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Escheriosome-mediated cytosolic delivery of PLK1-specific siRNA: potential in treatment of liver cancer in BALB/c mice Aim: In the present study, the anticancer efficacy of a novel escheriosome-based formulation of PLK1-specific siRNA was evaluated against liver cancer in BALB/c mice. Materials & methods: The escheriosome-based siRNA nanoparticles were prepared using lipids isolated from Escherichia coli. The escheriosomes were characterized for size, surface charge and stability. The anticancer potential of PLK1-specific siRNA formulation was ascertained on the basis of expression of pro-/anti-apoptotic factors and histopathological studies. Results: The escheriosome-entrapped siRNA was found to be released in surrounding milieu in a sustained manner. The nanoformulation was successful in modulating proapoptotic factors and eventually helped in better survival of the treated animals. Conclusion: Our data demonstrate the efficacy of systemically administered siRNA in the treatment of experimental liver cancer. This novel therapeutic strategy may be applicable to a broad range of cancers in patients with the obstinate form of the disease. KEYWORDS: apoptosis • DEN • escheriosome • liver cancer • nanoparticle • p53 • PLK1 • siRNA
RNA interference (RNAi) is a newly discovered cellular strategy for silencing genes in a sequence-specific manner. The introduction of cognate double-stranded siRNA can knock down the target on the basis of direct homology-dependent post-transcriptional gene silencing. The promise of specific RNA degradation has generated much excitement and opened new vistas for its use as a therapeutic modality in cancer therapy, since specific siRNAs can be designed to stifle oncogenes involved in proliferation, survival, invasion, angiogenesis, metastasis, as well as inhibition of apoptosis. The strategy can also be extended to regulate genes imparting resistance to chemo- or radio-therapy [1,2]. Polo-like kinases (PLKs), a family of conserved serine/threonine kinases, are important regulators of cell-cycle progression [3–5]. Among various members of the PLK family, PLK1 represents an ideal protein-kinase target for cancer drug development. PLK1 gene expression is tightly controlled with mRNA
10.2217/NNM.13.21 © 2014 Future Medicine Ltd
Arun Chauhan1, Swaleha Zubair2, Ahmad Nadeem3, Sajid Ali Ansari4, Mohammad Yunus Ansari1 & Owais Mohammad*1 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India 2 Women’s College, Aligarh Muslim University, Aligarh-202002, India 3 Amity University, Lucknow-34, India 4 Centre of Excellence in Material Sciences, Department of Applied Physics, Aligarh Muslim University, Aligarh, India *Author for correspondence: Tel.: +91 0571 2720388 Fax: +91 0571 2721776 [email protected] 1
accumulation beginning in the S phase, with peak levels detected at the G2/M transition and through mitosis with a corresponding increase in levels of expressed proteins [3]. PLK1 contributes to multiple processes, including centrosome maturation, bipolar spindle formation, cytokinesis, the activation of the Cdk1/cyclin B1 cascade by phosphorylating cyclin B1 and cdc25C, targeting them toward the nucleus and regulating anaphasepromoting complex-mediated degradation required for mitotic exit. Overexpression of PLK1 has been shown to result in the formation of abnormal centrosomes and mitotic spindle poles, which have been correlated with aneuploidy and chromosomal instability leading to tumor development [4,5]. Despite the considerable potential of RNAi in the treatment of cancer and various other disorders, several impediments demand appropriate attention before exogenous siRNA can be widely used as an effective anticancer agent. These include:
Nanomedicine (2014) 9(4), 407–420
part of
ISSN 1743-5889
407
Research Article Chauhan, Zubair, Nadeem, Ansari, Ansari & Mohammad • Rapid degradation of siRNA by serum nucleases • Poor membrane permeability ensuing in limited cellular uptake • The need for effective design of active siRNAs to ensure optimal gene-silencing activity, with minimal ‘off-target’ effects • The need to achieve efficient intracellular delivery to target cells in vivo Several delivery vehicles have been combined with siRNAs to improve their delivery in animal models [6,7], such as nanoparticles [8], aptamer–siRNA conjugates [9], nanoimmunoliposomes [10], cationic polymer and lipid-based siRNA complexes, among others [11]. Among various strategies, viral-based vectors are highly efficient delivery systems for siRNA; however, the potential for mutagenicity, limited loading capacity, high production costs and, most importantly, safety risks caused by their inflammatory and immunogenic effects severely limit the applicability of virosomes. These concerns have led to the pursuit of nonviral alternatives [6–9]. Unfortunately, most of the available nonviral delivery systems are also marred by several