Biomaterials 35 (2014) 10058e10069
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Highly effective inhibition of lung cancer growth and metastasis by systemic delivery of siRNA via multimodal mesoporous silica-based nanocarrier Yijie Chen a, b, Hongchen Gu a, b, *, Ding Sheng-Zi Zhang b, Fan Li a, b, Tengyuan Liu b, Weiliang Xia a, b, * a
State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China School of Biomedical Engineering & Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 August 2014 Accepted 1 September 2014 Available online 29 September 2014
Lung cancer has been the leading type of cancers with regard to mortality and mobility. New versions of RNAi-based therapy are greatly required to tackle the challenges of lung cancer. In this study, we developed a novel siRNA delivery vector based on our magnetic mesoporous silica nanoparticles (MMSNs) platform. This nanocarrier was constructed by loading siRNAs into the mesopores of M-MSNs, followed by polyethylenimine (PEI) capping, PEGylation and fusogenic peptide KALA modification. The resultant delivery system exhibited prolonged half-life in bloodstream, enhanced cell membrane translocation and endosomal escapablity, and favorable tissue biocompatibility and biosafety. Systemic application of vascular endothelial growth factor (VEGF) siRNA via this nanocarrier resulted in remarkable tumor suppression, both in subdermal and orthotopic lung cancer models, while tumor metastasis was also significantly reduced, overall leading to improved survival. In addition, the magnetic core of the particles and the functionalized fluorescence markers conveniently enabled in vivo imaging of target tissues. Taken together, this M-MSNs-based siRNA delivery vehicle has shown very favorable applicability for cancer therapy. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Systemic delivery siRNA VEGF Lung cancer therapy Mesoporous silica nanoparticles Metastasis
1. Introduction Lung cancer has been the leading cancer type for cancer related death of both sexes in the US [1]. Likewise, in Europe, lung cancer has overtaken breast cancer to be the top killer among common cancers in women [2]. Unfortunately, although numerous efforts in surgery, radiation therapy and chemotherapy have been devoted to the treatment of lung cancer, the efficacy evidenced by the 5-year survival was still below 15% [3]. It is clear that development of more effective therapies for lung cancer is mandatory. Small interfering RNA (siRNA) as therapeutics could be tailored to the treatment of disease of interest by triggering specific knockdown of target genes, thereby restoring the balance of the regulatory network that otherwise leads to the disease onset [4].
* Corresponding authors. School of Biomedical Engineering & Med-X Research Institute, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China. E-mail addresses: [email protected] (H. Gu), [email protected], weiliangxia@ gmail.com (W. Xia). http://dx.doi.org/10.1016/j.biomaterials.2014.09.003 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
Currently, few siRNA-based therapies reach the final stage of clinical trials; for cancer, only limited trials have been registered [4]. The biggest obstacle, however, is to deliver intact siRNAs to the target site efficiently in vivo without inducing adverse effects. Hence the design of a safe and efficient delivery system for siRNA is critical for translating siRNA-based therapy into the clinic [5,6]. Desired features of an ideal siRNA delivery system are effective protection of loaded nucleic acids, prolonged half-life in bloodstream, efficient cell membrane translocation, timely endosomal escape, and good tissue biocompatibility [6]. Additionally, noninvasive imaging modality is also favored for real-time assessment of siRNA delivery to optimize treatment strategies or for diagnosis/ prognosis [7]. To date a number of siRNA carriers for in vivo delivery in the laboratory settings have been reported, which include lipid-based agents [8,9], cholesterol conjugation systems [10,11], cationic delivery systems [12,13], as well as inorganic nanoparticles systems [14]. Recently, a new type of drug delivery vectors termed mesoporous silica nanoparticles (MSNs) have emerged as promising chemotherapeutics/nucleic acid carriers due to their noticeable
Y. Chen et al. / Biomaterials 35 (2014) 10058e10069
advantages which include pore-size tunability, large loading capacity, excellent biocompatibility and modifiability [15]. This type of delivery platform has been tested useful as siRNA nanocarrier to treat cancer [14,16e20], yet there is still much room to improve the anticancer efficacy for systemic delivery of therapeutic siRNAs. Therefore, new generation of siRNA delivery vectors with more thorough studies covering not only their therapeutic effects but also biosafety are in great need [4]. Herein we report the development of a novel magnetic mesoporous silica nanoparticles (M-MSNs)-based siRNA delivery system for lung cancer treatment, both in ectopic and orthotopic models, and evaluate tumor metastasis in these in vivo studies. We also report in detail the biocompatibility and biosafety of our nanocarrier, with the hope of translating the laboratory findings into the clinic. We choose vascular endothelial growth factor (VEGF) as the target gene for our study. Cancer cells acquire distinctive features that enable their growth, metastasis and resistance to treatment [21]. VEGF is overexpressed and secreted by most tumor cells, especially lung cancer, which stimulates the formation of new blood vessels through proliferation of endothelial cells [22e24]. Anti-angiogenesis therapy has already been widely used in the clinic and VEGF is often targeted to demonstrate RNAi-mediated tumor therapy [25]. Decrease of intratumoral microvessel density and restrain of tumor growth as a result of VEGF down-regulation are readily observed [18,26]. 2. Materials and methods 2.1. siRNA preparation Negative Control siRNA (denoted as NC siRNA, scrambled sequences) (sense, 50 UUC UCC GAA CGU GUC ACG UdTdT-30 ; antisense, 50 -ACG UGA CAC GUU CGG AGA AdTdT-30 ), fluorescein amidite (FAM) labeled N.C. siRNA (modified at the 50 end of the sense strand). VEGF siRNA [26] (sense, 50 -GGA GUA CCC UGA UGA GAU CdTdT; antisense, 50 -GAU CUC AUC AGG GUA CUC CdTdT), fluorescently labeled Cyanine Dyes 3 (Cy3) VEGF siRNA (modified at the 50 end of the sense strand) were synthesized by GenePharma Co. Ltd. (Shanghai, China).
