- Email: [email protected]
Potential of siRNA-albumin complex against cancer
Chemico-Biological Interactions xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Potential of siRNA-albumin complex against cancer Na Liua, Yan-Hua Qia, Chuan-Tao Chengb, Wen Bin Yangb, Anshoo Malhotrac, Qi Zhoua,∗ a b c
Department of Ultrasound, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710004, China Department of General Surgery, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710004, China Department of Biophysics, PGIMER, Chandigarh, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Albumin siRNA Colon cancer 99m Tc
RNA interference is a highly specific as well as efficient technology for gene therapy application in molecular oncology. The present study was planned to develop an efficient and stable tumor selective delivery mechanism for siRNA gene therapy for the purpose of both diagnosis as well as therapy. We have used 20 Male wistar rats for the formation of colon cancer model and utilized albumin as carrier molecule for the delivery of siRNA against vascular endothelial growth factor receptor 2 (VEGF R2). The study results confirmed efficient delivery of siRNA at tumor site as confirmed by tagging of siRNA-albumin complex with 99mTC. Moreover, the expression of VEGF also showed decline after efficient delivery of siRNA at tumor site. The study concluded that albumin is an efficient molecule for the efficient delivery of siRNA at tumor sites.
1. Introduction RNA interference or post-transcriptional gene silencing (PTGS) is one of the latest, innovative, highly specific and efficient technologies for gene therapy application in molecular oncology [1]. It is already a well-established research tool for analyses of molecular mechanisms for various diseases including cancer as it allows researchers to silence the expression of certain genes with great efficacy and specificity. Small interfering RNA (siRNA) is a class of double stranded RNA molecules that plays the major role in post transcriptional gene silencing by interfering with expressions of specific genes with complimentary nucleotide sequence [2]. However, for the proper utilization of this form of gene therapy, an efficient tumor specific in-vivo delivery mechanism is essential. Many scientific groups, companies are involved in the development of efficient in-vivo deliver mechanisms for siRNA. So, the prime aim of the present study was to develop an efficient as well as stable tumor selective delivery mechanism for siRNA gene therapy. The serum albumin is an endogenous nano biomolecule capable of binding to multiple endogenous metabolites, drugs and metal ions [3,4]. The biological application of nanoparticles is a rapidly developing area of nanotechnology that raises new possibilities in the diagnosis as well as treatment of cancer [5,6]. Albumin is emerging as a versatile protein carrier for drug targeting and for improving the pharmacokinetic profile of peptide- or protein-based drugs. Albumin is the most abundant plasma protein (35–50 g/L human serum) with a molecular weight of 66.5 kDa. Like most of the plasma proteins, albumin is synthesized in the liver where it is produced at a rate of
∗
approximately 0.7 mg/h for every gram of liver (i.e. 10–15 g daily); Human serum albumin (HSA) with an average half-life of 19 days exhibits multiple functions and has excellent binding properties. In this study, we utilized drug delivery efficacy of nano systems, sensitivity as well as therapeutic potential of RNA interference gene therapy. Moreover, we exploited radioisotope 99mTc for confirming the specificity of the newly formed complex. 2. Materials and methods 2.1. Chemicals All chemicals, reagents etc. were procured from Sigma Aldrich company [USA]. Bio vision kit (catalogue no. K5365) was utilized for the analyses of VEGF expression. siRNA specific for inhibiting expression of VEGF receptor-2 (VEGF R2) gene, was procured from the Thermo Fisher Scientific (USA) (Catalogue no. 145034). Caveolin-1 Colorimetric Cell-Based ELISA Kit (OKAG00588) was procured from Aviva Systems Biology. 2.2. Animals 20 Male Wistar rats in the weight range of 140–160 g were procured from the central animal house, PGIMER, Chandigarh, India. Another, set of 20 rats in the weight range of 140–160 g was procured for radiotagging experiment. All the animals were housed in polypropylene cages under hygienic conditions in the departmental animal house by
Corresponding author. E-mail address: [email protected] (Q. Zhou).
https://doi.org/10.1016/j.cbi.2018.04.028 Received 24 February 2018; Received in revised form 20 April 2018; Accepted 24 April 2018 0009-2797/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Liu, N., Chemico-Biological Interactions (2018), https://doi.org/10.1016/j.cbi.2018.04.028
Chemico-Biological Interactions xxx (xxxx) xxx–xxx
N. Liu et al.
2.7. Expression of VEGF
strictly following the guidelines as outlined by institutional ethical committee.
Further, to study the efficacy of siRNA therapy, we investigated VEGF protein expression by utilizing colorimetric kit. This expression was studied in the colon tissues of halves of DMH treated rats before treatment with this complex and in halves of the animals after 4 h of treatment with siRNA-albumin complex.
2.3. Experimental design for set 1 rats Animals (total 20 rats of Set 1) were segregated equally and randomly into 2 treatment groups. Animals in Group I served as normal controls. Animals in Group II were given (Dimethyl hydrazine) DMH injections subcutaneously at a dose rate of 30 mg/kg bodyweight/week for total duration of 20 weeks [7]. DMH is a carcinogen used for induction of colon cancers in rats. All the animals had free access to the diet and water and the treatments continued for a total duration of the study. After 20 weeks, half of Group II animals were treated with albumin-siRNA complex for the evaluation of efficacy of newly formed siRNA complex against VEGF expression.