limitations that pose hurdles in usage of siRNA for treatment of cancer in clinical settings [8,9]. We earlier demonstrated that the plasma membrane lipids of Escherichia coli have strong membrane–membrane fusion properties, and lipid vesicles formed thereof can deliver encapsulated material to the cytosol of the target cells [12,13]. In the present study, we developed siRNA-based fusogenic liposome (escheriosome [EC]) prepared with the plasma membrane lipid of E. coli to achieve cytosolic delivery of siRNA. The in-house developed nanoparticles were characterized for their size, surface properties and entrapment efficiency of encapsulated siRNA. Finally, we established the anticancer potential of siRNA-bearing EC nanoparticles in the treatment of liver cancer in model animals. Besides survival of cancer inflicted animals, the regulation of anti-/pro-apoptotic protein expression in cancer cells was used as a parameter to establish efficacy of siRNA-bearing ECs. Materials & methods Materials
E. coli K12 (nonpathogenic) strain was procured from the Institute of Microbial Technology (Chandigarh, India). Diethyl nitrosamine (DEN), RPMI-1640 medium and bicinchoninic acid protein estimation kit were purchased from Sigma-Aldrich (MO, USA). PLK1-specific siRNA was purchased from Santa Cruz Biotechnology, Inc. (CA, USA). The rest of the chemicals were of the highest purity available (Supplementary Material; see
408
Nanomedicine (2014) 9(4)
online at www.futuremedicine.com/doi/suppl/10.2217/ NNM.13.21). Animals
Female BALB/c mice of weight 20 ± 2 g (4–6 weeks old) were obtained from the Indian Veterinary Research Institute’s (Bareilly, India) animal house facility (Supplementary Material). Preparation of siRNA-bearing EC
Briefly, E. coli membrane lipids were isolated using published procedure as standardized in our laboratory [13]. The lipid solution was reduced to thin, dry film and hydrated followed by sonication under N2 atmosphere to get plain EC preparation (Supplementary Material). The empty ECs, prepared following the aforementioned method, were mixed at this stage with an equal volume of siRNA solution (10 mg/ml) [14]. The mixture was flash frozen and thawed (three cycles), and then lyophilized. The free-flowing, dried powder thus obtained was rehydrated and passed through a poly carbonate membrane filter to obtain a homogenous liposomal formulation. The final preparation was centrifuged at 14,000 × g and the pellet was further washed at least three times with normal saline to remove the traces of unentrapped siRNA. Entrapment efficiency of siRNA in EC nanoparticles
Entrapment efficacy of siRNA in ECs was assessed by dissolving an aliquot of the nanoparticles in HPLCgrade methanol followed by analysis of siRNA content by HPLC method (Supplementary Material) [15]. The percentage entrapment efficiency (%EE) was calculated using Equation 1: % EE = ^ Amount of siRNA entrapped h ^Total amount of siRNA used in the beginningh
# 100
(Equation 1)
Determination of zeta-potential of EC-based siRNA
Zeta-potential of EC-based siRNA (EC-siRNA) nano particles was measured by the Zetasizer Nano ZS instrument (Malvern Instrument Limited, Worcestershire, UK; Supplementary Material). Transmission electron microscopy
Transmission electron microscopy (TEM) was performed to characterize the size of EC-siRNA nanoparticles using the transmission electron microscope, model Zeiss EVO®40 electron microsope (Carl Zeiss, Oberkochen, Germany). An aliquot of EC formulation (a drop) was mounted on a clear glass stub, air dried and coated
future science group
siRNA nanoformulation against liver cancer
with gold–palladium alloy using a sputter coater. An accelerating voltage of 20.00 kV was used for imaging. NANOPHOX imaging
Research Article
Pi/200 µl per animal,) was administered intravenously once a day for 7 days consecutively. • Group 1: healthy control animals
The lyophilized preparation of EC-siRNA nanoparticles was suspended in distilled water (2 mg/ml) and a single drop was analyzed by Particle Size Analyzer NANOPHOX (Sympatec GmbH, Clausthal-Zefferfeld, Germany) (Supplementary Material).
• Group 2: untreated animals with liver cancer (DEN administered followed by no siRNA treatment)
Atomic force microscopy analysis
• Group 5: siRNA-bearing EC-treated animals
A PerkinElmer digital instrument multimode scanning probe microscope (PerkinElmer, Singapore) equipped with a nanoscope controller was used for atomic force microscopy (AFM) analysis. A drop of the formulation was mounted and used for drop coating onto a Si (III) disc. Samples were analyzed using contact mode AFM. In vitro release kinetics of active siRNA from EC nanoparticles
In order to assess the release kinetics of siRNA from ECs, multiple weighed aliquots of siRNA nanoparticles were dispensed in separate microvials. Various buffers of the desired pH range (pH 5, 7 or 9) were used to perform release kinetics studies (Supplementary Material).
