Preparation, characterization and immunological evaluation: canine parvovirus synthetic peptide loaded PLGA nanoparticles
© Derman et al. 2015
Received: 11 July 2015
Accepted: 6 October 2015
Published: 20 October 2015
Canine parvovirus 2 (CPV-2) remains a significant worldwide canine pathogen and the most common cause of viral enteritis in dogs. The 1 L15 and 7 L15 peptides overlap each other with QPDGGQPAV residues (7-15 of VP2 capsid protein of CPV) is shown to produce high immune response. PLGA nanoparticles were demonstrated to have special properties such as; controlled antigen release, protection from degradation, elimination of booster-dose and enhancing the cellular uptake by antigen presenting cells. Nevertheless, there is no study available in literature, about developing vaccine based on PLGA nanoparticles with adjuvant properties against CPV.
Thus, the aim of the present study was to synthesize and characterize high immunogenic W-1 L19 peptide (from the VP2 capsid protein of CPV) loaded PLGA nanoparticle and to evaluate their in vitro immunogenic activity.
PLGA nanoparticles were produced with 5.26 ± 0.05 % loading capacity and high encapsulation efficiency with 81.2 ± 3.1 %. Additionally, it was evaluated that free NPs and W-1 L19 peptide encapsulated PLGA nanoparticles have Z-ave of 183.9 ± 12.1 nm, 221.7 ± 15.8 nm and polydispersity index of 0.107 ± 0.08, 0.135 ± 0.12 respectively. It was determined that peptide loaded PLGA nanoparticles were successfully phagocytized by macrophage cells and increased NO production at 2-folds (*P < 0.05) in contrast to free peptide, and 3-folds (*P < 0.01) in contrast to control.
In conclusion, for the first time, W-1 L19 peptide loaded PLGA nanoparticles were successfully synthesized and immunogenic properties evaluated. Obtained results showed that PLGA nanoparticles enhanced the capacity of W-1 L19 peptide to induce nitric oxide production in vitro due to its adjuvant properties. Depend on the obtained results, these nanoparticles can be accepted as potential vaccine candidate against Canine Parvovirus. Studies targeting PLGA nanoparticles based delivery system must be maintained in near future in order to develop new and more effective nano-vaccine formulations.
Canine parvovirus (CPV) is a small, non-enveloped, autonomously replicating Single-strained DNA virus , remains a significant worldwide canine pathogen , is the cause enteric and myocardial disease in dogs [3, 4]. In experimentally affected dogs, mortality without treatment has been reported as high as 91 % [2, 5]. The two peptides overlap each other with the sequence QPDGGQPAV residues (7 to 15 of VP2) 1 L15 (MSDGAVQPDGGQPAV) and 7 L15 (QPDGGQPAVRNERAT), have different potencies in inducing virus-neutralizing antibodies, produce good immune response in mice and immunogenic in several animal species [6, 7]. Therefore, different approaches, particularly using these peptide sequences, are available for developing synthetic peptide based vaccines against Canine Parvovirus [6–9]. However, synthetic peptides when used as a vaccine, without a delivery system have been shown to be ineffective due to its rapid degradation by proteases, along with its poor cellular uptake and immunogenicity . In order to elicit a higher immune response and improve the efficiency of peptide-based vaccine, it is generally necessary to use a carrier system such as protein, polymer or nano-micro particles.
Nanoparticle based antigen delivery system is a rapidly developing area within nanotechnology. Especially nano sized particular system based on biodegradable polymers offer potential solution to disadvantages of the current vaccines . Poly(DL,lactic-co-glycolic acid) (PLGA) is approved by Food and Drug Administration (FDA) and widely used copolymer for nanoparticular delivery system, owing to its biodegradability and biocompatibility . Encapsulation of vaccine antigens using PLGA nanoparticles provides several advantage over the other antigen delivery systems, such as; (i) antigen can be controlled released over a longer period , (ii) antigen can be protected against degradation in the presence of proteolytic enzymes , (iii) eliminated the need for booster dosed , (iv) enhance the antigen cellular uptake by antigen presenting cells (APC) [13, 14]. Moreover, their submicron size and their large specific surface area favor their adsorption compared to larger carriers . According to these significant properties of PLGA polymer were studied in order to develop new peptide based vaccine delivery systems against infectious disease such as Bacillus anthracis , Hepatitis B [17, 18], Chlamiydia Trachomatis , and also against allergic asthma , melanoma cancer . However, to our knowledge, there is no study available in literature, in regards to develop vaccines based on PLGA nanoparticles with adjuvant properties against CPV.
The main goal of the present study was to synthesize and characterize PLGA nanoparticle loaded with high immunogenic W-1 L19 peptide sequences from the VP2 capsid protein of CPV and to evaluate their in vitro immunogenic activity. For this purpose, loading capacity, encapsulation efficiency, antigen release and morphological investigation of the nanoparticles were conducted. Additionally, in vitro cytotoxicity of nanoparticles was investigated on J-774 cell lines and finally potency of PLGA nanoparticles to induce NO production at non-toxic concentrations were evaluated in macrophages.
