Chitosan-coated poly(lactic-co-glycolic) acid nanoparticles as an efficient delivery system for Newcastle disease virus DNA vaccine

Abstract

We determined the efficacy and safety of chitosan (CS)-coated poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) as a delivery system for a vaccine to protect chickens against Newcastle disease virus (NDV). The newly constructed vaccine contained DNA (the F gene) of NDV. The Newcastle disease virus (NDV) F gene deoxyribonucleic acid (DNA) plasmid (pFDNA)-CS/PLGA-NPs were spherical (diameter =699.1±5.21 nm [mean ± standard deviation]) and smooth, with an encapsulation efficiency of 98.1% and a Zeta potential of +6.35 mV. An in vitro release assay indicated that CS controlled the burst release of plasmid DNA, such that up to 67.4% of the entire quantity of plasmid DNA was steadily released from the pFDNA-CS/PLGA-NPs. An in vitro expression assay indicated that the expression of nanoparticles (NPs) was maintained in the NPs. In an immunization test with specific pathogen-free chickens, the pFDNA-CS/PLGA-NPs induced stronger cellular, humoral, and mucosal immune responses than the plasmid DNA vaccine alone. The pFDNA-CS/PLGA-NPs did not harm 293T cells in an in vitro assay and did not harm chickens in an in vivo assay. Overall, the results indicated that CS-coated PLGA NPs can serve as an efficient and safe mucosal immune delivery system for NDV DNA vaccine.

Keywords:

• mucosal immune delivery system
• immune effect

Introduction

Virulent Newcastle disease virus (NDV) is the prototype of the paramixoviruses that causeNewcastle disease (ND). The virus, which causes high mortality among mature chickens and chicks,infects the respiratory tract, nerves, or intestines. NDV mainly expresseshemagglutinin-neuramindase and fusion (F) glycoproteins. In many countries, ND is a catastrophicproblem for the poultry industry.¹ Although thedisease is difficult to control, vaccines are available. The efficacy of NDV vaccines depends on theinduction of F glycoproteins.² Traditional NDVvaccines include two types: inactivated vaccines and attenuated live vaccines.³ Both of these traditional types of vaccines have important limitations,including reversion to virulence and induction of respiratory pathological changes. In addition, thedifficulty in differentiating between vaccinated chickens and naturally infected chickenscomplicates diagnosis.

In research by Robinson et al the injection of chickens with plasmid DNA provided a new way toprotect chickens from lethal influenza viruses.⁴ Inthe same year, Fynan et al⁵ reported that theinclusion of gold-encapsulated plasmid particles in influenza virus vaccines reduced the requirementfor DNA in the vaccines to 0.4 pg, which was 1/250th of the amount reported formerly. Reducing theDNA content was important because DNA-based vaccines can induce long-term cellular and humoralimmune reactions in animals and humans.^6–8

DNA vaccines have not been widely used for several other reasons. Some studies have shown thatthe vaccines, which are usually administered via intramuscular (IM) injection, can fail to reach theantigen-presenting cells and therefore fail to induce immune responses because of difficulty incrossing cell membranes.^9–11 Sun et al reported that effective immunization oflarge animals required large amounts of DNA.¹²Researchers have recently suggested several measures that could increase the efficacy of DNAvaccines. These measures include plasmid DNA optimization, improvement of delivery methods, thetargeting of the antigen-presenting cells, and the use of immunologic adjuvants.^13,14

Recent research has indicated that polymeric nanoparticles (NPs) can be used as potent adjuvantsas part of a “nano” mucosal immune delivery system. NPs are biodegradable andbiocompatible, have low toxicity, and protect the antigen or DNA from damage.^15–17 Amongall the polymers, polyesters based on polylactic acid, polyglycolic acid, and their copolymers,poly(lactic-co-glycolic) acids (PLGAs), have attracted the most attention and have been used ascarriers for a wide range of vaccines.^18–21