• Group 3: Sham-ECs (empty ECs) • Group 4: siRNA-only treated animals
• Group 6: scrambled siRNA-treated animals Histopathological studies
To examine the effect of EC-siRNA nanoparticles in the treatment of cancer, liver tissue samples were collected from control as well as various experimental groups, and histopathological studies were performed (Supplementary Material). Preparation of nuclear fraction
Potential of EC-encapsulated siRNA in the treatment of cancer
The liver tissues were removed from experimental mice using sharp scalpel blades. The tissue samples were placed on ice before processing [17], homogenized in a Teflon® homogenizer (Thomas Scientific, NJ, USA) in the presence of protease inhibitor cocktail, following published procedure as standardized in our laboratory (Supplementary Material) [17].
Induction & treatment of liver cancer in BALB/c mice
Western blotting
Liver cancer was induced in experimental mice by administering a single dose of DEN in 0.5 ml normal saline via the intraperitoneal route [16]. Establishment of liver cancer was confirmed by estimation of liver enzymes (alkaline phosphatase, aspartate transaminase and g-glutamyl transferase), and also by histopathological studies that were performed 1 month following DEN administration. At 1 month post-DEN treatment, siRNAbearing EC formulation (100 nM siRNA/500 nm lipid Pi/200 µl normal saline per animal) was administrated intravenously for 7 days consecutively. After a resting period of 10 days, mice were euthanized by CO2 inhalation, liver tissue from normal as well as cancer-inflicted mice were excised and portions of the tissues were prepared for further studies. Frozen tissues were stored at -80oC until further experimentation. Treatment schedule & assessment parameters
The animals with a liver tumor were pooled and randomized into five groups each comprising of ten animals. Normal, untreated mice were included in the control group. At 1 month post-DEN treatment, siRNAbearing EC formulation (100 nM siRNA/500 nm lipid
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Liver cancer cell lysate (30 µg protein) was resolved on 10% SDS-PAGE gel and electro blotted onto nitrocellulose membrane [18]. Employing an enhanced chemiluminiscence kit, the membrane was probed for the presence of p53, p53 mutant (p53mut), PLK1 and Bax using specific antibodies. Blots were reprobed with an antibody for b-actin and used as a control for equal protein loading and transfer. Densitometric values of protein bands were quantified using Alpha Image Analysis software (Proteinsimple, CA, USA) on Alpha Image Gel Documentation System. Determination of caspase-9 level by confocal microscopy
Liver cancer cells were isolated from various experimental groups (Sham-EC, free siRNA and ECsiRNA-treated cancerous mice) following protocol as standardized in our laboratory [19]. Confocal microscopy was performed using a Zeiss 510 Meta confocal microscope (Carl Zeiss) equipped with an argon ion laser mounted on an inverted microscope (Supplementary Material).
www.futuremedicine.com
409
Research Article Chauhan, Zubair, Nadeem, Ansari, Ansari & Mohammad Statistical analysis
Size analysis of EC-siRNA nanoparticles
The Kaplan–Meier analysis was employed to estimate survival of cancer-free animals and differences were analyzed by log-rank test.
Figure 2A
Results Entrapment efficiency, zeta-potential and release kinetics of EC-siRNA nanoparticles
EC-siRNA formulation showed approximately 25.4 ± 3.3 %EE of PLK1-specific siRNA. Furthermore, the siRNA-bearing nanoparticles were found to have a zeta-potential of -5.4 ± 0.6 mV, suggesting a net negative charge on the surface of vesicles. The release profile exhibited slow and sustained release of siRNA over an extended time period. The nanoparticles were found to withstand mild change in pH conditions for a period of more than 50 h (Figure 1). For the initial 10 h, only approximately 10–20% of total-loaded siRNA was released at pH 7 and 9. In the next 50 h, approximately 21 and 38% of siRNA was released from the formulation incubated at pH 9 and 7, respectively; whereas, 61% of siRNA was released at pH 5. Altogether, the delivery system was able to maintain slow and steady release of encapsulated siRNA for an extended time period.