The water-soluble synthetic peptide representing W-1 L19 from the VP2 capsid protein of Canine Parvovirus (W-MSDGAVQPDGGQPAVRNERA) and the non-immunogenic scrambled peptide (WMSDGAVQPDGGQPAVRNERA) were synthesized via solid phase peptide synthesis method by Caslo Laboratory ApS (Denmark). Tryptophan (W) amino acid was also added to the N-terminus of peptide sequences in order to provide UV-spectral analysis. PLGA (lactide:glicolide = 50:50; inherent viscosity 0.45-0.60 dL/g, Mw ~ 38-54 kDa P50/50), 3-(4,5-dimetil triazol-2-il)-2,5-difeniltetrazoliumbromid (MTT), Fluorescein isothiocyanate (FITC), dimethyl sulfoxide (DMSO), and polyvinyl alcohol were purchased from Sigma Aldrich (St. Louis, USA), dichloromethane (DCM) was purchased Ridel de Haen. Mouse J774 macrophage cell line was obtained from Histology and Embryology Department, Istanbul University, Istanbul, Turkey. Ultra-pure water was obtained from Millipore MilliQ Gradient system.
Preparation of polymeric nanoparticles
Canine Parvovirus W-1 L19 peptide was encapsulated as an antigen in PLGA nanoparticles by a modified water/oil/water double emulsion solvent evaporation method . Briefly, primary emulsion between internal aqueous phase containing peptide (5 mg/ml) and organic phase (75 mg/ml PLGA in dichloromethane) was prepared by sonication (55 W, amplitude of % 50, 2 min) (Bandelin Sonopuls, Germany) over an ice bath. Thereafter, the resulting primary emulsion (w/o) was added drop wise to external aqueous phase containing 4 ml PVA (% 2.5 w/v) and emulsified in an ice-water bath to form the double emulsion (w/o/w). The emulsifications were carried out using micro tip probe sonicator set at 55 W of energy output (Bandelin-sonopuls) for 2 min in an ice bath. The double emulsion (w/o/w) was diluted in 80 ml PVA (% 0.5 w/v) solution and the emulsion was stirred overnight on a magnetic stirrer plate at room temperature for evaporation of organic phase. The resulting particles were collected by centrifugation at 10.000 x g for 20 min (Sartorius-Biofuge), washed three times with ultra-pure water to remove excess PVA and then lyophilized. We also prepared FITC loaded nanoparticles, for morphological investigation with fluorescence microscopy and in vitro cellular uptake study. Fluorescent nanoparticles were fabricated in a similar method where FITC was used in place of peptide. An equivalent volume of ultra-pure water was similarly encapsulated in P50/50 to serve as a control (Free NP). All lyophilized nanoparticles were stored at −80 °C until used.
Encapsulation efficiency and peptide loading capacity
Peptide encapsulation efficiency (EE) was detected via indirect quantification methods by using UV-Vis Spectroscopy at 280 nm. EE was determined by measuring the concentration of free peptide in supernatant which obtained from the ultracentrifugation of nanoparticles. The peptide concentration in the supernatant was determined by comparing the concentration to a previously constructed standard calibration curve. The concentration of loading peptide was calculated indirectly by calculating the differences between the initial concentrations of the peptide used (5 mg/ml) and the concentration of free peptide in supernatant.
Where A is the total peptide amount, B is the free peptide amount, and C is the quantified nanoparticle weight . A standard calibration curve of the absorbance as a function of peptide concentration was studied at 280 nm. All measurements were performed in triplicate.
Atomic force microscopy (AFM), scanning electron microscopy (SEM), fluorescent microscopy (FM)
Both atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to ascertain surface morphology and size of nanoparticles (free and peptide loaded). AFM (Shimadzu SPM 9600, Japan) studies were performed as previously described . About 5 μl each of nanoparticle solution were dropped to freshly cleaved 1 cm2 mica surface and incubated for 5 min. Mica surface was rinsed with ultra-pure water and dried for 20 min, the morphological analysis was performed by dynamic mode in 2-dimensional (2-D) and 3- dimensional (3-D).
SEM was used to verify uniformity of nanoparticle shape and size as previously described . The fabricated nanoparticles were dropped onto black carbon tape with a double-side. After that, they were vacuum-coated with a platinum mixture for 45 s and morphologically analyzed with a FE-SEM (CamScan Apollo 300 Field-Emission SEM, UK) at 20 kV.
Fourier transform infrared (FT-IR) spectrometry
Infrared Spectroscopy of the samples was performed in IR-Prestige 21 FTIR Spectrophotometer (Shimadzu, Japan). FT-IR spectra were recorded for P50/50, W1L-19 peptide and peptide loaded nanoparticle in universal attenuation total reflectance (ATR) mode. The measurement range was 4000–750 cm−1, scan number for per sample was 16, and resolution was 4 cm−1.