PLGA is authorized by the US Food and Drug Administration (FDA), and PLGA NPs (microspheres) havebeen thoroughly studied as a protein or DNA vaccine mucosal delivery system that protects theencapsulated protein or DNA vaccine from enzyme digestion and that extends the release time of theprotein or DNA vaccine.^22,23 A number of studies have reported improved antibody responses whenantigens are orally administered in PLGA particles.^24–27 Nevertheless, PLGA NPs havelimited use in mucosal vaccination because of their poor mucoadhesivity and immunoenhancing ability.In recent years, chitosan (CS) has been used as a coating material for PLGA NPs because of itsbiological adhesive properties and ability to improve the immunological response to mucosalvaccination.^28,29 By modifying the surface of PLGA NPs, CS provides the followingadvantages: 1) it decreases the burst release of the encapsulated protein or DNA; 2) it increasesthe stability of biological macromolecules; 3) it enhances the inversion of Zeta potential, andpromotes cellular adhesion and retention of the delivery system at the target site; and 4) it offersthe possibility of conjugating targeting ligands to free amino groups on its surface.³⁰ Budhian et al reported that coating PLGA NPs with CSreduced the burst release of haloperidol from 70% to 36%.³¹ Tahara et al have also successfully developed gene delivery vectors using CS surfacemodification of PLGA NPs.^32,33

In this study, we prepared CS-coated PLGA NPs containing the F gene plasmid DNA of NDV(pFDNA-CS/PLGA-NPs) and assessed the ability of the preparation to induce immune responses andprotect specific pathogen-free (SPF) chickens from ND after intranasal (IN) administration. We alsoassessed the bioactivity and safety of the pFDNA-CS/PLGA-NPs with in vitro expression and cellcytotoxicity assays.

Materials and methods

Preparation of the pFDNA-CS/PLGA-NPs

The eukaryotic expression plasmid pVAX I-opti F DNA, which contains the F gene of NDV, wasencapsulated in PLGA NPs (pFNDV-PLGA-NPs) by a water/oil/water double emulsion-solvent evaporationmethod.³⁴ Previous research indicated that theoptimal conditions for preparation of these NPs were 50 watts (w) for 30 seconds for the primaryemulsion, 50 w for 60 seconds for the secondary emulsion, a DNA:PLGA ratio of 0.5:100, andcombination of 40 mg/mL PLGA with 2.0% polyvinyl alcohol (PVA).³⁵ Accordingly, pFDNA-CS/PLGA-NPs were prepared in three main steps.First, 40 mg of PLGA was dissolved in 1 mL of methylene chloride (oil phase), and 800 μg ofthe DNA solution was added with the primary emulsion. Second, 2 mL of 2% PVA and the secondaryemulsion (W₂) were added to the primary emulsion; the resulting compound emulsion(primary and secondary emulsions) was added in drops to the CS solutions, which contained 15 mL of0.5% PVA, and the preparation was shaken at 500 r/min for 5 hours. Third, the CS/PLGA NPs containingpFDNA were recovered by centrifugation (4,500 r/min, 10 minutes, 4°C), washed three timeswith sterilized deionized water, centrifuged, and freeze-dried. The resulting NPs were referred toas pFDNA-CS/PLGA-NPs.

Characterization of the pFDNA-CS/PLGA-NPs

We determined the effects of CS concentration (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) and CSvolume (0, 1.5, 3.0, 5.0, and 7.5 mL) on the characteristics of the pFDNA-CS/PLGA-NPs, respectively.Encapsulation efficiency (EE) was measured as previously described.³ The morphological and surface characteristics of the pFDNA-CS/PLGA-NPswere examined by JEM-200EX transmission electron microscopy (Hitachi Ltd., Tokyo, Japan). Theparticle size and Zeta potentials were measured using a Zetasizer 2000 from Malvern Instruments(Malvern, UK).

In vitro release of the pFDNA-CS/PLGA-NPs

An in vitro release assay was carried out to determine the release of plasmid DNA from theNPs.³ Briefly, samples were periodically collected(after 0, 6, 12, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216, and 240 hours) and centrifuged at10,000 r/min for 10 minutes at 4°C. The collected pFDNA-CS/PLGA-NPs were counted. Allexperiments were performed five times.