shows a representative TEM image of the siRNA-bearing EC nanoparticles. Formation of spherical nanoparticles with a size of 140 ± 10 nm is clearly revealed by TEM analysis. The dimensions of EC-siRNA nanoparticles were further established by NANOPHOX particle analyzer. The size distribution of the particles was determined by digital analysis of the image of counted particles. A sharp and predominant single peak approximately 150 ± 15 nm signifies size distribution, which is in concordance with the electron microscopically determined size of the in-house prepared nanoparticles (Figure 2B). A representative AFM image of nano drug formulation (Figure 2C) revealed a number of spherical nanoparticles. The liposomes were not overlapping with each other or in the form of clusters and assemblies. In Figure 2C, the height of the ECsiRNA nanoparticle is exaggerated for better perception of the fine structures as visualized with single-surface 3D AFM. Histopathological studies
The efficacy of various forms of PLK1-specific siRNA was established by histopathological studies. Liver tissue samples from mice belonging to various treated
70 EC-siRNA nanoparticle (pH 9) EC-siRNA nanoparticle (pH 7)
60
EC-siRNA nanoparticle (pH 5)
Release (%)
50
40
30
20
10
0 0
10
20
30
40
50
60
Time (h) Figure 1. Release profile of siRNA from escheriosome-based nanoparticles. siRNA-bearing EC formulation was incubated in sterile phosphate-buffered saline (5 mM PO4; 150 mM saline) of various pHs (pH 5, 7 and 9) for an extended time period. The released siRNA in the surrounding medium was quantified over time by HPLC, as described in the ‘Materials & methods’ section. Data are mean ± standard deviation of three independent experiments. EC: Escheriosome.
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Nanomedicine (2014) 9(4)
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Research Article
siRNA nanoformulation against liver cancer
Impact of EC-siRNA nanoparticles on apoptosis
Downregulation of p53 wild-type (p53wt) was observed upon exposure to DEN (control untreated cancerous mice; Figure 4A, lane 2) compared with healthy (normal) control mice (Figure 4A, lane 1). Treatment with Sham-EC did not show significant effect on upregulation of p53wt (Figure 4A , lane 3), whereas free siRNA treatment resulted in a slight increase in the expression of p53wt, which was not only used as a parameter to establish induction of liver cancer in the experimental animals, but also offered as a tool to ascertain efficacy of various siRNA formulations (Figure 4A, lane 4). Significant upregulation of p53wt was observed when cancerous mice were treated with EC-siRNA nanoparticle formulations (Figure 4A, lane 5). As depicted in Figure 4B,i, a comparatively upregulated expression (80%) of p53wt protein was recorded in mice treated with siRNA-bearing EC formulations compared with healthy control animals (100%). The groups of animals treated with Sham-EC (17%), free siRNA (31%) or untreated animals (18%) were not able to elevate the level of p53 in the treated animals. Exposure of animals to DEN ensued in upregulation of p53mut (Figure 4A, lane 2). Treatment with EC-siRNA nano particle formulations resulted in its downregulation
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A
500 nm
Density distribution q3*
B
275 250 225 200 175 150 125 100 75 50 25 0.0 0.51.0
5 10
50 100 500 1000 5000 10,000 Particle size (nm)
30 25 20 15 10 5 0
Particle size (µm)
C Particle size (nm)
groups were used for histopathological analysis. Figure 3 shows sections of liver cancer cells after treatment with free, as well as nanoparticle encapsulated, siRNA formulations. Control healthy liver tissue shows normal hepatic laminae, sinusoids and hepatocytes (Figure 3A). No congestion and infiltration was observed; whereas, cancerous liver (untreated control; Figure 3B) shows a loss of normal hepatic structure, including laminae and sinusoids, dilated central vein and widespread hepatocyte necrosis. The treatment with free siRNA, resulted in mild recovery of cancerous liver (Figure 3D), showing enlarged hepatocyte contour, narrowing of sinusoidal space, mild apoptosis and Kupffer cell prominence was still predominantly seen. By contrast, in the mice treated with siRNA-bearing EC formulation, noticeable tumor regression in comparison with free form siRNA and untreated control was observed. Maintained hepatic microstructure, focal necrosis and apoptosis were also detected. siRNA-bearing EC treatment showed the best regression where normal hepatic architecture was found to be maintained. Focal lytic apoptosis was also very much apparent. The group of animals treated with Sham-EC or scrambled siRNA showed clusters of centrilobular oxyphilic hepatocytes, which are presumably because of coagulative necrosis (Figure 3C). Moreover, hepatic microstructures were maintained to a good extent. Most of the hepatoctyes were accompanied with eosinophilic as well as abundant Kupffer cells.
0.8 0
0.2
0.4 0.6 0.8 1.0 Particle size (µm)
1.2
0
Figure 2. Escheriosome size determination. (A) Representative transmission electron microscope image of escheriosome-based siRNA nanoparticles. (B) Corresponding particle analyzer data of escheriosome-based siRNA nanoparticle as obtained by NANOPHOX (Sympatec GmbH, Clausthal-Zefferfeld, Germany) particle size analyzer. (C) Representative 3D atomic force microscope image of escheriosome-based siRNA nanoparticles.