Particle size, zeta potential and polydispersity index (PdI)
The intensity size distribution, the Z-average (Z-Ave), and PdI of nanoparticles were performed by using dynamic light scattering technique using a Zetasizer (Zetasizer Nano ZS, Malvern, UK) instrument equipped with 4.0 mV He-Ne laser (633 nm). Measurements were carried out at 25 ± 0.1 °C with using 0.8872 cP of viscosity and 1.330 of refractive index for the solutions. The number of runs and run durations were chose as automatically. Electrophoretic light scattering (ELS) is used for zeta potential (ζ) measurement of particles and carried out in the folded capillary cell at 25 ± 0.1 °C. The measurements were performed with the following parameters: viscosity, 0.8872 cP; dielectric constant, 79; f(ka), 1.50 (Smoluchowski). The measurement durations and voltage selections were set to automatic mode.
All samples were prepared by diluting with phosphate buffer saline (PBS), filtered with a 0.20 μm RC-membrane filter (Sartorius) before measurement, and all measurements were performed three times.
In vitro peptide release
The release of the W-1 L19 peptide in vitro from the peptide loaded nanoparticle were determined following the method of Dixit et al. . Briefly the peptide loaded nanoparticle aliquots suspended in PBS (pH 7.4) with % 0.01 sodium azide and the suspension were incubated at 37 °C in a shaking incubator (60 rpm). At predetermined time intervals (6 h, 12 h, 1, 3, 7, 14, 21, 28, 35, 42 days), tubes were centrifuged, and the supernatants were collected followed by resuspension pellet in fresh PBS. The peptide concentration in the supernatant was determined with UV-Vis Spectroscopy at 280 nm by comparing the concentration to a previously constructed standard calibration curve.
Morphological changes depend on the time
In order to examine morphological changes depending on the time (during the degradation of nanoparticles), the nanoparticle suspension in PBS were incubated at 37° and samples were analyzed in 30 and 60 days with AFM.
Cell viability assay
Cell viability assays of nanoparticles and peptides were performed on J774 cell lines by MTT method. Briefly, 3 × 104 cells/ml were seeded into microplates and were incubated overnight. After incubation, different concentrations of nanoparticles and peptides ranging from 1 mg/ml to 0.01 mg/ml in PBS were added onto cells. Following to 48 h incubation at 37 °C, 10 μl MTT reactant 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (10 mg/ml) were included into all wells of microplates in order to assess susceptibility of cells against agents. When formazan crystals that are signature of viability were detected, these crystals were dissolved by DMSO. Then colorimetric density in microplates was read by using a Microplate Reader at 570 nm.
Cellular uptake study
In order to observe uptake of nanoparticles into macrophages, 5 × 104 cells/ml were seeded into each wells of a 6 well-plate. Following to one night incubation, nanoparticles at the concentrations of 0.5 mg/ml, which was detected as non-toxic concentration for macrophages were put in each wells of the plate. Cellular uptake was visualized by a florescence microscope thanks to fluorescein molecule which was embedded into nanoparticles. Photographs related to uptake were taken by Olympus C-5050 digital camera.
Quantification of nitrix oxide (NO) level
In experiments, in order to evaluate nitric oxide production following to nanoparticle and peptide formulations, we used non-toxic concentration (500 μg/ml) of both nanoparticles and peptide. In concentrations higher than 500 μg/ml, there was a sharp decline in viability values of J774 cells in regardless of nanoparticle and peptide. Therefore, a concentration of 500 μg/ml was chosen both nanoparticles and peptides. For this experiment, 5 × 104 J774 macrophage cells were seeded into 96 well microplate. After overnight incubation, macrophage cells were exposed to free NP, peptide loaded NP and peptide with the concentrations of 500 μg/ml, while only PBS was used in control group. Following to 48 h incubation, supernatants above the cells were picked up and 50 μl of supernatants from each group were transferred to another microplate. In order to observe colorimetric reaction and evaluate nitric oxide production, 50 μl of griess reactive was transferred to all wells of new microplate. On the other side, different concentrations of nitride were used as control. After 30 min incubation at room temperature, microplate was read at 540 nm absorbance by using a Microplate-Reader.
All experiments were repeated at least three times in triplicate wells. Data were expressed as mean ± standard deviation. All statistical analyses were performed using SPSS 15.0 . Non parametric analysis with Mann-Whitney U-test was carried out on the data of the biological variables to examine differences between the groups. P value less than 0.05 (p < 0.05) was accepted as significant.
Results and discussion
Encapsulation efficiency and loading capacity
The w/o/w double emulsion solvent evaporation method was used for fabrication of W-1 L19 peptide loaded PLGA nanoparticles. The ratio of aqueous phase (containing W-1 L19 peptide) to organic phase (containing PLGA) was kept low for production of nano-size particles as this ratio directed affects the particle size. First, we determined process yield, peptide loading capacity, and encapsulation efficiency. Lyophilized nanoparticles were weighed and yield of the process was estimated to be 81.2 ± 3.1 %. Peptide loading capacity and encapsulation efficiency were determined by measuring the concentration of free peptide in supernatant which obtained from the ultracentrifugation of nanoparticles. Our results from indirect methods employed for calculate the EE and LC and they were found 85.3 ± 2.2 % and 5.26 ± 0.05 % respectively.