In vitro transfection and western blot analysis of the pFDNA-CS/PLGA-NPs

293T cells were grown in poly-lysine-treated 6-well plates and cultured at 37°C in aCO₂ incubator. An in vitro transfection experiment was carried out according to theinstructions from the Lipofectamine™ 2000 reagent kit (Invitrogen™; LifeTechnologies Corp, Carlsbad, CA, USA), using four groups: 1) the naked plasmid DNA group; 2) thepFDNA-CS/PLGA-NP transfected group; 3) the blank CS/PLGA-NP group; and 4) the negative cell controlgroup. Western blot analysis was carried out as previously described.³⁶ Briefly, after 72 hours of transfection, the 293T cells werecollected and disrupted using radioim-munoprecipitation assay solution (50 mmol/L Tris-HCl[pH 8.5], 5 mmol/L 2-hydroxy-1-ethanethiol, 100 mmo1/L KCl, 1 mmol/Lphenylmethanesulfonyl fluoride, and 1% Nonidet P-40 (octylphenoxypolyethoxyethanol). The lysate wascentrifuged at 14,000 r/min at 4°C, and the supernatant was subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis. Proteins were transferred to a nitrocellulose membrane(Amersham; GE Healthcare, Little Chalfont, UK) using a BioRad semidry unit. The membrane was washedwith phosphate-buffered saline (PBS), blocked with 5% fat-free milk overnight, and then incubatedwith an NDV-positive serum at a 1:500 dilution for 1 hour. After the membrane was washed three timeswith PBS with Tween^® (phosphate buffered saline with Tween-20), fluoresceinisothiocyanate labeled goat anti-chicken secondary antibody was added at a dilution of 1:5,000 for 1hour. The image was acquired with an Odyssey infrared imaging system (LI-COR Odyssey; LI-CORBiosciences Inc., Lincoln, NE, USA).

The safety of pFDNA-CS/PLGA-NPs

Cell Counting Kit-8 reagent was used to evaluate the in vitro cytotoxicity, and optical densityat 450 nm (OD₄₅₀) was measured to determine the survival rate of 293T cells. For the invivo assay, 30 4-week-old SPF chickens from the Harbin Veterinary Research Institute LaboratoryAnimal Center were randomly assigned to two groups. The in vitro and in vivo cytotoxicity of thepFDNA-CS/PLGA-NPs were evaluated as previously described.³⁶ Chickens in one group were immunized, by IN route, with 0.2 mL of the pFDNA-CS/PLGA-NPs,which contained a total of 200 μg of plasmid DNA. Chickens in the other group wereimmunized, by IM route, with 0.2 mL of the naked plasmid DNA. The chickens were continuouslyobserved for 21 days, and any abnormalities were noted.

Immunization of SPF chickens

A total of 120 4-week-old SPF chickens were randomly assigned to six groups. Group 1 was treatedwith PBS buffer IM. Groups 2 and 3 were treated with blank CS/PLGA-NPs IM or IN, respectively. Group4 was treated with 200 μg of naked plasmid DNA (0.1 mL) IM. Groups 5 and 6 were treated with0.2 mL pFDNA-CS/PLGA-NPs (containing 200 μg of plasmid DNA) IM or IN, respectively. Alltreatments were repeated 14 days later.

Detection of immunoglobulin antibody in the serum of immunized chickens

Blood samples were collected from the wing veins of the six groups of chickens at 7, 14, 21, 28,35, and 42 days after the first treatment. The serum was obtained by centrifugation at 3,000 r/minfor 10 minutes at 4°C. Enzyme-linked immunosorbent assay (ELISA) was performed to assess thetiters of the NDV-specific immunoglobulin G (IgG) in the sera, using a NDV IgG ELISA Kit (RapidbioCo., Ltd., West Hills, CA, USA) according to the instruction manual.

Detection of IgA antibody in mucosa extracts of immunized chickens

Serum, tears, bile, and tracheal fluid were collected from two chickens from each of the sixgroups of treated (immunized) chickens, at 7, 14, 21, 28, 35, and 42 days after the first treatment.IgA antibody was evaluated with a NDV IgA ELISA Kit.

Lymphocyte proliferation in immunized chickens

To assess the cell-mediated immune responses of immunized chickens at 14, 28, and 42 days afterthe first treatment, we measured lymphocyte proliferation with the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay, as previouslydescribed.³⁷ This was done with ten chickens fromeach of the six groups. The stimulation index (SI) was determined using the following formula:³⁸$\text{SI}={\text{OD}}_{570}\mathrm{T}/{\text{OD}}_{570}\mathrm{C},$(1)

where T is the mean value (number of OD at 570 nm) of the test group (pFDNA-CS/PLGA-NPs IN,pFDNA-CS/PLGA-NPs IM, pVAX I-opti F IM, blank CS/PLGA-NPs IN, or blank CS/PLGA-NPs IM), andOD₅₇₀ C is the mean value of the control (PBS) group.

