a moderate-power laser was evaluated by the MTT assay, cell apoptotic detection, and
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western blotting for caspase-3.
In the MTT assay, the Cal-27 cells on 96-well plates
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were either left untreated or treated with fresh mediea containing siRNA oligos, GNRs, and GNRs-siRNA at the siRNA concentration of 0.28 pM and GNRs concentration of 97.5 pM for 24 h.
In the positive group, 0.375 pM siRNA delivered
by lipofectamine 2000 was added to the cells, incubated for 6 h at 37 °C, and then was replaced by the 97.5 pM GNRs medium.
810 nm laser irradiation with a power
density of 600 J·cm-2 was performed on most cells at the end of incubation. cells without treatment were used as a control. 15
The
After incubation for 24 h, the cell
ACCEPTED MANUSCRIPT viability was assessed by the MTT assay as previously described.
In analyzing the
apoptotic response, the cells were treated in the same manner as in the MTT assay and harvested for apoptosis and necrosis assay and western blotting for caspase-3.
The
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apoptosis and necrosis assay was performed as previously reported and finally analyzed on a flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using the Cell
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Quest software (Becton Dickinson) [27].
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2.12 Western blots
The harvested cells were lysed in M-PER (Pierce Chemical, Rockford, IL) containing Halt Protease Inhibitor cocktail (Pierce).
The denatured protein was
collected and the concentration was determined by the BCA Protein Assay Kit Subsequently, 20 µg of protein were separated using a 10% sodium
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(Pierce).
dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Billerica, MA).
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The membranes were blocked by 5% non-fat dry milk for 1 h under room temperature The blocked membranes were incubated overnight
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to prevent nonspecific binding.
at 4 °C with the primary anti-HSP27 (1:2000, A0240, ABclonal), anti-HSP60 (1:2000, A0969, ABclonal), anti-HSP70 (1:2000, A0284, ABclonal), anti-HSP90 (1:2000, A0365, ABclonal), anti-BAG3 (1:2000, 10599-1-AP, proteintech), cleaved PARP (1:1000, #5625, CST), and anti-GAPDH (1:5000, sc-365062, Santa Cruz) antibodies. The immunoblots were probed with Horseradish peroxidase-conjugated goat anti-rabbit or antimouse secondary antibodies (Pierce) for 1 h at room temperature. 16
ACCEPTED MANUSCRIPT Finally, the protein were visualized with a chemiluminescence kit (Pierce) and photographed.
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2.13 RNA extraction and real-time quantitative PCR The total RNA was isolated from the cells using TRIzol Reagent (Invitrogen). 1 µg total RNA from each sample was reverse transcribed to cDNA (20 µl) primed by
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oligo (dT) using M-MuLV reverse transcriptase (Fermentas, Glen Burnie, MD).
The quantitative PCR
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mRNA was analyzed quantitatively by real-time PCR.
The
reaction was carried out in a 20 µl reaction volume containing 2 µl cDNA as a template, 10 µl 2 × Maxima SYBR Green PCR master mix (Takara, Kyoto, Japan), and 0.8 µl of the primer mix (10 µm forward primer, 10 µm reverse primer).
All the
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reactions were conducted in triplicate on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA).
The expression was normalized to GAPDH
which was used as the endogenous control in each experiment.
The primer
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nucleotide sequences for PCR are presented in Table S2.
2.14 Establishment of Tumor Xenografts Model The male BALB/c nude mice (4-6 weeks old, 18-20 g, 40 animals) were
obtained from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). animals were housed in sterilized cages and a 12 h light/dark cycle.
The
All the
experiments were approved by the ethics committee of Wuhan University and were performed according to institutional animal use and care regulations. 17
2 × 107 Cal-27
ACCEPTED MANUSCRIPT cells in 200 µl PBS were subcutaneously injected into the right flank of mice to initiate tumor growth.
The longest and shortest dimension of tumors were detected
by a digital calipers.
After the longest dimension of the tumor reached 5 mm, the
2.15 Distribution of GNR-siRNA inside the tumor
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tumor bearing mice were chosen for in vivo treatment.
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Twelve mice with established tumors were randomly divided into 4 groups (n = In the
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3 per group): control group, siRNA group, GNRs group, GNRs-siRNA group.
control group, the mouse was intratumorally injected with sterilized 10 mM PBS solusion.
In the other three group, the mice were intratumorally injected with the
siRNAFAM dispersion (15.2 pM siRNAFAM), GNRs dispersion (5.3 nM GNRs), and
respectively.
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GNR-siRNAFAM dispersion (containing 5.3 nM GNRs and 15.2 pM siRNAFAM), The injectied volume of each type of dispersion was 200 µL per 50
mm3 tumor volume.