2.2. Preparation of M-MSN_siRNA@PEI-PEG-KALA delivery vehicles M-MSN_siRNA@PEI was synthesized in accordance with previous work [27]. In brief, 50 mL water solution of siRNA was added into 2 mL centrifuge tube with 1 mg M-MSN, then 50 mL 4 M guanidine hydrochloride solution and 200 mL ethanol was added. The mixture was well dispersed by vortex for 30 s and then was continuously shaken at 270 rpm at 25 C for 1 h. Finally, free siRNA in supernatant was eliminated through centrifugation at 12,000 rpm, and the precipitation was resuspended in ethanol. Thereafter, the siRNA-encapsulated M-MSNs were dropwise added into the PEI solution to form M-MSN_siRNA@PEI through electrostatic interaction under ultrasonication. Such M-MSN_siRNA@PEI was further washed in deionized water (pH 5.5) and eventually was dispersed in ethanol. Meanwhile, we employed a novel modification of PEI-coated M-MSN_siRNA, in which a KALA peptide related to membrane transduction and endosomal escape was coupled to PEI through a heterobifunctional polyethylene glycol (NHS-PEG-Mal, 3.5 kDa) to form an M-MSN_siRNA@PEI-PEG-KALA delivery system. First, 250 mL NHS-PEG-Mal (dissolved in DMSO, 8 mg/mL), in which the maleimide group was covalently reacted with cysteine within 50 mL KALA (dissolved in water, 0.5 mg/mL) under 37 C for 30 min resulting in an NHS-PEG-KALA conjugate. Then 1 mg prepared M-MSN_siRNA@PEI dispersed in ethanol was added into the described NHSPEG-KALA solution at room temperature (RT). After 30 min reaction, the mixture was centrifuged at 13,000 rpm to obtain the desired vector in the pellet, which were washed with deionized water and dispersed in saline at various concentrations (Fig. 1A).
2.3. Cell lines and cultures A549 cells (human alveolar basal epithelial adenocarcinomic cell line), L02 cells (human normal liver cell line), PC-3 (human prostate cancer cell line) and HCCLM-3 (human hepatocellular carcinoma cell line) were obtained from Cell Bank of Chinese Academy of Science (Shanghai, China), and cultured in DMEM (A549&HCCLM-3) or RMPI1640 (L02&PC-3) supplemented with 10% fetal bovine serum (FBS; v/v) and 2% penicillin-streptomycin (PS; v/v). The cells were incubated at 37 C with 5% CO2 in a humid cell incubator.
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2.4. Cytotoxicity assessment Cell viability of A549 and L02 cells were measured using the CCK-8 assay. Cells were seeded at a density of 1 104 cells per well on 96-well plates, and the following experiments was conducted until the 80% confluence. After removing the culture medium, the fresh medium was supplemented containing 10% FBS and various concentrations (20e400 mg/mL) of M-MSN_NC siRNA@PEI-PEG-KALA or M-MSN_NC siRNA@PEI. A negative control group was treated with equivalent volume of saline mixed with culture medium. Cells were coincubated with nanoparticles for 24 h, and subjected to CCK-8 assay following vendor's protocol. All experiments were performed in hexaplicate, and the averaged absorbance values were normalized to negative control. 2.5. In vitro RNAi experiment to determine VEGF knockdown by M-MSN_VEGF siRNA@PEI-PEG-KALA A549 cells were grown in 24-well plates to ~80% confluency, and each well was added with increasing amount of M-MSN_VEGF siRNA@PEI-PEG-KALA (equivalent siRNA dosage ranging from 37.5 nM to 187.5 nM), or with 80 mg/mL nanocarriers (equivalent siRNA dosage of 150 nM). After incubation with various siRNA vectors for 24 h, A549 cells were replenished with fresh medium and cultured for another 24 h. Then the conditioned medium was collected, centrifuged and subjected to ELISA to determine VEGF levels. PC-3 cells and HCCLM-3 cells were performed to determine the VEGF levels with 80 mg/mL nanocarriers (equivalent siRNA dosage of 150 nM) according with the experimental process in A549 cells. nM ¼ nmol/L ¼ pmol/mL. 2.6. Biodistribution study For biodistribution studies, 2 mg M-MSN_NC siRNA@PEI-PEG-KALA dispersed in 200 mL saline was i.v. injected into each tumor-bearing nude mouse (20 ± 2 g). These mice were randomly divided into three groups with each terminated at 1 day, 3 days or 7 days after injection and major organs (heart, liver, spleen, lung, kidney, brain, tumor, stomach) were collected. Dissected organs were weighed and dissolved in strong acid solution (HNO3:HF:H2SO4 ¼ 1:1:1) at 60 C for 24 h. To determine the Fe content in various organs, atomic absorption analysis (AAS) was performed (n ¼ 3/ group). Mice administrated with saline served as negative control (n ¼ 3). The Fe content in tissues was presented in the unit of the percentage of injected dose per gram of organ (%ID/g). 