2.8. Radiolabeling of siRNA-albumin complex with
99m
Tc
For confirmation of specify of siRNA-albumin complex, we tagged TC radioisotope with albumin in Si-RNA-albumin complex following the method of Djokic et al. [8].
99m
2.9. Treatment of set 2 of rats with
99m
TC labeled siRNA-albumin complex
Animals (total 20 rats of Set 2) were segregated equally and randomly into 2 treatment groups. Animals in Group A served as normal controls. Animals in Group B were given (Dimethyl hydrazine) DMH injections subcutaneously at a dose rate of 30 mg/kg bodyweight/week for total duration of 20 weeks [7]. Both the groups viz. control as well as DMH treated subjected to 99mTC labeled siRNA complex by giving intravenous injections. The rats were sacrificed after 4 Hr. of treatment and the radioisotope counts were recorded in important organs by scintillation counter.
2.4. Formation of siRNA-albumin complex and treatment of rats We developed formulations of siRNA, and albumin complex using variable concentrations. The binding was confirmed by the gel filtration chromatography using prepacked GE lifescience PD10 G-25 chromatography columns. Quality control tests for physicochemical checks and biological stability were also performed for confirmation of stability. The complex was prepared in DNAse-RNAse free water (GIBCO, Germany). The human serum albumin (HSA, 67 KDa) solution were prepared in DNAse-RNAse free water and filtered with a 0.22 μm cellulose acetate filter (Corning,NY) immediately prior to use. The complexation reaction (step) was performed with a fixed siRNA concentration of 22 μg/ml. HSA was added at a final concentration of 0,125 mg/ml and the system was allowed to react for a further 30 min. The rats from treatment group were subjected to siRNA complex treatment by giving intravenous injection of 0.2 ml of synthesized formulations. The rats were sacrificed after the 4 h of treatment for further experiments.
2.10. Statistical analyses The statistical significance of the data has been determined using one-way analysis of variance [ANOVA] followed a multiple post-hoc least significant difference test. The results are represented as Means ± S.D. The software used was SPSS 20. 3. Results The results obtained from various experiments conducted in this study are shown in Tables 1–3. The data from treatment group have been compared with the normal control animals.
2.5. Isolation of cells from colon Incubated the entire rat colon in 25 ml of 5 mM EDTA in HBSS (room temperature) in a 50 ml conical tube. Placed the conical tube in a 37 °C shaking air bath (250 rpm) for 15 min. After 15 min, carefully poured off the fluid and refilled the tube with 25 ml of 5 mM EDTA. Repeat the above provess for a total of 5 washe. Rinsed the colon twice in 20 ml of ice-cold PBS. Placed the mouse colon tissue in a new 50 ml conical tube containing 20 ml of RPMI-5, 10 U of dispase, and 2000 U of collagenase D (room temperature). Placed the tube in a 37 °C shaking air bath oriented vertically. Shook at 250 rpm for up to 60 min. After exposure to the dispase and collagenase D, the colon tissue showed digestion and the colon tissue apeared stringy. Shook each tube up and down 2–4 times to break up the tissue. Pelleted the tissue at 200 × g in a tabletop centrifuge (4 °C) for 5 min. Resuspended each pellet by adding 10 ml ACK lysis buffer (47 °C). Centrifuged again at 200 × g for 5 min (4 °C), poured off and discarded the supernatant. Passed half of the cell suspension (5 ml) through a 70 μm mesh strainer using a 10 ml pipette into a 100 mm TC-treated dish. Then added an additional 15 ml of RPMI-5. Placed the tissue culture dishes in a 10% CO2 incubator at 37 °C. The culture conditions were the same for mouse and human primary cells.
3.1. VEGF protein expression in the colons The VEGF protein expression was initially significantly higher in the colons of DMH treated rats as compared to controls (Table 1). However, after treatment with newly formed -albumin-siRNA complex, a significant decrease in the expression of VEGF in the colons of treatment (group 2) animals were noticed in comparison to controls. 3.2. Caveolin-1 protein expression The caviolin-1 protein expression was noticed to be significantly higher in the colons of DMH treated rats in comparison to controls Table 1 VEGF protein expression.
2.6. Expression of Caveolin-1
Groups
VEGF Expression before treatment with albuminsiRNA (pg/mL)
VEGF Expression after 4 h of treatment with albuminsiRNA (pg/mL)
Control DMH Treated (Colon cancer model)
6.18 ± 0.05 12.13 ± 0.02c
6.05 ± 0.01 6.15 ± 0.05
n = 5 per group. Data are expressed in Mean ± S.D. c P ≤ 0.001 by Least Significance Difference test when values are compared with normal control group.
The expression of Caveolin-1 gene was studied by using Colorimetric Cell-Based kit of caveolin gene accoriding to the instructions provided. 2
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N. Liu et al.
