International Journal of Biological Macromolecules 63 (2014) 154–157
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Short communication
Amidase encapsulated O-carboxymethyl chitosan nanoparticles for vaccine delivery K.T. Smitha 1 , M. Sreelakshmi 1 , N. Nisha, R. Jayakumar, Raja Biswas ∗ Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi 682041, India
a r t i c l e
i n f o
Article history: Received 28 September 2013 Received in revised form 14 October 2013 Accepted 28 October 2013 Available online 5 November 2013 Keywords: Amidase encapsulation Immunogenicity Staphylococcus aureus
a b s t r a c t This work reports the development of amidase encapsulated O-carboxymethyl chitosan nanoparticles (Ami-O-CMC NPs) of 300 ± 50 nm size by ionic cross-linking method. The prepared Ami-O-CMC NPs had an encapsulation efficiency of 55.39%. Haemolysis assay and cytotoxicity studies proved the hemocompatibility and cytocompatibility of the prepared NPs. The sustained release of Ami from the NPs is expected to prolong its immunogenicity and in turn lead to development of better protective immunity against Staphylococcus aureus infections. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Staphylococcus aureus, a gram-positive opportunistic pathogenic bacterium, is one of the leading causes of a wide variety of infections including pneumonia, endocarditis, osteomylitis, septic arthritis, toxic-shock syndrome, scalded-skin syndrome and food poisoning [1,2]. Worldwide emergence of methicillin (MRSA), vancomycin (VRSA) and other drug resistant strains of S. aureus necessitate the need to find alternative treatment strategies against this pathogen [3]. An effective vaccine could be an alternative to prevent infections caused by S. aureus. Till date several strategies have been employed to generate an effective vaccine against S. aureus. Whole-cell live or killed vaccines; purified type 5 and 8 capsular polysaccharide; poly-N-acetyl glucosamine; individual covalently cell wall linked surface antigens like clumping factor A, clumping factor B, iron-regulated surface determinant B, or fibronectin-binding protein failed to provide full protection against S. aureus challenge [4]. So far every effort to design vaccine against S. aureus failed in clinical trials [5]. We speculate that an efficient vaccine delivery system would enhance the antigenicity and immunogencity of the candidate vaccine antigens. Hence, we conducted this study.
∗ Corresponding author at: Centre for Nanoscience and Molecular medicine, Amrita Institute of Medical Sciences, AIMS–Ponekkara, Edapally, Cochin, Kerala, India- 682041. Tel.: +91 484 4001234x8764. E-mail addresses: [email protected], [email protected] (R. Biswas). 1 1 Two authors contributed equally. 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.045
Several nano and micro-particles were prepared in the past for delivering antigens and to enhance their immunogenic properties. Murillo et al., demonstrated that subcutaneous delivery of Brucella ovis antigens encapsulated poly (caprolactone) (PCL) microparticles induced a robust Th1/Th2 cellular immune response [6]. Florindoa et al., showed that intranasal administration of Streptococcus equi antigen encapsulated PCL nanospheres leads to an enhanced humoral, cellular and mucosal immune responses in mice against S. equi infections [7]. Chitosan is produced by deacetylation of chitin which is abundant in crustacean shells. Chemically chitosan is a linear polymer of ˇ (1–4)-linked D-glucosamine and N-acetyl-D-glucosamine units. Chitosan is a non-toxic, biodegradable and biocompatible polymer of high molecular weight. Chitosan derivatives are used in biomedical applications due to its bio-compatibility, bio-degradability and non-toxicity. O-carboxymethyl chitosan (O-CMC), a water soluble chitosan derivative, has favourable biocompatibility as chitosan [8,9]. In this study, we identified amidase (Ami) as a vaccine candidate and encapsulated this protein in O-CMC NPs. Ami is a 62 kDa protein present in S. aureus in the N-terminal part of the bifunctional protein major autolysin. Ami is ubiquitously present in all staphylococcal strains, cleaves the peptidoglycan layer and helps in daughter cell separation after cell division [10,11]. We assume that O-CMC NPs could act as a potential carrier for Ami protein as the encapsulation of Ami within O-CMC would prevent rapid degradation and elimination of this protein from the bloodstream. Moreover, the controlled and sustained release of Ami from O-CMC NPs would help in prolonging the antigenicity of Ami and in turn provide protective immunity towards S. aureus infections.