2.7. Pharmacokinetics study M-MSN_Cy3-siRNA@PEI-PEG-KALA or naked-Cy3-siRNA were prepared and injected intravenously into 3 six-week-old nude mice (20 ± 2 g) at a dose of 1.5 mg/ kg Cy3-labeled siRNA either as free form or within nanoparticles. At various time points after injection, 20 mL of blood was collected and dissolved in 1 mL of lysis buffer (1% SDS, 1% Triton X-100 and 40 mM Tris acetate). The Cy3 fluorescence intensity was measured by LS 55 Luminescence Spectrometer (Perkin Elmer, USA). The Cy3-siRNA content in the blood was presented in the unit of the percentage of injected dose per gram of blood (%ID/g). Total blood volume was defined as 5% of body weight. The obtained data was analyzed using WinNolin software, and the non-compartment model was fit to the data. The area under curve (AUC), plasma clearance (CI) and mean retention time (MRT) was determined. 2.8. Subdermal and orthotopic models of human lung cancer and therapy Six-week-old athymic nude mice were purchased from B&K Universal Group Limited, Shanghai, China. They were housed in sterile isolated cages with a 12 h light/dark cycle at constant temperatures (24e26 C) and had free access to food and water. Animal procedures were carried out according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Shanghai Jiao Tong University, Shanghai, China. To construct a subcutaneous lung cancer model, a total of 5 106 A549 cells mixed with Matrigel™ (BD, America) at the ratio of 1:1 (total volume 100 mL) were s.c. injected into the right flank region of female nude mice (18 ± 2 g), which were randomly divided into four groups (n ¼ 5/group). Therapy started when the xenograft tumors appeared to reach an average size of 50 mm3. Thereafter, the asdescribed siRNA formulations were administered through tail vein using 29-gauge needle syringe, at a dose of 100 mg/kg M-MSNs containing 3.5 nmol siRNA and dispersed in 200 mL saline, following the regimen described in Fig. 4A. Tumor growth was measured every five days by using the digital calipers, and the tumor volume was calculated based on the formula: tumor volume ¼ (major axis) (minor axis)2/ 2. Fifteen days after the final injection, tumors were harvested and subjected to various tests. To construct an orthotopic lung cancer model, a total of 3 106 A549 cells resuspended in Matrigel™ (BD, America) at the ratio of 1:1 (total volume 50 mL) were transthoracically injected into the upper lobe of right lung of male nude mice (23 ± 1 g) by 29-gauge needle syringe under a Leica MZ-16 dissecting microscope (MicroOptics of Florida, Davie, FL). A small wound made to facilitate transthoracic injection was closed by sutures. Then, all mice were randomly divided into four groups (n ¼ 6/group). The as-described siRNA formulations were administered through tail vein using 29-gauge needle syringe, at a dose of 100 mg/kg M-MSNs
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Y. Chen et al. / Biomaterials 35 (2014) 10058e10069
Fig. 1. Schematic representation of the design of this study: (A) synthesis of M-MSN_siRNA@PEI-PEG-KALA. (B) Transmission electron microscope (TEM) image of M-MSN_NC siRNA@PEI-PEG-KALA (bar ¼ 50 nm). (C) Dynamic light scattering (DLS) measurements of size distribution for M-MSN_NC siRNA@PEI-PEG-KALA (dispersed in saline). (D) TGA (thermogravimetric analysis) curves of various types of M-MSNs that were loaded with siRNA. The red dotted line is the replicated assay for different types of M-MSNs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) containing 3.5 nmol siRNA and dispersed in 200 mL saline, following the regimen described in Fig. 5A. After the survival rate of the saline group dropped to below 30%, all remaining mice were terminated by cervical dislocation, and lungs and livers were harvested for following experiments. Weight was closely monitored and measurement was carried out throughout experimental process every five days.
harvested, fixed in 4% paraformaldehyde, and histological sections (10 mm in thickness) were achieved using Freezing Microtome, stained with hematoxylin and eosin (H&E) and examined by a digital microscope (Leica, Germany).
2.9. T2-weighted MRI imaging
Data are expressed as means ± standard deviations (SD). The significance of difference was analyzed by one- or two-way analysis of variance (ANOVA), and the survival was analyzed by survival curve using the GraphPad Prism 5 software. Differences with p < 0.05 were considered statistically significant.
T2-weighted imaging was performed using MesoMR23-060HeI imaging instrument (Shanghai Niumag Corporation). Tumor-bearing mice was anaesthetized with ketamine (10 mL/kg). Imaging was performed before, and at intervals after i.v. injection of M-MSN_NC siRNA@PEI-PEG-KALA at a dose of 100 mg/kg. The instrumental parameters were set as follows: a 0.55 T magnet, K space ¼ 192 256 mm, section thickness ¼ 3 mm, TE ¼ 60 ms, TR ¼ 2000 ms, FOVRead ¼ 100 mm, FOVPhase ¼ 100 mm, and number of scans (NS) ¼ 4.
