healthmedicinet

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
12]. 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 22] 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 30
^th
days, while nanoparticles were totally lost their compact structures at the end of
60
^th
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 40]. 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 19]. 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 11]. 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 39]. 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 14]. 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 47]. 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 ?g/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 ?g/ml were chosen as macrophage cells stayed compact and showed no cytotoxicity
on J774 macrophage cells, while at concentrations higher than 500 ?g/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 ?m-10 ?m) macro-pinocytosis (particle size:
100 nm-5 ?m), 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 52].

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 55]. 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 27]. 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.