Accepted Article
Received Date : 27-Nov-2013 Revised Date : 11-Feb-2014 Accepted Date : 13-Feb-2014 Article type
: Research Article
Immunocompatibility and Toxicity Studies of Poly-L-Lysine Nanocapsules in Sprague Dawely Rats for Drug Delivery Applications Janeesh P.A1, Haider Sami3, Dhanya C.R1, Sri Sivakumar2 and Annie Abraham1* 1
*Department of Biochemistry, University of Kerala, Kariavattom campus, Trivandrum, Kerala, India.
2
Unit of Excellence on Soft Nanofabrication, Department of Chemical Engineering, Indian Institute of Technology Kanpur (IIT), Uttar Pradesh, India.
3
Department of Biological Sciences and Bioengineering, Indian Institute of Technology (IIT) Kanpur, Uttar Pradesh, India.
Correspondence author* Dr. Annie Abraham* Professor of Biochemistry Director, School of Life sciences Department of Biochemistry University of Kerala, Kariavattom Campus, Trivandrum - 695 581, Kerala, India Email: [email protected] Phone: Off: +91-471-2308078 Mob: +91-9447246692 Fax: +91-471-2307158
Running Head: Immunocompatibility and Toxicity Studies of PLL This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12313 This article is protected by copyright. All rights reserved.
Accepted Article
Abstract Poly-L-Lysine (PLL) Nanocapsules are the emerging drug delivery vehicle for the therapeutics of targeted diseases. The study was designed for the synthesis and characterisation of PLL nanocapsules and to know its immunocompatibility and toxicity behaviour for in vivo drug delivery applications. Alteration in haematological parameters, Immunomodulatory gene expression by RT-PCR studies, toxicity markers status, immunobloting of major inflammatory marker proteins and histopathological studies from major tissues of rat after intravenous administration of PLL nanocapsules after 30 days were assessed. In vivo toxicity markers activity, haematological parameters alteration and RT-PCR analysis of important immunomodulatory genes like monocyte chemotactic protein-1(MCP 1), tumor necrosis factor-alpha (TNF-α), Intercellular adhesion molecule-1 (ICAM-1) and interleukin-6 (IL-6) showed least changes when compared with control. The immunoblotting of major inflammatory markers like cyclooxygenase-2 (COX-2), lipoxygenase-15 (LOX-15) and
nitric
oxide
synthase
(NOS)
found
have
least
expression
showing
the
immunocompatibility of PLL nanocapsules. Histopathological studies of important tissues showed almost normal architecture after treatment using different concentration of PLL nanocapsules after the experimental period. The results showed a promising outcome and further confirmed the immunocompatibility and non toxicity of PLL nanocapsules in vivo for drug delivery applications. Key words: Immunocompatibility, Poly-L-Lysine (PLL) Nanocapsules, Immunomodulatory genes, Inflammatory markers.
Introduction Polymeric nanocapsules are widely used as drug delivery vehicle owning to their mechanistic property. It is fabricated through step-wise adsorption of polymers using electrostatics, Hbonding, covalent chemistry, etc as driving force, followed by the dissolution of the core template (1-2). Recently, we have synthesised and characterised Poly-L-Lysine (PLL) nanocapsules by layer-by-layer (LbL) assembly of polymers on silica templates and coating with Poly-L-Lysine. Nanodelivery systems offer potential advantages like site specific delivery of drugs, peptides, and genes, improved in vitro and in vivo stability and reduced side effect profile. However, because nanoparticles are often picked up by the phagocytic cells of the immune system and
This article is protected by copyright. All rights reserved.
Accepted Article
there may be undesirable interactions between nanoparticles and immune system which may promote inflammatory responses (3-4). The influence of size, solubility and surface modification on the biocompatibility of nanoparticles and their use in biological applications is well known. However, the effects of nanoparticles properties on immune system are still being explored and immunological studies of various nanopreparations are very much increasing (5). The main challenge faced in the field of drug delivery is the immune response to these nanomaterials and in vivo toxicity, so suitable study should be done before administration of nanomaterials for in vivo drug delivery applications. In general, cationic (positively-charged) particles are more likely to induce inflammatory reactions than anionic (negatively charged) and neutral species (6). PLL nanocapsules were positively charged and we would like to know immunocompactibity and toxicity of PLL nanocapsules in in vivo rat model. Hence, our study was designed for the synthesis, characterisation, immunocompatibility and toxicity studies of PLL nanocapsules for understanding its suitability for in vivo drug delivery applications.