2.11. Statistical analysis
3. Results
2.10. Blood analysis and histology
3.1. Preparation and characterization of a novel siRNA nanocarrier: M-MSN_siRNA@PEI-PEG-KALA
All blood analyses were performed by Shanghai Research Center for Biomodel Organisms, Shanghai, China. Measurements for major hematology and immunogenicity related markers were performed on whole blood, and various biochemistry parameters were tested on mouse serum 48 h after the final injection of five repeated doses of 100 mg/kg M-MSN_NC siRNA@PEI-PEG-KALA every five days. The control group was saline without nanocarriers treated in exactly the same regimen. All blood samples were stabilized with heparin (10 U/500 mL whole blood). After collection of blood, animals were killed by cervical dislocation under deep ketamine anesthesia. Major organs including liver, spleen, lung and kidney were
In this study, we developed a novel type of siRNA nanocarrier, MMSN_siRNA@PEI-PEG-KALA, which is composed of several key units: an M-MSN core containing the cargo siRNA, a polyethylenimine (PEI) layer coated for the following functionalization steps that include anti-fouling coating with polyethylene glycol (PEG), and conjugation of a fusogenic KALA peptide with endosomolytic function [28] (Fig. 1A). M-MSNs were synthesized to be of ~50 nm in diameter
Y. Chen et al. / Biomaterials 35 (2014) 10058e10069
(Supplementary Fig. 1A) and ~3.6 nm in pore size (Supplementary Fig. 1B). Then, siRNA was loaded into M-MSNs under such solution condition as 0.67 M guanidine hydrochloride (Guan-HCl) plus 66.7% ethanol (v/v). Due to the charge screening by Guan-HCl, siRNA could flow into the pores of M-MSNs and the dehydrated effect mediated by organic solvent resulted in the encapsulation of siRNA into the mesopores [27]. The amount of siRNA loaded in M-MSNs could be regulated through increasing the concentration of siRNA without volume change in the loading solution. These siRNA-loaded M-MSNs were coated with PEI to form M-MSN_siRNA@PEI composite through electrostatic interaction. Then a biocompatible macromolecule Nhydroxy-succinimide-poly (ethylene glycol)-maleimide (NHS-PEGMal, 3.5 kDa; PEG refers to this size thereafter) [29] containing maleimide moieties was firstly reacted with the thiol groups in KALA peptide to form NHS-PEG-KALA complex, which were grafted onto the surface of M-MSN_siRNA@PEI via the reaction between amino groups and NHS. The resultant siRNA nanocarrier was denoted as MMSN_siRNA@PEI-PEG-KALA, whose physical/chemical properties were characterized. We used negative control siRNA (NC siRNA, scrambled sequences) in these experiments. In this work, the loading amount of siRNA in vector was ~30 mg siRNA/g M-MSNs (1.875 nmol/mg M-MSNs) and ~28 mg siRNA/g M-MSNs (1.75 nmol/ mg M-MSNs) for in vitro gene silencing and in vivo cancer therapy applications, respectively. TEM image of M-MSN_NC siRNA@PEI-PEG-KALA showed that the nanoparticle size was estimated to be ~50 nm (Fig. 1B), with Zeta potential of þ23.6 ± 1.2 mV (Supplementary Fig. 2A). Dynamic light scattering (DLS) analysis then showed that the M-MSN_NC siRNA@PEI-PEG-KALA had a larger diameter at 154.4 ± 1 nm (Fig. 1C) than those of M-MSN_NC siRNA@PEI (142.6 ± 1 nm) and M-MSNs (89.6 ± 0.7 nm) (Supplementary Fig. 3A). These results indicated that the particle size increased because of the modification of polymer coating. In order to demonstrate that M-MSNs had been modified with polymer successfully, a positive staining reagent phosphotungstic acid (PTA) was introduced [18]. Consequently, TEM showed that the contrast of the amine groups in PEI and KALA peptides in M-MSN_NC siRNA@PEI-PEG-KALA was enhanced through PTA staining, but the size was also estimated to be ~50 nm (Supplementary Fig. 3B). In another word, M-MSN and M-MSN@PEI-PEG-KALA exhibited similar sizes by TEM experiments, which failed to distinguish the polymer thickness in these particles. We then performed thermogravimetric analysis (TGA) to estimate the amount (weight ratio) of loaded/modified molecules in the nanocarrier (Fig. 1D). The weight loss for M-MSN_NC siRNA, M-MSN_NC siRNA@PEI and M-MSN_NC siRNA@PEI-PEG-KALA were 8.9 ± 0.141%, 22.35 ± 0.071% and 30.66 ± 0.198% of the total, respectively (Fig. 1D). Based on these results, the relative mass ratios of PEI and PEG-KALA in the M-MSN_NC siRNA@PEI-PEG-KALA were ~14.6% and ~11.0%, respectively. The ratio of grafted KALA was ~17.4% of total KALA, as was determined by fluorescent intensities using FITC-labeled KALA (Supplementary Fig. 2B). The transverse relaxation time (T2) of M-MSN_siRNA@PEI-PEG-KALA was also measured at 0.5 T with a spin-echo pulse sequence. The drastically decreased signal intensity of T2-weighted images was observed as Fe concentration (correspond to nanoparticle concentration) increased ( Supplementary Fig. 2C). The T2 relaxation rate (1/T2) and Fe concentration rendered good linearity indicating M-MSN_siRNA@PEI-PEG-KALA as a contrast agent with the r2 value of 226.92 mM1s1 (Supplementary Fig. 2D). 3.2. Cytotoxicity and gene silencing effectiveness of MMSN_siRNA@PEI-PEG-KALA in vitro Cytotoxicity is a primary concern in development of a novel drug delivery system. The cell viability of A549 (an adenocarcinomic
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human alveolar basal epithelial cell line) and L02 (human normal hepatic cell line) incubated with various concentrations of MMSN_NC siRNA@PEI-PEG-KALA for 24 h was measured by the CCK-8 assay (Fig. 2A). These nanocarriers showed negligible cytotoxicity even at 200 mg/mL for both A549 and L02 cell lines (Fig. 2A). By contrast, M-MSN_NC siRNA@PEI exhibited severe cytotoxicity in A549 and L02 cell lines even at a much lower concentration (50 mg/ mL) (Supplementary Fig. 4). To further assess their biocompatibility, we conducted experiments to characterize the interactions of various functionalized M-MSNs with red blood cells (RBCs). Hemolytic activities of M-MSN_NC siRNA@PEI without modification of PEG-KALA conjugation (>85%, normalized to positive control) and M-MSN_NC siRNA@PEI-PEG-KALA (
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Highly effective inhibition of lung cancer growth and metastasis by systemic delivery of siRNA via multimodal mesoporous silica-based nanocarrier Yijie Chen a, b, Hongchen Gu a, b, *, Ding Sheng-Zi Zhang b, Fan Li a, b, Tengyuan Liu b, Weiliang Xia a, b, * a
