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

Figures & data

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.

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.

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.

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.

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.

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.

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.