Experimental Section Materials All reagents used were of analytical grade. Tetraethoxysilane (TES 28 SQ), nOctadecyltrimethoxysilane (91.6%), aqueous ammonia
(reagent grade, 32 wt.%),
Glutaraldehyde Poly-L-Lysine and Histopaque 1077 purchased from Sigma-Aldrich co, St. Louis, USA. All chemicals and reagents used for cell culture analysis experiments were purchased from Sigma, Aldrich, USA. Antibody against COX-2, LOX-15, NOS (Sigma Aldrich, USA). RT-PCR kit were purchased from Qiagen India and primers were purchased from Sigma Aldrich respectively. Rest of the chemicals and solvents used were purchased from SRL and spectrochem, India.
Methods Synthesis of Silica template for Layer-by-Layer (LbL) assembly The synthesis of Silica template for LbL assembly has been carried out as described by Buchel et al. (7). Absolute ethanol (58.5 g, 1.27 mol), deionized water (10 g), and aqueous ammonia (32 wt.%, 2.82 g, 0.17 mol) were mixed in a flask. After heating the mixture to 300C, tetraethoxysilane (5.6 g, 0.026 mol) was added rapidly and mixed for 5s by shaking to This article is protected by copyright. All rights reserved.
Accepted Article
ensure homogeneity. After 1h, a mixture of teraethoxysilane (4.67 g, 0.022 mol) and noctadecyltrimethoxysilane was added drop by drop over a period of 20 min while stirring with a magnetic stirrer and then kept at ambient temperature for 1h. The solvent was removed in vacuum at 600C using a rotary evaporator and the resulting white powder was dried overnight at 1000C. To remove the porogen, the powder was subjected to calcination in air for a period of 6h at 5500C. Infiltration method for synthesis of Poly-L-Lysine (PLL) nanocapsules PLL nanocapsules were prepared as described by Sivakumar et al. (8) and characterised by Scanning electron microscopy (SEM). Briefly, APTES-modified mesoporous silica (10 mg) was taken and polymer was infiltrated into the mesoporous shell by incubating the template particles with PLL solution (5 mg/ml in 0.2 M NaCl; pH 8.5) overnight with gentle mixing. Post infiltration, the unbound polymer was washed off thrice by water. The polymer was then cross-linked by incubating with glutaraldehyde for 20 min and then washed with water. The template was etched out using 2 M HF: 8 M NH4F (pH 5) to get nanocapsules. Animal experiments Sprague Dawely rats were obtained from Department of Biochemistry, University of Kerala, India for specific study. All ethical guidelines were followed for the conduct of animal experiments in strict compliance with the Institutional animal ethical committee and committee for the purpose of control and supervision of experiments on animals (CPCSEA) government of India and ethical sanction no.IAEU-KU-24/2011-12-BC.AA (22) for the conduction of animal experiment. In vivo Study Sprague Dawely male rats were randomly divided into 6 groups of 6 animals each and experimental period up to 30 days. Group-I Control, Group-II Saline treated (4ml/kg) body weight, Group -III- PLL nanocapsules treated with (1.5 x 1012 PLL nanocapsules /Kg) body weight, Group -IV- PLL nanocapsules treated with (3.0 x 1012PLL nanocapsules /Kg) body weight, Group-V- PLL nanocapsules treated with (4.5 x 1012 PLL nanocapsules /Kg) body weight. Group-VI- Silica particle (177.5 mg/kg) body weight. In vivo toxicity was also carried out by intravenous administration of nanocapsules by tail vein injection in Sprague Dawely rat. Isolation of Rat peripheral blood mononuclear cells (PBMCs) and Cell culture Peripheral blood mononuclear cells were isolated from rat as previously described (9). The isolated cells were cultured in collagen I coated plates and maintained at 370C with 5% CO2 in RPMI as cultured medium supplemented with 10% heat inactivated FBS, 1% l-glutamine This article is protected by copyright. All rights reserved.