Physicochemical properties of nanoparticles
Fourier transform infrared (FT-IR) analysis
Particle size, zeta potential and PdI measurements
Size distribution, zeta potential and PdI values of nanoparticles
Zeta potential (mV)
183.9 ± 12.1
0.107 ± 0.08
−36.8 ± 3.5
Peptide loaded nanoparticle
221.7 ± 15.8
0.135 ± 0.12
−35.1 ± 2.9
The free and peptide loaded nanoparticles showed a negative surface charges of around 36.8 ± 3.5 and 35.1 ± 2.9 mV, respectively which means that they were stable in dispersion state (Table 1). Further, PdI values are 0.107 ± 0.08 and 0.135 ± 0.12 for free and peptide loaded nanoparticle, respectively. The nanoparticles exhibited a relatively narrow PdI (less than 0.15) indicating the monodisperse formulation, which is useful for treatment effect .
In vitro release study of peptide loaded nanoparticles
Morphological changes depend on the time
Especially AFM views related to morphology of nanoparticles at the day 60 demonstrated that nanoparticles lost their spherical shapes and this can be explained by the hydrolysis of PLGA nanoparticles. In Fig. 6c and d, the area outside of nanoparticles within release medium were observed as “foggy”. This turbidity may be explained by hydrolysis of nanoparticles and release of peptide antigen to the medium.
Cell viability assay
Quantification of nitrix oxide (NO) Level
Poly(DL,lactic-co-glycolic acid) is approved by Food and Drug Administration (FDA) and widely used copolymer for nanoparticular delivery system, owing to its biodegradability and biocompatibility . In the previous studies demonstrate that, PLGA nanoparticles have adjuvant effect for various vaccine antigens [17, 33–35]. However, we could not find any study in literature investigating immunogenic features of PLGA nanoparticles as adjuvants against Canine Parvovirus.
In this study, for the first time, W-1 L19 peptide loaded PLGA nanoparticles were successfully synthesized by using water/oil/water double emulsion solvent evaporation method. Results of particle characterization with SEM, AFM, FT-IR and zetasizer demonstrated that synthesized particles were nano-sized, narrow sized distributed and smooth spherical shaped. Moreover, controlled release of W-1 L19 peptide from the particles were observed under physiological pH (7.4). According to biocompatibility tests of nanoparticles that were maintained on J774 cell lines, non-toxic concentrations of W-1 L19 peptide loaded PLGA nanoparticles were found and their high immunogenic features were determined by evaluation of nitric oxide amounts in macrophages cells.
As it is known, in vaccine delivery researches based on PLGA nanoparticles, especially 200-500 nm ranged particles are preferred [11, 27, 28, 36, 37]. That’s why at these dimensions, PLGA nanoparticles can easily activate dendritic cells, antigen specific T helper cells and cytotoxic T lymphocyte cells in order to generate high humoral and cellular immune response, they can be endocytosed by antigen presenting cells (APCs) as well [11, 14, 37].
In the present study, we encapsulated Canine Parvovirus W-1 L19 antigenic peptide to PLGA (50:50) nanoparticles by double emulsion solvent evaporation method  with small modifications. PLGA nanoparticles were produced with 5.26 ± 0.05 % loading capacity and high encapsulation efficiency with 81.2 ± 3.1. Additionally, it was evaluated that free NPs and W-1 L19 peptide encapsulated PLGA nanoparticles have Z-ave of 183.9 ± 12.1 nm and 221.7 ± 15.8 nm, respectively. It can be thought that synthesized nanoparticles are small enough to interact with APCs and induce cellular and humoral immune response.
Zeta potential is the essential particle characteristic and affecting particle stability, all of studies about zeta potential of PLGA nanoparticle resulted that PLGA nanoparticles which were prepared with PVA as a surfactant has a negative surface charge [14, 19, 20, 31, 38, 39]. Similarly in our study, the zeta potential of free NPs and peptide loaded NPs was −36.8 ± 3.5 mV and −35.1 ± 2.9 mV respectively, indicating a high stability due to the high repulsion between nanoparticles.
Characterization of W-1 L19 peptide encapsulated PLGA nanoparticles with AFM and SEM exhibited that synthesized nanoparticles were smooth surfaced and spherical in shape. In several studies, uses of smooth and spherical nanoparticles were suggested as well adjuvant activity for antigens and there was no requirement to apply booster doses of vaccines since they provide opportunity to controlled release by degradation of PLGA nanoparticles [11, 27, 36]. In general biodegradation of nanoparticulate vaccine delivery systems are investigated by evaluating release kinetics. For the first time, in this study, we visualized nanoparticulate system for 30 and 60 days in physiological conditions (pH 7.4 and 37 C) by using AFM. According to AFM images, we determined that nanoparticles protected their spherical shapes at the end of 30th days, while nanoparticles were totally lost their compact structures at the end of 60th days showing that PLGA nanoparticles released high amounts of W-1 L19 antigens to the medium.