4. Discussion
Table 2 Caveolin-1 protein expression. Groups
Caveolin-1 protein expression (pg/mL)
Control DMH Treated Colon cancer model
7.28 ± 0.01 15.21 ± 0.02b
RNA interference therapy has ability to silence the expression of any disease-related gene in a selective and sequence-dependent manner. However, there is a major hurdle that prevents its use as the goldstandard therapy for cancer. The major barrier is the specific delivery of siRNAs to the desired site. To overcome the major problem of siRNA delivery, multiple researchers have worked on variable approaches of efficient in-vivo delivery of siRNA. These approaches included both synthetic as well as natural delivery systems. Synthetic delivery systems included physical methods, conjugation methods, etc. and natural carriers involved viruses and bacteria [9]. We have utilized albumin as a delivery molecule for siRNA in this study. The serum albumin is an endogenous nano-particle and is known for its binding properties to various endogenous metabolites, drugs and metal ions [10]. The biological application of nanoparticles is a rapidly developing area of nanotechnology that raises new possibilities in the diagnosis as well as treatment of human cancers. Albumin is emerging as a versatile protein carrier for drug targeting and for improving the pharmacokinetic profile of various drugs. In this study, we utilized drug delivery efficacy of nano systems, sensitivity as well as therapeutic potential of RNA interference gene therapy. The serum albumin is an efficient nano sized drug delivery system that offered preferential and specific target oriented delivery of siRNA against VEGF R2 at tumor site. The serum albumin is equipped with molecular machinery for various transporting mechanisms. The main transport mechanism would most likely be Caveolin-mediated endocytosis coupled with endosomal escape to release the siRNA intracellular for action. As gp60 (albodin) receptors are expressed in high numbers in tumors due to high expression of cavelion-1 gene as observed in the present study in the colons of DMH treated animals [11–15]. So, high concentrations of albumin receptors at tumor site significantly increased the uptake of the formed complex in the tumorous colons of group 2 animals (Fig. 1). Similar results have been already reported with use of albumin with paclitaxel [14,15]. The secreted Protein Acidic and Rich in Cysteine factor (SPARC) is another important factor responsible for increased target specific localization of albumin-siRNA in colons of treated animals due to albumin binding. The SPARC is basically a secreted protein acidic and rich in cysteine, which is expressed in variety of tumors,
n = 5 per group. Data are expressed in Mean ± S.D. b P ≤ 0.001 by Least Significance Difference test when values are compared with normal control group. Table 3 Animal Biodistribution studies of
99m
Tc-albumin-SiRNA complex.
Organs
Uptake after 4hr. (Control) Group A
Uptake after 4hr. (Colon cancer model) Group B
Colon Liver Heart Lungs Blood Muscle Spleen Intestine Bone Thyroid
3.06 ± 0.97 ± 0.08 ± 0.45 ± 0.23 ± 0.01% 1.12 ± 0.19 ± 0.00% 0.18 ±
8.36a ± 0.17% 0.94 ± 0.12% 0.07 ± 0.02% 0.43 ± 0.12% 0.19 ± 0.08% 0.01% 1.1 ± 0.20% 0.15 ± 0.03% 0.00% 0.19 ± 0.04%
0.65% 0.12% 0.02% 0.12% 0.08% 0.20% 0.03% 0.04%
n = 10 per group. Data are expressed in Mean ± S.D. ‘%’ in the table describes the mean %ID/g ± SD. a p < 0.05.
(Table 2).
3.3. Animal biodistribution studies of
99m
Tc-albumin-SiRNA
The results of the bio-distribution of 99mTc-albumin-SiRNA complex expressed as %ID/g of organs after 4 h of injection are presented in Table 3. The results indicated that signfincant rise in the uptakes of complex in the colons of group B animals.
Fig. 1. Graphic representation of the mechanism of action of siRNA-albumin complex in control as well as cancer cell. 3
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N. Liu et al.
References
including lung cancer, colon cancer [16]. So, this factor created a sink for albumin at tumor site as it has got high affinity for albumin. These entire factors collectively contributed towards increased and specific localization of the albumin bound siRNA-albumin complex. Moreover, radiolabeling with 99mTC also confirmed the specificity of the newly formed albumin-siRNA complex. Recent study by Sarett et al. also utilized long-lived endogenous serum albumin as a carrier for siRNA [17]. Another study exploited cationic bovine serum albumin (CBSA)-based self-assembled nanoparticles for the delivery of siRNA during lung metastatic cancer [18]. We also noticed significant decline in the expression of VEGF proteins in comparison to controls after 4 h of treatment with the newly formed complex. This could be owed to the specific deliver of siRNA against vascular endothelial growth factor receptor-2 (VEGF R2) gene, by albumin in cancer cells. The siRNA against VEGF receptor 2 significantly prevented the expression of VEGF, thereby directly affected angiogenesis process in cancer cells. The above observation could be justified by the fact that autocrine feed-forward loop in tumor cells stimulates VEGF production via VEGFR2-dependent activation of mTOR as observeed in an earlier report [19] The prime limitations of this study included lack of in-vitro validation of the specificity of VEGF R2 siRNA. Also, we could not study the expression of genes at mRNA levlels. The present study showed for the first time that albumin acted as an efficient target specific carrier for siRNA at tumor site for successful inhibition of the expression of VEGF R2 gene. In this way, this strategy could be extended to the clinical settings for the future development of advanced cancer therapeutics.