Accepted Article
1% HEPES and 100 U/ml penicillin and 100 μg/ml streptomycin streptomycin. The cells were dispersed in culture plates (1 x104 cells per well) and incubated with different concentrations of PAH nanocapsules for 24hr at identical environment. RT-PCR study mRNA was isolated from PBMCs and RT-PCR analysis of important immunomodulatory genes coding for monocyte chemotactic protein-1(MCP 1), tumor necrosis factor-alpha (TNF-α), Intercellular adhesion molecule-1 (ICAM-1) and interleukin-6 (IL-6) was carried out using Trizol reagent for RNA isolation from Medox Biotech India PVT, Ltd. RT-PCR and PCR amplification were carried out using the RT-PCR kit from QIAGEN, India. Initial PCR activation for 15 min at 950C followed by 3 steps of cycling process. Each cycle consists of denaturation for 1min at 940C, annealing for 1min at 650C, extension for 1min at 720C, repeated for 37 cycles and final extension for 10min at 720C. The PCR products were run on 0.8 % Agarose gels and stained with Ethidium Bromide and visualized with a UVtransilluminator. SDS−PAGE and Western Blotting Studies The western blotting of major inflammatory markers like cyclooxygenase-2 (COX-2), lipoxygenase-15 (LOX-15) and nitric oxide synthase (NOS) were performed as described previously by Peter et al. (10). Primary antibody of COX-2, LOX-15, NOS (Sigma, India) was added at the indicated dilution in blocking buffer and incubated on a rocking platform for 1 hour at room temperature. Binding was detected by incubation with peroxidase-conjugated secondary antibody (Sigma), diluted 1:1000 in blocking buffer, and visualized by chemiluminescense
Toxicity markers status in vivo Activity of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) as per the method of Reitman and Frankel (11), serum creatine kinase (CK) as per the method of Rosalki (12) and serum alkaline phosphatase (ALP) as per the method of Varle et al. (13) were assayed using established procedure. Hematological study in vivo Hematological parameters such as haemoglobin content, total White Blood Cell (WBC) count, RBC count, neutrophil, Lymphocyte and blood urea nitrogen of the normal and nanocapsule injected rat were analysed using a semi-auto analyzer.
This article is protected by copyright. All rights reserved.
Accepted Article
Histological Analysis of Rat tissue Tissues (Liver, Kidney, Lungs and Spleen) were fixed with Bouin’s fluid and processed for sectioning following standard techniques of Lamberg and Rothstein (14). Tissues were embedded in paraffin wax (58-60°C), sectioned at 5-6μm thickness on a microtome (York Inc. USA) and stained with Harris’ Hematoxylin-Aqueous Eosin stain and observed for histological changes under light microscope. Statistical analysis All statistical calculations were carried out with the statistical package for social sciences (SPSS) software program. The values are expressed as the mean ± SD. The data were statistically analyzed using analysis of variance (ANOVA) and significant difference of means was determined using Duncan’s multiple range tests at the level of p
Received Date : 27-Nov-2013 Revised Date : 11-Feb-2014 Accepted Date : 13-Feb-2014 Article type
: Research Article
Immunocompatibility and Toxicity Studies of Poly-L-Lysine Nanocapsules in Sprague Dawely Rats for Drug Delivery Applications Janeesh P.A1, Haider Sami3, Dhanya C.R1, Sri Sivakumar2 and Annie Abraham1* 1
*Department of Biochemistry, University of Kerala, Kariavattom campus, Trivandrum, Kerala, India.
2
Unit of Excellence on Soft Nanofabrication, Department of Chemical Engineering, Indian Institute of Technology Kanpur (IIT), Uttar Pradesh, India.
3
Department of Biological Sciences and Bioengineering, Indian Institute of Technology (IIT) Kanpur, Uttar Pradesh, India.
Correspondence author* Dr. Annie Abraham* Professor of Biochemistry Director, School of Life sciences Department of Biochemistry University of Kerala, Kariavattom Campus, Trivandrum - 695 581, Kerala, India Email: [email protected] Phone: Off: +91-471-2308078 Mob: +91-9447246692 Fax: +91-471-2307158
Running Head: Immunocompatibility and Toxicity Studies of PLL This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12313 This article is protected by copyright. All rights reserved.
Accepted Article
Abstract Poly-L-Lysine (PLL) Nanocapsules are the emerging drug delivery vehicle for the therapeutics of targeted diseases. The study was designed for the synthesis and characterisation of PLL nanocapsules and to know its immunocompatibility and toxicity behaviour for in vivo drug delivery applications. Alteration in haematological parameters, Immunomodulatory gene expression by RT-PCR studies, toxicity markers status, immunobloting of major inflammatory marker proteins and histopathological studies from major tissues of rat after intravenous administration of PLL nanocapsules after 30 days were assessed. In vivo toxicity markers activity, haematological parameters alteration and RT-PCR analysis of important immunomodulatory genes like monocyte chemotactic protein-1(MCP 1), tumor necrosis factor-alpha (TNF-α), Intercellular adhesion molecule-1 (ICAM-1) and interleukin-6 (IL-6) showed least changes when compared with control. The immunoblotting of major inflammatory markers like cyclooxygenase-2 (COX-2), lipoxygenase-15 (LOX-15) and
nitric
oxide
synthase
(NOS)
found
have
least
expression
showing
the
immunocompatibility of PLL nanocapsules. Histopathological studies of important tissues showed almost normal architecture after treatment using different concentration of PLL nanocapsules after the experimental period. The results showed a promising outcome and further confirmed the immunocompatibility and non toxicity of PLL nanocapsules in vivo for drug delivery applications. Key words: Immunocompatibility, Poly-L-Lysine (PLL) Nanocapsules, Immunomodulatory genes, Inflammatory markers.