In studies targeting development of vaccine delivery systems, long-time release of antigens from nanoparticles is crucial as they can provide long-term-protection against diseases [11, 14, 40]. This may also reduce quantities of immunization process and increase the antigen presentations to APCs . Depending on their biodegradable features which lead to long-term controlled release as well as biocompatibility, PLGA nanoparticles have been widely studied in vaccine development especially against infectious diseases [11, 18, 25] and cancer [21, 41–43]. Taha et al exhibited that PLGA nanoparticles caused the 20 % release of Major Outer Membrane Protein (MOMP) at one day and 48.6, 70 and 100 % of antigen were released at first, second and third week, respectively . Manish et al studied on protective efficacies of Immunogenic Domain 4 of Protective Antigen (PAD4) loaded PLGA nanoparticles against Bacillus anthracis. This group demonstrated that 50 % of PAD4 antigens released from nanoparticles during first 24 h. Totally 75 % of PAD4 antigens released at the end of 4 weeks . In the another study, Primard et al, prepared multifunctional PLGA nanoparticles by encapsulating an immunomodulator Imiquimod (IMQ) and BSA as an antigen in order to target Toll-like Receptor 7. In these study, it was shown that PLGA nanoparticles rapidly released % 40 of IMQ at 24 h . As it is clearly seen, in most of studies, PLGA nanoparticles showed high burst-release kinetics which is identified as high amounts of antigens’ release in 24 h [11, 19, 39]. However, in some studies it was pointed out that high burst release especially in 24 h is not preferred since it leads to low T cell response and antigen encapsulated nanoparticles with low burst release features may demonstrate better vaccine activity. For that reason, Silva and colleagues synthesized 24-residue long synthetic antigenic peptide of Ovalbumin (OVA24) peptide encapsulated PLGA nanoparticles with w/o/w double emulsion method and studied on diminishing burst release of antigens from nanoparticles by changing first and second emulsion medium. These group compared low (<10 %) and high (75 %) burst release in terms of immunogenic properties and found out that low burst release resulted in higher T cell response . Similarly, in our study, only 7 % of antigens were released at first 24 h which indicates sustained slow release. Our results overlapped with similar studies [14, 44–46] since antigen release from PLGA nanoparticles were shown to be biphasic release character. These results show that our synthetized particles might be attractive candidate for further vaccine studies.
One of the most important properties of PLGA nanoparticles is improving biocompatibility and bioavailability of biologically active molecules such as peptides, drugs, proteins etc . In several studies, it has been shown that encapsulation of antigenic molecules into PLGA nanoparticles decreased their toxicity as well as enhancing their bioavailability [11, 14, 17–21, 25, 38, 39, 48]. Our findings demonstrated that IC50 values of free NP, peptide loaded NP and peptides were assessed as 750, 740 and 650 μg/ml, respectively. In all of studied concentrations, encapsulation of peptides into PLGA nanoparticles increased applied dosages of peptides since viability amounts of macrophages that were exposed to free peptides were lower than macrophages that were exposed to peptide loaded PLGA nanoparticles [11, 14, 17–19, 21, 39, 48]. This is related to special features of PLGA nanoparticles that enhance biocompatibility of used antigens. For nitric oxide production and cellular uptake studies, concentrations of 500 μg/ml were chosen as macrophage cells stayed compact and showed no cytotoxicity on J774 macrophage cells, while at concentrations higher than 500 μg/ml macrophage cells started to lose their compact morphology and their viability values decreased sharply.
As it is known, phagocytosis of a particle into macrophage cells is influenced by the size, shape and surface properties. In various studies, it was demonstrated that PLGA micro/nanoparticles were internalized by macrophages with different pathways such as phagocytosis (particle size: 0.5 μm-10 μm) macro-pinocytosis (particle size: 100 nm-5 μm), clathrin-mediated pinocytosis (particle size: approximately 120 nm), caveolin-mediated pinocytosis (particle size: approximately 80 nm), clathrin- and caveolin-independent pinocytosis (particle size: approximately 50 nm) [49–51]. However, accurate uptake mechanisms of PLGA nanoparticles have not been understood clearly, anymore. According to literature data, we can think that our nanoparticles which was sized as 221.7 ± 15.8 nm, may internalize into macrophages by using macro-pinocytosis pathway .
Nitric Oxide (NO) is one of the most important immune-effector molecules in the body, playing role in host defense in bacteria, fungi, parasites and viruses [53, 54]. NO can enhance immune response again infections by stimulating cytokine production and leading macrophages to kill intracellular pathogens . Therefore, in vaccine studies, determination of increased NO levels following to exposure to applied immunogenic molecule is substantial since it is the sign of augmented immune response. Accordingly, in our study, we studied on production of NO by macrophages after treatment with control (PBS), free PLGA nanoparticles, peptide alone and peptide loaded PLGA nanoparticles. Due to the results, it was determined that peptide loaded PLGA nanoparticles increased NO production at 2-folds (∗P < 0.05) in contrast to free peptide, and 3-folds (∗P < 0.01) in contrast to control and free PLGA nanoparticles. The significant difference especially between peptide loaded nanoparticles and free nanoparticles can be explained by high adjuvant features of PLGA nanoparticles . We think that PLGA nanoparticles enhanced antigenicity of peptides due to their special properties while it did not stimulate any immune response as a good adjuvant need to do. This implicated that peptide loaded PLGA nanoparticles were good at enhancing immune response and may also stimulate the other immunological pathways.