[1] X. Chen, L.S. Mangala, C. Rodriguez-Aguayo, X. Kong, G. Lopez-Berestein, A.K. Sood, RNA interference-based therapy and its delivery systems, Canc. Metastasis Rev. 37 (1) (2018 Mar) 107–124. [2] C.L. Liu, Z.Y. Deng, E.R. Du, C.S. Xu, Long non-coding RNA BC168687 small interfering RNA reduces high glucose and high free fatty acid-induced expression of P2X7 receptors in satellite glial cell, Mol. Med. Rep. 17 (4) (2018 Feb 1) 5851–5859. [3] A. Malhotra, B.R. Mittal, SiRNA gene therapy using albumin as a carrier, Pharmacogenetics Genom. 24 (12) (2014 Dec) 582–587. [4] H. Lestradet, The antagonistic action of albumin against the activity of insulin, Presse Med. 28 (2018) 69 1961 Oct. [5] B.L. Ye, R. Zheng, X.J. Ruan, Z.H. Zheng, H.J. Cai, Chitosan-coated doxorubicin nano-particles drug delivery system inhibits cell growth of liver cancervia p53/ PRC1 pathwa, Biochem Biophys Res Commun 495 (1) (2018 Jan 1) 414–420. [6] L. Mary Lazer, B. Sadhasivam, K. Palaniyandi, T. Muthuswamy, I. Ramachandran, A. Balakrishnan, S. Pathak, S. Narayan, S. Ramalingam, Chitosan-based nano-formulation enhances the anticancer efficacy of hesperetin, Int. J. Biol. Macromol. 107 (Pt B) (2018 Feb) 1988–1998. [7] X. Yuan, S. Naguib, Z. Wu, Recent advances of siRNA delivery by nanoparticles, Expet Opin. Drug Deliv. 8 (2011) 521–536. [8] A.P. Soler, R.D. Miller, K.V. Laughlin, N.Z. Carp, D.M. Klurfeld, J.M. Mullin, Increased tight junctional permeability is associated with the development of colon cancer, Carcinogenesis 20 (1999) 1425–1431. [9] D. Djokic, D. Jankovic, Tatjana Maksin, Radiochemical purity and particles number determination of modified 99mTC-macroagreagated albumin, J Serb chem 67 (2002) 573–579. [10] Y. Fan, J. Yi, Y. Zhang, W. Yokoyama, Fabrication of curcumin-loaded bovine serum albumin (BSA)-dextran nanoparticles and the cellular antioxidant activity, Food Chem. 239 (2018 Jan 15) 121. [11] K.Y.Y. Fung, G.D. Fairn, W.L. Lee, Transcellular vesicular transport in epithelial and endothelial cells: challenges and opportunities, Traffic 19 (1) (2018 Jan) 5–18. [12] C. Chanthick, A. Suttitheptumrong, N. Rawarak, S.N. Pattanakitsakul, Transcytosis Involvement in Transport System and Endothelial Permeability of Vascular Leakage during Dengue Virus Infection, Viruses 10 (2) (2018 Feb 8) E69. [13] L. Mocan, C. Matea, F.A. Tabaran, O. Mosteanu, T. Pop, T. Mocan, C. Iancu, Photothermal treatment of liver cancer with albumin-conjugated gold nanoparticles initiates Golgi Apparatus-ER dysfunction and caspase-3 apoptotic pathway activation by selective targeting of Gp60 receptor, Int. J. Nanomed. 10 (2015 Aug 26) 5435–5445. [14] W.J. Gradishar, S. Tjulandin, N. Davidson, H. Shaw, N. Desai, P. Bhar, et al., Phase III trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil-based paclitaxel in women with breast cancer, J. Clin. Oncol. 23 (2005) 7794–7803. [15] N. Desai, V. Trieu, Z. Yao, L. Louie, S. Ci, A. Yang, et al., Increased antitumor activity, intratumor paclitaxel concentrations, and endothelial cell transport of cremophor-free, albumin-bound paclitaxel, ABI-007, compared with cremophor-based paclitaxel, Clin. Canc. Res. 12 (2006) 1317–1324. [16] Porter, et al., J. Histochem. Cytochem. 43 (1995) 791–800. [17] S.M. Sarett, T.A. Werfel, L. Lee, M.A. Jackson, K.V. Kilchrist, D. Brantley-Sieders, C.L. Duvall, Lipophilic siRNA targets albumin in situ and promotes bioavailability, tumor penetration, and Carrier-free gene silencing, Proc. Natl. Acad. Sci. U. S. A. 114 (32) (2017 Aug 8) E6490–E6497. [18] J. Han, Q. Wang, Z. Zhang, T. Gong, X. Sun, Cationic bovine serum albumin based self-assembled nanoparticles as siRNA delivery vector for treating lung metastatic cancer, Small 10 (2014) 524–535. [19] S. Chatterjee, C. Lukas, Heukamp, Siobal Maike, Schöttle Jakob, Wieczorek Caroline, Peifer Martin, Frasca Davide, Koker Mirjam, König Katharina, Meder Lydia, Rauh Daniel, Buettner Reinhard, Wolf Jürgen, A.Brekken Rolf, Neumaier Bernd, Christofori Gerhard, K. Thomas Roman, T. Roland, Ullrich1,4Tumor VEGF: VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer, J. Clin. Invest. 123 (4) (2013 Apr 1) 1732–1740.
5. Conclusion The present study concluded that albumin is an efficient carrier for siRNA delivery. However, future studies are essential for extension of this technology for clinical applications. Acknowledgement We are thankful to Shaanxi Academy of Social Science and Technology Development Fund. S2017-ZDYF-YBXM-SF-0503. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.cbi.2018.04.028. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2018.04.028.