Protection against NDV strain F48E9

The ability of pFDNA-CS/PLGA-NPs to protect chickens against NDV strainF₄₈E₉ was determined. Strain F₄₈E₉ is highly virulent(mean death time ≤60 hours, intracerebral pathogenicity index >1.6). Protection wasdetermined as previously described.³ Briefly, whenthe level of ND serum antibody of every immune group increased to 6.0 log 2, eight chickens wereselected at random from the six groups and infected by IM route with 0.1 mL of the highly virulentNDV strain F₄₈E₉, for challenge studies with a viral titer of 10^4.5egg infectious dose (EID)₅₀/0.1 mL. Clinical signs of disease and mortality weremonitored on a daily basis, and continuously observed for 14 days. The infected chickens andcorresponding negative control chickens were euthanized, and the glandular stomach, duodenum, andbursa of Fabricius were collected for examination by histological staining.

Statistical analysis

Unless noted otherwise, all experiments were repeated three times, and each value was measured intriplicate. Data are presented as means ± standard deviation. Means were compared usingone-sided Student’s t-tests. Differences were considered to bestatistically significant at P<0.05.

Results

Preparation and characterization of the pFDNA-CS/PLGA-NPs

Based on EE, the optimal CS concentration and volume for preparation of pFDNA-CS/PLGA-NPs was 0.6mg/mL (Table 1) and 1.5 mL (Table 2), respectively. With this concentration and volume, the mean EEwas about 98.7%. The pFDNA-CS/PLGA-NPs were spheres with smooth surfaces. They did not aggregate,and they did not suffer from subsidence damage (Figure1). The mean diameter was 699.1±5.21 nm, particle-size dispersity was 0.005 (Figure 2A), and Zeta potential was 6.35±2.75 mV(Figure 2B). Using the optimal CS concentration of0.6 mg/mL and volume of 1.5 mL, we prepared and assessed five independent batches ofpFDNA-CS/PLGA-NPs. The EE values did not significantly differ (P>0.05)among the five batches, indicating that the preparation procedure was reproducible and reliable.

Table 1 Encapsulation efficiency and diameter of the pFDNA-CS/PLGA-NPs as affected by chitosanconcentration

Chitosan concentration (mg/mL)	Encapsulation efficiency (%)	Diameter (nm)
0	83.8±1.84	370.7
0.2	88.8±2.19	520.9
0.4	94.1±2.12	616.0
0.6	98.7±0.75	699.1
0.8	93.4±1.36	723.7
1.0	89.8±1.68	736.5

Notes: Chitosan concentration was evaluated with a chitosan volume of 1.5 mL. Valuesfor encapsulation efficiency are expressed as mean ± SD; n=5.

Abbreviations: pFDNA-CS/PLGA-NPs, chitosan-coated Newcastle disease virus F geneencapsulated in poly(lactic-co-glycolic) acid nanoparticles; SD, standard deviation; n, number oftest.

Table 2 Encapsulation efficiency of the pFDNA-CS/PLGA-NPs as affected by chitosan volume

Chitosan volume (mL)	Encapsulation efficiency (%)
0	87.3±0.82
1.5	98.7±0.87
3.0	92.8±1.52
5.0	85.9±1.84
7.5	81.2±2.06

Notes: Chitosan volume was evaluated with a chitosan concentration of 0.6 mg/mL.Values are expressed as mean ± SD; n=5.

Abbreviation: pFDNA-CS/PLGA-NPs, chitosan-coated Newcastle disease virus F geneencapsulated in poly(lactic-co-glycolic) acid nanoparticles; SD, standard deviation; n, number oftest.

Figure 1 Transmission electron micrographs of the pFDNA-CS/PLGA-NPs at (A) 1,000×magnification and (B) 3,000× magnification.

Abbreviation: pFDNA-CS/PLGA-NPs, chitosan-coated Newcastle disease virus F geneencapsulated in poly(lactic-co-glycolic) acid nanoparticles.

Figure 2 Size and Zeta potential of the pFDNA-CS/PLGA-NPs.

Notes: (A) Size distribution. The mean (± SD) diameter was699.1±5.21 nm. (B) The Zeta potential was 6.35±2.75 mV.

Abbreviations: pFDNA-CS/PLGA-NPs, chitosan-coated Newcastle disease virus F geneencapsulated in poly(lactic-co-glycolic) acid nanoparticles; SD, standard deviation.

In vitro release of the pFDNA-CS/PLGA-NPs

The plasmid DNA encapsulated in the CS/PLGA-NPs was gradually released throughout the assay,although the release was faster during the first 120 hours than during the subsequent 120 hours(Figure 3). The rate of release was relatively slowand sustained, apparently because the CS reduced desorption and diffusion.