In conclusion, to our knowledge this is the first study to synthesis and characterization and in vitro evaluation of W-1 L19 peptide encapsulated PLGA50:50 nanoparticles and its immunostimulating effect on J774 Murine macrophage-like cells. Both particles size distribution, zeta potential and sustained slow release of antigenic peptide from nanoparticles together with accomplishments to induce significantly higher NO production than free peptide, propose that nanoparticular system can be interesting vaccine candidate against Canine parvovirus infections. However, we think that much more efforts must be performed especially on the subject of in vitro stimulation of immune response following to W-1 L19 peptide encapsulated PLGA50:50 nanoparticles exposure. Moreover, obtained data is promising to test the immunogenicity and efficacy of W-1 L19 as a nanovaccine candidate against Canine Parvovirus in mice.
The authors thanks the Yildiz Technical University Scientific Research Projects Coordination Department (YTU BAP, Project Number: 2011-07-04-DOP01) and Scientific and Technological Research Council of Turkey (TUBITAK, Grant Number: 2211) for financial support of this work. They also thank Dr. Seyhun Kipcak and Dr. Yeliz Basaran Elalmis for their contribution to SEM and AFM studies, respectively.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Derman S, Kizilbey K, Mansuroglu B, Mustafaeva Z. Synthesıs and characterızatıon of canıne parvovırus (CPV) VP2 W-7L-20 synthetıc peptıde for synthetıc vaccıne. Fresenıus Envıronmental Bull. 2014;23(2 A):558–66.Google Scholar
- Prittie J. Canine parvoviral enteritis: a review of diagnosis, management, and prevention. J Vet Emerg Crit Care. 2004;14(3):167–76.View ArticleGoogle Scholar
- de Turiso JL, Cortes E, Martinez C, de Ybanez RR, Simarro I, Vela C, et al. Recombinant vaccine for canine parvovirus in dogs. J Virol. 1992;66(5):2748–53.Google Scholar
- Vihinen-Ranta M, Lindfors E, Heiska L, Veijalainen P, Vuento M. Detection of canine parvovirus antigens with antibodies to synthetic peptides. Arch Virol. 1996;141(9):1741–8.View ArticlePubMedGoogle Scholar
- Kariuki NM, Nyaga P, Buoro I, Gathumbi P. Effectiveness of fluids and antibiotics as supportive therapy of canine parvovirus-2 enteritis in puppies. Bull Anim Health Prod Afr. 1990;38:379–89.Google Scholar
- Casal JI, Langeveld J, Cortes E, Schaaper W, van Dijk E, Vela C, et al. Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence. J Virol. 1995;69(11):7274–7.PubMed CentralPubMedGoogle Scholar
- Langeveld J, Casal JI, Osterhaus A, Cortes E, De Swart R, Vela C, et al. First peptide vaccine providing protection against viral infection in the target animal: studies of canine parvovirus in dogs. J Virol. 1994;68(7):4506–13.PubMed CentralPubMedGoogle Scholar
- Alvarez JIC, Olmo CV, Langeveld JPM, Meloen RH, Dalsgaard K. Veterinary medicine. Google Patents. 1998.Google Scholar
- Langeveld JP, Martinez-Torrecuadrada J, Boshuizen RS, Meloen RH, Casal JI. Characterisation of a protective linear B cell epitope against feline parvoviruses. Vaccine. 2001;19(17):2352–60.View ArticlePubMedGoogle Scholar
- Dai C, Wang B, Zhao H. Microencapsulation peptide and protein drugs delivery system. Colloids Surf B Biointerfaces. 2005;41(2):117–20.View ArticlePubMedGoogle Scholar
- Manish M, Rahi A, Kaur M, Bhatnagar R, Singh S. A single-dose PLGA encapsulated protective antigen domain 4 nanoformulation protects mice against Bacillus anthracis spore challenge. PLoS One. 2013;8(4), e61885.PubMed CentralView ArticlePubMedGoogle Scholar
- Sen GP. Fabrication of Poly (DL-Lactic-Co-Glycolic Acid) Nanoparticles and Synthetic Peptide Drug Conjugate for Anti-cancer Drug Delivery. Middle East Technical University. Ankara, Turkey; 2009.Google Scholar
- Gerdts V, Mutwiri G, Richards J, Hurk SDL-v, Potter AA. Carrier molecules for use in veterinary vaccines. Vaccine. 2013;31(4):596–602.View ArticlePubMedGoogle Scholar
- Silva A, Rosalia R, Sazak A, Carstens M, Ossendorp F, Oostendorp J, et al. Optimization of encapsulation of a synthetic long peptide in PLGA nanoparticles: Low-burst release is crucial for efficient CD8 < sup > +</sup > T cell activation. Eur J Pharm Biopharm. 2013;83(3):338–45.View ArticlePubMedGoogle Scholar
- Rajapaksa TE, Lo DD. Microencapsulation of vaccine antigens and adjuvants for mucosal targeting. Curr Immunol Rev. 2010;6(1):29–37.View ArticleGoogle Scholar