(Figure 4A, lane 5). As depicted in Figure 4B,ii, the level of p53mut was reduced to normal levels in the animals treated with siRNA-bearing EC (20%) compared with the untreated control (92%). Treatment with free siRNA was not as effective in downregulation of p53mut (81%) compared with its nanoparticle encapsulated form (p
Escheriosome-mediated cytosolic delivery of PLK1-specific siRNA: potential in treatment of liver cancer in BALB/c mice Aim: In the present study, the anticancer efficacy of a novel escheriosome-based formulation of PLK1-specific siRNA was evaluated against liver cancer in BALB/c mice. Materials & methods: The escheriosome-based siRNA nanoparticles were prepared using lipids isolated from Escherichia coli. The escheriosomes were characterized for size, surface charge and stability. The anticancer potential of PLK1-specific siRNA formulation was ascertained on the basis of expression of pro-/anti-apoptotic factors and histopathological studies. Results: The escheriosome-entrapped siRNA was found to be released in surrounding milieu in a sustained manner. The nanoformulation was successful in modulating proapoptotic factors and eventually helped in better survival of the treated animals. Conclusion: Our data demonstrate the efficacy of systemically administered siRNA in the treatment of experimental liver cancer. This novel therapeutic strategy may be applicable to a broad range of cancers in patients with the obstinate form of the disease. KEYWORDS: apoptosis • DEN • escheriosome • liver cancer • nanoparticle • p53 • PLK1 • siRNA
RNA interference (RNAi) is a newly discovered cellular strategy for silencing genes in a sequence-specific manner. The introduction of cognate double-stranded siRNA can knock down the target on the basis of direct homology-dependent post-transcriptional gene silencing. The promise of specific RNA degradation has generated much excitement and opened new vistas for its use as a therapeutic modality in cancer therapy, since specific siRNAs can be designed to stifle oncogenes involved in proliferation, survival, invasion, angiogenesis, metastasis, as well as inhibition of apoptosis. The strategy can also be extended to regulate genes imparting resistance to chemo- or radio-therapy [1,2]. Polo-like kinases (PLKs), a family of conserved serine/threonine kinases, are important regulators of cell-cycle progression [3–5]. Among various members of the PLK family, PLK1 represents an ideal protein-kinase target for cancer drug development. PLK1 gene expression is tightly controlled with mRNA
10.2217/NNM.13.21 © 2014 Future Medicine Ltd
Arun Chauhan1, Swaleha Zubair2, Ahmad Nadeem3, Sajid Ali Ansari4, Mohammad Yunus Ansari1 & Owais Mohammad*1 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India 2 Women’s College, Aligarh Muslim University, Aligarh-202002, India 3 Amity University, Lucknow-34, India 4 Centre of Excellence in Material Sciences, Department of Applied Physics, Aligarh Muslim University, Aligarh, India *Author for correspondence: Tel.: +91 0571 2720388 Fax: +91 0571 2721776 [email protected] 1
accumulation beginning in the S phase, with peak levels detected at the G2/M transition and through mitosis with a corresponding increase in levels of expressed proteins [3]. PLK1 contributes to multiple processes, including centrosome maturation, bipolar spindle formation, cytokinesis, the activation of the Cdk1/cyclin B1 cascade by phosphorylating cyclin B1 and cdc25C, targeting them toward the nucleus and regulating anaphasepromoting complex-mediated degradation required for mitotic exit. Overexpression of PLK1 has been shown to result in the formation of abnormal centrosomes and mitotic spindle poles, which have been correlated with aneuploidy and chromosomal instability leading to tumor development [4,5]. Despite the considerable potential of RNAi in the treatment of cancer and various other disorders, several impediments demand appropriate attention before exogenous siRNA can be widely used as an effective anticancer agent. These include:
Nanomedicine (2014) 9(4), 407–420
part of
ISSN 1743-5889
407
Research Article Chauhan, Zubair, Nadeem, Ansari, Ansari & Mohammad • Rapid degradation of siRNA by serum nucleases • Poor membrane permeability ensuing in limited cellular uptake • The need for effective design of active siRNAs to ensure optimal gene-silencing activity, with minimal ‘off-target’ effects • The need to achieve efficient intracellular delivery to target cells in vivo Several delivery vehicles have been combined with siRNAs to improve their delivery in animal models [6,7], such as nanoparticles [8], aptamer–siRNA conjugates [9], nanoimmunoliposomes [10], cationic polymer and lipid-based siRNA complexes, among others [11]. Among various strategies, viral-based vectors are highly efficient delivery systems for siRNA; however, the potential for mutagenicity, limited loading capacity, high production costs and, most importantly, safety risks caused by their inflammatory and immunogenic effects severely limit the applicability of virosomes. These concerns have