Figure 3 In vitro release profiles of the plasmid DNA pVAX I-opti F from pFDNA-CS/PLGA-NPs.

Note: Values are expressed as means ± SD (n=3).

Abbreviations: pFDNA-CS/PLGA-NPs, chitosan-coated Newcastle disease virus F geneencapsulated in poly(lactic-co-glycolic) acid nanoparticles; SD, standard deviation; n, number oftest; pVAX I-optiF, eukaryotic expression plasmids.

In vitro expression of the pFDNA-CS/PLGA-NPs

Specific fluorescence was detected in the naked plasmid DNA group (Figure 4A) and the pFDNA-CS/PLGA-NP transfected group (Figure 4B) but not in the blank CS/PLGA-NPs group (Figure 4C) or in the negative cell control group (Figure 4D). Expression of the antigen was furtherdemonstrated by western blot (Figure 4E), whichindicated that the expected 58 kDa antigen was expressed in 293T cells transfected withpFDNA-CS/PLGA-NPs and naked plasmid DNA but not in cells treated with the blank CS/PLGA-NPs or inthe negative cell control group. These results proved that the pFDNA-CS/PLGA-NPs could express andprotect the antigen in vitro.

Figure 4 In vitro expression of the pFDNA-CS/PLGA-NPs in 293T cells as indicated by indirectimmunofluorescence analysis at ×40 (A–D) and westernblotting (E).

Notes: (A) The naked plasmid DNA group; (B)pFDNA-CS/PLGA-NP-transfected group; (C) blank CS/PLGA-NP group; and (D)293T cell group as the negative control. (E): From left to right are lanes: 1, M, 2, 3,4. Lane 1: naked plasmid DNA group. M: protein marker. Lane 2: 293T cells as the negative control.Lane 3: pFDNA-CS/PLGA-NP-transfected group. Lane 4: blank CS/PLGA-NP group.

Abbreviations: CS/PLGA-NP, chitosan-coated poly(lactic-co-glycolic) acidnanoparticle; pFDNA-CS/PLGA-NP, chitosan-coated Newcastle disease virus F gene encapsulated inpoly(lactic-co-glycolic) acid nanoparticle.

Safety of the pFDNA-CS/PLGA-NPs

The survival rate of 293T cells was 85.14%±8.13% when treated with pFDNA-CS/PLGA-NPs andwas 84.72%±6.04% for nontreated control cells; the difference was not statisticallysignificant (P>0.05). Cell morphology was similar for cells in thepFDNA-CS/PLGA-NPs and control groups. Chickens immunized with either the pFDNA-CS/PLGA-NPs or thenaked plasmid DNA did not exhibit nervous signs, clinical symptoms, or necropsy lesions within the 3weeks following treatment. These in vitro and in vivo results showed that the pFDNA-CS/PLGA-NPs weresafe.

Immunization of SPF chickens with pFDNA-CS/PLGA-NPs

IgG antibody in serum

IgG antibody titers in sera peaked on day 28 for chickens immunized IM with naked plasmid DNA butpeaked on day 35 for chickens immunized IM or IN with pFDNA-CS/PLGA-NPs; the titer on day 35 and day42 was greater (P<0.05) with pFDNA-CS/PLGA-NPs than with naked plasmid DNA(Figure 5). IgG antibody titer did not increase inchickens treated with PBS alone or with blank CS/PLGA-NPs.

Figure 5 IgG antibody titers in the serum of SPF chickens immunized with PBS (IM), blank CS/PLGA-NPs (IM),blank CS/PLGA-NPs (IN), pVAX I-opti F (the naked plasmid DNA) (IM), pFDNA-CS/PLGA-NPs (IM), andpFDNA-CS/PLGA-NPs (IN).

Note: Values are expressed as mean ± SD (n=5).

Abbreviations: CS/PLGA-NPs, chitosan-coated poly(lactic-co-glycolic) acidnanoparticles; Ig, immunoglobulin; IM, intramuscular; IN, intranasal; OD₄₅₀, opticaldensity at 450 nm; PBS, phosphate buffered saline; pFDNA-CS/PLGA-NPs, chitosan-coated Newcastledisease virus F gene encapsulated in poly(lactic-co-glycolic) acid nanoparticles; SPF, specificpathogen-free; SD, standard deviation; pVAX I-optiF, eukaryotic expression plasmids.

