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Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs
Accepted Manuscript Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs Li Fan, Yongsheng Zhang, Fuli Wang, Qian Yang, Jiali Tan, Grifantini Renata, Hong Wu, Chaojun Song, Boquan Jin PII:
S0142-9612(15)00879-0
DOI:
10.1016/j.biomaterials.2015.10.069
Reference:
JBMT 17166
To appear in:
Biomaterials
Received Date: 9 September 2015 Revised Date:
20 October 2015
Accepted Date: 26 October 2015
Please cite this article as: Fan L, Zhang Y, Wang F, Yang Q, Tan J, Renata G, Wu H, Song C, Jin B, Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs, Biomaterials (2015), doi: 10.1016/j.biomaterials.2015.10.069. 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 Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs Li Fan1, 2, 3*, Yongsheng Zhang4*, Fuli Wang5*, Qian Yang6, Jiali Tan7, Grifantini Renata8,
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Hong Wu1†, Chaojun Song3‡, Boquan Jin3§
1
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Department of pharmaceutical analysis, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710032 2 Department of Physics, Chinese University of Hong Kong, Shatin, HongKong 3 Department of Immunology, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710032 4 Tangdu hosipital, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710038 5 Department of Urology, Xijing Hospital, Xi’an, Shaanxi, China, 710032 6 Institute of Materia Medica, School of Pharmacy, The Fourth Military Medical University, Xian, Shaanxi, China, 710032 7 Department of Orthodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University & Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, 510055, China. 8 Externautics SpA, Siena, SI, 53100, Italy
These authors contributed equally to this work. Corresponding author, Department of pharmaceutical analysis, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710032, email: [email protected] ‡ Corresponding author, Department of Immunology, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710032, email: [email protected] § Corresponding author, Department of Immunology, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710032, email: [email protected] *
†
ACCEPTED MANUSCRIPT List of abbreviations
Dox HCPT
Dimethyl sulfoxide phosphate buffered saline fast protein liquid chromatography 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide Doxorubicin 10-hydroxycamptothecin
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Abstract
N-hydroxysuccinimide Fetal bovine serum
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NHS FBS DMSO PBS FPLC MTT
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DCM UV-Vis GSH Mes EDC
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N2
Transmission electron microscopy Dynamic light scattering Tetraethyl orthosilicate (3-Aminopropyl) triethoxysilane Poly (ethylene glycol) 2-mercaptoethyl ether acetic acid Nitrogen Dichloromethane ultraviolet-visible Glutathione 2-(N-morpholino)ethanesulfonic acid 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
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TEM DLS TEOS APTES COOH-PEG5000-SH
To achieve active tumor targeting and sequential release of 3 drugs to a tumor site in
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one nanoparticulate system,
self-decomposable SiO2 nanoparticles modified by
3-aminopropyltriethoxysilane (APTS) as their inner structure were used to double load HCPT (in the NP core) and Dox (on the NP surface). Meanwhile, monoclonal antibodies (mAb 198.3) against the FAT1 antigen and Bcl-2 siRNA were conjugated onto PEGylated Au-PEG-COOH nanoparticles. The obtained drug-loaded SiO2 nanoparticles were coated with the Au-PEG-mAb.198.3/siRNA nanoparticles through electrostatic interaction to form the SiO2@AuNP sequential drug delivery system, which featured the controlled and
ACCEPTED MANUSCRIPT sequential release of siRNA, Dox and HCPT step by step to maximize its anticancer efficacy. The results revealed that the SiO2@AuNP sequential drug delivery system specifically targeted tumor cells and was internalize rapidly, followed by endosome escape and
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sequential drug release. Importantly, the sustainable release characteristics of SiO2 made the Tmax difference between HCPT and Dox approximately 8-12 h, and this enhanced the sensitizing efficiency of HCPT on Dox compared with co-administration. The in vivo
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antitumor results demonstrated that the tumor size after SiO2@AuNP treatment is 1/400
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compared with the saline control group and approximately 1/40 of the HCPT/Dox co-treatment group without any noticeable systemic toxicity.
Keywords: Self-decomposable nanoparticles, HCPT, Dox, Bcl-2, Sequential drug release
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Introduction
Multifunctional nanoparticulate drug delivery systems (NDDSs) have made a great impact on overcoming the limitations of conventional chemotherapies by active or passive
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targeting[1]. NDDSs combine at least two different functions, such as longevity and targetability[2], and are loaded with more than one drug or gene therapy-related material,
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such as antisense oligonucleotides or small interfering RNAs (siRNAs)[3]. To lengthen the circulation time, NDDSs are usually coated with hydrophilic polymers, such as poly (ethylene glycol) (PEG)[4]. For example, PEG modification stabilized nanorods, enabling long-lasting circulation in blood due to a stealth character[5]. After that, active targeting can be achieved by attaching targeting ligands, such as PEGylated doxorubicin (Dox)-loaded liposomes with
ACCEPTED MANUSCRIPT HER2 antibodies attached, successfully targeted HER2-overexpressing SK-BR3 cells in mice [6]
.
Although NDDSs can preferentially accumulate in tumors, drug resistance is another
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major problem in current cancer treatment. Consequently, many current research studies aim to combine chemotherapy with RNA interference (RNAi) to specifically knock down the
to the anti-cancer drug
[7]
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expression of the drug resistance gene, making resistant cells transiently become sensitized . For example, the sequential delivery of siRNA and Dox by the
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PEI-GO nanocarrier shows a synergistic effect, which leads to significantly improved chemotherapy efficacy [8]. Among several combinations of chemotherapeutics, Bcl-2 specific siRNA has been considered an attractive choice to avoid the Dox-activated cellular defense mechanism of anti-apoptosis, which prevents cell death
[9, 10]
. Moreover, a co-delivery
reported
that
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strategy has been proposed to achieve the synergistic effect for cancer therapies. It was co-delivery
of
Dox
and
10-hydroxycamptothecin
(HCPT)
using
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MPEG-b-PAMAMG2.5 NPs significantly increased the in vitro cytotoxicity in resistant cancer cells and improved tumor growth inhibition
[11]
. Moreover, in our recent study, the
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administration of HCPT and Dox at 12 h intervals has better synergistic effects on Colo 205 cancer cells.
Therefore, to realize the multiple functions mentioned above, scientists have developed nanoparticles that deliver one or two chemotherapy drugs, but it has been a huge challenge to integrate these functions in one nanoparticle, especially the step-by-step release of multiple drugs from a single nanoparticle scaffold
[12]
. This has, until now,
ACCEPTED MANUSCRIPT hampered the development of smart and versatile “all-in-one” nanoparticles for tumor treatment.
In our previous work, we built up self-decomposable nanoparticles based on silicon
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dioxide that could realize controlled release and carrier decomposition simultaneously
[13]
.
They can also double load two drugs via different loading mechanisms, i.e., “grown-in” and
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“absorbed”. The first load of the NPs was realized by introducing the first molecules during the SiO2 NP growth, and the second load was carried out by soaking the loaded NPs in a
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solution containing the second molecules. In this study, self-decomposable SiO2 nanoparticles modified by 3-aminopropyltriethoxysilane (APTS) as their inner structure were used to double load HCPT (in the NP core) and Dox (on the NP surface). Meanwhile, monoclonal antibodies (mAb 198.3) against the FAT1 antigen and Bcl-2 siRNA were
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conjugated onto PEGylated Au-PEG-COOH nanoparticles. The obtained drug-loaded SiO2 nanoparticles were coated by Au-PEG-mAb.198.3/siRNA nanoparticles through an
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electrostatic interaction to form the SiO2@AuNP sequential drug delivery system, which featured the controlled and sequential release of siRNA, Dox and HCPT step by step to
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maximize its anticancer efficacy (Fig. 1). Results and Discussions
HCPT and Dox double-loaded SiO2 nanoparticles (HCPT-SiO2-Dox) were synthesized according to our previous method[13] via two different loading mechanisms. HCPT was first “grown-in” during the preparation of the SiO2 NPs[13]. After that, HCPT-SiO2 NPs were washed in ethanol and MilliQ water to remove ungrown-in HCPT. In such NPs, the drug was most concentrated in the center of the nanoparticle with a radial concentration gradient,
ACCEPTED MANUSCRIPT which made the NPs self-decomposable. APTS as a conventional silane coupling agent was then reacted with purified HCPT-SiO2 NPs to form a positively charged surface for further adsorption. Dox was then loaded on the NP surface by absorbing. Schematics to illustrate
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the configuration of the HCPT-SiO2-Dox NPs, Au-PEG-198.3/siRNA NPs and SiO2@AuNP system were shown in Fig. 2a, c, and e, respectively.
By TEM measurement, the size of the HCPT-SiO2-Dox NPs with spherical morphology is
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approximately 20-50 nm (Fig. 2b). Au-PEG-198.3/siRNA NPs was approximately 2-5 nm, and
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the size distribution was highly monodisperse (Fig. 2d). Au-PEG-198.3/siRNA NPs were attached on the HCPT-SiO2-Dox NP surface via electrostatic interactions. The obtained SiO2@AuNP system showed sphere morphology with diameter in 50 nm. Gold nanoparticles (Au-PEG-mAb198.3/siRNA) can be clearly observed as small dots on the SiO2 NP surface (Fig.
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2f).
Flow cytometry analysis demonstrated that the SiO2@AuNP system could bind to native FAT1 molecules on the surface of Colo205 cells (Fig. 1 in [23]), and this result was
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consistent with our previous work on free mAb198.3_Cy5 and Au-PEG-(Cy5) _mAb198.3 [14].