4
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Potential of siRNA-albumin complex against cancer Na Liua, Yan-Hua Qia, Chuan-Tao Chengb, Wen Bin Yangb, Anshoo Malhotrac, Qi Zhoua,∗ a b c
Department of Ultrasound, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710004, China Department of General Surgery, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710004, China Department of Biophysics, PGIMER, Chandigarh, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Albumin siRNA Colon cancer 99m Tc
RNA interference is a highly specific as well as efficient technology for gene therapy application in molecular oncology. The present study was planned to develop an efficient and stable tumor selective delivery mechanism for siRNA gene therapy for the purpose of both diagnosis as well as therapy. We have used 20 Male wistar rats for the formation of colon cancer model and utilized albumin as carrier molecule for the delivery of siRNA against vascular endothelial growth factor receptor 2 (VEGF R2). The study results confirmed efficient delivery of siRNA at tumor site as confirmed by tagging of siRNA-albumin complex with 99mTC. Moreover, the expression of VEGF also showed decline after efficient delivery of siRNA at tumor site. The study concluded that albumin is an efficient molecule for the efficient delivery of siRNA at tumor sites.
1. Introduction RNA interference or post-transcriptional gene silencing (PTGS) is one of the latest, innovative, highly specific and efficient technologies for gene therapy application in molecular oncology [1]. It is already a well-established research tool for analyses of molecular mechanisms for various diseases including cancer as it allows researchers to silence the expression of certain genes with great efficacy and specificity. Small interfering RNA (siRNA) is a class of double stranded RNA molecules that plays the major role in post transcriptional gene silencing by interfering with expressions of specific genes with complimentary nucleotide sequence [2]. However, for the proper utilization of this form of gene therapy, an efficient tumor specific in-vivo delivery mechanism is essential. Many scientific groups, companies are involved in the development of efficient in-vivo deliver mechanisms for siRNA. So, the prime aim of the present study was to develop an efficient as well as stable tumor selective delivery mechanism for siRNA gene therapy. The serum albumin is an endogenous nano biomolecule capable of binding to multiple endogenous metabolites, drugs and metal ions [3,4]. The biological application of nanoparticles is a rapidly developing area of nanotechnology that raises new possibilities in the diagnosis as well as treatment of cancer [5,6]. Albumin is emerging as a versatile protein carrier for drug targeting and for improving the pharmacokinetic profile of peptide- or protein-based drugs. Albumin is the most abundant plasma protein (35–50 g/L human serum) with a molecular weight of 66.5 kDa. Like most of the plasma proteins, albumin is synthesized in the liver where it is produced at a rate of
∗
approximately 0.7 mg/h for every gram of liver (i.e. 10–15 g daily); Human serum albumin (HSA) with an average half-life of 19 days exhibits multiple functions and has excellent binding properties. In this study, we utilized drug delivery efficacy of nano systems, sensitivity as well as therapeutic potential of RNA interference gene therapy. Moreover, we exploited radioisotope 99mTc for confirming the specificity of the newly formed complex. 2. Materials and methods 2.1. Chemicals All chemicals, reagents etc. were procured from Sigma Aldrich company [USA]. Bio vision kit (catalogue no. K5365) was utilized for the analyses of VEGF expression. siRNA specific for inhibiting expression of VEGF receptor-2 (VEGF R2) gene, was procured from the Thermo Fisher Scientific (USA) (Catalogue no. 145034). Caveolin-1 Colorimetric Cell-Based ELISA Kit (OKAG00588) was procured from Aviva Systems Biology. 2.2. Animals 20 Male Wistar rats in the weight range of 140–160 g were procured from the central animal house, PGIMER, Chandigarh, India. Another, set of 20 rats in the weight range of 140–160 g was procured for radiotagging experiment. All the animals were housed in polypropylene cages under hygienic conditions in the departmental animal house by
Corresponding author. E-mail address: [email protected] (Q. Zhou).
https://doi.org/10.1016/j.cbi.2018.04.028 Received 24 February 2018; Received in revised form 20 April 2018; Accepted 24 April 2018 0009-2797/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Liu, N., Chemico-Biological Interactions (2018), https://doi.org/10.1016/j.cbi.2018.04.028
Chemico-Biological Interactions xxx (xxxx) xxx–xxx
N. Liu et al.
2.7. Expression of VEGF
strictly following the guidelines as outlined by institutional ethical committee.
Further, to study the efficacy of siRNA therapy, we investigated VEGF protein expression by utilizing colorimetric kit. This expression was studied in the colon tissues of halves of DMH treated rats before treatment with this complex and in halves of the animals after 4 h of treatment with siRNA-albumin complex.
2.3. Experimental design for set 1 rats Animals (total 20 rats of Set 1) were segregated equally and randomly into 2 treatment groups. Animals in Group I served as normal controls. Animals in Group II were given (Dimethyl hydrazine) DMH injections subcutaneously at a dose rate of 30 mg/kg bodyweight/week for total duration of 20 weeks [7]. DMH is a carcinogen used for induction of colon cancers in rats. All the animals had free access to the diet and water and the treatments continued for a total duration of the study. After 20 weeks, half of Group II animals were treated with albumin-siRNA complex for the evaluation of efficacy of newly formed siRNA complex against VEGF expression.