K.T. Smitha et al. / International Journal of Biological Macromolecules 63 (2014) 154–157
155
2. Materials and methods
2.6. Encapsulation efficiency and in vitro Ami release
2.1. Materials
The absorbance of the prepared Ami-O-CMC NPs, bare O-CMC NPs and Ami was analysed at 280 nm in Nanodrop spectrophotometer. Absorbance of bare O-CMC NPs was reduced from Ami O-CMC NPs to find the final concentration of Ami in the NPs. The encapsulation efficiency was determined by
O-CMC (12 kDa, degree of deacetylation: 61.8% and degree of substitution: 0.54) was purchased from Koyo chemical Co. Ltd., Japan. Calcium chloride and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Sigma–Aldrich. L929 and VERO cell lines were purchased from NCCS, India. All the other chemicals used were of analytical grade.
2.2. Protein purification Amidase (Ami) gene was previously cloned and expressed in pQE-30 (pQE-Ami) plasmid as N-terminal histidine tagged protein. E. coli strain M15 (pQE-Ami) were cultured in LB media with ampicillin (100 g/ml). The cells were allowed to grow till the mid exponential phase (O.D ∼ 0.5) and the protein expression was induced by adding 1 mM IPTG to the culture. The cells were allowed to grow further for 6 h and harvested by centrifugation. Ami was then purified using Ni-NTA chromatography under denaturing conditions as described earlier [10] and analyzed in 15% SDS-PAGE. The purified Ami protein was then used for encapsulation within O-CMC NPs. It was dialysed against PBS before using for NP preparation.
2.3. Antigenicity testing To determine the antigenicity of Ami, we used serum samples from S. aureus infected patients. Sera samples from three S. aureus septic patients were obtained after proper approval from Institutional ethical committee and informed consent from these patients. Serum fractions were collected by centrifuging the blood samples at 3500 rpm for 15 min. Antigenicity of Ami was determined using ELISA and Western blot. For ELISA, the 96-well plates were coated with purified 6X histidine Ami (5 g/well). The patient sera (1:50 dilution) were used as a source of primary antibody and Peroxidase conjugated goat anti-human secondary IgG (1:5000) was used as a secondary antibody. The Ami and antibody interactions were detected at 450 nm and using 3, 3 , 5, 5 -Tetramethylbenzidine (TMB) as a substrate. To perform Western blot analysis, Ami, after gel electrophoresis, was transferred electrophoretically to a nitrocellulose (NC) membrane using a Minigel system (Bio-Rad). The NC membrane was blocked, incubated with primary and secondary antibody as described above and developed using ECL reagent as described by the manufacturer (Amersham).
2.4. Preparation of amidase loaded O-CMC nanoparticles (Ami-O-CMC NPs) 0.1% O-CMC solution was prepared by dissolving it in distilled water. Ami protein (0.5 mg/ml) was then added to O-CMC solution under constant stirring. 1% CaCl2 was added drop wise to this solution until turbidity was observed, which indicates the formation of Ami-O-CMC NPs. The NPs were collected by centrifugation at 20,000 rpm for 20 min. The pellet obtained was washed thrice with milliQ water and used for further studies.
2.5. Physicochemical characterization of Ami-O-CMC NPs Particle size distribution of Ami-O-CMC NPs was studied using dynamic light scattering (DLS Malvern Particle size analyser). The size as well as surface morphology was determined by scanning electron microscopy (SEM JEOL JSM-6490LA).