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This indicated that the SiO2@AuNP system has the FAT1 targeting capability. After binding to membranes, SiO2@AuNP started its endocytosis process involving the accumulation in membrane-sunken vesicles, transfer to early and late endosomes, and fusion to become late endosomes/lysosomes. Usually, nanoparticle-based drug delivery systems remain trapped inside endosomes/lysosomes, followed by degradation or exocytosis. Therefore, endosome/lysosome escape is of crucial importance to increase the therapeutic efficacy for nanoparticle-based drug delivery
[15]
. In our study, the
ACCEPTED MANUSCRIPT time-dependent co-localization of SiO2@AuNPs and lysosomes (stained by LysoTracker) was investigated by confocal laser scanning microscope as well as TEM to demonstrate the particle intracellular fate, especially whether and when these particles escape endocytic
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vesicles to access the cytosol. It can be seen from Fig. 3a-h that SiO2@AuNPs completed the internalizing process and entered the endosomal vesicles. The high overlapping rate (over 78.29%) of SiO2@AuNPs and lysosomes/endosomes at 6 h demonstrated that the
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endocytosis process of SiO2@AuNP occurred by phagocytosis and the nanoparticles were
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located in the endocytic vesicles. After that, the particles gradually escaped from the endosomes/lysosomes, presenting decreasing overlapping rates in the following 6 h. After 24 h of incubation, the diffusion of the bright, spot-shaped green fluorescence revealed that the endosome/lysosome membranes were disrupted, and there is little overlap between
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red and green fluorescence (25.93%), demonstrating that most of the particles have translocated into the cytoplasm. From the TEM results (Fig. 3i-l), we also found the clear progress of SiO2@AuNPs from endocytosis to endosome/lysosome escape. Most of the
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SiO2@AuNPs entered clathrin-coated pits, which evolved to primary endocytic vesicles at 3
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h incubation (Fig. 3i). Gradually, these primary vesicles fused together or with lysosomes (Fig. 3j). Discontinuous membranes of endocytic vesicles followed (Fig. 3k), which subsequently promoted SiO2@AuNP escape. After 24 h incubation, scattered SiO2@AuNPs were easily found in the cytoplasm (Fig. 3l) demonstrating the successful escape of the particles from the lysosomes/endosomes. It is worth noting that after 24 h of incubation, the SiO2@AuNPs became hollow structural particles, revealing that almost all the drugs grown in or absorbed on had been released. This result was consistent with drug release
ACCEPTED MANUSCRIPT profiles in cytoplasm. The high contrast margin created by the AuNPs demonstrated that SiO2@AuNPs still remained whole structures before self-decomposing the SiO2. That is, siRNA on AuNPs was also translocated into the cytoplasm, which is the premise of knocking
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down the Bcl-2 protein in the cytoplasm.
The drug release behavior in the cytoplasm of the SiO2@AuNP system could be seen as
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two sequential release parts of the outer AuNP layer and inner self-decomposable SiO2 core. On the surface of the outer AuNP layer, siRNA and mAb198.3 were simultaneously attached
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via thiol-terminated PEG chains. In our previous study, we visually investigated the targeting efficiency of AuNPs in vivo. Free gold nanoparticles without any modification or isotype control antibodies attached showed weak fluorescence, mainly due to the enhanced permeability and retention effect [16](EPR effect) of AuNPs. The fluorescence intensity of the
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mAb198.3-attached AuNP treatment group is higher than that of free AuNPs and isotype-attached AuNPs [14].
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After endocytosis, mediated by mAb198.3, the siRNA release process was illustrated by Fig. 4A and demonstrated by Fig. 4B-C. SiRNA was released by the place exchange of [17]
(Fig. 4A). Different band shifts on the denatured polyacrylamide gel
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glutathione (GSH)
page (Fig. 4B) demonstrated the process of GSH-triggered siRNA release. The bands place-exchanged by GSH run to the same level as HS-siRNA, indicating that the siRNA release was accomplished in the presence of GSH. As shown in the western blot result (Fig. 4C), the Bcl-2 protein in the colo-205 cells has been knocked down approximately 50% after the administration of Au-PEG-siRNA/mAb198.3. The release behavior of the inner self-decomposable SiO2 NPs was also investigated in
ACCEPTED MANUSCRIPT Mes buffer (pH 5.5, at 37 C, simulating the endosome/lysosome pH environment) and in the 。
cytoplasm of colo-205 cells. The UV absorption spectra were taken from the supernatant to measure the amount of HCPT and Dox released at certain time intervals. Fig. 5A and 5B
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plotted the cumulative release profiles of HCPT and Dox in Mes buffer (pH 5.5) and cytoplasm. In Fig. 5A, after an initial quick rise in the amount of Dox released at 12 h, an obvious plateau appeared in the profile and lasted until 48 h. The release of HCPT took 24 h
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to reach the plateau, and a stable HCPT concentration level was maintained afterward. In
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the cytoplasm, the release characteristics of Dox and HCPT have similar Tmax intervals (12 h) to that in Mes buffer; however, both Dox and HCPT present slower release profiles (Fig. 5B). Together with the drug release, the continuous morphological evolution of the inner self-decomposable SiO2 NPs was also observed using TEM (Fig. 2 in [23]). Most of the NPs
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remained intact at the first 4 h, while rough edges appeared after 6 h of immersion in deionized water. This is mainly due to the release of Dox absorbed on the NP surface. With the elongation of the immersed hours, obvious hollow features gradually appeared in the
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center of the NPs due to the release of the “grown-in” HCPT. Such uniform center-hollow
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features continued to enlarge over 48 h, leaving a spherical, thin, discontinuous shell. It was reported that co-delivered Dox and HCPT significantly increased the in vitro cytotoxicity and improved tumor growth inhibition in vivo [11]. HCPT is considered one of the most promising anticancer drugs that target the nuclear enzyme topoisomerase I. In our study, we also investigated the synergetic effects of HCPT and Dox in colo-205 cells with an MTT assay. It was demonstrated by a drug release study that the Tmax between HCPT and Dox is approximately 8-12 h. The results hinted that the synergetic effect of HCPT on Dox
ACCEPTED MANUSCRIPT should be investigated upon co-administration and after 12 h. Indeed, in Colo 205 cells, the cytotoxicity of Dox was significantly potentiated by HCPT co-treatment simultaneously and after 12 h (Fig. 4D). The potentiation of Dox cytotoxicity was most pronounced at 0.5 μg/mL,
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where HCPT treatment at 12 h caused a double reduction of the cell viability (Fig. 6). Similarly, a moderate HCPT-induced potentiation of Dox cytotoxicity by co-treatment was observed at 5 μg/mL (Fig. 6).
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Based on the results in vitro above, we evaluated the antitumor efficacy of SiO2@AuNP
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sequential drug delivery system in a xenograft mouse model by i.v. injection. The SiO2@AuNPs significantly delayed subcutaneous colo-205 tumor growth as demonstrated by tumor weight at a dose of 10 mg/kg every three days with i.v. injection (Fig. 7A-B). We also tested the effect of both the outer AuNP layer and the inner SiO2 layer in different
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combinations with mAb198.3/siRNA used at the same dose; they also delayed the tumor growth at different levels (Fig. 7A-B). The delaying effect on the tumor growth was primarily due to three factors: the targeted therapy of mAb198.3, Bcl-2 protein inhibition of siRNA
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and synergistic effects of HCPT and Dox. After the outer AuNP (Au-198.3/siRNA) treatment,
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the average tumor size was less than 1/4 of that in the saline control group. The tumor inhibitory rate of the HCPT-SiO2-Dox treatment group was similar to the co-administration of HCPT and Dox after 12 h, and this result is consistent with the in vitro findings that the cell viability of HCPT and Dox in the 12 h treatment group was much lower than that of the free Dox group and HCPT+Dox co-treatment group. This result also demonstrated that co-delivered HCPT and Dox using a self-decomposable SiO2 particle system could maximize the synergy of HCPT and Dox. At the end point of the study with SiO2@AuNPs, the tumor
ACCEPTED MANUSCRIPT weight decreased 400-fold. On the other hand, changes in body weights were also investigated to evaluate the safety of the SiO2@AuNPs. No significant decreases in the body weights were observed in all the tumor-bearing mice compared with the saline control from
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4 weeks after initial administration (Fig. 7C). These results indicated that the SiO2@AuNP treatment at 10 mg/kg exhibited a remarkable anticancer effect and did not lead to marked toxicity in the experimental mice.
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Conclusion
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Herein, tumor-homing and biocompatible SiO2@AuNP-based nanoparticles can serve as a platform vehicle to load both chemotherapeutics and siRNA to achieve optimal efficacy. Additionally, SiO2@AuNPs for the sequential delivery of Bcl-2 siRNA, Dox, and HCPT may maximize antitumor efficacy. Significantly enhanced antitumor efficacy was achieved due to
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both the targeting function and the programmed sequential release of different drugs. Additionally, such a nanoparticle drug system was also found to largely reduce the systemic toxicity.