Introduction Polymeric nanocapsules are widely used as drug delivery vehicle owning to their mechanistic property. It is fabricated through step-wise adsorption of polymers using electrostatics, Hbonding, covalent chemistry, etc as driving force, followed by the dissolution of the core template (1-2). Recently, we have synthesised and characterised Poly-L-Lysine (PLL) nanocapsules by layer-by-layer (LbL) assembly of polymers on silica templates and coating with Poly-L-Lysine. Nanodelivery systems offer potential advantages like site specific delivery of drugs, peptides, and genes, improved in vitro and in vivo stability and reduced side effect profile. However, because nanoparticles are often picked up by the phagocytic cells of the immune system and
This article is protected by copyright. All rights reserved.
Accepted Article
there may be undesirable interactions between nanoparticles and immune system which may promote inflammatory responses (3-4). The influence of size, solubility and surface modification on the biocompatibility of nanoparticles and their use in biological applications is well known. However, the effects of nanoparticles properties on immune system are still being explored and immunological studies of various nanopreparations are very much increasing (5). The main challenge faced in the field of drug delivery is the immune response to these nanomaterials and in vivo toxicity, so suitable study should be done before administration of nanomaterials for in vivo drug delivery applications. In general, cationic (positively-charged) particles are more likely to induce inflammatory reactions than anionic (negatively charged) and neutral species (6). PLL nanocapsules were positively charged and we would like to know immunocompactibity and toxicity of PLL nanocapsules in in vivo rat model. Hence, our study was designed for the synthesis, characterisation, immunocompatibility and toxicity studies of PLL nanocapsules for understanding its suitability for in vivo drug delivery applications.
Experimental Section Materials All reagents used were of analytical grade. Tetraethoxysilane (TES 28 SQ), nOctadecyltrimethoxysilane (91.6%), aqueous ammonia
(reagent grade, 32 wt.%),
Glutaraldehyde Poly-L-Lysine and Histopaque 1077 purchased from Sigma-Aldrich co, St. Louis, USA. All chemicals and reagents used for cell culture analysis experiments were purchased from Sigma, Aldrich, USA. Antibody against COX-2, LOX-15, NOS (Sigma Aldrich, USA). RT-PCR kit were purchased from Qiagen India and primers were purchased from Sigma Aldrich respectively. Rest of the chemicals and solvents used were purchased from SRL and spectrochem, India.
Methods Synthesis of Silica template for Layer-by-Layer (LbL) assembly The synthesis of Silica template for LbL assembly has been carried out as described by Buchel et al. (7). Absolute ethanol (58.5 g, 1.27 mol), deionized water (10 g), and aqueous ammonia (32 wt.%, 2.82 g, 0.17 mol) were mixed in a flask. After heating the mixture to 300C, tetraethoxysilane (5.6 g, 0.026 mol) was added rapidly and mixed for 5s by shaking to This article is protected by copyright. All rights reserved.