Encapsulation efficiency(%)
=
Initial concentration of Ami − Final concentration of Ami Initial concentration of Ami
× 100
500 l of Ami-O-CMC NPs was incubated in 37 ◦ C shaker and the release was estimated at different time points. 50 l of the sample was taken at each time interval and centrifuged at 14,000 rpm for 10 min. The supernatant was then analyzed in Nanodrop Spectrophotometer to estimate the protein concentration at 280 nm. Release was quantified as Release(%) =
Released Ami Total Ami
× 100
2.7. Hemolysis assay Fresh human blood sample was diluted 5 times using PBS. 100 l of different concentrations of the prepared Ami-O-CMC NPs (50, 100 and 200 g/ml) were added to 200 l of diluted blood and incubated at 37 ◦ C for 24 h. Saline and PBS treated blood were treated as negative and positive controls respectively. The samples were centrifuged at 3,000 rpm for 15 min and the absorbance of supernatants was measured at 540 nm. 2.8. Cytotoxicity evaluation- MTT assay Cytotoxicity of the prepared NPs was studied against L929 and VERO cell lines in 96 well microtiter plates (10,000 cells/well). Increasing concentrations of Ami-O-CMC NPs (50, 100 and 200 g/ml) were added into the wells in triplicates. Untreated cells were used as positive controls and triton-X treated cells as negative controls. After 24 h MTT assay was performed using standard protocols [9]. 3. Results and discussion 3.1. Protein purification and antigenicity testing Ami is a non-covalently cell wall associated protein of S. aureus and is vital in cell separation. Plasmid map of pQE-Ami is depicted in Fig. 1A. Protein bands of SDS-PAGE visualized with Coomassie brilliant blue R-250 showed bands in gel corresponding to 62 kDa in size indicating the presence of Ami as shown by arrow mark in Fig. 1C. The purified Ami protein was used for ELISA and Western blot against S. aureus infected patient’s sera. The ELISA result showed that the Ami protein was antigenic in nature against all the sera tested (Fig. 2 A). To reconfirm the ELISA result, we tested the antigenicity of Ami using western blot. Equal amounts of Ami protein were loaded in each lane of the SDS-PAGE gel (Fig. 2B, C). Half of the gel was stained with coomassie and the rest half was used for Western blot. Western blot analysis reconfirmed the antigenic nature of Ami protein. Although Ami was antigenic in nature, the antigenicity of Ami differed among patients. It showed maximum antigenicity against sera from patient 2 and least antigenicity from patient 3 (Fig. 2A, C). Our results showed that Ami is an antigenic protein of S. aureus and can be used as a vaccine candidate to evoke protective immunity against S. aureus.
156
K.T. Smitha et al. / International Journal of Biological Macromolecules 63 (2014) 154–157
Fig. 5. Cell viability of (A) L929 and (B) VERO cells treated with Ami-O-CMC NPs (24 h).
3.2. Nanoparticle preparation and physico-chemical characterization
Fig. 1. (A) Plasmid map of pQE-Ami (B) Schematic representation of protein purification by Ni-NTA chromatography (C) 15% SDS gel showing the hourly over-expression of Ami protein in E. coli.
Ami-O-CMC NPs were prepared by ionic cross-linking of O-CMC solution using CaCl2 as cross-linker. DLS results showed that the NPs were in the size range of 300 ± 50 nm. This was reconfirmed by SEM (Fig. 3A). Also the particles were observed to be spherical in nature. 3.3. Encapsulation efficiency and in vitro amidase release
Fig. 2. Antigenicity of amidase (Ami) determined using (A) ELISA; (B, C) 15% SDSPAGE and corresponding Western blot demonstrating the antigenicity of Ami against 3 MRSA infected patient serum.