2.8. Radiolabeling of siRNA-albumin complex with
99m
Tc
For confirmation of specify of siRNA-albumin complex, we tagged TC radioisotope with albumin in Si-RNA-albumin complex following the method of Djokic et al. [8].
99m
2.9. Treatment of set 2 of rats with
99m
TC labeled siRNA-albumin complex
Animals (total 20 rats of Set 2) were segregated equally and randomly into 2 treatment groups. Animals in Group A served as normal controls. Animals in Group B were given (Dimethyl hydrazine) DMH injections subcutaneously at a dose rate of 30 mg/kg bodyweight/week for total duration of 20 weeks [7]. Both the groups viz. control as well as DMH treated subjected to 99mTC labeled siRNA complex by giving intravenous injections. The rats were sacrificed after 4 Hr. of treatment and the radioisotope counts were recorded in important organs by scintillation counter.
2.4. Formation of siRNA-albumin complex and treatment of rats We developed formulations of siRNA, and albumin complex using variable concentrations. The binding was confirmed by the gel filtration chromatography using prepacked GE lifescience PD10 G-25 chromatography columns. Quality control tests for physicochemical checks and biological stability were also performed for confirmation of stability. The complex was prepared in DNAse-RNAse free water (GIBCO, Germany). The human serum albumin (HSA, 67 KDa) solution were prepared in DNAse-RNAse free water and filtered with a 0.22 μm cellulose acetate filter (Corning,NY) immediately prior to use. The complexation reaction (step) was performed with a fixed siRNA concentration of 22 μg/ml. HSA was added at a final concentration of 0,125 mg/ml and the system was allowed to react for a further 30 min. The rats from treatment group were subjected to siRNA complex treatment by giving intravenous injection of 0.2 ml of synthesized formulations. The rats were sacrificed after the 4 h of treatment for further experiments.
2.10. Statistical analyses The statistical significance of the data has been determined using one-way analysis of variance [ANOVA] followed a multiple post-hoc least significant difference test. The results are represented as Means ± S.D. The software used was SPSS 20. 3. Results The results obtained from various experiments conducted in this study are shown in Tables 1–3. The data from treatment group have been compared with the normal control animals.
2.5. Isolation of cells from colon Incubated the entire rat colon in 25 ml of 5 mM EDTA in HBSS (room temperature) in a 50 ml conical tube. Placed the conical tube in a 37 °C shaking air bath (250 rpm) for 15 min. After 15 min, carefully poured off the fluid and refilled the tube with 25 ml of 5 mM EDTA. Repeat the above provess for a total of 5 washe. Rinsed the colon twice in 20 ml of ice-cold PBS. Placed the mouse colon tissue in a new 50 ml conical tube containing 20 ml of RPMI-5, 10 U of dispase, and 2000 U of collagenase D (room temperature). Placed the tube in a 37 °C shaking air bath oriented vertically. Shook at 250 rpm for up to 60 min. After exposure to the dispase and collagenase D, the colon tissue showed digestion and the colon tissue apeared stringy. Shook each tube up and down 2–4 times to break up the tissue. Pelleted the tissue at 200 × g in a tabletop centrifuge (4 °C) for 5 min. Resuspended each pellet by adding 10 ml ACK lysis buffer (47 °C). Centrifuged again at 200 × g for 5 min (4 °C), poured off and discarded the supernatant. Passed half of the cell suspension (5 ml) through a 70 μm mesh strainer using a 10 ml pipette into a 100 mm TC-treated dish. Then added an additional 15 ml of RPMI-5. Placed the tissue culture dishes in a 10% CO2 incubator at 37 °C. The culture conditions were the same for mouse and human primary cells.
3.1. VEGF protein expression in the colons The VEGF protein expression was initially significantly higher in the colons of DMH treated rats as compared to controls (Table 1). However, after treatment with newly formed -albumin-siRNA complex, a significant decrease in the expression of VEGF in the colons of treatment (group 2) animals were noticed in comparison to controls. 3.2. Caveolin-1 protein expression The caviolin-1 protein expression was noticed to be significantly higher in the colons of DMH treated rats in comparison to controls Table 1 VEGF protein expression.
2.6. Expression of Caveolin-1
Groups
VEGF Expression before treatment with albuminsiRNA (pg/mL)
VEGF Expression after 4 h of treatment with albuminsiRNA (pg/mL)
Control DMH Treated (Colon cancer model)
6.18 ± 0.05 12.13 ± 0.02c
6.05 ± 0.01 6.15 ± 0.05
n = 5 per group. Data are expressed in Mean ± S.D. c P ≤ 0.001 by Least Significance Difference test when values are compared with normal control group.