State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China School of Biomedical Engineering & Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 August 2014 Accepted 1 September 2014 Available online 29 September 2014
Lung cancer has been the leading type of cancers with regard to mortality and mobility. New versions of RNAi-based therapy are greatly required to tackle the challenges of lung cancer. In this study, we developed a novel siRNA delivery vector based on our magnetic mesoporous silica nanoparticles (MMSNs) platform. This nanocarrier was constructed by loading siRNAs into the mesopores of M-MSNs, followed by polyethylenimine (PEI) capping, PEGylation and fusogenic peptide KALA modification. The resultant delivery system exhibited prolonged half-life in bloodstream, enhanced cell membrane translocation and endosomal escapablity, and favorable tissue biocompatibility and biosafety. Systemic application of vascular endothelial growth factor (VEGF) siRNA via this nanocarrier resulted in remarkable tumor suppression, both in subdermal and orthotopic lung cancer models, while tumor metastasis was also significantly reduced, overall leading to improved survival. In addition, the magnetic core of the particles and the functionalized fluorescence markers conveniently enabled in vivo imaging of target tissues. Taken together, this M-MSNs-based siRNA delivery vehicle has shown very favorable applicability for cancer therapy. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Systemic delivery siRNA VEGF Lung cancer therapy Mesoporous silica nanoparticles Metastasis
1. Introduction Lung cancer has been the leading cancer type for cancer related death of both sexes in the US [1]. Likewise, in Europe, lung cancer has overtaken breast cancer to be the top killer among common cancers in women [2]. Unfortunately, although numerous efforts in surgery, radiation therapy and chemotherapy have been devoted to the treatment of lung cancer, the efficacy evidenced by the 5-year survival was still below 15% [3]. It is clear that development of more effective therapies for lung cancer is mandatory. Small interfering RNA (siRNA) as therapeutics could be tailored to the treatment of disease of interest by triggering specific knockdown of target genes, thereby restoring the balance of the regulatory network that otherwise leads to the disease onset [4].
* Corresponding authors. School of Biomedical Engineering & Med-X Research Institute, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China. E-mail addresses: [email protected] (H. Gu), [email protected], weiliangxia@ gmail.com (W. Xia). http://dx.doi.org/10.1016/j.biomaterials.2014.09.003 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
Currently, few siRNA-based therapies reach the final stage of clinical trials; for cancer, only limited trials have been registered [4]. The biggest obstacle, however, is to deliver intact siRNAs to the target site efficiently in vivo without inducing adverse effects. Hence the design of a safe and efficient delivery system for siRNA is critical for translating siRNA-based therapy into the clinic [5,6]. Desired features of an ideal siRNA delivery system are effective protection of loaded nucleic acids, prolonged half-life in bloodstream, efficient cell membrane translocation, timely endosomal escape, and good tissue biocompatibility [6]. Additionally, noninvasive imaging modality is also favored for real-time assessment of siRNA delivery to optimize treatment strategies or for diagnosis/ prognosis [7]. To date a number of siRNA carriers for in vivo delivery in the laboratory settings have been reported, which include lipid-based agents [8,9], cholesterol conjugation systems [10,11], cationic delivery systems [12,13], as well as inorganic nanoparticles systems [14]. Recently, a new type of drug delivery vectors termed mesoporous silica nanoparticles (MSNs) have emerged as promising chemotherapeutics/nucleic acid carriers due to their noticeable
Y. Chen et al. / Biomaterials 35 (2014) 10058e10069
advantages which include pore-size tunability, large loading capacity, excellent biocompatibility and modifiability [15]. This type of delivery platform has been tested useful as siRNA nanocarrier to treat cancer [14,16e20], yet there is still much room to improve the anticancer efficacy for systemic delivery of therapeutic siRNAs. Therefore, new generation of siRNA delivery vectors with more thorough studies covering not only their therapeutic effects but also biosafety are in great need [4]. Herein we report the development of a novel magnetic mesoporous silica nanoparticles (M-MSNs)-based siRNA delivery system for lung cancer treatment, both in ectopic and orthotopic models, and evaluate tumor metastasis in these in vivo studies. We also report in detail the biocompatibility and biosafety of our nanocarrier, with the hope of translating the laboratory findings into the clinic. We choose vascular endothelial growth factor (VEGF) as the target gene for our study. Cancer cells acquire distinctive features that enable their growth, metastasis and resistance to treatment [21]. VEGF is overexpressed and secreted by most tumor cells, especially lung cancer, which stimulates the formation of new blood vessels through proliferation of endothelial cells [22e24]. Anti-angiogenesis therapy has already been widely used in the clinic and VEGF is often targeted to demonstrate RNAi-mediated tumor therapy [25]. Decrease of intratumoral microvessel density and restrain of tumor growth as a result of VEGF down-regulation are readily observed [18,26]. 2. Materials and methods 2.1. siRNA preparation Negative Control siRNA (denoted as NC siRNA, scrambled sequences) (sense, 50 UUC UCC GAA CGU GUC ACG UdTdT-30 ; antisense, 50 -ACG UGA CAC GUU CGG AGA AdTdT-30 ), fluorescein amidite (FAM) labeled N.C. siRNA (modified at the 50 end of the sense strand). VEGF siRNA [26] (sense, 50 -GGA GUA CCC UGA UGA GAU CdTdT; antisense, 50 -GAU CUC AUC AGG GUA CUC CdTdT), fluorescently labeled Cyanine Dyes 3 (Cy3) VEGF siRNA (modified at the 50 end of the sense strand) were synthesized by GenePharma Co. Ltd. (Shanghai, China).