led to the pursuit of nonviral alternatives [6–9]. Unfortunately, most of the available nonviral delivery systems are also marred by several limitations that pose hurdles in usage of siRNA for treatment of cancer in clinical settings [8,9]. We earlier demonstrated that the plasma membrane lipids of Escherichia coli have strong membrane–membrane fusion properties, and lipid vesicles formed thereof can deliver encapsulated material to the cytosol of the target cells [12,13]. In the present study, we developed siRNA-based fusogenic liposome (escheriosome [EC]) prepared with the plasma membrane lipid of E. coli to achieve cytosolic delivery of siRNA. The in-house developed nanoparticles were characterized for their size, surface properties and entrapment efficiency of encapsulated siRNA. Finally, we established the anticancer potential of siRNA-bearing EC nanoparticles in the treatment of liver cancer in model animals. Besides survival of cancer inflicted animals, the regulation of anti-/pro-apoptotic protein expression in cancer cells was used as a parameter to establish efficacy of siRNA-bearing ECs. Materials & methods Materials
E. coli K12 (nonpathogenic) strain was procured from the Institute of Microbial Technology (Chandigarh, India). Diethyl nitrosamine (DEN), RPMI-1640 medium and bicinchoninic acid protein estimation kit were purchased from Sigma-Aldrich (MO, USA). PLK1-specific siRNA was purchased from Santa Cruz Biotechnology, Inc. (CA, USA). The rest of the chemicals were of the highest purity available (Supplementary Material; see
408
Nanomedicine (2014) 9(4)
online at www.futuremedicine.com/doi/suppl/10.2217/ NNM.13.21). Animals
Female BALB/c mice of weight 20 ± 2 g (4–6 weeks old) were obtained from the Indian Veterinary Research Institute’s (Bareilly, India) animal house facility (Supplementary Material). Preparation of siRNA-bearing EC
Briefly, E. coli membrane lipids were isolated using published procedure as standardized in our laboratory [13]. The lipid solution was reduced to thin, dry film and hydrated followed by sonication under N2 atmosphere to get plain EC preparation (Supplementary Material). The empty ECs, prepared following the aforementioned method, were mixed at this stage with an equal volume of siRNA solution (10 mg/ml) [14]. The mixture was flash frozen and thawed (three cycles), and then lyophilized. The free-flowing, dried powder thus obtained was rehydrated and passed through a poly carbonate membrane filter to obtain a homogenous liposomal formulation. The final preparation was centrifuged at 14,000 × g and the pellet was further washed at least three times with normal saline to remove the traces of unentrapped siRNA. Entrapment efficiency of siRNA in EC nanoparticles
Entrapment efficacy of siRNA in ECs was assessed by dissolving an aliquot of the nanoparticles in HPLCgrade methanol followed by analysis of siRNA content by HPLC method (Supplementary Material) [15]. The percentage entrapment efficiency (%EE) was calculated using Equation 1: % EE = ^ Amount of siRNA entrapped h ^Total amount of siRNA used in the beginningh
# 100
(Equation 1)
Determination of zeta-potential of EC-based siRNA
Zeta-potential of EC-based siRNA (EC-siRNA) nano particles was measured by the Zetasizer Nano ZS instrument (Malvern Instrument Limited, Worcestershire, UK; Supplementary Material). Transmission electron microscopy
Transmission electron microscopy (TEM) was performed to characterize the size of EC-siRNA nanoparticles using the transmission electron microscope, model Zeiss EVO®40 electron microsope (Carl Zeiss, Oberkochen, Germany). An aliquot of EC formulation (a drop) was mounted on a clear glass stub, air dried and coated
future science group
siRNA nanoformulation against liver cancer
with gold–palladium alloy using a sputter coater. An accelerating voltage of 20.00 kV was used for imaging. NANOPHOX imaging
Research Article
Pi/200 µl per animal,) was administered intravenously once a day for 7 days consecutively. • Group 1: healthy control animals
The lyophilized preparation of EC-siRNA nanoparticles was suspended in distilled water (2 mg/ml) and a single drop was analyzed by Particle Size Analyzer NANOPHOX (Sympatec GmbH, Clausthal-Zefferfeld, Germany) (Supplementary Material).
• Group 2: untreated animals with liver cancer (DEN administered followed by no siRNA treatment)
Atomic force microscopy analysis
• Group 5: siRNA-bearing EC-treated animals
A PerkinElmer digital instrument multimode scanning probe microscope (PerkinElmer, Singapore) equipped with a nanoscope controller was used for atomic force microscopy (AFM) analysis. A drop of the formulation was mounted and used for drop coating onto a Si (III) disc. Samples were analyzed using contact mode AFM. In vitro release kinetics of active siRNA from EC nanoparticles
In order to assess the release kinetics of siRNA from ECs, multiple weighed aliquots of siRNA nanoparticles were dispensed in separate microvials. Various buffers of the desired pH range (pH 5, 7 or 9) were used to perform release kinetics studies (Supplementary Material).