The encapsulation efficiency of Ami-O-CMC NPs was found to be 55.39%. The increase in concentration of Ami resulted in poor encapsulation as Ami tends to precipitate. The release profile of Ami from Ami-O-CMC NPs with respect to time was obtained (Fig. 3B). It showed a sustained release of Ami with 80% release at 48 h. The initial burst release of Ami can be attributed to release of Ami adsorbed onto the surface of NPs. The slow and sustained release of Ami would help in sustaining the antigenicity of Ami thus making Ami-O-CMC NPs a potential candidate for vaccine development. 3.4. Hemolysis assay The hemocompatability of the prepared Ami-O-CMC NPs were analysed by hemolysis assay. Fig. 4A shows the photograph of blood treated with the Ami-O-CMC NPs and Fig. 4B represents the graph showing percentage haemolysis of red blood cells. Results indicate a less than 12.5% haemolysis by all the three samples of Ami-O-CMC NPs. 3.5. Cytocompatability assessment
Fig. 3. (A) SEM image of Ami-O-CMC NPs (B) Release profile of Ami from Ami-O-CMC NPs in PBS (pH 7.4) at 37 ◦ C.
The Fig. 5 represents the cell viability plot of L929 and VERO cell lines after treatment with different concentrations of the prepared
Fig. 4. (A) Photograph of human blood treated with Ami-O-CMC NPs (B) Hemolysis assay plot.
K.T. Smitha et al. / International Journal of Biological Macromolecules 63 (2014) 154–157
NPs for 24 h. At all the concentrations tested, the cell viability was above 75% confirming the cytocompatibility of Ami-O-CMC NPs towards L929 and VERO cells.
157
is supported by SRF fellowship from ICMR, India. The authors are thankful to Mr Sajin P. Ravi for SEM. We thank Amrita Centre for Nanoscience and Molecular Medicine, Kochi for infrastructural support.
4. Conclusion References Ami protein was purified and used for developing Ami-O-CMC NPs via ionic cross-linking method. This non-haemolytic and nontoxic Ami-O-CMC NPs showed a sustained release of Ami which is thought to enhance the immunogenicity of Ami and in turn act as a better vaccine candidate to provide protection against S. aureus infections. This study could pave way for its application in animal models for the better understanding of how encapsulation of an antigen leads to increased antigenicity and eventually to a better vaccine development. Acknowledgement Raja Biswas is a Ramalingaswami fellow from DBT. The work in his laboratory is supported by grants from DBT, India. N. Nisha
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
G.L. Archer, Clin. Infect. Dis. 26 (1998) 1179–1181. J.K. Todd, Pediatr. Rev. 26 (2005) 444–450. H.F. Chambers, F.R. DeLeo, Nat. Rev. Microbiol. 7 (2009) 629–641. Y.K. Stranger-Jones, T. Bae, O. Schneewind, Proc. Natl. Acad. Sci. USA 103 (2006) 16942–16947. F. Bagnoli, S. Bertholet, G. Grandi, Front. Cell. Infect. Microbiol. 2 (2012) 16. M. Murillo, M.J. Grillo, J. Rene, C.M. Marın, M. Barberan, M.M. Goni, J.M. Blasco, J.M. Irache, C. Gamazo, Vaccine 19 (2001) 4099–4106. H.F. Florindoa, S. Pandit, L. Lacerda, L.M.D. Goncalves, H.O. Alpar, A.J. Almeida, Biomaterials 30 (2009) 879–891. A. Anitha, V.V. Divya Rani, R. Krishna, V. Sreeja, N. Selvamurugan, S.V. Nair, H. Tamura, R. Jayakumar, Carbohydr. Polymers 78 (2009) 672–677. A. Anitha, S. Maya, N. Deepa, K.P. Chennazhi, S.V. Nair, H. Tamura, R. Jayakumar, Carbohydr. Polymers 83 (2011) 452–461. T. Baba, O. Schneewind, EMBO J. 17 (1998) 4639–4646. R. Biswas, L. Voggu, U.K. Simon, P. Hentschel, G. Thumm, F. Gotz, FEMS Microbiol Lett. 259 (2006) 260–268.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Short communication
Amidase encapsulated O-carboxymethyl chitosan nanoparticles for vaccine delivery K.T. Smitha 1 , M. Sreelakshmi 1 , N. Nisha, R. Jayakumar, Raja Biswas ∗ Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi 682041, India
a r t i c l e
i n f o
Article history: Received 28 September 2013 Received in revised form 14 October 2013 Accepted 28 October 2013 Available online 5 November 2013 Keywords: Amidase encapsulation Immunogenicity Staphylococcus aureus