IgA antibody in mucosa extracts

The IgA antibody content was significantly higher (P<0.05) in thepFDNA-CS/PLGA-NPs IN group than in the other groups, for tears (Figure 6B), tracheal fluid (Figure 6C), and bile (Figure 6D). In serum,the IgA antibody content did not differ between the naked plasmid DNA IM group and thepFDNA-CS/PLGA-NPs IN group during day 7 to day 28 (Figure6A) but was higher in the latter group on day 35 and day 42. These results indicated that thepFDNA-CS/PLGA-NPs induced a stronger and more sustained mucosal immune response than the nakedplasmid DNA.

Figure 6 IgA antibody content in serum (A), tears (B), tracheal fluid(C), and bile (D) of SPF chickens immunized with PBS (IM), blankCS/PLGA-NPs (IM), blank CS/PLGA-NPs (IN), pVAX I-opti F (the naked plasmid DNA) (IM),pFDNA-CS/PLGA-NPs (IM), and pFDNA-CS/PLGA-NPs (IN).

Note: Values are expressed as mean ± SD (n=5).

Abbreviations: CS/PLGA-NPs, chitosan-coated poly(lactic-co-glycolic) acidnanoparticles; Ig, immunoglobulin; IM, intramuscular; IN, intranasal; PBS, phosphate bufferedsaline; pFDNA-CS/PLGA-NPs, chitosan-coated Newcastle disease virus F gene encapsulated inpoly(lactic-co-glycolic) acid nanoparticles; SPF, specific pathogen-free; SD, standard deviation; n,number of test; pVAX I-optiF, eukaryotic expression plasmids.

Lymphocyte proliferation

At 42 days after the first treatment, the SI was significantly higher(P<0.01) for chickens immunized with pFDNA-CS/PLGA-NPs (either IM or IN)than with the plasmid DNA or with blank CS/PLGA-NPs. The SI was significantly higher(P<0.05) with IN than with IM application of pFDNA-CS/PLGA-NPs. The resultsshowed that the pFDNA-CS/PLGA-NPs significantly enhanced the immune function of T lymphocytes (Table 3).

Table 3 The stimulation index of T lymphocyte proliferation in SPF chickens after immunization

Treatment	Stimulation index
	14 dpi	28 dpi	42 dpi
pFDNA-CS/PLGA-NPs IN	2.137±0.042^a	3.136±0.044^a	4.440±0.008^a
pFDNA-CS/PLGA-NPs IM	1.506±0.036^b	2.926±0.055^a	3.538±0.004^b
pVAX I-opti F IM	2.064±0.078^a	3.330±0.009^a	2.816±0.030^c
Blank CS/PLGA-NPs IN	1.182±0.018^c	1.297±0.059^b	1.446±0.011^d
Blank CS/PLGA-NPs IM	1.094±0.018^c	1.233±0.014^b	1.298±0.014^d
PBS	1.034±0.012^c	1.080±0.006^b	1.106±0.004^d

Notes: Values are expressed as mean ± SD; n=5. Values within the same columnwith the different lower case letter (a–d) in the superscript are significantly different(P<0.05; Student’s t-test).

Abbreviations: CS, chitosan; dpi, days after the first immunization treatment; IM,intramuscular; IN, intranasal; PBS, phosphate-buffered saline; pFDNA-CS/PLGA-NPs, chitosan-coatedNewcastle disease virus F gene encapsulated in poly(lactic-co-glycolic) acid nanoparticles; SD,standard deviation; SPF, specific pathogen-free; pVAX I-optiF, eukaryotic expression plasmids;CS/PLGA-NPs, CS-coated PLGA NPs; NPs, nanoparticles.

Protection against NDV strain F₄₈E₉

Within 2 to 5 days, the highly virulent NDV strain F₄₈E₉ had killed allchickens treated with PBS or blank CS/PLGA-NPs (Table4). In contrast, strain F₄₈E₉ killed only 12.5%, 0%, and 50% of thechickens treated with pFDNA-CS/PLGA-NPs IM, pFDNA-CS/PLGA-NPs IN, and the naked plasmid DNA,respectively (Table 4). Feeding, drinking, andother aspects of behavior were normal for chickens treated with pFDNA-CS/PLGA-NPs IM and IN but weresomewhat abnormal for chickens treated with the naked plasmid DNA. Pathological andhistopathological changes were not evident in chickens treated with pFDNA-CS/PLGA-NPs IN (Figure 7G), but small histopathological changes wereevident in chickens treated with pFDNA-CS/PLGA-NPs IM (Figure 7F) and naked plasmid DNA (Figure7E). Histopathological changes typical of ND were evident in chickens treated with PBS andblank CS/PLGA-NPs (Figure 7B–D). Thesefindings demonstrated that treatment with the pFDNA-CS/PLGA-NPs IN induces an effective mucosalimmune response and protects chickens against ND.