Accepted Article
ensure homogeneity. After 1h, a mixture of teraethoxysilane (4.67 g, 0.022 mol) and noctadecyltrimethoxysilane was added drop by drop over a period of 20 min while stirring with a magnetic stirrer and then kept at ambient temperature for 1h. The solvent was removed in vacuum at 600C using a rotary evaporator and the resulting white powder was dried overnight at 1000C. To remove the porogen, the powder was subjected to calcination in air for a period of 6h at 5500C. Infiltration method for synthesis of Poly-L-Lysine (PLL) nanocapsules PLL nanocapsules were prepared as described by Sivakumar et al. (8) and characterised by Scanning electron microscopy (SEM). Briefly, APTES-modified mesoporous silica (10 mg) was taken and polymer was infiltrated into the mesoporous shell by incubating the template particles with PLL solution (5 mg/ml in 0.2 M NaCl; pH 8.5) overnight with gentle mixing. Post infiltration, the unbound polymer was washed off thrice by water. The polymer was then cross-linked by incubating with glutaraldehyde for 20 min and then washed with water. The template was etched out using 2 M HF: 8 M NH4F (pH 5) to get nanocapsules. Animal experiments Sprague Dawely rats were obtained from Department of Biochemistry, University of Kerala, India for specific study. All ethical guidelines were followed for the conduct of animal experiments in strict compliance with the Institutional animal ethical committee and committee for the purpose of control and supervision of experiments on animals (CPCSEA) government of India and ethical sanction no.IAEU-KU-24/2011-12-BC.AA (22) for the conduction of animal experiment. In vivo Study Sprague Dawely male rats were randomly divided into 6 groups of 6 animals each and experimental period up to 30 days. Group-I Control, Group-II Saline treated (4ml/kg) body weight, Group -III- PLL nanocapsules treated with (1.5 x 1012 PLL nanocapsules /Kg) body weight, Group -IV- PLL nanocapsules treated with (3.0 x 1012PLL nanocapsules /Kg) body weight, Group-V- PLL nanocapsules treated with (4.5 x 1012 PLL nanocapsules /Kg) body weight. Group-VI- Silica particle (177.5 mg/kg) body weight. In vivo toxicity was also carried out by intravenous administration of nanocapsules by tail vein injection in Sprague Dawely rat. Isolation of Rat peripheral blood mononuclear cells (PBMCs) and Cell culture Peripheral blood mononuclear cells were isolated from rat as previously described (9). The isolated cells were cultured in collagen I coated plates and maintained at 370C with 5% CO2 in RPMI as cultured medium supplemented with 10% heat inactivated FBS, 1% l-glutamine This article is protected by copyright. All rights reserved.
Accepted Article
1% HEPES and 100 U/ml penicillin and 100 μg/ml streptomycin streptomycin. The cells were dispersed in culture plates (1 x104 cells per well) and incubated with different concentrations of PAH nanocapsules for 24hr at identical environment. RT-PCR study mRNA was isolated from PBMCs and RT-PCR analysis of important immunomodulatory genes coding for monocyte chemotactic protein-1(MCP 1), tumor necrosis factor-alpha (TNF-α), Intercellular adhesion molecule-1 (ICAM-1) and interleukin-6 (IL-6) was carried out using Trizol reagent for RNA isolation from Medox Biotech India PVT, Ltd. RT-PCR and PCR amplification were carried out using the RT-PCR kit from QIAGEN, India. Initial PCR activation for 15 min at 950C followed by 3 steps of cycling process. Each cycle consists of denaturation for 1min at 940C, annealing for 1min at 650C, extension for 1min at 720C, repeated for 37 cycles and final extension for 10min at 720C. The PCR products were run on 0.8 % Agarose gels and stained with Ethidium Bromide and visualized with a UVtransilluminator. SDS−PAGE and Western Blotting Studies The western blotting of major inflammatory markers like cyclooxygenase-2 (COX-2), lipoxygenase-15 (LOX-15) and nitric oxide synthase (NOS) were performed as described previously by Peter et al. (10). Primary antibody of COX-2, LOX-15, NOS (Sigma, India) was added at the indicated dilution in blocking buffer and incubated on a rocking platform for 1 hour at room temperature. Binding was detected by incubation with peroxidase-conjugated secondary antibody (Sigma), diluted 1:1000 in blocking buffer, and visualized by chemiluminescense
Toxicity markers status in vivo Activity of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) as per the method of Reitman and Frankel (11), serum creatine kinase (CK) as per the method of Rosalki (12) and serum alkaline phosphatase (ALP) as per the method of Varle et al. (13) were assayed using established procedure. Hematological study in vivo Hematological parameters such as haemoglobin content, total White Blood Cell (WBC) count, RBC count, neutrophil, Lymphocyte and blood urea nitrogen of the normal and nanocapsule injected rat were analysed using a semi-auto analyzer.
This article is protected by copyright. All rights reserved.
Accepted Article
Histological Analysis of Rat tissue Tissues (Liver, Kidney, Lungs and Spleen) were fixed with Bouin’s fluid and processed for sectioning following standard techniques of Lamberg and Rothstein (14). Tissues were embedded in paraffin wax (58-60°C), sectioned at 5-6μm thickness on a microtome (York Inc. USA) and stained with Harris’ Hematoxylin-Aqueous Eosin stain and observed for histological changes under light microscope. Statistical analysis All statistical calculations were carried out with the statistical package for social sciences (SPSS) software program. The values are expressed as the mean ± SD. The data were statistically analyzed using analysis of variance (ANOVA) and significant difference of means was determined using Duncan’s multiple range tests at the level of p