a b s t r a c t This work reports the development of amidase encapsulated O-carboxymethyl chitosan nanoparticles (Ami-O-CMC NPs) of 300 ± 50 nm size by ionic cross-linking method. The prepared Ami-O-CMC NPs had an encapsulation efficiency of 55.39%. Haemolysis assay and cytotoxicity studies proved the hemocompatibility and cytocompatibility of the prepared NPs. The sustained release of Ami from the NPs is expected to prolong its immunogenicity and in turn lead to development of better protective immunity against Staphylococcus aureus infections. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Staphylococcus aureus, a gram-positive opportunistic pathogenic bacterium, is one of the leading causes of a wide variety of infections including pneumonia, endocarditis, osteomylitis, septic arthritis, toxic-shock syndrome, scalded-skin syndrome and food poisoning [1,2]. Worldwide emergence of methicillin (MRSA), vancomycin (VRSA) and other drug resistant strains of S. aureus necessitate the need to find alternative treatment strategies against this pathogen [3]. An effective vaccine could be an alternative to prevent infections caused by S. aureus. Till date several strategies have been employed to generate an effective vaccine against S. aureus. Whole-cell live or killed vaccines; purified type 5 and 8 capsular polysaccharide; poly-N-acetyl glucosamine; individual covalently cell wall linked surface antigens like clumping factor A, clumping factor B, iron-regulated surface determinant B, or fibronectin-binding protein failed to provide full protection against S. aureus challenge [4]. So far every effort to design vaccine against S. aureus failed in clinical trials [5]. We speculate that an efficient vaccine delivery system would enhance the antigenicity and immunogencity of the candidate vaccine antigens. Hence, we conducted this study.
∗ Corresponding author at: Centre for Nanoscience and Molecular medicine, Amrita Institute of Medical Sciences, AIMS–Ponekkara, Edapally, Cochin, Kerala, India- 682041. Tel.: +91 484 4001234x8764. E-mail addresses: [email protected], [email protected] (R. Biswas). 1 1 Two authors contributed equally. 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.045
Several nano and micro-particles were prepared in the past for delivering antigens and to enhance their immunogenic properties. Murillo et al., demonstrated that subcutaneous delivery of Brucella ovis antigens encapsulated poly (caprolactone) (PCL) microparticles induced a robust Th1/Th2 cellular immune response [6]. Florindoa et al., showed that intranasal administration of Streptococcus equi antigen encapsulated PCL nanospheres leads to an enhanced humoral, cellular and mucosal immune responses in mice against S. equi infections [7]. Chitosan is produced by deacetylation of chitin which is abundant in crustacean shells. Chemically chitosan is a linear polymer of ˇ (1–4)-linked D-glucosamine and N-acetyl-D-glucosamine units. Chitosan is a non-toxic, biodegradable and biocompatible polymer of high molecular weight. Chitosan derivatives are used in biomedical applications due to its bio-compatibility, bio-degradability and non-toxicity. O-carboxymethyl chitosan (O-CMC), a water soluble chitosan derivative, has favourable biocompatibility as chitosan [8,9]. In this study, we identified amidase (Ami) as a vaccine candidate and encapsulated this protein in O-CMC NPs. Ami is a 62 kDa protein present in S. aureus in the N-terminal part of the bifunctional protein major autolysin. Ami is ubiquitously present in all staphylococcal strains, cleaves the peptidoglycan layer and helps in daughter cell separation after cell division [10,11]. We assume that O-CMC NPs could act as a potential carrier for Ami protein as the encapsulation of Ami within O-CMC would prevent rapid degradation and elimination of this protein from the bloodstream. Moreover, the controlled and sustained release of Ami from O-CMC NPs would help in prolonging the antigenicity of Ami and in turn provide protective immunity towards S. aureus infections.