2.2. Preparation of M-MSN_siRNA@PEI-PEG-KALA delivery vehicles M-MSN_siRNA@PEI was synthesized in accordance with previous work [27]. In brief, 50 mL water solution of siRNA was added into 2 mL centrifuge tube with 1 mg M-MSN, then 50 mL 4 M guanidine hydrochloride solution and 200 mL ethanol was added. The mixture was well dispersed by vortex for 30 s and then was continuously shaken at 270 rpm at 25 C for 1 h. Finally, free siRNA in supernatant was eliminated through centrifugation at 12,000 rpm, and the precipitation was resuspended in ethanol. Thereafter, the siRNA-encapsulated M-MSNs were dropwise added into the PEI solution to form M-MSN_siRNA@PEI through electrostatic interaction under ultrasonication. Such M-MSN_siRNA@PEI was further washed in deionized water (pH 5.5) and eventually was dispersed in ethanol. Meanwhile, we employed a novel modification of PEI-coated M-MSN_siRNA, in which a KALA peptide related to membrane transduction and endosomal escape was coupled to PEI through a heterobifunctional polyethylene glycol (NHS-PEG-Mal, 3.5 kDa) to form an M-MSN_siRNA@PEI-PEG-KALA delivery system. First, 250 mL NHS-PEG-Mal (dissolved in DMSO, 8 mg/mL), in which the maleimide group was covalently reacted with cysteine within 50 mL KALA (dissolved in water, 0.5 mg/mL) under 37 C for 30 min resulting in an NHS-PEG-KALA conjugate. Then 1 mg prepared M-MSN_siRNA@PEI dispersed in ethanol was added into the described NHSPEG-KALA solution at room temperature (RT). After 30 min reaction, the mixture was centrifuged at 13,000 rpm to obtain the desired vector in the pellet, which were washed with deionized water and dispersed in saline at various concentrations (Fig. 1A).
2.3. Cell lines and cultures A549 cells (human alveolar basal epithelial adenocarcinomic cell line), L02 cells (human normal liver cell line), PC-3 (human prostate cancer cell line) and HCCLM-3 (human hepatocellular carcinoma cell line) were obtained from Cell Bank of Chinese Academy of Science (Shanghai, China), and cultured in DMEM (A549&HCCLM-3) or RMPI1640 (L02&PC-3) supplemented with 10% fetal bovine serum (FBS; v/v) and 2% penicillin-streptomycin (PS; v/v). The cells were incubated at 37 C with 5% CO2 in a humid cell incubator.
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2.4. Cytotoxicity assessment Cell viability of A549 and L02 cells were measured using the CCK-8 assay. Cells were seeded at a density of 1 104 cells per well on 96-well plates, and the following experiments was conducted until the 80% confluence. After removing the culture medium, the fresh medium was supplemented containing 10% FBS and various concentrations (20e400 mg/mL) of M-MSN_NC siRNA@PEI-PEG-KALA or M-MSN_NC siRNA@PEI. A negative control group was treated with equivalent volume of saline mixed with culture medium. Cells were coincubated with nanoparticles for 24 h, and subjected to CCK-8 assay following vendor's protocol. All experiments were performed in hexaplicate, and the averaged absorbance values were normalized to negative control. 2.5. In vitro RNAi experiment to determine VEGF knockdown by M-MSN_VEGF siRNA@PEI-PEG-KALA A549 cells were grown in 24-well plates to ~80% confluency, and each well was added with increasing amount of M-MSN_VEGF siRNA@PEI-PEG-KALA (equivalent siRNA dosage ranging from 37.5 nM to 187.5 nM), or with 80 mg/mL nanocarriers (equivalent siRNA dosage of 150 nM). After incubation with various siRNA vectors for 24 h, A549 cells were replenished with fresh medium and cultured for another 24 h. Then the conditioned medium was collected, centrifuged and subjected to ELISA to determine VEGF levels. PC-3 cells and HCCLM-3 cells were performed to determine the VEGF levels with 80 mg/mL nanocarriers (equivalent siRNA dosage of 150 nM) according with the experimental process in A549 cells. nM ¼ nmol/L ¼ pmol/mL. 2.6. Biodistribution study For biodistribution studies, 2 mg M-MSN_NC siRNA@PEI-PEG-KALA dispersed in 200 mL saline was i.v. injected into each tumor-bearing nude mouse (20 ± 2 g). These mice were randomly divided into three groups with each terminated at 1 day, 3 days or 7 days after injection and major organs (heart, liver, spleen, lung, kidney, brain, tumor, stomach) were collected. Dissected organs were weighed and dissolved in strong acid solution (HNO3:HF:H2SO4 ¼ 1:1:1) at 60 C for 24 h. To determine the Fe content in various organs, atomic absorption analysis (AAS) was performed (n ¼ 3/ group). Mice administrated with saline served as negative control (n ¼ 3). The Fe content in tissues was presented in the unit of the percentage of injected dose per gram of organ (%ID/g). 