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Experimental section
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Synthesis and Characterization of Dox and HCPT dual loaded SiO2 NPs (SiO2-HCPT/Dox), Au-PEG-198.3/siRNA NPs and SiO2@AuNP NPs sequential drug delivery system The SiO2@AuNP NP sequential drug delivery system is composed of inner dual loaded SiO2 NPs and an outer Au-PEG-198.3/siRNA layer. The inner Dox and HCPT dual loading SiO2 NPs were prepared by first encapsulating HCPT, then absorbing Dox. The SiO2-HCPT/Dox was synthesized following the method by Zhang et. al. [13] with some modifications. In brief, HCPT (4 mg) was dissolved in ethanol-aqueous ammonia solution (aqueous ammonia, 3.4
ACCEPTED MANUSCRIPT mL, 25%, v/v was mixed with 75 mL ethanol), followed by adding 200 μL of TEOS (Tetraethyl orthosilicate). The solution was stirred for 24 h in darkness and then centrifuged at 15,000 rpm for 10 min. The precipitate (SiO2-HCPT NPs) was purified by washing and centrifuged at
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15,000 rpm 3 times to remove free HCPT and excess TEOS. The purified SiO2-HCPT NPs (1 mg) were then suspended in ethanol (3 mL), followed by adding (3-Aminopropyl) triethoxysilane (APTES) (40 μL). The mixture was stirred in a water bath (60°C) for 3 hours.
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The obtained NH2-SiO2-HCPT NPs were purified by washing and centrifuged at 12,000 rpm 3
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times. Finally, NH2-SiO2-HCPT NPs (10 mg) were suspended in Dox solution (2 mg/mL, 1 mL) overnight in darkness to absorb Dox on the particle surface. The obtained SiO2-HCPT/Dox NPs were then centrifuged to remove unloaded Dox.
The outer Au-PEG-198.3/siRNA layer was synthesized according to the conventional
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Brust-Schiffrin method [18], a place exchange reaction and a carbodiimide reaction. In brief, 2 nm thiol-derivative gold nanoparticles (C5-Au) were first synthesized. An aqueous solution of hydrogen tetrachloroaurate (1 g, 250 ml) was mixed with a solution of
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tetraoctylammonium bromide in toluene (2.1 g, 500 ml). The two-phase mixture was
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vigorously stirred until all the tetrachloroaurate was transferred into the organic layer and C5SH (0.337 ml) was then added to the organic phase. A freshly prepared aqueous solution of sodium borohydride (2 g with a small amount of water to dissolve) was quickly added with vigorous stirring. After stirring overnight, the organic phase was separated, evaporated with a rotary evaporator and mixed with 400 ml ethanol to remove excess thiol. The mixture was kept at -18°C and the dark brown precipitate was filtered off and washed with ethanol many times. The mole ratio of HAuCl4: TOAB: C5SH: NaBH4=1:1.5:1.2: 20
ACCEPTED MANUSCRIPT Poly (ethylene glycol) 2-mercaptoethyl ether acetic acid (COOH-PEG5000-SH)-capped gold nanoparticles (Au-PEG-COOH) were prepared via a place exchange reaction. Briefly, 20 mg of C5-Au NPs and 80 mg of COOH-PEG5000-SH were weighed in two separate vials, and 5
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ml dry dichloromethane (DCM) was added to each of the vials. COOH-PEG5000-SH solution was added dropwise to the C5-Au NP solution and stirred for 2 days under N2 protection. The obtained Au-PEG-COOH was further washed with Hexane/DCM twice to remove free
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ligands, dried under reduced pressure and solubilized in distilled water. After 2 days of
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dialysis, the Au-PEG-COOH NPs were lyophilized and re-dissolved in MilliQ water. The attachment of siRNA to the Au-PEG-COOH was performed using the same place exchange protocols. Thiol-derivative siRNA was purchased from Guangzhou Ribobio CO., LTD. 10 mg of C5-Au NPs, 20 mg of COOH-PEG5000-SH, and 10 nmol thiol-derivative siRNA
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were used for the place exchange process. The obtained Au-PEG-siRNA NPs were lyophilized and re-dissolved in MilliQ water. Agarose gel electrophoresis was utilized to determine the siRNA and AuNP binding ratio. The results revealed that the molar ratio of
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AuNPs to siRNA molecules is approximately 1/2.5.
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The attachment of mAb198.3 to Au-PEG-siRNA NPs was performed using standard 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) coupling chemistry. The molar ratio of Au-PEG-siRNA NPs: EDC: NHS: mAb198.3 was 1:10:10:5. The carboxyl groups on the particle surface are first activated by reaction with EDC and NHS in 0.1 M MES buffer (pH 5.5) for 1 h. Then, the mAb198.3 attachment was performed by adding the concentrated activated nanoparticles to a dilute MES solution containing mAb198.3 at pH 5.5. The solution was mixed well and shaken gently overnight at
ACCEPTED MANUSCRIPT 4°C in the dark. After the reaction, Au-PEG-198.3/siRNA NPs were separated by fast protein liquid chromatography (FPLC) to remove free antibody and dialyzed to remove NHS and EDC. The mobile phase was MilliQ water at a flow rate of 5 mL/min. The ratio of mAb198.3
Spectrophotometer (Thermo Scientific) at 280 nm.
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conjugated onto AuNPs is approximately 1/3, which is determined by a NanoDrop 2000c
The SiO2@AuNP sequential drug delivery system was prepared by simply mixing
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Au-PEG-198.3/siRNA NPs with SiO2-HCPT/Dox NPs under stirring. Au-PEG-198.3/siRNA NPs
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rapidly binds to SiO2-HCPT/Dox NPs due to electrostatic interactions.
Au-PEG-198.3/siRNA NPs, SiO2-HCPT/Dox NPs and the SiO2@AuNP sequential drug delivery system were analyzed by ultraviolet-visible (UV-Vis) spectrum (HitachiU-3501 UV-Vis NIR spectrophotometer) and transmission electron microscopy (TEM) (PhilipsCM120)
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for the general morphology and size distribution.
Characterizations of the Drug Release and Carrier Decomposition Process The in vitro targeting efficiency of the Au-PEG-198.3/siRNA NP layer was determined
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by flow cytometry analysis in vitro. The targeting and internalizing efficacy in vivo were
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evaluated by a fluorescence imaging system (Cambridge Research & Instrumentation, Inc., Maestro EX) with Ex=640 nm. Glutathione (GSH) mediated thiol-siRNA release from Au-PEG-198.3/siRNA NPs were evaluated by denaturing polyacrylamide gel electrophoresis. A 10% polyacrylamide gel was used to visualize the siRNA release process. Au-PEG-198.3/siRNA NPs were incubated for 2 h with 5 mM GSH, and the gel electrophoresis was performed at 125 V. All siRNA bands were visualized using an AlphaImager ultraviolet imaging system (Biosciences).
ACCEPTED MANUSCRIPT The Bcl-2 silencing effect of Au-PEG-198.3/siRNA NPs on Colo-205 cells was studied by Western blot analysis using primary antibodies specific for Bcl-2 and secondary anti-rabbit IgG-HRP, followed by a visualization by enhanced chemiluminescence. Colo-205 cells (5×106)
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were resuspended in Nucleofector solution (Nucleofector kit V) with 3 μg Bcl-2 siRNA+freestyle Max, Au-PEG-siRNA or control siRNA+freestyle Max using the Amaxa Nucleofector apparatus and program U-16. Immediately after transfection, the cells were
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transferred into RPMI 1640 medium with 10% FBS and incubated for 48 hours.
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The drug release profiles of the inner SiO2-HCPT/Dox NP layer in water, 2-(N-morpholino)ethanesulfonic acid (Mes) buffer (pH 5.5) and medium with serum were determined by UV-Vis and fluorescence spectra. Equal amounts of SiO2-HCPT/Dox NPs (1 mg/mL) were dispersed in 10 mL deionized water, Mes buffer, or Medium with 10% fetal
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bovine serum (FBS), separately. Each sample was centrifuged at a different time point and the supernatant was separated by centrifugal filtration (molecular weight cut off: 30 000 Da, Millipore). The SiO2-HCPT/Dox NPs were dried and re-dispersed in deionized water. UV-Vis
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absorption and fluorescence spectra were taken from both re-dispersed particles solution
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and supernatants using a Hitachi U-3501 UV-Vis NIR spectrophotometer and Hitachi F7000 fluorescence spectrophotometer. The degradation of the SiO2 carrier was monitored by a morphology investigation using TEM. We also examined the drug release kinetics in the cytoplasm using a Colo-205 cell line, which was cultured in RPMI 1640 medium supplemented 10% heat-inactivated FBS, 1% streptomycin, and 1% penicillin. The cells were maintained in a standard cell culture incubator at 37°C in a humidified atmosphere with 5% CO2. All of the NPs were sterilized via
ACCEPTED MANUSCRIPT cobalt-60 (NPs in powder form) for 24 h and dispersed in the medium by ultrasonication for at least 20 min immediately before their introduction to the cells. Cells were seeded at initial densities of 5×104 cells/mL in 25 mm2 flasks (for TEM samples) or dishes (for confocal
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samples) with 2 ml of medium each and incubated for 24 h, and the medium was changed with NP medium. Different feeding time intervals were adopted, as specified in individual experimental results.