K.T. Smitha et al. / International Journal of Biological Macromolecules 63 (2014) 154–157
155
2. Materials and methods
2.6. Encapsulation efficiency and in vitro Ami release
2.1. Materials
The absorbance of the prepared Ami-O-CMC NPs, bare O-CMC NPs and Ami was analysed at 280 nm in Nanodrop spectrophotometer. Absorbance of bare O-CMC NPs was reduced from Ami O-CMC NPs to find the final concentration of Ami in the NPs. The encapsulation efficiency was determined by
O-CMC (12 kDa, degree of deacetylation: 61.8% and degree of substitution: 0.54) was purchased from Koyo chemical Co. Ltd., Japan. Calcium chloride and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Sigma–Aldrich. L929 and VERO cell lines were purchased from NCCS, India. All the other chemicals used were of analytical grade.
2.2. Protein purification Amidase (Ami) gene was previously cloned and expressed in pQE-30 (pQE-Ami) plasmid as N-terminal histidine tagged protein. E. coli strain M15 (pQE-Ami) were cultured in LB media with ampicillin (100 g/ml). The cells were allowed to grow till the mid exponential phase (O.D ∼ 0.5) and the protein expression was induced by adding 1 mM IPTG to the culture. The cells were allowed to grow further for 6 h and harvested by centrifugation. Ami was then purified using Ni-NTA chromatography under denaturing conditions as described earlier [10] and analyzed in 15% SDS-PAGE. The purified Ami protein was then used for encapsulation within O-CMC NPs. It was dialysed against PBS before using for NP preparation.
2.3. Antigenicity testing To determine the antigenicity of Ami, we used serum samples from S. aureus infected patients. Sera samples from three S. aureus septic patients were obtained after proper approval from Institutional ethical committee and informed consent from these patients. Serum fractions were collected by centrifuging the blood samples at 3500 rpm for 15 min. Antigenicity of Ami was determined using ELISA and Western blot. For ELISA, the 96-well plates were coated with purified 6X histidine Ami (5 g/well). The patient sera (1:50 dilution) were used as a source of primary antibody and Peroxidase conjugated goat anti-human secondary IgG (1:5000) was used as a secondary antibody. The Ami and antibody interactions were detected at 450 nm and using 3, 3 , 5, 5 -Tetramethylbenzidine (TMB) as a substrate. To perform Western blot analysis, Ami, after gel electrophoresis, was transferred electrophoretically to a nitrocellulose (NC) membrane using a Minigel system (Bio-Rad). The NC membrane was blocked, incubated with primary and secondary antibody as described above and developed using ECL reagent as described by the manufacturer (Amersham).
2.4. Preparation of amidase loaded O-CMC nanoparticles (Ami-O-CMC NPs) 0.1% O-CMC solution was prepared by dissolving it in distilled water. Ami protein (0.5 mg/ml) was then added to O-CMC solution under constant stirring. 1% CaCl2 was added drop wise to this solution until turbidity was observed, which indicates the formation of Ami-O-CMC NPs. The NPs were collected by centrifugation at 20,000 rpm for 20 min. The pellet obtained was washed thrice with milliQ water and used for further studies.
2.5. Physicochemical characterization of Ami-O-CMC NPs Particle size distribution of Ami-O-CMC NPs was studied using dynamic light scattering (DLS Malvern Particle size analyser). The size as well as surface morphology was determined by scanning electron microscopy (SEM JEOL JSM-6490LA).