2.7. Pharmacokinetics study M-MSN_Cy3-siRNA@PEI-PEG-KALA or naked-Cy3-siRNA were prepared and injected intravenously into 3 six-week-old nude mice (20 ± 2 g) at a dose of 1.5 mg/ kg Cy3-labeled siRNA either as free form or within nanoparticles. At various time points after injection, 20 mL of blood was collected and dissolved in 1 mL of lysis buffer (1% SDS, 1% Triton X-100 and 40 mM Tris acetate). The Cy3 fluorescence intensity was measured by LS 55 Luminescence Spectrometer (Perkin Elmer, USA). The Cy3-siRNA content in the blood was presented in the unit of the percentage of injected dose per gram of blood (%ID/g). Total blood volume was defined as 5% of body weight. The obtained data was analyzed using WinNolin software, and the non-compartment model was fit to the data. The area under curve (AUC), plasma clearance (CI) and mean retention time (MRT) was determined. 2.8. Subdermal and orthotopic models of human lung cancer and therapy Six-week-old athymic nude mice were purchased from B&K Universal Group Limited, Shanghai, China. They were housed in sterile isolated cages with a 12 h light/dark cycle at constant temperatures (24e26 C) and had free access to food and water. Animal procedures were carried out according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Shanghai Jiao Tong University, Shanghai, China. To construct a subcutaneous lung cancer model, a total of 5 106 A549 cells mixed with Matrigel™ (BD, America) at the ratio of 1:1 (total volume 100 mL) were s.c. injected into the right flank region of female nude mice (18 ± 2 g), which were randomly divided into four groups (n ¼ 5/group). Therapy started when the xenograft tumors appeared to reach an average size of 50 mm3. Thereafter, the asdescribed siRNA formulations were administered through tail vein using 29-gauge needle syringe, at a dose of 100 mg/kg M-MSNs containing 3.5 nmol siRNA and dispersed in 200 mL saline, following the regimen described in Fig. 4A. Tumor growth was measured every five days by using the digital calipers, and the tumor volume was calculated based on the formula: tumor volume ¼ (major axis) (minor axis)2/ 2. Fifteen days after the final injection, tumors were harvested and subjected to various tests. To construct an orthotopic lung cancer model, a total of 3 106 A549 cells resuspended in Matrigel™ (BD, America) at the ratio of 1:1 (total volume 50 mL) were transthoracically injected into the upper lobe of right lung of male nude mice (23 ± 1 g) by 29-gauge needle syringe under a Leica MZ-16 dissecting microscope (MicroOptics of Florida, Davie, FL). A small wound made to facilitate transthoracic injection was closed by sutures. Then, all mice were randomly divided into four groups (n ¼ 6/group). The as-described siRNA formulations were administered through tail vein using 29-gauge needle syringe, at a dose of 100 mg/kg M-MSNs
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Fig. 1. Schematic representation of the design of this study: (A) synthesis of M-MSN_siRNA@PEI-PEG-KALA. (B) Transmission electron microscope (TEM) image of M-MSN_NC siRNA@PEI-PEG-KALA (bar ¼ 50 nm). (C) Dynamic light scattering (DLS) measurements of size distribution for M-MSN_NC siRNA@PEI-PEG-KALA (dispersed in saline). (D) TGA (thermogravimetric analysis) curves of various types of M-MSNs that were loaded with siRNA. The red dotted line is the replicated assay for different types of M-MSNs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) containing 3.5 nmol siRNA and dispersed in 200 mL saline, following the regimen described in Fig. 5A. After the survival rate of the saline group dropped to below 30%, all remaining mice were terminated by cervical dislocation, and lungs and livers were harvested for following experiments. Weight was closely monitored and measurement was carried out throughout experimental process every five days.
harvested, fixed in 4% paraformaldehyde, and histological sections (10 mm in thickness) were achieved using Freezing Microtome, stained with hematoxylin and eosin (H&E) and examined by a digital microscope (Leica, Germany).
2.9. T2-weighted MRI imaging
Data are expressed as means ± standard deviations (SD). The significance of difference was analyzed by one- or two-way analysis of variance (ANOVA), and the survival was analyzed by survival curve using the GraphPad Prism 5 software. Differences with p < 0.05 were considered statistically significant.
T2-weighted imaging was performed using MesoMR23-060HeI imaging instrument (Shanghai Niumag Corporation). Tumor-bearing mice was anaesthetized with ketamine (10 mL/kg). Imaging was performed before, and at intervals after i.v. injection of M-MSN_NC siRNA@PEI-PEG-KALA at a dose of 100 mg/kg. The instrumental parameters were set as follows: a 0.55 T magnet, K space ¼ 192 256 mm, section thickness ¼ 3 mm, TE ¼ 60 ms, TR ¼ 2000 ms, FOVRead ¼ 100 mm, FOVPhase ¼ 100 mm, and number of scans (NS) ¼ 4.