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Furthermore, due to the specific features of the siRNA, it could not pass through
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endosome membrane itself to locate in the cytoplasm after endocytosis, so the delivery system should provide a mechanism to escape the fate of endosomal-lysosomal degradation and ultimately facilitate the cytosolic release of the siRNA into the target cells [19-21]
. Live cell confocal microscopy was used to assess the cellular uptake and intracellular
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fate of the SiO2@AuNP sequential drug delivery system. To acquire the fluorescence signal of lysosome escape, LysoTracker® Green DND-26 (Life Technologies, Cat. No. L-7526) was utilized (excitation/emission: 488/500-550 nm). Nucleuses were stained by DAPI
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(ThermoFisher Scientific D1306) with excitation/emission ~ 358⁄461 nm. Another
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fluorescence signal of HCPT was detected at 500-600 nm with an excitation at 405 nm. A quantitative analysis of co-localization has been performed using Matlab [22]. Approximately 5×104 Colo-205 cells were seeded onto 35 mm confocal imaging dish (ibidi, Cat. No. 81156). After that, the cells were treated with SiO2@AuNP (40 μg/ml) in FBS free medium for 3 h, 6 h, 12 h and 24 h. Cells were then collected by centrifuged at 1,000 rpm, washed by phosphate buffered saline (PBS) and incubated with 0.5 μM LysoTracker for 1 h. Then 300 nM DAPI stain solution was add to cover the cells, then incubated for 1-5 minutes, protected from light.
ACCEPTED MANUSCRIPT After that, remove the stain solution and wash the cells 2-3 times in PBS. Images were taken using a Leica TCS SP5 Confocal Microscope at different time intervals. To
confirm
the
synergistic
effect
of
HCPT
and
Dox,
a
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3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to evaluate cell viability after treatment. Colo-205 cells (5×104 cells/well) seeded in 96-well plate were cultured in RPMI1640 with 10% FBS at 37°C under 5% CO2 in an incubator. HCPT
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and Dox were diluted in culture medium to obtain the desired concentrations (50 ng/mL,
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0.5 μg/mL, 5 μg/mL and 50 μg/mL). After 1 day of culture time, the plate was centrifuged at 1,500 rpm for 5 min and the culture medium from each well was discarded. Then, the cells we separately treated with 0.1 mL of HCPT, Dox, HCPT+ Dox, and HCPT+ Dox (12 h interval administration) medium solutions in a series of concentrations. After 24 h of incubation, the
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culture medium was removed by centrifugation and replaced with 100 μL of the new culture medium containing 10% MTT reagent. The cells were then incubated for 4 h at 37°C to allow the formazan dye to form. The culture medium in each well was then removed, and
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dimethyl sulfoxide (DMSO) (200 μL/well) was added for an additional 30 min of incubation.
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The quantification determining the cell viability was performed using optical absorbance (490 nm) and an ELISA plate reader. Antitumor efficacy evaluation in vivo All animal experiments were approved by the Animal Experiment Administration Committee of The Fourth Military Medical University. Female BALB/C nude mice (6-8 weeks) were purchased from Hunan SJA Laboratory Animal Co., Ltd. Colo-205 cells (5×106 cells,
ACCEPTED MANUSCRIPT total volume 0.1 mL) were injected into mice legs subcutaneously. All animals were monitored for activity, physical condition, body weight, and tumor growth.
Ten days after the implantation of the cells, tumor mass formation was observed in
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each mouse. The mice bearing cancer xenografts were randomly divided into 10 treatment groups and a saline control group (5 mice/group). Twenty milligrams of nanoparticles were
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dissolved in 1 ml of saline. One hundred microliters of nanoparticle solutions were administered by tail intravenous (i.v.) injection every 3 days for each mouse. Free drugs
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were also dissolved in saline and administered by tail i.v. injection every 3 days for 3 weeks. The dose conversion was calculated at 10 mg/kg (both Dox and HCPT).
After that, cancerous mice were sacrificed for the collection of the tumors. The full
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tumor from each mouse was removed and weighed. Each experiment was performed in triplicate. All of the data are reported as the mean ± S.D.
Acknowledgement
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This work was partially funded by grants from the National Natural Science Foundation
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of China (No. 81201179, No. 30901358, No. 81271687, No. 81571786, No. 81570803, No. 31440044) and Hong Kong scholarship and Postdoctoral Science Foundation of China.
References
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ACCEPTED MANUSCRIPT 2003;2:214-21. [5] Niidome T. 9. Diagnostic and therapeutic applications of biocompatible gold nanorods: Original research article: PEG-modified gold nanorods with a stealth character for in vivo applications, 2006. Journal of controlled release : official journal of the Controlled Release Society. 2014;190:48-50. [6] Gao J, Zhong W, He J, Li H, Zhang H, Zhou G, et al. Tumor-targeted PE38KDEL delivery via PEGylated anti-HER2 immunoliposomes. International journal of pharmaceutics. 2009;374:145-52. [7] Tsouris V, Joo MK, Kim SH, Kwon IC, Won YY. Nano carriers that enable co-delivery of
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[9] Tekedereli I, Alpay SN, Akar U, Yuca E, Ayugo-Rodriguez C, Han HD, et al. Therapeutic Silencing of Bcl-2 by Systemically Administered siRNA Nanotherapeutics Inhibits Tumor Growth by Autophagy
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and Apoptosis and Enhances the Efficacy of Chemotherapy in Orthotopic Xenograft Models of ER (-) and ER (+) Breast Cancer. Molecular therapy Nucleic acids. 2013;2:e121.
[10] Yoon HY, Son S, Lee SJ, You DG, Yhee JY, Park JH, et al. Glycol chitosan nanoparticles as
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specialized cancer therapeutic vehicles: sequential delivery of doxorubicin and Bcl-2 siRNA. Scientific reports. 2014;4:6878.
[11] Zhang Y, Xiao C, Li M, Chen J, Ding J, He C, et al. Co-delivery of 10-hydroxycamptothecin with doxorubicin conjugated prodrugs for enhanced anticancer efficacy. Macromolecular bioscience. 2013;13:584-94.
[12] Liao L, Liu J, Dreaden EC, Morton SW, Shopsowitz KE, Hammond PT, et al. A convergent synthetic platform for single-nanoparticle combination cancer therapy: ratiometric loading and
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controlled release of cisplatin, doxorubicin, and camptothecin. J Am Chem Soc. 2014;136:5896-9. [13] Zhang S, Chu Z, Yin C, Zhang C, Lin G, Li Q. Controllable drug release and simultaneously carrier decomposition of SiO2-drug composite nanoparticles. Journal of the American Chemical Society. 2013;135:5709-16.
[14] Fan L, Campagnoli S, Wu H, Grandi A, Parri M, De Camilli E, et al. Negatively charged AuNP
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modified with monoclonal antibody against novel tumor antigen FAT1 for tumor targeting. Journal of experimental & clinical cancer research : CR. 2015;34:103. [15] Hu J, Liu G, Wang C, Liu T, Zhang G, Liu S. Spatiotemporal monitoring endocytic and cytosolic pH
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gradients with endosomal escaping pH-responsive micellar nanocarriers. Biomacromolecules. 2014;15:4293-301.
[16] Greish K. Enhanced permeability and retention (EPR) effect for anticancer nanomedicine drug targeting. Methods in molecular biology. 2010;624:25-37. [17] Hong R, Han G, Fernandez JM, Kim BJ, Forbes NS, Rotello VM. Glutathione-mediated delivery and release using monolayer protected nanoparticle carriers. J Am Chem Soc. 2006;128:1078-9. [18] Li Y, Zaluzhna O, Xu B, Gao Y, Modest JM, Tong YJ. Mechanistic insights into the Brust-Schiffrin two-phase synthesis of organo-chalcogenate-protected metal nanoparticles. J Am Chem Soc. 2011;133:2092-5. [19] Juliano R, Alam MR, Dixit V, Kang H. Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res. 2008;36:4158-71. [20] Li T, Wu M, Zhu YY, Chen J, Chen L. Development of RNA interference-based therapeutics and application of multi-target small interfering RNAs. Nucleic Acid Ther. 2014;24:302-12.
ACCEPTED MANUSCRIPT [21] Gujrati M, Malamas A, Shin T, Jin E, Sun Y, Lu ZR. Multifunctional cationic lipid-based nanoparticles facilitate endosomal escape and reduction-triggered cytosolic siRNA release. Molecular pharmaceutics. 2014;11:2734-44. [22] Kreft M, Milisav I, Potokar M, Zorec R. Automated high through-put colocalization analysis of multichannel confocal images. Comput Methods Programs Biomed. 2004;74:63-7. [23] Wu G, Song C, Renata G, Fan L, Wu H, Jin B. Supporting Data for Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs. Data in
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Brief. 2015; Submitted.
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Figures
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Figure 1 Illustrative schematic representing the preparation of the SiO2@AuNP delivery system and its endocytosis after binding to cell surface targets. Following escape from the
ACCEPTED MANUSCRIPT endosomes/lysosomes, the drugs or siRNAs were sequentially released in cytoplasm to
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eliminate cancer cells.
Figure 2 Illustration and characterization of HCPT-SiO2-Dox NPs, Au-PEG-198.3/siRNA NPs and SiO2@AuNP. Structure of HCPT-SiO2-Dox NPs, Au-PEG-198.3/siRNA NPs and SiO2@AuNP were illustrated in (a), (c) and (e). The morphology and size of NPs were characterized by TEM (b, d and f). Lyophilized nanoparticles were re-dispersed in water by
ACCEPTED MANUSCRIPT sonication at room temperature. TEM images were recorded at 120 kV. The dispersion was
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placed on a copper grid and air-dried before taking the images.