Encapsulation efficiency(%)
=
Initial concentration of Ami − Final concentration of Ami Initial concentration of Ami
× 100
500 l of Ami-O-CMC NPs was incubated in 37 ◦ C shaker and the release was estimated at different time points. 50 l of the sample was taken at each time interval and centrifuged at 14,000 rpm for 10 min. The supernatant was then analyzed in Nanodrop Spectrophotometer to estimate the protein concentration at 280 nm. Release was quantified as Release(%) =
Released Ami Total Ami
× 100
2.7. Hemolysis assay Fresh human blood sample was diluted 5 times using PBS. 100 l of different concentrations of the prepared Ami-O-CMC NPs (50, 100 and 200 g/ml) were added to 200 l of diluted blood and incubated at 37 ◦ C for 24 h. Saline and PBS treated blood were treated as negative and positive controls respectively. The samples were centrifuged at 3,000 rpm for 15 min and the absorbance of supernatants was measured at 540 nm. 2.8. Cytotoxicity evaluation- MTT assay Cytotoxicity of the prepared NPs was studied against L929 and VERO cell lines in 96 well microtiter plates (10,000 cells/well). Increasing concentrations of Ami-O-CMC NPs (50, 100 and 200 g/ml) were added into the wells in triplicates. Untreated cells were used as positive controls and triton-X treated cells as negative controls. After 24 h MTT assay was performed using standard protocols [9]. 3. Results and discussion 3.1. Protein purification and antigenicity testing Ami is a non-covalently cell wall associated protein of S. aureus and is vital in cell separation. Plasmid map of pQE-Ami is depicted in Fig. 1A. Protein bands of SDS-PAGE visualized with Coomassie brilliant blue R-250 showed bands in gel corresponding to 62 kDa in size indicating the presence of Ami as shown by arrow mark in Fig. 1C. The purified Ami protein was used for ELISA and Western blot against S. aureus infected patient’s sera. The ELISA result showed that the Ami protein was antigenic in nature against all the sera tested (Fig. 2 A). To reconfirm the ELISA result, we tested the antigenicity of Ami using western blot. Equal amounts of Ami protein were loaded in each lane of the SDS-PAGE gel (Fig. 2B, C). Half of the gel was stained with coomassie and the rest half was used for Western blot. Western blot analysis reconfirmed the antigenic nature of Ami protein. Although Ami was antigenic in nature, the antigenicity of Ami differed among patients. It showed maximum antigenicity against sera from patient 2 and least antigenicity from patient 3 (Fig. 2A, C). Our results showed that Ami is an antigenic protein of S. aureus and can be used as a vaccine candidate to evoke protective immunity against S. aureus.
156
K.T. Smitha et al. / International Journal of Biological Macromolecules 63 (2014) 154–157
Fig. 5. Cell viability of (A) L929 and (B) VERO cells treated with Ami-O-CMC NPs (24 h).
3.2. Nanoparticle preparation and physico-chemical characterization
Fig. 1. (A) Plasmid map of pQE-Ami (B) Schematic representation of protein purification by Ni-NTA chromatography (C) 15% SDS gel showing the hourly over-expression of Ami protein in E. coli.
Ami-O-CMC NPs were prepared by ionic cross-linking of O-CMC solution using CaCl2 as cross-linker. DLS results showed that the NPs were in the size range of 300 ± 50 nm. This was reconfirmed by SEM (Fig. 3A). Also the particles were observed to be spherical in nature. 3.3. Encapsulation efficiency and in vitro amidase release
Fig. 2. Antigenicity of amidase (Ami) determined using (A) ELISA; (B, C) 15% SDSPAGE and corresponding Western blot demonstrating the antigenicity of Ami against 3 MRSA infected patient serum.