The expression of Caveolin-1 gene was studied by using Colorimetric Cell-Based kit of caveolin gene accoriding to the instructions provided. 2
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4. Discussion
Table 2 Caveolin-1 protein expression. Groups
Caveolin-1 protein expression (pg/mL)
Control DMH Treated Colon cancer model
7.28 ± 0.01 15.21 ± 0.02b
RNA interference therapy has ability to silence the expression of any disease-related gene in a selective and sequence-dependent manner. However, there is a major hurdle that prevents its use as the goldstandard therapy for cancer. The major barrier is the specific delivery of siRNAs to the desired site. To overcome the major problem of siRNA delivery, multiple researchers have worked on variable approaches of efficient in-vivo delivery of siRNA. These approaches included both synthetic as well as natural delivery systems. Synthetic delivery systems included physical methods, conjugation methods, etc. and natural carriers involved viruses and bacteria [9]. We have utilized albumin as a delivery molecule for siRNA in this study. The serum albumin is an endogenous nano-particle and is known for its binding properties to various endogenous metabolites, drugs and metal ions [10]. The biological application of nanoparticles is a rapidly developing area of nanotechnology that raises new possibilities in the diagnosis as well as treatment of human cancers. Albumin is emerging as a versatile protein carrier for drug targeting and for improving the pharmacokinetic profile of various drugs. In this study, we utilized drug delivery efficacy of nano systems, sensitivity as well as therapeutic potential of RNA interference gene therapy. The serum albumin is an efficient nano sized drug delivery system that offered preferential and specific target oriented delivery of siRNA against VEGF R2 at tumor site. The serum albumin is equipped with molecular machinery for various transporting mechanisms. The main transport mechanism would most likely be Caveolin-mediated endocytosis coupled with endosomal escape to release the siRNA intracellular for action. As gp60 (albodin) receptors are expressed in high numbers in tumors due to high expression of cavelion-1 gene as observed in the present study in the colons of DMH treated animals [11–15]. So, high concentrations of albumin receptors at tumor site significantly increased the uptake of the formed complex in the tumorous colons of group 2 animals (Fig. 1). Similar results have been already reported with use of albumin with paclitaxel [14,15]. The secreted Protein Acidic and Rich in Cysteine factor (SPARC) is another important factor responsible for increased target specific localization of albumin-siRNA in colons of treated animals due to albumin binding. The SPARC is basically a secreted protein acidic and rich in cysteine, which is expressed in variety of tumors,
n = 5 per group. Data are expressed in Mean ± S.D. b P ≤ 0.001 by Least Significance Difference test when values are compared with normal control group. Table 3 Animal Biodistribution studies of
99m
Tc-albumin-SiRNA complex.
Organs
Uptake after 4hr. (Control) Group A
Uptake after 4hr. (Colon cancer model) Group B
Colon Liver Heart Lungs Blood Muscle Spleen Intestine Bone Thyroid
3.06 ± 0.97 ± 0.08 ± 0.45 ± 0.23 ± 0.01% 1.12 ± 0.19 ± 0.00% 0.18 ±
8.36a ± 0.17% 0.94 ± 0.12% 0.07 ± 0.02% 0.43 ± 0.12% 0.19 ± 0.08% 0.01% 1.1 ± 0.20% 0.15 ± 0.03% 0.00% 0.19 ± 0.04%
0.65% 0.12% 0.02% 0.12% 0.08% 0.20% 0.03% 0.04%
n = 10 per group. Data are expressed in Mean ± S.D. ‘%’ in the table describes the mean %ID/g ± SD. a p < 0.05.
(Table 2).
3.3. Animal biodistribution studies of
99m
Tc-albumin-SiRNA
The results of the bio-distribution of 99mTc-albumin-SiRNA complex expressed as %ID/g of organs after 4 h of injection are presented in Table 3. The results indicated that signfincant rise in the uptakes of complex in the colons of group B animals.
Fig. 1. Graphic representation of the mechanism of action of siRNA-albumin complex in control as well as cancer cell. 3
Chemico-Biological Interactions xxx (xxxx) xxx–xxx
N. Liu et al.
References
including lung cancer, colon cancer [16]. So, this factor created a sink for albumin at tumor site as it has got high affinity for albumin. These entire factors collectively contributed towards increased and specific localization of the albumin bound siRNA-albumin complex. Moreover, radiolabeling with 99mTC also confirmed the specificity of the newly formed albumin-siRNA complex. Recent study by Sarett et al. also utilized long-lived endogenous serum albumin as a carrier for siRNA [17]. Another study exploited cationic bovine serum albumin (CBSA)-based self-assembled nanoparticles for the delivery of siRNA during lung metastatic cancer [18]. We also noticed significant decline in the expression of VEGF proteins in comparison to controls after 4 h of treatment with the newly formed complex. This could be owed to the specific deliver of siRNA against vascular endothelial growth factor receptor-2 (VEGF R2) gene, by albumin in cancer cells. The siRNA against VEGF receptor 2 significantly prevented the expression of VEGF, thereby directly affected angiogenesis process in cancer cells. The above observation could be justified by the fact that autocrine feed-forward loop in tumor cells stimulates VEGF production via VEGFR2-dependent activation of mTOR as observeed in an earlier report [19] The prime limitations of this study included lack of in-vitro validation of the specificity of VEGF R2 siRNA. Also, we could not study the expression of genes at mRNA levlels. The present study showed for the first time that albumin acted as an efficient target specific carrier for siRNA at tumor site for successful inhibition of the expression of VEGF R2 gene. In this way, this strategy could be extended to the clinical settings for the future development of advanced cancer therapeutics.