Figure 3 Intracellular co-localization of SiO2@AuNP with lysosomes. Colo-205 cells were treated with SiO2@AuNP for different time intervals from 3-24 h and then stained with LysoTracker for lysosomes (in green). SiO2@AuNP was detected by HCPT signal at excitation wavelength 405 nm and emission wavelength 500-600 nm (in red). LysoTracker is green fluorescent dye that stains acidic compartments (lysosomes) in live cells with excitation/emission ~488/500-550 nm. Nucleus localization was detected by DAPI with excitation/emission ~358⁄461 nm (in blue). Overlapping rate of SiO2@AuNP and LysoTracker
ACCEPTED MANUSCRIPT were calculated using Matlab software (A–H). TEM was also used for observing the intracellular fate of the SiO2@AuNPs at different time intervals (E-H). TEM images were
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recorded at 120 kV.
Figure 4 SiRNA release procedure of outer AuNP layer. Schematic illustration of siRNA
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release procedures via GSH place exchange (A), confirmed by denatured SDS page (B) and
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western blot (C).
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Figure 5 Cumulative release profiles of Dox and HCPT from inner self-decomposable SiO2 core in Mes buffer (pH 5.5) (A) and cytoplasm (B). All of the data are reported as the means ± S.D. Sigmoidal fittings were performed by GraphPad Prism 5.01 software.
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*** ***
50ng/ml 0.5ug/ml 5ug/ml 50ug/ml
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0.8 0.6 0.4 0.2
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Figure 6 An MTT assay was carried out to demonstrate the synergetic effect of HCPT and Dox. Each experiment was repeated six times. All of the data are reported as the means ± S.D. and analyzed by one-way ANOVA statistical analysis with Newman-Keuls Multiple
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Comparison Test (GraphPad Prism 5.01 software) to determine the differences between
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groups. Significance was defined as P < 0.0001 (***).
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Figure 7 The antitumor effect of SiO2@AuNP on nude mice bearing Colo-205 cells subcutaneously was studied in vivo. Values of tumor (A) and body weight (C) changes are expressed as mean ± SD (g, n=5). Dissected tumor tissues from the nude mice (B). The nude mice were administered SiO2@AuNP via i.v. injection every 3 days.
S0142-9612(15)00879-0
DOI:
10.1016/j.biomaterials.2015.10.069
Reference:
JBMT 17166
To appear in:
Biomaterials
Received Date: 9 September 2015 Revised Date:
20 October 2015
Accepted Date: 26 October 2015
Please cite this article as: Fan L, Zhang Y, Wang F, Yang Q, Tan J, Renata G, Wu H, Song C, Jin B, Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs, Biomaterials (2015), doi: 10.1016/j.biomaterials.2015.10.069. 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 Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs Li Fan1, 2, 3*, Yongsheng Zhang4*, Fuli Wang5*, Qian Yang6, Jiali Tan7, Grifantini Renata8,
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Hong Wu1†, Chaojun Song3‡, Boquan Jin3§
1
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Department of pharmaceutical analysis, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710032 2 Department of Physics, Chinese University of Hong Kong, Shatin, HongKong 3 Department of Immunology, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710032 4 Tangdu hosipital, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710038 5 Department of Urology, Xijing Hospital, Xi’an, Shaanxi, China, 710032 6 Institute of Materia Medica, School of Pharmacy, The Fourth Military Medical University, Xian, Shaanxi, China, 710032 7 Department of Orthodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University & Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, 510055, China. 8 Externautics SpA, Siena, SI, 53100, Italy
These authors contributed equally to this work. Corresponding author, Department of pharmaceutical analysis, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710032, email: [email protected] ‡ Corresponding author, Department of Immunology, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710032, email: [email protected] § Corresponding author, Department of Immunology, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710032, email: [email protected] *
†
ACCEPTED MANUSCRIPT List of abbreviations
Dox HCPT
Dimethyl sulfoxide phosphate buffered saline fast protein liquid chromatography 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide Doxorubicin 10-hydroxycamptothecin
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Abstract
N-hydroxysuccinimide Fetal bovine serum
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NHS FBS DMSO PBS FPLC MTT
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DCM UV-Vis GSH Mes EDC
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N2
Transmission electron microscopy Dynamic light scattering Tetraethyl orthosilicate (3-Aminopropyl) triethoxysilane Poly (ethylene glycol) 2-mercaptoethyl ether acetic acid Nitrogen Dichloromethane ultraviolet-visible Glutathione 2-(N-morpholino)ethanesulfonic acid 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
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TEM DLS TEOS APTES COOH-PEG5000-SH
To achieve active tumor targeting and sequential release of 3 drugs to a tumor site in
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one nanoparticulate system,
self-decomposable SiO2 nanoparticles modified by
3-aminopropyltriethoxysilane (APTS) as their inner structure were used to double load HCPT (in the NP core) and Dox (on the NP surface). Meanwhile, monoclonal antibodies (mAb 198.3) against the FAT1 antigen and Bcl-2 siRNA were conjugated onto PEGylated Au-PEG-COOH nanoparticles. The obtained drug-loaded SiO2 nanoparticles were coated with the Au-PEG-mAb.198.3/siRNA nanoparticles through electrostatic interaction to form the SiO2@AuNP sequential drug delivery system, which featured the controlled and
ACCEPTED MANUSCRIPT sequential release of siRNA, Dox and HCPT step by step to maximize its anticancer efficacy. The results revealed that the SiO2@AuNP sequential drug delivery system specifically targeted tumor cells and was internalize rapidly, followed by endosome escape and
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sequential drug release. Importantly, the sustainable release characteristics of SiO2 made the Tmax difference between HCPT and Dox approximately 8-12 h, and this enhanced the sensitizing efficiency of HCPT on Dox compared with co-administration. The in vivo
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antitumor results demonstrated that the tumor size after SiO2@AuNP treatment is 1/400
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compared with the saline control group and approximately 1/40 of the HCPT/Dox co-treatment group without any noticeable systemic toxicity.
Keywords: Self-decomposable nanoparticles, HCPT, Dox, Bcl-2, Sequential drug release
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Introduction
Multifunctional nanoparticulate drug delivery systems (NDDSs) have made a great impact on overcoming the limitations of conventional chemotherapies by active or passive
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targeting[1]. NDDSs combine at least two different functions, such as longevity and targetability[2], and are loaded with more than one drug or gene therapy-related material,
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such as antisense oligonucleotides or small interfering RNAs (siRNAs)[3]. To lengthen the circulation time, NDDSs are usually coated with hydrophilic polymers, such as poly (ethylene glycol) (PEG)[4]. For example, PEG modification stabilized nanorods, enabling long-lasting circulation in blood due to a stealth character[5]. After that, active targeting can be achieved by attaching targeting ligands, such as PEGylated doxorubicin (Dox)-loaded liposomes with
ACCEPTED MANUSCRIPT HER2 antibodies attached, successfully targeted HER2-overexpressing SK-BR3 cells in mice [6]
.
Although NDDSs can preferentially accumulate in tumors, drug resistance is another
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major problem in current cancer treatment. Consequently, many current research studies aim to combine chemotherapy with RNA interference (RNAi) to specifically knock down the
to the anti-cancer drug
[7]
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expression of the drug resistance gene, making resistant cells transiently become sensitized . For example, the sequential delivery of siRNA and Dox by the
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PEI-GO nanocarrier shows a synergistic effect, which leads to significantly improved chemotherapy efficacy [8]. Among several combinations of chemotherapeutics, Bcl-2 specific siRNA has been considered an attractive choice to avoid the Dox-activated cellular defense mechanism of anti-apoptosis, which prevents cell death
[9, 10]
. Moreover, a co-delivery
reported
that
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strategy has been proposed to achieve the synergistic effect for cancer therapies. It was co-delivery
of
Dox
and
10-hydroxycamptothecin
(HCPT)
using
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MPEG-b-PAMAMG2.5 NPs significantly increased the in vitro cytotoxicity in resistant cancer cells and improved tumor growth inhibition
[11]
. Moreover, in our recent study, the
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administration of HCPT and Dox at 12 h intervals has better synergistic effects on Colo 205 cancer cells.
Therefore, to realize the multiple functions mentioned above, scientists have developed nanoparticles that deliver one or two chemotherapy drugs, but it has been a huge challenge to integrate these functions in one nanoparticle, especially the step-by-step release of multiple drugs from a single nanoparticle scaffold
[12]
. This has, until now,
ACCEPTED MANUSCRIPT hampered the development of smart and versatile “all-in-one” nanoparticles for tumor treatment.
In our previous work, we built up self-decomposable nanoparticles based on silicon
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dioxide that could realize controlled release and carrier decomposition simultaneously
[13]
.