After 6 h post-administration, all the mice were sacrificed and The ex vivo tumors were subsequently
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the tumors were extracted immediately.
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embedded in the Tissue-Tek OCT (Sakura Finetek), frozen in liquid nitrogen, and cut into sections 5 µm thick on the CM1850 UV cryostat (Leica Microsystems).
The
sections were mounted with DAPI containing mounting solution (Invitrogen) and imaged by the Leica DM4000B fluorescence microscope (Leica, Nussloch, Germany) at excitation wavelengths of 405 nm (DAPI) and 488 nm (siRNAFAM).
The cell
uptake of siRNAFAM in vivo was quantified by calculating the percent of the cells that took up siRNA among the total cells in each group. 18
ACCEPTED MANUSCRIPT 2.16 In vivo photothermal treatment After the longest dimension of the inoculated tumors reached ~5 mm, the mice were randomized into 5 groups (n = 10 per group).
The mice in groups 3 and 4 were
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the naïve state and served as the control.
The mice in group 1 were kept in
intratumorally injected with the siRNA solution (15.2 pM , 200 µL per 50 mm3 tumor volume) and GNRs solution (5.3 nM, 200 µL per 50mm3 tumor volume), respectively.
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The mice in groups 2 and 5 were intratumorally injected with the prepared
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GNRs-siRNA solution (200 µL per 50 mm3 tumor volume) at the same concentration of siRNA and GNRs as in the group 3 and group 4, respectively.
After 24 h
post-injection, the mice in groups 3, 4, and 5 were irradiated by the 810 nm near-infrared laser (Gigaa Optronics Technology Co., Ltd., Wuhan, China) with a Aftewards, the mice were returned to the animal
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power density of 600 J·cm-2.
housing and 24 h later, three mice in each group were randomly selected for the BAG3 immunohistochemistry and TUNNEL assay.
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to measure the tumor growth volume.
The remaining mice were used
The largest and shortest tumor diameters were
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determined every other day with the digital caliper.
The formula: V = 1/2 (L × W2)
was used to calculate the tumor volume, where L and W were the length (longest diameter) and width (shortest diameter), respectively.
The increased tumor percent
(Ti) was calculated according to the formula: Ti (%) = (DP - DC) / DC × 100%, where DC and DP were the tumor volumes before and after treatment, respectively. At day 18 after the treatment, the mice were euthanatized and the tumors were extracted and photographed. 19
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2.17 Immunohistochemistry and TUNEL assay Twenty four hours after the treatment, the collected tumors were fixed in 4%
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paraffin or frozen in liquid nitrogen for subsequent BAG3 immunohistochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay.
In BAG3 immunohistochemistry, the slices were stained
A streptavidin-biotin-peroxidase complex with diaminobenzidine (DAB) as
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4 °C.
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with the rabbit anti-BAG3 polyclonal antibody (1:1000, Proteintech) overnight at
the peroxidase substrate was used to visualize immunoreactivity in accordance with the
manufacturer's
instructions
Peroxidase/DAB+, Rabbit/Mouse).
REAL
TM
Detection
System,
The stained sections were counterstained with
In the TUNEL assay, the slides were stained with the TUNEL
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hematoxylin.
(Dako
technique on the in situ cell death detection kit (7 sea pharmtech, Shanghai, China) according to the manufacture’s instructions.
The fluorescent images were acquired
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on the Leica DM4000B fluorescence microscope (Leica, Nussloch, Germany).
2.18 Statistical analysis All the data were expressed as means ± standard deviations from three
independent experiments.
The One-way ANOVA and Student-Newman-Keuls were
used in the statistical analysis with p < 0.05 being considered to be statistically significant.
20
ACCEPTED MANUSCRIPT 3. Results and Discussion The CTAB-coated GNRs with an aspect ratio of about 4.2 were synthesized by a seed-mediated growth method [58].
Negatively-charged PSS was introduced to the
PSS-coated
GNRs
(GNRs/PSS)
[59].
Afterwards,
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surface of the GNRs followed by absorption of positively-charged PDDAC onto the the
positively-charged
GNRs/PSS/PDDAC possesses the ability to bind with negatively-charged BAG3 The optimal binding ratio of GNRs to BAG3 siRNA is
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siRNA electrostatically.
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investigated by the agarose gel electrophoresis assay (see Figure S1).
Figure 1 shows the characteristics of the products in the GNRs-siRNA synthesis. As shown in Figure 1a, the original CTAB-GNRs exhibit two SPR bands: a weak transverse SPR (TSPR) band at about 510 nm and a strong longitudinal SPR (LSPR) A slight red-shift is observed from the LSPR band in both the
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band at about 808 nm.