[1] X. Chen, L.S. Mangala, C. Rodriguez-Aguayo, X. Kong, G. Lopez-Berestein, A.K. Sood, RNA interference-based therapy and its delivery systems, Canc. Metastasis Rev. 37 (1) (2018 Mar) 107–124. [2] C.L. Liu, Z.Y. Deng, E.R. Du, C.S. Xu, Long non-coding RNA BC168687 small interfering RNA reduces high glucose and high free fatty acid-induced expression of P2X7 receptors in satellite glial cell, Mol. Med. Rep. 17 (4) (2018 Feb 1) 5851–5859. [3] A. Malhotra, B.R. Mittal, SiRNA gene therapy using albumin as a carrier, Pharmacogenetics Genom. 24 (12) (2014 Dec) 582–587. [4] H. Lestradet, The antagonistic action of albumin against the activity of insulin, Presse Med. 28 (2018) 69 1961 Oct. [5] B.L. Ye, R. Zheng, X.J. Ruan, Z.H. Zheng, H.J. Cai, Chitosan-coated doxorubicin nano-particles drug delivery system inhibits cell growth of liver cancervia p53/ PRC1 pathwa, Biochem Biophys Res Commun 495 (1) (2018 Jan 1) 414–420. [6] L. Mary Lazer, B. Sadhasivam, K. Palaniyandi, T. Muthuswamy, I. Ramachandran, A. Balakrishnan, S. Pathak, S. Narayan, S. Ramalingam, Chitosan-based nano-formulation enhances the anticancer efficacy of hesperetin, Int. J. Biol. Macromol. 107 (Pt B) (2018 Feb) 1988–1998. [7] X. Yuan, S. Naguib, Z. Wu, Recent advances of siRNA delivery by nanoparticles, Expet Opin. Drug Deliv. 8 (2011) 521–536. [8] A.P. Soler, R.D. Miller, K.V. Laughlin, N.Z. Carp, D.M. Klurfeld, J.M. Mullin, Increased tight junctional permeability is associated with the development of colon cancer, Carcinogenesis 20 (1999) 1425–1431. [9] D. Djokic, D. Jankovic, Tatjana Maksin, Radiochemical purity and particles number determination of modified 99mTC-macroagreagated albumin, J Serb chem 67 (2002) 573–579. [10] Y. Fan, J. Yi, Y. Zhang, W. Yokoyama, Fabrication of curcumin-loaded bovine serum albumin (BSA)-dextran nanoparticles and the cellular antioxidant activity, Food Chem. 239 (2018 Jan 15) 121. [11] K.Y.Y. Fung, G.D. Fairn, W.L. Lee, Transcellular vesicular transport in epithelial and endothelial cells: challenges and opportunities, Traffic 19 (1) (2018 Jan) 5–18. [12] C. Chanthick, A. Suttitheptumrong, N. Rawarak, S.N. Pattanakitsakul, Transcytosis Involvement in Transport System and Endothelial Permeability of Vascular Leakage during Dengue Virus Infection, Viruses 10 (2) (2018 Feb 8) E69. [13] L. Mocan, C. Matea, F.A. Tabaran, O. Mosteanu, T. Pop, T. Mocan, C. Iancu, Photothermal treatment of liver cancer with albumin-conjugated gold nanoparticles initiates Golgi Apparatus-ER dysfunction and caspase-3 apoptotic pathway activation by selective targeting of Gp60 receptor, Int. J. Nanomed. 10 (2015 Aug 26) 5435–5445. [14] W.J. Gradishar, S. Tjulandin, N. Davidson, H. Shaw, N. Desai, P. Bhar, et al., Phase III trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil-based paclitaxel in women with breast cancer, J. Clin. Oncol. 23 (2005) 7794–7803. [15] N. Desai, V. Trieu, Z. Yao, L. Louie, S. Ci, A. Yang, et al., Increased antitumor activity, intratumor paclitaxel concentrations, and endothelial cell transport of cremophor-free, albumin-bound paclitaxel, ABI-007, compared with cremophor-based paclitaxel, Clin. Canc. Res. 12 (2006) 1317–1324. [16] Porter, et al., J. Histochem. Cytochem. 43 (1995) 791–800. [17] S.M. Sarett, T.A. Werfel, L. Lee, M.A. Jackson, K.V. Kilchrist, D. Brantley-Sieders, C.L. Duvall, Lipophilic siRNA targets albumin in situ and promotes bioavailability, tumor penetration, and Carrier-free gene silencing, Proc. Natl. Acad. Sci. U. S. A. 114 (32) (2017 Aug 8) E6490–E6497. [18] J. Han, Q. Wang, Z. Zhang, T. Gong, X. Sun, Cationic bovine serum albumin based self-assembled nanoparticles as siRNA delivery vector for treating lung metastatic cancer, Small 10 (2014) 524–535. [19] S. Chatterjee, C. Lukas, Heukamp, Siobal Maike, Schöttle Jakob, Wieczorek Caroline, Peifer Martin, Frasca Davide, Koker Mirjam, König Katharina, Meder Lydia, Rauh Daniel, Buettner Reinhard, Wolf Jürgen, A.Brekken Rolf, Neumaier Bernd, Christofori Gerhard, K. Thomas Roman, T. Roland, Ullrich1,4Tumor VEGF: VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer, J. Clin. Invest. 123 (4) (2013 Apr 1) 1732–1740.
5. Conclusion The present study concluded that albumin is an efficient carrier for siRNA delivery. However, future studies are essential for extension of this technology for clinical applications. Acknowledgement We are thankful to Shaanxi Academy of Social Science and Technology Development Fund. S2017-ZDYF-YBXM-SF-0503. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.cbi.2018.04.028. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2018.04.028.
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