Table 4 Protection of the immunized SPF chickens after challenge with the highly virulent NDV strainF₄₈E₉

Treatment	Number of dead chickens/total number of chickens	Mortality (%)	Protection (%)
PBS IM	8/8	100	0
Blank CS/PLGA-NPs IM	8/8	100	0
Blank CS/PLGA-NPs IN	8/8	100	0
pVAX I-opti F IM	4/8	50	50
pFDNA-CS/PLGA-NPs IM	1/8	12.5	87.5
pFDNA-CS/PLGA-NPs IN	0/8	0	100

Abbreviations: CS, chitosan; IM, intramuscular; IN, intranasal; NDV, Newcastledisease virus; PBS, phosphate-buffered saline; pFDNA-CS/PLGA-NPs, chitosan-coated Newcastle diseasevirus F gene encapsulated in poly(lactic-co-glycolic) acid nanoparticles; SPF, specificpathogen-free; pVAX I-optiF, eukaryotic expression plasmids; CS/PLGA-NPs, CS-coated PLGA NPs.

Figure 7 Histopathology of the glandular stomach, duodenum, and bursa of Fabricius of normal chickens(A) and of chickens challenged with the highly virulent NDV strainF₄₈E₉ after treatment with PBS IM (B); blank CS/PLGA-NPs IM(C); blank CS/PLGA-NPs IN (D); the naked plasmid DNA IM (E);pFDNA-CS/PLGA-NPs IM (F); or pFDNA-CS/PLGA-NPs IN (G).

Notes: Tissues are indicated above the three columns of micrographs, and treatmentsare indicated on the left side of each row of micrographs. (A1–A3) normal tissues of theglandular stomach, duodenum, and bursa of Fabricius; (B1, C1, D1, E1, F1 and G1) tissues of theglandular stomach PBS (IM), blank CS/PLGA (IM), blank CS/PLGA (IN), and the naked plasmid DNA (IM),pFDNA-CS/PLGAs (IM), pFDNA-CS/PLGAs (IM); (B2, C2, D2, E2, F2 and G2) tissues of the duodenum PBS(IM), blank CS/PLGA (IM), blank CS/PLGA (IN), and the naked plasmid DNA (IM), pFDNA-CS/PLGA (IM),pFDNA-CS/PLGA (IN); (B3, C3, D3, E3, F3 and G3) tissues of the bursa of Fabricius PBS (IM), blankCS/PLGA (IM), blank CS/PLGA (IN), and the naked plasmid DNA (IM), pFDNA-CS/PLGA (IM), pFDNA-CS/PLGA(IN).

Abbreviations: CS/PLGA-NPs, chitosan-coated poly(lactic-co-glycolic) acidnanoparticles; IM, intramuscular; IN, intranasal; PBS, phosphate-buffered saline; pFDNA-CS/PLGA-NPs,chitosan-coated Newcastle disease virus F gene encapsulated in poly(lactic-co-glycolic) acidnanoparticles; NDV, Newcastle disease virus.

Discussion

Polymeric NPs have been widely used to deliver drugs because they can increase drug stability,are easily absorbed by cells, have good adhesive properties, are able to target specific organs, andcan bind ligands on their surfaces.³⁹ Inparticular, biodegradable NPs are available that deliver medicines to target sites and that are thendegraded after passing the target site.⁴⁰ Wetherefore determined whether biodegradable polymers could be used to deliver newly developed DNAvaccines. Zhao et al previously reported that pFNDV-PLGA-NPs that were prepared with awater/oil/water double emulsion-solvent evaporation method did not change the encapsulated plasmidDNA but promoted the sustained release of the plasmid DNA and induced stronger mucosal immuneresponses than for nonencapsulated plasmid DNA.³⁵Because other researchers showed that CS NPs could sustain release and stabilize the plasmidDNA,⁴¹ in the current study, we investigatedwhether CS could improve the performance of PLGA-NPs.