- des Rieux A, Fievez V, Garinot M, Schneider Y-J, Préat V. Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J Control Release. 2006;116(1):1–27.View ArticlePubMedGoogle Scholar
- Chong CS, Cao M, Wong WW, Fischer KP, Addison WR, Kwon GS, et al. Enhancement of T helper type 1 immune responses against hepatitis B virus core antigen by PLGA nanoparticle vaccine delivery. J Control Release. 2005;102(1):85–99.View ArticlePubMedGoogle Scholar
- Jaganathan K, Vyas SP. Strong systemic and mucosal immune responses to surface-modified PLGA microspheres containing recombinant hepatitis B antigen administered intranasally. Vaccine. 2006;24(19):4201–11.View ArticlePubMedGoogle Scholar
- Taha MA, Singh SR, Dennis VA. Biodegradable PLGA85/15 nanoparticles as a delivery vehicle for Chlamydia trachomatis recombinant MOMP-187 peptide. Nanotechnology. 2012;23(32):325101.View ArticlePubMedGoogle Scholar
- Xiao X, Zeng X, Zhang X, Ma L, Liu X, Yu H, et al. Effects of Caryota mitis profilin-loaded PLGA nanoparticles in a murine model of allergic asthma. Int J Nanomedicine. 2013;8:4553.PubMed CentralPubMedGoogle Scholar
- Ma W, Chen M, Kaushal S, McElroy M, Zhang Y, Ozkan C, et al. PLGA nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses. Int J Nanomedicine. 2012;7:1475.PubMed CentralView ArticlePubMedGoogle Scholar
- Yin Y, Chen D, Qiao M, Wei X, Hu H. Lectin-conjugated PLGA nanoparticles loaded with thymopentin:< i > Ex vivo</i > bioadhesion and < i > in vivo</i > biodistribution. J Control Release. 2007;123(1):27–38.View ArticlePubMedGoogle Scholar
- Topuzogullari M, Bulmus V, Dalgakiran E, Dincer S. pH-and temperature-responsive amphiphilic diblock copolymers of 4-vinylpyridine and oligoethyleneglycol methacrylate synthesized by RAFT polymerization. Polymer. 2014;55(2):525–34.View ArticleGoogle Scholar
- Keum C-G, Noh Y-W, Baek J-S, Lim J-H, Hwang C-J, Na Y-G, et al. Practical preparation procedures for docetaxel-loaded nanoparticles using polylactic acid-co-glycolic acid. Int J Nanomedicine. 2011;6:2225.PubMed CentralPubMedGoogle Scholar
- Dixit S, Singh SR, Yilma AN, Agee II RD, Taha M, Dennis VA. Poly (lactic acid)–poly (ethylene glycol) nanoparticles provide sustained delivery of a < i > Chlamydia trachomatis</i > recombinant MOMP peptide and potentiate systemic adaptive immune responses in mice. Nanomedicine: NBM. 201410(6):1311–1321.Google Scholar
- Inc S. SPSS for Windows version 15.0. Chicago, Illinois, USA: SPSS Inc; 2006.Google Scholar
- Akagi T, Baba M, Akashi M. Biodegradable nanoparticles as vaccine adjuvants and delivery systems: regulation of immune responses by nanoparticle-based vaccine. Polymers in Nanomedicine. Springer; Berlin, Germany. 2012, pp. 31–64.Google Scholar
- Anderson JM, Shive MS. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev. 2012;64:72–82.View ArticleGoogle Scholar
- Li X, Sloat BR, Yanasarn N, Cui Z. Relationship between the size of nanoparticles and their adjuvant activity: data from a study with an improved experimental design. Eur J Pharm Biopharm. 2011;78(1):107–16.PubMed CentralView ArticlePubMedGoogle Scholar
- Waeckerle-Men Y., Gander B, Groettrup M. Delivery of tumor antigens to dendritic cells using biodegradable microspheres. Adoptive Immunotherapy: Methods and Protocols. Springer; Zurich, Switzerland 2005. pp. 35–46Google Scholar
- Yan F, Zhang C, Zheng Y, Mei L, Tang L, Song C, et al. The effect of poloxamer 188 on nanoparticle morphology, size, cancer cell uptake, and cytotoxicity. Nanomedicine: NBM. 2010;6(1):170–8.View ArticleGoogle Scholar
- Li W, Joshi MD, Singhania S, Ramsey KH, Murthy AK. Peptide Vaccine: Progress and Challenges. Vaccine. 2014;2(3):515–36.View ArticleGoogle Scholar
- Feng L, Qi XR, Zhou XJ, Maitani Y, Cong WS, Jiang Y, et al. Pharmaceutical and immunological evaluation of a single-dose hepatitis B vaccine using PLGA microspheres. J Control Release. 2006;112(1):35–42.View ArticlePubMedGoogle Scholar
- Moore A, McGuirk P, Adams S, Jones WC, Paul MGJ, O'Hagan DT, et al. Immunization with a soluble recombinant HIV protein entrapped in biodegradable microparticles induces HIV-specific CD8 < sup > +</sup > cytotoxic T lymphocytes and CD4 < sup > +</sup > Th1 cells. Vaccine. 1995;13(18):1741–9.View ArticlePubMedGoogle Scholar