• Group 3: Sham-ECs (empty ECs) • Group 4: siRNA-only treated animals
• Group 6: scrambled siRNA-treated animals Histopathological studies
To examine the effect of EC-siRNA nanoparticles in the treatment of cancer, liver tissue samples were collected from control as well as various experimental groups, and histopathological studies were performed (Supplementary Material). Preparation of nuclear fraction
Potential of EC-encapsulated siRNA in the treatment of cancer
The liver tissues were removed from experimental mice using sharp scalpel blades. The tissue samples were placed on ice before processing [17], homogenized in a Teflon® homogenizer (Thomas Scientific, NJ, USA) in the presence of protease inhibitor cocktail, following published procedure as standardized in our laboratory (Supplementary Material) [17].
Induction & treatment of liver cancer in BALB/c mice
Western blotting
Liver cancer was induced in experimental mice by administering a single dose of DEN in 0.5 ml normal saline via the intraperitoneal route [16]. Establishment of liver cancer was confirmed by estimation of liver enzymes (alkaline phosphatase, aspartate transaminase and g-glutamyl transferase), and also by histopathological studies that were performed 1 month following DEN administration. At 1 month post-DEN treatment, siRNAbearing EC formulation (100 nM siRNA/500 nm lipid Pi/200 µl normal saline per animal) was administrated intravenously for 7 days consecutively. After a resting period of 10 days, mice were euthanized by CO2 inhalation, liver tissue from normal as well as cancer-inflicted mice were excised and portions of the tissues were prepared for further studies. Frozen tissues were stored at -80oC until further experimentation. Treatment schedule & assessment parameters
The animals with a liver tumor were pooled and randomized into five groups each comprising of ten animals. Normal, untreated mice were included in the control group. At 1 month post-DEN treatment, siRNAbearing EC formulation (100 nM siRNA/500 nm lipid
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Liver cancer cell lysate (30 µg protein) was resolved on 10% SDS-PAGE gel and electro blotted onto nitrocellulose membrane [18]. Employing an enhanced chemiluminiscence kit, the membrane was probed for the presence of p53, p53 mutant (p53mut), PLK1 and Bax using specific antibodies. Blots were reprobed with an antibody for b-actin and used as a control for equal protein loading and transfer. Densitometric values of protein bands were quantified using Alpha Image Analysis software (Proteinsimple, CA, USA) on Alpha Image Gel Documentation System. Determination of caspase-9 level by confocal microscopy
Liver cancer cells were isolated from various experimental groups (Sham-EC, free siRNA and ECsiRNA-treated cancerous mice) following protocol as standardized in our laboratory [19]. Confocal microscopy was performed using a Zeiss 510 Meta confocal microscope (Carl Zeiss) equipped with an argon ion laser mounted on an inverted microscope (Supplementary Material).
www.futuremedicine.com
409
Research Article Chauhan, Zubair, Nadeem, Ansari, Ansari & Mohammad Statistical analysis
Size analysis of EC-siRNA nanoparticles
The Kaplan–Meier analysis was employed to estimate survival of cancer-free animals and differences were analyzed by log-rank test.
Figure 2A
Results Entrapment efficiency, zeta-potential and release kinetics of EC-siRNA nanoparticles
EC-siRNA formulation showed approximately 25.4 ± 3.3 %EE of PLK1-specific siRNA. Furthermore, the siRNA-bearing nanoparticles were found to have a zeta-potential of -5.4 ± 0.6 mV, suggesting a net negative charge on the surface of vesicles. The release profile exhibited slow and sustained release of siRNA over an extended time period. The nanoparticles were found to withstand mild change in pH conditions for a period of more than 50 h (Figure 1). For the initial 10 h, only approximately 10–20% of total-loaded siRNA was released at pH 7 and 9. In the next 50 h, approximately 21 and 38% of siRNA was released from the formulation incubated at pH 9 and 7, respectively; whereas, 61% of siRNA was released at pH 5. Altogether, the delivery system was able to maintain slow and steady release of encapsulated siRNA for an extended time period.