In our first experiments, we found that the EE was highest when NPs were prepared with a CSvolume of 1.5 mL and a CS concentration of 0.6 mg/mL. The EE of NPs determines the effectiveness ofthe gene delivery and subsequent expression of encoded genes in vitro and in vivo.⁴² In this study, the use of 1.5 mL of CS at 0.6 mg/mLof CS produced an EE of 98.7%, which was higher than the previously reported EE for thepFNDV-PLGA-NPs without CS.³⁵ When CS was used atthis volume and concentration, the resulting pFD-NA-CS/PLGA-NPs had an appropriate size andmaintained the biological activity of the encapsulated plasmid DNA. In addition to not aggregating,the pFDNA-CS/PLGA-NPs had a core–shell structure, a diameter of 699.1±5.21 nm, goodstability, a Zeta potential of 6.35±2.75 mV, and a polydispersity index of 0.005.

We also conducted an in vitro assay of the release of the DNA encapsulated in pFDNA-CS/PLGA-NPs.Hu et al⁴³ showed that pH could influence therelease of plasmid DNA from NPs and that NPs are more swollen at pH 7.0–7.4 than at pH 4.0.Thus, our assay used pFDNA-CS/PLGA-NPs that were swollen in PBS at pH 7.4. The results of the assayrevealed a gradual and sustained release of DNA from the pFDNA-CS/PLGA-NPs, suggesting a strongphysical interaction between the drug and polysaccharide layer on the NPs’ surface. The CSslightly reduced the DNA burst release effect in the first 24 hours, indicating that the CS layersurrounding the PLGA-NPs acts as a physical barrier that slows the release of DNA from thePLGA-NPs.

The cellular uptake of the NPs was visualized by indirect immunofluorescence. The in vitro celltoxicity and expression studies indicated that the pFDNA-CS/PLGA-NPs did not damage cells andmaintained DNA bioactivity. The survival rate was higher than the previously reported 80.14% forpFNDV-PLGA-NPs without CS.³⁵ In vivo cytotoxicityassay showed that the pFDNA-CS/PLGA-NPs did not have pathological effects on chickens. Overall, theresults showed that the DNA in the NPs was not altered and that the NPs containing DNA and coatedwith CS would not harm chickens.

Analyses of IgG and IgA antibody responses revealed that IN immunization with pFDNA-CS/PLGA-NPsinduced stronger responses than immunization with naked plasmid DNA. The IgA antibody contents ofgroup pFDNA-CS/PLGA-NPs IN were much higher than the previously reported 800–1,000 ng/mL(P<0.05).³⁵ INimmunization was very effective in eliciting mucosal and systemic immune responses.^44,45Our findings suggest that the nasal administration of pFDNA-CS/PLGA-NPs is effective in inducing theimmune response against ND, probably because the CS coating changes the surface charge of thePLGA-NPs and prolongs the antigen contact time with mucosal surfaces.⁴⁶

In summary, our comparison of the pFDNA-CS/PLGA-NPs and naked plasmid DNA showed that theimmunogenicity and protective immunity can be improved by encapsulating the plasmid DNA intoCS-coated PLGA NPs.

Although this study demonstrates the potential of CS-modified PLGA NPs as an efficient deliverysystem for NDV DNA vaccine in mucosal immunization, a number of challenges must be addressed. First,the trace amounts of initiator, toxic organics, and other impurities in the polymer must be removedbefore a vaccine is commercialized. Toxic solvent remaining in the natural polymer during NPpreparation must also be removed. Second, the cost of preparation must be reduced. Finally, thecontrolled and targeted release of NPs must be improved. We suspect that these challenges can be metby technological advancements in the biomedical and material sciences.

Acknowledgments

We gratefully acknowledge the Key Laboratory of Functional Inorganic Material Chemistry(Heilongjiang University), the Ministry of Education and Engineering Research Center of AgriculturalMicrobiology Technology, and the Ministry of Education for providing the facilities to carry outthis work. This work was supported, in part, by the National Natural Science Foundation of thePeople’s Republic of China (grant number 31072119), the Key Project of the Chinese Ministryof Education (grant number 212048), the Program for New Century Excellent Talents in University(grant number NCET-12-0707), the Innovative Research Team for Agricultural Microbiology FermentationTechnology at Heilongjiang Provincial University (grant number 2012td009), the Changjiang ScholarCandidates Program for Provincial Universities in Heilongjiang (grant number 2014CJHB005), theScientific and Technological Key Project of Heilongjiang Province (GC13B403), the Early Research andDevelopment Cultivation Project of Scientific and Technological Achievements Industrialization forProvincial Universities in Hei-longjiang (grant number 1253CGZH10), and the Innovation Foundation ofHarbin (grant number 2013RFQXJ030).

Disclosure

The authors report no conflicts of interest in this work.