2.11. Statistical analysis
3. Results
2.10. Blood analysis and histology
3.1. Preparation and characterization of a novel siRNA nanocarrier: M-MSN_siRNA@PEI-PEG-KALA
All blood analyses were performed by Shanghai Research Center for Biomodel Organisms, Shanghai, China. Measurements for major hematology and immunogenicity related markers were performed on whole blood, and various biochemistry parameters were tested on mouse serum 48 h after the final injection of five repeated doses of 100 mg/kg M-MSN_NC siRNA@PEI-PEG-KALA every five days. The control group was saline without nanocarriers treated in exactly the same regimen. All blood samples were stabilized with heparin (10 U/500 mL whole blood). After collection of blood, animals were killed by cervical dislocation under deep ketamine anesthesia. Major organs including liver, spleen, lung and kidney were
In this study, we developed a novel type of siRNA nanocarrier, MMSN_siRNA@PEI-PEG-KALA, which is composed of several key units: an M-MSN core containing the cargo siRNA, a polyethylenimine (PEI) layer coated for the following functionalization steps that include anti-fouling coating with polyethylene glycol (PEG), and conjugation of a fusogenic KALA peptide with endosomolytic function [28] (Fig. 1A). M-MSNs were synthesized to be of ~50 nm in diameter
Y. Chen et al. / Biomaterials 35 (2014) 10058e10069
(Supplementary Fig. 1A) and ~3.6 nm in pore size (Supplementary Fig. 1B). Then, siRNA was loaded into M-MSNs under such solution condition as 0.67 M guanidine hydrochloride (Guan-HCl) plus 66.7% ethanol (v/v). Due to the charge screening by Guan-HCl, siRNA could flow into the pores of M-MSNs and the dehydrated effect mediated by organic solvent resulted in the encapsulation of siRNA into the mesopores [27]. The amount of siRNA loaded in M-MSNs could be regulated through increasing the concentration of siRNA without volume change in the loading solution. These siRNA-loaded M-MSNs were coated with PEI to form M-MSN_siRNA@PEI composite through electrostatic interaction. Then a biocompatible macromolecule Nhydroxy-succinimide-poly (ethylene glycol)-maleimide (NHS-PEGMal, 3.5 kDa; PEG refers to this size thereafter) [29] containing maleimide moieties was firstly reacted with the thiol groups in KALA peptide to form NHS-PEG-KALA complex, which were grafted onto the surface of M-MSN_siRNA@PEI via the reaction between amino groups and NHS. The resultant siRNA nanocarrier was denoted as MMSN_siRNA@PEI-PEG-KALA, whose physical/chemical properties were characterized. We used negative control siRNA (NC siRNA, scrambled sequences) in these experiments. In this work, the loading amount of siRNA in vector was ~30 mg siRNA/g M-MSNs (1.875 nmol/mg M-MSNs) and ~28 mg siRNA/g M-MSNs (1.75 nmol/ mg M-MSNs) for in vitro gene silencing and in vivo cancer therapy applications, respectively. TEM image of M-MSN_NC siRNA@PEI-PEG-KALA showed that the nanoparticle size was estimated to be ~50 nm (Fig. 1B), with Zeta potential of þ23.6 ± 1.2 mV (Supplementary Fig. 2A). Dynamic light scattering (DLS) analysis then showed that the M-MSN_NC siRNA@PEI-PEG-KALA had a larger diameter at 154.4 ± 1 nm (Fig. 1C) than those of M-MSN_NC siRNA@PEI (142.6 ± 1 nm) and M-MSNs (89.6 ± 0.7 nm) (Supplementary Fig. 3A). These results indicated that the particle size increased because of the modification of polymer coating. In order to demonstrate that M-MSNs had been modified with polymer successfully, a positive staining reagent phosphotungstic acid (PTA) was introduced [18]. Consequently, TEM showed that the contrast of the amine groups in PEI and KALA peptides in M-MSN_NC siRNA@PEI-PEG-KALA was enhanced through PTA staining, but the size was also estimated to be ~50 nm (Supplementary Fig. 3B). In another word, M-MSN and M-MSN@PEI-PEG-KALA exhibited similar sizes by TEM experiments, which failed to distinguish the polymer thickness in these particles. We then performed thermogravimetric analysis (TGA) to estimate the amount (weight ratio) of loaded/modified molecules in the nanocarrier (Fig. 1D). The weight loss for M-MSN_NC siRNA, M-MSN_NC siRNA@PEI and M-MSN_NC siRNA@PEI-PEG-KALA were 8.9 ± 0.141%, 22.35 ± 0.071% and 30.66 ± 0.198% of the total, respectively (Fig. 1D). Based on these results, the relative mass ratios of PEI and PEG-KALA in the M-MSN_NC siRNA@PEI-PEG-KALA were ~14.6% and ~11.0%, respectively. The ratio of grafted KALA was ~17.4% of total KALA, as was determined by fluorescent intensities using FITC-labeled KALA (Supplementary Fig. 2B). The transverse relaxation time (T2) of M-MSN_siRNA@PEI-PEG-KALA was also measured at 0.5 T with a spin-echo pulse sequence. The drastically decreased signal intensity of T2-weighted images was observed as Fe concentration (correspond to nanoparticle concentration) increased ( Supplementary Fig. 2C). The T2 relaxation rate (1/T2) and Fe concentration rendered good linearity indicating M-MSN_siRNA@PEI-PEG-KALA as a contrast agent with the r2 value of 226.92 mM1s1 (Supplementary Fig. 2D). 3.2. Cytotoxicity and gene silencing effectiveness of MMSN_siRNA@PEI-PEG-KALA in vitro Cytotoxicity is a primary concern in development of a novel drug delivery system. The cell viability of A549 (an adenocarcinomic
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human alveolar basal epithelial cell line) and L02 (human normal hepatic cell line) incubated with various concentrations of MMSN_NC siRNA@PEI-PEG-KALA for 24 h was measured by the CCK-8 assay (Fig. 2A). These nanocarriers showed negligible cytotoxicity even at 200 mg/mL for both A549 and L02 cell lines (Fig. 2A). By contrast, M-MSN_NC siRNA@PEI exhibited severe cytotoxicity in A549 and L02 cell lines even at a much lower concentration (50 mg/ mL) (Supplementary Fig. 4). To further assess their biocompatibility, we conducted experiments to characterize the interactions of various functionalized M-MSNs with red blood cells (RBCs). Hemolytic activities of M-MSN_NC siRNA@PEI without modification of PEG-KALA conjugation (>85%, normalized to positive control) and M-MSN_NC siRNA@PEI-PEG-KALA (