PSS and PDDAC modification process.
Following complexation with BAG3 siRNA,
a further red-shift of about 4 nm in the LSPR band can be observed.
It is well
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known that the LSPR band peak shift from the GNRs results from the local refractive
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index around the GNRs, which is sensitive to the changes on the surface of the GNRs [61].
Here, the redshift of the LSPR band is probably due to binding of siRNA with
the GNRs/PSS/PDDAC forming GNRs/PSS/PDDAC-siRNA (named GNRs-siRNA). Similar results have been reported showing that binding of biological molecules can induce the LSPR band shift from the GNRs [62,63].
In the process, no obvious
broadening in the LSPR band can be found thus demonstrating good dispersion of the products.
The zeta potentials in Figure 1b present more evidence about successful 21
ACCEPTED MANUSCRIPT modification of the GNRs in different stages, i.e. +43.9 mV (CTAB-GNRs), -33.0 mV (GNRs/PSS), and +38.0 mV (GNRs/PSS/PDDAC).
After incubation of the cationic
GNRs/PSS/PDDAC substrate with anionic BAG3 siRNA, the zeta potentials reverse
GNRs in the final GNRs-siRNA.
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from positive (+38.0 mV) to negative (-24.2 mV), indicating binding of siRNA with The TEM image of GNRs-siRNA in Figure 1c
shows that the GNR morphology is almost unchanged and no aggregation can be
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observed, further confirming that the modification and conjugation processes do not
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affect the dispersion status of the GNRs.
Figure 1. Characteristics of the synthesis of GNRs-siRNA nanoplex. spectra
and
(b)
Zeta
potential
of
original
(a) Absorption
CTAB-GNRs,
GNRs/PSS/PDDAC, and GNRs/PSS/PDDAC-siRNA (GNRs-siRNA). 22
GNRs/PSS, (c) TEM
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(d) Temperature change curves of the aqueous solutions
containing CTAB-GNRs and GNRs-siRNA, and pure water irradiated by the 810 nm
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laser for 20 min.
The photothermal conversion ability of the GNRs-siRNA under 810 nm laser irradiation is examined by using the original CTAB-GNRs and pure water as the As shown in Figure 1d, the temperature of the CTAB-GNRs and
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control samples.
respectively.
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GNRs-siRNA increases by 38.1 oC and 37.5 °C after NIR light irradiation for 20 min, In contrast, the temperature of pure water only increases by 3.4 °C.
The results indicate that the GNRs-siRNA retains the good photothermal performance of the original GNRs.
The
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The heat shock response in oral cancer cells induced by PTT is investigated.
photothermal effect of the GNRs on Cal-27 cells is first assessed by the MTT assay (see Figure S2).
The cytotoxic effect of the GNRs on cancer cells is hampered at an
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irradiation power density of 600 J·cm-2 (3.3 W·cm-2 for 3 mins), suggesting that the
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heat shock response may be induced under these conditions.
The expression of heat
shock response related genes in the GNRs-treated cancer cells under NIR laser irradiation at 600 J·cm-2 is evaluated on both the transcription level (mRNA) and protein level by real time-PCR and western blots (see Figure 2).
As shown in Figure
2a, the mRNA expressions of HSP27, HSP60, HSP70, HSP90, and BAG3 increase significantly after irradiation for 2 h and then decrease gradually within 24 h.
BAG3
shows the largest relative increase in the mRNA expression in the Cal-27 cells and it 23
ACCEPTED MANUSCRIPT is almost ten times that of the control cells at 2 h.
The western blots results (see
Figure 2b) show that the protein expression of BAG3 is largely increased in the GNRs treated cancer cells at 4 h post irradiation, although the protein expression of HSP27 The results indicate
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shows the largest increase among the detected HSP proteins.
that the GNRs-mediated PTT induces obvious heat shock response in the cancer cells and BAG3 is the most obviously induced on the mRNA level and also increases
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largely on the protein level.
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BAG3 is chosen as the target gene for the following reasons.
First of all, among
the detected heat shock related proteins, the expression of BAG3 is strongly induced on the mRNA and increases greatly on the protein level, indicating its critical role in the heat shock response at least in PTT of oral cancer cells.
The RNA interference Here,
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approach is based on the endogenous degradation of mRNA of the target gene.
our results demonstrate that BAG3 mRNA is the most induced among all the selected genes.
Although the HSP27 protein expression is higher than BAG3 in the GNRs
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treated cancer cells after laser irradiation, BAG3 is selected as the silencing target Secondly, BAG3 as an antiapoptotic protein
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gene due to the better silencing effects.
is important to the pleiotropic effects [50-52].