They can also double load two drugs via different loading mechanisms, i.e., “grown-in” and
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“absorbed”. The first load of the NPs was realized by introducing the first molecules during the SiO2 NP growth, and the second load was carried out by soaking the loaded NPs in a
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solution containing the second molecules. In this study, self-decomposable SiO2 nanoparticles modified by 3-aminopropyltriethoxysilane (APTS) as their inner structure were used to double load HCPT (in the NP core) and Dox (on the NP surface). Meanwhile, monoclonal antibodies (mAb 198.3) against the FAT1 antigen and Bcl-2 siRNA were
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conjugated onto PEGylated Au-PEG-COOH nanoparticles. The obtained drug-loaded SiO2 nanoparticles were coated by Au-PEG-mAb.198.3/siRNA nanoparticles through an
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electrostatic interaction to form the SiO2@AuNP sequential drug delivery system, which featured the controlled and sequential release of siRNA, Dox and HCPT step by step to
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maximize its anticancer efficacy (Fig. 1). Results and Discussions
HCPT and Dox double-loaded SiO2 nanoparticles (HCPT-SiO2-Dox) were synthesized according to our previous method[13] via two different loading mechanisms. HCPT was first “grown-in” during the preparation of the SiO2 NPs[13]. After that, HCPT-SiO2 NPs were washed in ethanol and MilliQ water to remove ungrown-in HCPT. In such NPs, the drug was most concentrated in the center of the nanoparticle with a radial concentration gradient,
ACCEPTED MANUSCRIPT which made the NPs self-decomposable. APTS as a conventional silane coupling agent was then reacted with purified HCPT-SiO2 NPs to form a positively charged surface for further adsorption. Dox was then loaded on the NP surface by absorbing. Schematics to illustrate
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the configuration of the HCPT-SiO2-Dox NPs, Au-PEG-198.3/siRNA NPs and SiO2@AuNP system were shown in Fig. 2a, c, and e, respectively.
By TEM measurement, the size of the HCPT-SiO2-Dox NPs with spherical morphology is
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approximately 20-50 nm (Fig. 2b). Au-PEG-198.3/siRNA NPs was approximately 2-5 nm, and
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the size distribution was highly monodisperse (Fig. 2d). Au-PEG-198.3/siRNA NPs were attached on the HCPT-SiO2-Dox NP surface via electrostatic interactions. The obtained SiO2@AuNP system showed sphere morphology with diameter in 50 nm. Gold nanoparticles (Au-PEG-mAb198.3/siRNA) can be clearly observed as small dots on the SiO2 NP surface (Fig.
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2f).
Flow cytometry analysis demonstrated that the SiO2@AuNP system could bind to native FAT1 molecules on the surface of Colo205 cells (Fig. 1 in [23]), and this result was
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consistent with our previous work on free mAb198.3_Cy5 and Au-PEG-(Cy5) _mAb198.3 [14].
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This indicated that the SiO2@AuNP system has the FAT1 targeting capability. After binding to membranes, SiO2@AuNP started its endocytosis process involving the accumulation in membrane-sunken vesicles, transfer to early and late endosomes, and fusion to become late endosomes/lysosomes. Usually, nanoparticle-based drug delivery systems remain trapped inside endosomes/lysosomes, followed by degradation or exocytosis. Therefore, endosome/lysosome escape is of crucial importance to increase the therapeutic efficacy for nanoparticle-based drug delivery
[15]
. In our study, the
ACCEPTED MANUSCRIPT time-dependent co-localization of SiO2@AuNPs and lysosomes (stained by LysoTracker) was investigated by confocal laser scanning microscope as well as TEM to demonstrate the particle intracellular fate, especially whether and when these particles escape endocytic
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vesicles to access the cytosol. It can be seen from Fig. 3a-h that SiO2@AuNPs completed the internalizing process and entered the endosomal vesicles. The high overlapping rate (over 78.29%) of SiO2@AuNPs and lysosomes/endosomes at 6 h demonstrated that the
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endocytosis process of SiO2@AuNP occurred by phagocytosis and the nanoparticles were
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located in the endocytic vesicles. After that, the particles gradually escaped from the endosomes/lysosomes, presenting decreasing overlapping rates in the following 6 h. After 24 h of incubation, the diffusion of the bright, spot-shaped green fluorescence revealed that the endosome/lysosome membranes were disrupted, and there is little overlap between
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red and green fluorescence (25.93%), demonstrating that most of the particles have translocated into the cytoplasm. From the TEM results (Fig. 3i-l), we also found the clear progress of SiO2@AuNPs from endocytosis to endosome/lysosome escape. Most of the
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SiO2@AuNPs entered clathrin-coated pits, which evolved to primary endocytic vesicles at 3
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h incubation (Fig. 3i). Gradually, these primary vesicles fused together or with lysosomes (Fig. 3j). Discontinuous membranes of endocytic vesicles followed (Fig. 3k), which subsequently promoted SiO2@AuNP escape. After 24 h incubation, scattered SiO2@AuNPs were easily found in the cytoplasm (Fig. 3l) demonstrating the successful escape of the particles from the lysosomes/endosomes. It is worth noting that after 24 h of incubation, the SiO2@AuNPs became hollow structural particles, revealing that almost all the drugs grown in or absorbed on had been released. This result was consistent with drug release
ACCEPTED MANUSCRIPT profiles in cytoplasm. The high contrast margin created by the AuNPs demonstrated that SiO2@AuNPs still remained whole structures before self-decomposing the SiO2. That is, siRNA on AuNPs was also translocated into the cytoplasm, which is the premise of knocking
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down the Bcl-2 protein in the cytoplasm.
The drug release behavior in the cytoplasm of the SiO2@AuNP system could be seen as
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two sequential release parts of the outer AuNP layer and inner self-decomposable SiO2 core. On the surface of the outer AuNP layer, siRNA and mAb198.3 were simultaneously attached
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via thiol-terminated PEG chains. In our previous study, we visually investigated the targeting efficiency of AuNPs in vivo. Free gold nanoparticles without any modification or isotype control antibodies attached showed weak fluorescence, mainly due to the enhanced permeability and retention effect [16](EPR effect) of AuNPs. The fluorescence intensity of the
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mAb198.3-attached AuNP treatment group is higher than that of free AuNPs and isotype-attached AuNPs [14].
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After endocytosis, mediated by mAb198.3, the siRNA release process was illustrated by Fig. 4A and demonstrated by Fig. 4B-C. SiRNA was released by the place exchange of [17]
(Fig. 4A). Different band shifts on the denatured polyacrylamide gel
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glutathione (GSH)
page (Fig. 4B) demonstrated the process of GSH-triggered siRNA release. The bands place-exchanged by GSH run to the same level as HS-siRNA, indicating that the siRNA release was accomplished in the presence of GSH. As shown in the western blot result (Fig. 4C), the Bcl-2 protein in the colo-205 cells has been knocked down approximately 50% after the administration of Au-PEG-siRNA/mAb198.3. The release behavior of the inner self-decomposable SiO2 NPs was also investigated in
ACCEPTED MANUSCRIPT Mes buffer (pH 5.5, at 37 C, simulating the endosome/lysosome pH environment) and in the 。
cytoplasm of colo-205 cells. The UV absorption spectra were taken from the supernatant to measure the amount of HCPT and Dox released at certain time intervals. Fig. 5A and 5B
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plotted the cumulative release profiles of HCPT and Dox in Mes buffer (pH 5.5) and cytoplasm. In Fig. 5A, after an initial quick rise in the amount of Dox released at 12 h, an obvious plateau appeared in the profile and lasted until 48 h. The release of HCPT took 24 h
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to reach the plateau, and a stable HCPT concentration level was maintained afterward. In
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the cytoplasm, the release characteristics of Dox and HCPT have similar Tmax intervals (12 h) to that in Mes buffer; however, both Dox and HCPT present slower release profiles (Fig. 5B). Together with the drug release, the continuous morphological evolution of the inner self-decomposable SiO2 NPs was also observed using TEM (Fig. 2 in [23]). Most of the NPs
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remained intact at the first 4 h, while rough edges appeared after 6 h of immersion in deionized water. This is mainly due to the release of Dox absorbed on the NP surface. With the elongation of the immersed hours, obvious hollow features gradually appeared in the
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center of the NPs due to the release of the “grown-in” HCPT. Such uniform center-hollow
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features continued to enlarge over 48 h, leaving a spherical, thin, discontinuous shell. It was reported that co-delivered Dox and HCPT significantly increased the in vitro cytotoxicity and improved tumor growth inhibition in vivo [11]. HCPT is considered one of the most promising anticancer drugs that target the nuclear enzyme topoisomerase I. In our study, we also investigated the synergetic effects of HCPT and Dox in colo-205 cells with an MTT assay. It was demonstrated by a drug release study that the Tmax between HCPT and Dox is approximately 8-12 h. The results hinted that the synergetic effect of HCPT on Dox
ACCEPTED MANUSCRIPT should be investigated upon co-administration and after 12 h. Indeed, in Colo 205 cells, the cytotoxicity of Dox was significantly potentiated by HCPT co-treatment simultaneously and after 12 h (Fig. 4D). The potentiation of Dox cytotoxicity was most pronounced at 0.5 μg/mL,
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where HCPT treatment at 12 h caused a double reduction of the cell viability (Fig. 6). Similarly, a moderate HCPT-induced potentiation of Dox cytotoxicity by co-treatment was observed at 5 μg/mL (Fig. 6).