The encapsulation efficiency of Ami-O-CMC NPs was found to be 55.39%. The increase in concentration of Ami resulted in poor encapsulation as Ami tends to precipitate. The release profile of Ami from Ami-O-CMC NPs with respect to time was obtained (Fig. 3B). It showed a sustained release of Ami with 80% release at 48 h. The initial burst release of Ami can be attributed to release of Ami adsorbed onto the surface of NPs. The slow and sustained release of Ami would help in sustaining the antigenicity of Ami thus making Ami-O-CMC NPs a potential candidate for vaccine development. 3.4. Hemolysis assay The hemocompatability of the prepared Ami-O-CMC NPs were analysed by hemolysis assay. Fig. 4A shows the photograph of blood treated with the Ami-O-CMC NPs and Fig. 4B represents the graph showing percentage haemolysis of red blood cells. Results indicate a less than 12.5% haemolysis by all the three samples of Ami-O-CMC NPs. 3.5. Cytocompatability assessment
Fig. 3. (A) SEM image of Ami-O-CMC NPs (B) Release profile of Ami from Ami-O-CMC NPs in PBS (pH 7.4) at 37 ◦ C.
The Fig. 5 represents the cell viability plot of L929 and VERO cell lines after treatment with different concentrations of the prepared
Fig. 4. (A) Photograph of human blood treated with Ami-O-CMC NPs (B) Hemolysis assay plot.
K.T. Smitha et al. / International Journal of Biological Macromolecules 63 (2014) 154–157
NPs for 24 h. At all the concentrations tested, the cell viability was above 75% confirming the cytocompatibility of Ami-O-CMC NPs towards L929 and VERO cells.
157
is supported by SRF fellowship from ICMR, India. The authors are thankful to Mr Sajin P. Ravi for SEM. We thank Amrita Centre for Nanoscience and Molecular Medicine, Kochi for infrastructural support.
4. Conclusion References Ami protein was purified and used for developing Ami-O-CMC NPs via ionic cross-linking method. This non-haemolytic and nontoxic Ami-O-CMC NPs showed a sustained release of Ami which is thought to enhance the immunogenicity of Ami and in turn act as a better vaccine candidate to provide protection against S. aureus infections. This study could pave way for its application in animal models for the better understanding of how encapsulation of an antigen leads to increased antigenicity and eventually to a better vaccine development. Acknowledgement Raja Biswas is a Ramalingaswami fellow from DBT. The work in his laboratory is supported by grants from DBT, India. N. Nisha
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
G.L. Archer, Clin. Infect. Dis. 26 (1998) 1179–1181. J.K. Todd, Pediatr. Rev. 26 (2005) 444–450. H.F. Chambers, F.R. DeLeo, Nat. Rev. Microbiol. 7 (2009) 629–641. Y.K. Stranger-Jones, T. Bae, O. Schneewind, Proc. Natl. Acad. Sci. USA 103 (2006) 16942–16947. F. Bagnoli, S. Bertholet, G. Grandi, Front. Cell. Infect. Microbiol. 2 (2012) 16. M. Murillo, M.J. Grillo, J. Rene, C.M. Marın, M. Barberan, M.M. Goni, J.M. Blasco, J.M. Irache, C. Gamazo, Vaccine 19 (2001) 4099–4106. H.F. Florindoa, S. Pandit, L. Lacerda, L.M.D. Goncalves, H.O. Alpar, A.J. Almeida, Biomaterials 30 (2009) 879–891. A. Anitha, V.V. Divya Rani, R. Krishna, V. Sreeja, N. Selvamurugan, S.V. Nair, H. Tamura, R. Jayakumar, Carbohydr. Polymers 78 (2009) 672–677. A. Anitha, S. Maya, N. Deepa, K.P. Chennazhi, S.V. Nair, H. Tamura, R. Jayakumar, Carbohydr. Polymers 83 (2011) 452–461. T. Baba, O. Schneewind, EMBO J. 17 (1998) 4639–4646. R. Biswas, L. Voggu, U.K. Simon, P. Hentschel, G. Thumm, F. Gotz, FEMS Microbiol Lett. 259 (2006) 260–268.