- Vordermeier H, Coombes A, Jenkins P, McGee J, O'Hagan D, Davis S, et al. Synthetic delivery system for tuberculosis vaccines: immunological evaluation of the < i > M. tuberculosis</i > 38 kDa protein entrapped in biodegradable PLG microparticles. Vaccine. 1995;13(16):1576–82.View ArticlePubMedGoogle Scholar
- Makadia HK, Siegel SJ. Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers. 2011;3(3):1377–97.PubMed CentralView ArticlePubMedGoogle Scholar
- Sneh-Edri H, Likhtenshtein D, Stepensky D. Intracellular targeting of PLGA nanoparticles encapsulating antigenic peptide to the endoplasmic reticulum of dendritic cells and its effect on antigen cross-presentation in vitro. Mol Pharm. 2011;8(4):1266–75.View ArticlePubMedGoogle Scholar
- Fairley SJ, Singh SR, Yilma AN, Waffo AB, Subbarayan P, Dixit S, et al. Chlamydia trachomatis recombinant MOMP encapsulated in PLGA nanoparticles triggers primarily T helper 1 cellular and antibody immune responses in mice: a desirable candidate nanovaccine. Int J Nanomedicine. 2013;8:2085.PubMed CentralPubMedGoogle Scholar
- Primard C, Poecheim J, Heuking S, Sublet E, Esmaeili F, Borchard G. Multifunctional PLGA-based nanoparticles encapsulating simultaneously hydrophilic antigen and hydrophobic immunomodulator for mucosal immunization. Mol Pharm. 2013;10(8):2996–3004.View ArticlePubMedGoogle Scholar
- Haddadi A, Hamdy S, Ghotbi Z, Samuel J, Lavasanifar A. Immunoadjuvant activity of the nanoparticles’ surface modified with mannan. Nanotechnology. 2014;25(35):355101.View ArticlePubMedGoogle Scholar
- Fonseca C, Simoes S, Gaspar R. Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity. J Control Release. 2002;83(2):273–86.View ArticlePubMedGoogle Scholar
- Gupta PN, Jain S, Nehate C, Alam N, Khare V, Dubey RD, et al. Development and evaluation of paclitaxel loaded PLGA: poloxamer blend nanoparticles for cancer chemotherapy. Int J Biol Macromol. 2014;69:393–399.View ArticlePubMedGoogle Scholar
- Löw K, Knobloch T, Wagner S, Wiehe A, Engel A, Langer K, et al. Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells. Nanotechnology. 2011;22(24):245102.View ArticlePubMedGoogle Scholar
- Manchanda R, Nagesetti A, Fernandez-Fernandez A, McGoron A. Development of a PLGA Nanoparticle Drug Delivery System Containing Imaging/Hyperthermia and Chemotherapy Agents, 25th Southern Biomedical Engineering Conference 2009, 15–17 May 2009. Miami, Florida, USA: Springer; 2009. p. 183–4.Google Scholar
- Ramchandani M, Robinson D. In vitro and in vivo release of ciprofloxacin from PLGA 50: 50 implants. J Control Release. 1998;54(2):167–75.View ArticlePubMedGoogle Scholar
- Soni A, Gadad A, Dandagi P, Mastiholimath V. Simvastatin-loaded PLGA nanoparticles for improved oral bioavailability and sustained release: effect of formulation variables. Asian J Pharm. 2011;5(2):57.View ArticleGoogle Scholar
- Bala I, Hariharan S, Kumar MR. PLGA nanoparticles in drug delivery: the state of the art. Critical Reviews™ in Therapeutic Drug Carrier Systems. 2004;21(5):387–422.Google Scholar
- Pawar D, Mangal S, Goswami R, Jaganathan K. Development and characterization of surface modified PLGA nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity. Eur J Pharm Biopharm. 2013;85(3):550–9.View ArticlePubMedGoogle Scholar
- Fröhlich E. The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int J Nanomedicine. 2012;7:5577.PubMed CentralView ArticlePubMedGoogle Scholar
- Hirota K, Terada H. Endocytosis of particle formulations by macrophages and its application to clinical treatment. INTECH Open Access Publisher; Rijeko, Croatia. 2012.Google Scholar
- Kettler K, Veltman K, van de Meent D, van Wezel A, Hendriks AJ. Cellular uptake of nanoparticles as determined by particle properties, experimental conditions, and cell type. Environ Toxicol Chem. 2014;33(3):481–92.View ArticlePubMedGoogle Scholar
- Weissleder R, Nahrendorf M, Pittet MJ. Imaging macrophages with nanoparticles. Nat Mater. 2014;13(2):125–38.View ArticlePubMedGoogle Scholar
- Akaike T, Maeda H. Nitric oxide and virus infection. Immunology. 2000;101(3):300–8.PubMed CentralView ArticlePubMedGoogle Scholar
- James SL. Role of nitric oxide in parasitic infections. Microbiol Rev. 1995;59(4):533–47.PubMed CentralPubMedGoogle Scholar
- Xing Z, Zganiacz A, Santosuosso M. Role of IL-12 in macrophage activation during intracellular infection: IL-12 and mycobacteria synergistically release TNF-α and nitric oxide from macrophages via IFN-γ induction. J Leukoc Biol. 2000;68(6):897–902.PubMedGoogle Scholar