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Based on the results in vitro above, we evaluated the antitumor efficacy of SiO2@AuNP
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sequential drug delivery system in a xenograft mouse model by i.v. injection. The SiO2@AuNPs significantly delayed subcutaneous colo-205 tumor growth as demonstrated by tumor weight at a dose of 10 mg/kg every three days with i.v. injection (Fig. 7A-B). We also tested the effect of both the outer AuNP layer and the inner SiO2 layer in different
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combinations with mAb198.3/siRNA used at the same dose; they also delayed the tumor growth at different levels (Fig. 7A-B). The delaying effect on the tumor growth was primarily due to three factors: the targeted therapy of mAb198.3, Bcl-2 protein inhibition of siRNA
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and synergistic effects of HCPT and Dox. After the outer AuNP (Au-198.3/siRNA) treatment,
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the average tumor size was less than 1/4 of that in the saline control group. The tumor inhibitory rate of the HCPT-SiO2-Dox treatment group was similar to the co-administration of HCPT and Dox after 12 h, and this result is consistent with the in vitro findings that the cell viability of HCPT and Dox in the 12 h treatment group was much lower than that of the free Dox group and HCPT+Dox co-treatment group. This result also demonstrated that co-delivered HCPT and Dox using a self-decomposable SiO2 particle system could maximize the synergy of HCPT and Dox. At the end point of the study with SiO2@AuNPs, the tumor
ACCEPTED MANUSCRIPT weight decreased 400-fold. On the other hand, changes in body weights were also investigated to evaluate the safety of the SiO2@AuNPs. No significant decreases in the body weights were observed in all the tumor-bearing mice compared with the saline control from
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4 weeks after initial administration (Fig. 7C). These results indicated that the SiO2@AuNP treatment at 10 mg/kg exhibited a remarkable anticancer effect and did not lead to marked toxicity in the experimental mice.
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Conclusion
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Herein, tumor-homing and biocompatible SiO2@AuNP-based nanoparticles can serve as a platform vehicle to load both chemotherapeutics and siRNA to achieve optimal efficacy. Additionally, SiO2@AuNPs for the sequential delivery of Bcl-2 siRNA, Dox, and HCPT may maximize antitumor efficacy. Significantly enhanced antitumor efficacy was achieved due to
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both the targeting function and the programmed sequential release of different drugs. Additionally, such a nanoparticle drug system was also found to largely reduce the systemic toxicity.
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Experimental section
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Synthesis and Characterization of Dox and HCPT dual loaded SiO2 NPs (SiO2-HCPT/Dox), Au-PEG-198.3/siRNA NPs and SiO2@AuNP NPs sequential drug delivery system The SiO2@AuNP NP sequential drug delivery system is composed of inner dual loaded SiO2 NPs and an outer Au-PEG-198.3/siRNA layer. The inner Dox and HCPT dual loading SiO2 NPs were prepared by first encapsulating HCPT, then absorbing Dox. The SiO2-HCPT/Dox was synthesized following the method by Zhang et. al. [13] with some modifications. In brief, HCPT (4 mg) was dissolved in ethanol-aqueous ammonia solution (aqueous ammonia, 3.4
ACCEPTED MANUSCRIPT mL, 25%, v/v was mixed with 75 mL ethanol), followed by adding 200 μL of TEOS (Tetraethyl orthosilicate). The solution was stirred for 24 h in darkness and then centrifuged at 15,000 rpm for 10 min. The precipitate (SiO2-HCPT NPs) was purified by washing and centrifuged at
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15,000 rpm 3 times to remove free HCPT and excess TEOS. The purified SiO2-HCPT NPs (1 mg) were then suspended in ethanol (3 mL), followed by adding (3-Aminopropyl) triethoxysilane (APTES) (40 μL). The mixture was stirred in a water bath (60°C) for 3 hours.
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The obtained NH2-SiO2-HCPT NPs were purified by washing and centrifuged at 12,000 rpm 3
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times. Finally, NH2-SiO2-HCPT NPs (10 mg) were suspended in Dox solution (2 mg/mL, 1 mL) overnight in darkness to absorb Dox on the particle surface. The obtained SiO2-HCPT/Dox NPs were then centrifuged to remove unloaded Dox.
The outer Au-PEG-198.3/siRNA layer was synthesized according to the conventional
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Brust-Schiffrin method [18], a place exchange reaction and a carbodiimide reaction. In brief, 2 nm thiol-derivative gold nanoparticles (C5-Au) were first synthesized. An aqueous solution of hydrogen tetrachloroaurate (1 g, 250 ml) was mixed with a solution of
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tetraoctylammonium bromide in toluene (2.1 g, 500 ml). The two-phase mixture was
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vigorously stirred until all the tetrachloroaurate was transferred into the organic layer and C5SH (0.337 ml) was then added to the organic phase. A freshly prepared aqueous solution of sodium borohydride (2 g with a small amount of water to dissolve) was quickly added with vigorous stirring. After stirring overnight, the organic phase was separated, evaporated with a rotary evaporator and mixed with 400 ml ethanol to remove excess thiol. The mixture was kept at -18°C and the dark brown precipitate was filtered off and washed with ethanol many times. The mole ratio of HAuCl4: TOAB: C5SH: NaBH4=1:1.5:1.2: 20
ACCEPTED MANUSCRIPT Poly (ethylene glycol) 2-mercaptoethyl ether acetic acid (COOH-PEG5000-SH)-capped gold nanoparticles (Au-PEG-COOH) were prepared via a place exchange reaction. Briefly, 20 mg of C5-Au NPs and 80 mg of COOH-PEG5000-SH were weighed in two separate vials, and 5
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ml dry dichloromethane (DCM) was added to each of the vials. COOH-PEG5000-SH solution was added dropwise to the C5-Au NP solution and stirred for 2 days under N2 protection. The obtained Au-PEG-COOH was further washed with Hexane/DCM twice to remove free
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ligands, dried under reduced pressure and solubilized in distilled water. After 2 days of
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dialysis, the Au-PEG-COOH NPs were lyophilized and re-dissolved in MilliQ water. The attachment of siRNA to the Au-PEG-COOH was performed using the same place exchange protocols. Thiol-derivative siRNA was purchased from Guangzhou Ribobio CO., LTD. 10 mg of C5-Au NPs, 20 mg of COOH-PEG5000-SH, and 10 nmol thiol-derivative siRNA
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were used for the place exchange process. The obtained Au-PEG-siRNA NPs were lyophilized and re-dissolved in MilliQ water. Agarose gel electrophoresis was utilized to determine the siRNA and AuNP binding ratio. The results revealed that the molar ratio of
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AuNPs to siRNA molecules is approximately 1/2.5.
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The attachment of mAb198.3 to Au-PEG-siRNA NPs was performed using standard 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) coupling chemistry. The molar ratio of Au-PEG-siRNA NPs: EDC: NHS: mAb198.3 was 1:10:10:5. The carboxyl groups on the particle surface are first activated by reaction with EDC and NHS in 0.1 M MES buffer (pH 5.5) for 1 h. Then, the mAb198.3 attachment was performed by adding the concentrated activated nanoparticles to a dilute MES solution containing mAb198.3 at pH 5.5. The solution was mixed well and shaken gently overnight at
ACCEPTED MANUSCRIPT 4°C in the dark. After the reaction, Au-PEG-198.3/siRNA NPs were separated by fast protein liquid chromatography (FPLC) to remove free antibody and dialyzed to remove NHS and EDC. The mobile phase was MilliQ water at a flow rate of 5 mL/min. The ratio of mAb198.3
Spectrophotometer (Thermo Scientific) at 280 nm.
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conjugated onto AuNPs is approximately 1/3, which is determined by a NanoDrop 2000c
The SiO2@AuNP sequential drug delivery system was prepared by simply mixing
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Au-PEG-198.3/siRNA NPs with SiO2-HCPT/Dox NPs under stirring. Au-PEG-198.3/siRNA NPs
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rapidly binds to SiO2-HCPT/Dox NPs due to electrostatic interactions.
Au-PEG-198.3/siRNA NPs, SiO2-HCPT/Dox NPs and the SiO2@AuNP sequential drug delivery system were analyzed by ultraviolet-visible (UV-Vis) spectrum (HitachiU-3501 UV-Vis NIR spectrophotometer) and transmission electron microscopy (TEM) (PhilipsCM120)
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for the general morphology and size distribution.
Characterizations of the Drug Release and Carrier Decomposition Process The in vitro targeting efficiency of the Au-PEG-198.3/siRNA NP layer was determined
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by flow cytometry analysis in vitro. The targeting and internalizing efficacy in vivo were
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evaluated by a fluorescence imaging system (Cambridge Research & Instrumentation, Inc., Maestro EX) with Ex=640 nm. Glutathione (GSH) mediated thiol-siRNA release from Au-PEG-198.3/siRNA NPs were evaluated by denaturing polyacrylamide gel electrophoresis. A 10% polyacrylamide gel was used to visualize the siRNA release process. Au-PEG-198.3/siRNA NPs were incubated for 2 h with 5 mM GSH, and the gel electrophoresis was performed at 125 V. All siRNA bands were visualized using an AlphaImager ultraviolet imaging system (Biosciences).
ACCEPTED MANUSCRIPT The Bcl-2 silencing effect of Au-PEG-198.3/siRNA NPs on Colo-205 cells was studied by Western blot analysis using primary antibodies specific for Bcl-2 and secondary anti-rabbit IgG-HRP, followed by a visualization by enhanced chemiluminescence. Colo-205 cells (5×106)
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were resuspended in Nucleofector solution (Nucleofector kit V) with 3 μg Bcl-2 siRNA+freestyle Max, Au-PEG-siRNA or control siRNA+freestyle Max using the Amaxa Nucleofector apparatus and program U-16. Immediately after transfection, the cells were
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transferred into RPMI 1640 medium with 10% FBS and incubated for 48 hours.