shows a representative TEM image of the siRNA-bearing EC nanoparticles. Formation of spherical nanoparticles with a size of 140 ± 10 nm is clearly revealed by TEM analysis. The dimensions of EC-siRNA nanoparticles were further established by NANOPHOX particle analyzer. The size distribution of the particles was determined by digital analysis of the image of counted particles. A sharp and predominant single peak approximately 150 ± 15 nm signifies size distribution, which is in concordance with the electron microscopically determined size of the in-house prepared nanoparticles (Figure 2B). A representative AFM image of nano drug formulation (Figure 2C) revealed a number of spherical nanoparticles. The liposomes were not overlapping with each other or in the form of clusters and assemblies. In Figure 2C, the height of the ECsiRNA nanoparticle is exaggerated for better perception of the fine structures as visualized with single-surface 3D AFM. Histopathological studies
The efficacy of various forms of PLK1-specific siRNA was established by histopathological studies. Liver tissue samples from mice belonging to various treated
70 EC-siRNA nanoparticle (pH 9) EC-siRNA nanoparticle (pH 7)
60
EC-siRNA nanoparticle (pH 5)
Release (%)
50
40
30
20
10
0 0
10
20
30
40
50
60
Time (h) Figure 1. Release profile of siRNA from escheriosome-based nanoparticles. siRNA-bearing EC formulation was incubated in sterile phosphate-buffered saline (5 mM PO4; 150 mM saline) of various pHs (pH 5, 7 and 9) for an extended time period. The released siRNA in the surrounding medium was quantified over time by HPLC, as described in the ‘Materials & methods’ section. Data are mean ± standard deviation of three independent experiments. EC: Escheriosome.
410
Nanomedicine (2014) 9(4)
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Research Article
siRNA nanoformulation against liver cancer
Impact of EC-siRNA nanoparticles on apoptosis
Downregulation of p53 wild-type (p53wt) was observed upon exposure to DEN (control untreated cancerous mice; Figure 4A, lane 2) compared with healthy (normal) control mice (Figure 4A, lane 1). Treatment with Sham-EC did not show significant effect on upregulation of p53wt (Figure 4A , lane 3), whereas free siRNA treatment resulted in a slight increase in the expression of p53wt, which was not only used as a parameter to establish induction of liver cancer in the experimental animals, but also offered as a tool to ascertain efficacy of various siRNA formulations (Figure 4A, lane 4). Significant upregulation of p53wt was observed when cancerous mice were treated with EC-siRNA nanoparticle formulations (Figure 4A, lane 5). As depicted in Figure 4B,i, a comparatively upregulated expression (80%) of p53wt protein was recorded in mice treated with siRNA-bearing EC formulations compared with healthy control animals (100%). The groups of animals treated with Sham-EC (17%), free siRNA (31%) or untreated animals (18%) were not able to elevate the level of p53 in the treated animals. Exposure of animals to DEN ensued in upregulation of p53mut (Figure 4A, lane 2). Treatment with EC-siRNA nano particle formulations resulted in its downregulation
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groups were used for histopathological analysis. Figure 3 shows sections of liver cancer cells after treatment with free, as well as nanoparticle encapsulated, siRNA formulations. Control healthy liver tissue shows normal hepatic laminae, sinusoids and hepatocytes (Figure 3A). No congestion and infiltration was observed; whereas, cancerous liver (untreated control; Figure 3B) shows a loss of normal hepatic structure, including laminae and sinusoids, dilated central vein and widespread hepatocyte necrosis. The treatment with free siRNA, resulted in mild recovery of cancerous liver (Figure 3D), showing enlarged hepatocyte contour, narrowing of sinusoidal space, mild apoptosis and Kupffer cell prominence was still predominantly seen. By contrast, in the mice treated with siRNA-bearing EC formulation, noticeable tumor regression in comparison with free form siRNA and untreated control was observed. Maintained hepatic microstructure, focal necrosis and apoptosis were also detected. siRNA-bearing EC treatment showed the best regression where normal hepatic architecture was found to be maintained. Focal lytic apoptosis was also very much apparent. The group of animals treated with Sham-EC or scrambled siRNA showed clusters of centrilobular oxyphilic hepatocytes, which are presumably because of coagulative necrosis (Figure 3C). Moreover, hepatic microstructures were maintained to a good extent. Most of the hepatoctyes were accompanied with eosinophilic as well as abundant Kupffer cells.
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Figure 2. Escheriosome size determination. (A) Representative transmission electron microscope image of escheriosome-based siRNA nanoparticles. (B) Corresponding particle analyzer data of escheriosome-based siRNA nanoparticle as obtained by NANOPHOX (Sympatec GmbH, Clausthal-Zefferfeld, Germany) particle size analyzer. (C) Representative 3D atomic force microscope image of escheriosome-based siRNA nanoparticles.
(Figure 4A, lane 5). As depicted in Figure 4B,ii, the level of p53mut was reduced to normal levels in the animals treated with siRNA-bearing EC (20%) compared with the untreated control (92%). Treatment with free siRNA was not as effective in downregulation of p53mut (81%) compared with its nanoparticle encapsulated form (p