Generally, BAG3 can bind with
HSP70 to inhibit the chaperone activity of HSP70 [50].
By means of BAG3 and
HSP70 multiprotein complex, many antiapoptotic modulators (such as anti-apoptotic Bcl-2 family members, Bcl-XL) as HSP70 client proteins can be stabilized and protected from proteasome-mediated degradation, thereby mitigating stress-induced apoptosis and sustaining cell survival to induce thermoresistance of the cancer cells. 24
ACCEPTED MANUSCRIPT There is emerging evidence that BAG3 also increases after chemotherapy to play a protective role against apoptosis and it therefore has been used as a potential therapeutic target to enhance chemotherapy [50].
In addition, BAG3 has been
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demonstrated to be involved in the stress response to X-ray along with HSP70 and p63 [51] indicating the role in the apoptosis resistance in radiotherapy.
Therefore, as
a versatile anti-apoptotic effector, silencing the expression or inhibiting the activity of
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BAG3 provides an effective strategy to improve the apoptotic response to Thirdly,
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hyperthermia [52], chemotherapy [53], and even radiotherapy [54].
elevated BAG3 expressions can be detected from some malignant tumors [52,55-57]. The specific expression and activity of BAG3 in neoplastic cells versus normal tissues have been observed [58].
In our study, a moderately higher BAG3 gene expression
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in the oral cancer cells compared to normal cells is observed (data not shown) and hence, BAG3 is a favorable therapeutic candidate to photo-sensitize cancer cells in
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order to improve the therapeutic efficacy.
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Figure 2. Heat shock response induced by PTT with GNRs.
(a) Relative mRNA
expression and (b) Protein expressions of HSP27, HSP60, HSP70, HSP90 and BAG3
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in Cal-27 cells for the indicated time after treatment with 97.5 pM GNRs for 24 h
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followed by irradiation by the 810 nm laser at a power density of 600 J·cm-2.
Cell imaging experiments are performed to investigate the uptake ability of Cal-27
cancer cells for GNRs-siRNA (see Figure 3a).
To visualize the GNRs-siRNA
nanoplexes by fluorescence microscopy, siRNA labeled with the green fluorescent FAM (siRNAFAM) is used to conjugate the GNRs.
The bare GNRs
(GNRs/PSS/PDDAC) and siRNAs are the negative control samples whereas the lipo-siRNAFAM (siRNAFAM delivered by lipofectamine) are the positive samples. 26
ACCEPTED MANUSCRIPT Although GNRs are well-known fluorescence quenchers, it has been demonstrated that attachment of fluorescent dyes through polyelectrolyte spacer can produce obvious fluorescence [38,46,49].
In the present study, the GNRs used for binding
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siRNAFAM are coated with PSS and PDDAC layer-by-layer, and the emission spectrum of FAM partly overlaps the weak TSPR band of the GNRs. obtained GNRs-siRNA nanoplexes can emit green fluorescence.
Therefore, the
As shown in Figure
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3, such green fluorescence can be observed from the cancer cells incubated with the
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GNRs-siRNAFAM for 6 h indicating effective delivery of siRNAFAM into the cells. The uptake of GNRs is manifested as particulates with a large thickness contrast in the GNRs and GNRs-siRNAFAM treated cells in the bright-field images. According to the merged images, the green fluorescence overlaps the location of the
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GNRs in the GNRs-siRNAFAM treated cells furnishing further evidence of delivery of siRNA by the GNRs into the cells.
As the control, the cells treated with siRNAFAM
alone only show weak green fluorescent signals and as the positive group, the
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lipo-siRNAFAM-treated cells show considerable green fluorescent dots.
Flow
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cytometry is employed to quantify the amount of siRNAFAM delivered to the cancer cells (shown in Figure 3b).
Compared to the untreated cells (blank group), about
87.6% of cells exhibit green fluorescence after incubating with the GNRs-siRNAFAM and it is comparable to that of cells treated with Lipo-siRNAFAM of about 84.9%. Since lipofectamine is a classical and recommended gene delivery vector, the results suggest that the GNRs are promising nanocarriers for the delivery of siRNA to cancer cells. 27
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Figure 3. Cell uptake of GNRs-siRNA by Cal-27 cells.
(a) Bright field, fluorescent,
and merged optical images of the untreated Cal-27 cells (denoting as blank) and cells
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incubated with GNRs, siRNA, GNRs-siRNA, and Lipo-siRNA, respectively.
The
siRNA is labeled with the green fluorescent FAM and nuclei are stained blue with (b) Flow cytometric analysis of green fluorescent FAM.
The results
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DAPI.
represent three independent experiments with mean ± SEM and **p