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The drug release profiles of the inner SiO2-HCPT/Dox NP layer in water, 2-(N-morpholino)ethanesulfonic acid (Mes) buffer (pH 5.5) and medium with serum were determined by UV-Vis and fluorescence spectra. Equal amounts of SiO2-HCPT/Dox NPs (1 mg/mL) were dispersed in 10 mL deionized water, Mes buffer, or Medium with 10% fetal
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bovine serum (FBS), separately. Each sample was centrifuged at a different time point and the supernatant was separated by centrifugal filtration (molecular weight cut off: 30 000 Da, Millipore). The SiO2-HCPT/Dox NPs were dried and re-dispersed in deionized water. UV-Vis
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absorption and fluorescence spectra were taken from both re-dispersed particles solution
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and supernatants using a Hitachi U-3501 UV-Vis NIR spectrophotometer and Hitachi F7000 fluorescence spectrophotometer. The degradation of the SiO2 carrier was monitored by a morphology investigation using TEM. We also examined the drug release kinetics in the cytoplasm using a Colo-205 cell line, which was cultured in RPMI 1640 medium supplemented 10% heat-inactivated FBS, 1% streptomycin, and 1% penicillin. The cells were maintained in a standard cell culture incubator at 37°C in a humidified atmosphere with 5% CO2. All of the NPs were sterilized via
ACCEPTED MANUSCRIPT cobalt-60 (NPs in powder form) for 24 h and dispersed in the medium by ultrasonication for at least 20 min immediately before their introduction to the cells. Cells were seeded at initial densities of 5×104 cells/mL in 25 mm2 flasks (for TEM samples) or dishes (for confocal
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samples) with 2 ml of medium each and incubated for 24 h, and the medium was changed with NP medium. Different feeding time intervals were adopted, as specified in individual experimental results.
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Furthermore, due to the specific features of the siRNA, it could not pass through
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endosome membrane itself to locate in the cytoplasm after endocytosis, so the delivery system should provide a mechanism to escape the fate of endosomal-lysosomal degradation and ultimately facilitate the cytosolic release of the siRNA into the target cells [19-21]
. Live cell confocal microscopy was used to assess the cellular uptake and intracellular
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fate of the SiO2@AuNP sequential drug delivery system. To acquire the fluorescence signal of lysosome escape, LysoTracker® Green DND-26 (Life Technologies, Cat. No. L-7526) was utilized (excitation/emission: 488/500-550 nm). Nucleuses were stained by DAPI
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(ThermoFisher Scientific D1306) with excitation/emission ~ 358⁄461 nm. Another
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fluorescence signal of HCPT was detected at 500-600 nm with an excitation at 405 nm. A quantitative analysis of co-localization has been performed using Matlab [22]. Approximately 5×104 Colo-205 cells were seeded onto 35 mm confocal imaging dish (ibidi, Cat. No. 81156). After that, the cells were treated with SiO2@AuNP (40 μg/ml) in FBS free medium for 3 h, 6 h, 12 h and 24 h. Cells were then collected by centrifuged at 1,000 rpm, washed by phosphate buffered saline (PBS) and incubated with 0.5 μM LysoTracker for 1 h. Then 300 nM DAPI stain solution was add to cover the cells, then incubated for 1-5 minutes, protected from light.
ACCEPTED MANUSCRIPT After that, remove the stain solution and wash the cells 2-3 times in PBS. Images were taken using a Leica TCS SP5 Confocal Microscope at different time intervals. To
confirm
the
synergistic
effect
of
HCPT
and
Dox,
a
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3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to evaluate cell viability after treatment. Colo-205 cells (5×104 cells/well) seeded in 96-well plate were cultured in RPMI1640 with 10% FBS at 37°C under 5% CO2 in an incubator. HCPT
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and Dox were diluted in culture medium to obtain the desired concentrations (50 ng/mL,
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0.5 μg/mL, 5 μg/mL and 50 μg/mL). After 1 day of culture time, the plate was centrifuged at 1,500 rpm for 5 min and the culture medium from each well was discarded. Then, the cells we separately treated with 0.1 mL of HCPT, Dox, HCPT+ Dox, and HCPT+ Dox (12 h interval administration) medium solutions in a series of concentrations. After 24 h of incubation, the
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culture medium was removed by centrifugation and replaced with 100 μL of the new culture medium containing 10% MTT reagent. The cells were then incubated for 4 h at 37°C to allow the formazan dye to form. The culture medium in each well was then removed, and
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dimethyl sulfoxide (DMSO) (200 μL/well) was added for an additional 30 min of incubation.
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The quantification determining the cell viability was performed using optical absorbance (490 nm) and an ELISA plate reader. Antitumor efficacy evaluation in vivo All animal experiments were approved by the Animal Experiment Administration Committee of The Fourth Military Medical University. Female BALB/C nude mice (6-8 weeks) were purchased from Hunan SJA Laboratory Animal Co., Ltd. Colo-205 cells (5×106 cells,
ACCEPTED MANUSCRIPT total volume 0.1 mL) were injected into mice legs subcutaneously. All animals were monitored for activity, physical condition, body weight, and tumor growth.
Ten days after the implantation of the cells, tumor mass formation was observed in
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each mouse. The mice bearing cancer xenografts were randomly divided into 10 treatment groups and a saline control group (5 mice/group). Twenty milligrams of nanoparticles were
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dissolved in 1 ml of saline. One hundred microliters of nanoparticle solutions were administered by tail intravenous (i.v.) injection every 3 days for each mouse. Free drugs
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were also dissolved in saline and administered by tail i.v. injection every 3 days for 3 weeks. The dose conversion was calculated at 10 mg/kg (both Dox and HCPT).
After that, cancerous mice were sacrificed for the collection of the tumors. The full
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tumor from each mouse was removed and weighed. Each experiment was performed in triplicate. All of the data are reported as the mean ± S.D.
Acknowledgement
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This work was partially funded by grants from the National Natural Science Foundation
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of China (No. 81201179, No. 30901358, No. 81271687, No. 81571786, No. 81570803, No. 31440044) and Hong Kong scholarship and Postdoctoral Science Foundation of China.
References
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ACCEPTED MANUSCRIPT [21] Gujrati M, Malamas A, Shin T, Jin E, Sun Y, Lu ZR. Multifunctional cationic lipid-based nanoparticles facilitate endosomal escape and reduction-triggered cytosolic siRNA release. Molecular pharmaceutics. 2014;11:2734-44. [22] Kreft M, Milisav I, Potokar M, Zorec R. Automated high through-put colocalization analysis of multichannel confocal images. Comput Methods Programs Biomed. 2004;74:63-7. [23] Wu G, Song C, Renata G, Fan L, Wu H, Jin B. Supporting Data for Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs. Data in
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Brief. 2015; Submitted.
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Figures
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Figure 1 Illustrative schematic representing the preparation of the SiO2@AuNP delivery system and its endocytosis after binding to cell surface targets. Following escape from the
ACCEPTED MANUSCRIPT endosomes/lysosomes, the drugs or siRNAs were sequentially released in cytoplasm to
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eliminate cancer cells.
Figure 2 Illustration and characterization of HCPT-SiO2-Dox NPs, Au-PEG-198.3/siRNA NPs and SiO2@AuNP. Structure of HCPT-SiO2-Dox NPs, Au-PEG-198.3/siRNA NPs and SiO2@AuNP were illustrated in (a), (c) and (e). The morphology and size of NPs were characterized by TEM (b, d and f). Lyophilized nanoparticles were re-dispersed in water by
ACCEPTED MANUSCRIPT sonication at room temperature. TEM images were recorded at 120 kV. The dispersion was
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placed on a copper grid and air-dried before taking the images.
Figure 3 Intracellular co-localization of SiO2@AuNP with lysosomes. Colo-205 cells were treated with SiO2@AuNP for different time intervals from 3-24 h and then stained with LysoTracker for lysosomes (in green). SiO2@AuNP was detected by HCPT signal at excitation wavelength 405 nm and emission wavelength 500-600 nm (in red). LysoTracker is green fluorescent dye that stains acidic compartments (lysosomes) in live cells with excitation/emission ~488/500-550 nm. Nucleus localization was detected by DAPI with excitation/emission ~358⁄461 nm (in blue). Overlapping rate of SiO2@AuNP and LysoTracker
ACCEPTED MANUSCRIPT were calculated using Matlab software (A–H). TEM was also used for observing the intracellular fate of the SiO2@AuNPs at different time intervals (E-H). TEM images were
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recorded at 120 kV.
Figure 4 SiRNA release procedure of outer AuNP layer. Schematic illustration of siRNA
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release procedures via GSH place exchange (A), confirmed by denatured SDS page (B) and
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western blot (C).
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Figure 5 Cumulative release profiles of Dox and HCPT from inner self-decomposable SiO2 core in Mes buffer (pH 5.5) (A) and cytoplasm (B). All of the data are reported as the means ± S.D. Sigmoidal fittings were performed by GraphPad Prism 5.01 software.
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50ng/ml 0.5ug/ml 5ug/ml 50ug/ml
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Cell viability (*100%)
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Figure 6 An MTT assay was carried out to demonstrate the synergetic effect of HCPT and Dox. Each experiment was repeated six times. All of the data are reported as the means ± S.D. and analyzed by one-way ANOVA statistical analysis with Newman-Keuls Multiple
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Comparison Test (GraphPad Prism 5.01 software) to determine the differences between
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groups. Significance was defined as P < 0.0001 (***).
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Figure 7 The antitumor effect of SiO2@AuNP on nude mice bearing Colo-205 cells subcutaneously was studied in vivo. Values of tumor (A) and body weight (C) changes are expressed as mean ± SD (g, n=5). Dissected tumor tissues from the nude mice (B). The nude mice were administered SiO2@AuNP via i.v. injection every 3 days.