Synthesis, characterization, and immune efficacy of layered double hydroxide@SiO₂ nanoparticles with shell-core structure as a delivery carrier for Newcastle disease virus DNA vaccine

Figures & data

Figure 1 Transmission electron microscopy photomicrograph (magnification 50,000×) (A) and particle size distribution (B) of the pFDNA-SiO₂-NPs.

Abbreviation: pFDNA-SiO₂-NPs, Newcastle disease virus F gene encapsulated in the SiO₂ nanoparticles.

Figure 2 Detection of SiO₂ nanoparticles loaded with plasmid DNA for connection, release, and protection properties.

Notes: (A) Agarose gel electrophoresis of the amino-modified silica-DNA complexes. Maker (M): DL 15,000 marker. Lane 1: plasmid DNA. Lanes 2–8: the mass ratios of SiO₂ to plasmid DNA were 1:5, 1:1, 5:1, 10:1, 20:1, 30:1, and 40:1, respectively. (B) Stability analysis of the plasmid DNA binding with SiO₂ nanoparticles. (C) Analysis of protection of the plasmid DNA from DNase I digestion. Maker (M): DL 15,000 marker. Lane 1: plasmid DNA. Lanes 2–8: the mass ratios of SiO₂ to plasmid DNA were 1:5, 1:1, 5:1, 10:1, 20:1, 30:1, and 40:1, respectively.

Figure 3 Transmission electron photomicrographs of the pFDNA-SiO₂-NPs (A), LDH (B), and pFDNA-LDH@SiO₂-NPs (C–E). Note: Magnification: 100,000×.

Abbreviations: LDH, layered double hydroxide; NPs, nanoparticles; pFDNA-LDH@SiO₂-NPs, Newcastle disease virus F gene encapsulated in LDH@SiO₂-NPs; pFDNA-SiO₂-NPs, Newcastle disease virus F gene encapsulated in the SiO₂ nanoparticles.

Figure 4 Size distribution of LDH (A) and pFDNA-LDH@SiO₂-NPs (B).

Notes: Measurement of these particles showed a narrow distribution pattern of LDH and pFDNA-LDH@SiO₂-NPs, and their average diameters were 99.1 nm and 371.93 nm, respectively.

Abbreviations: LDH, layered double hydroxide; NPs, nanoparticles; pFDNA-LDH@SiO₂-NPs, Newcastle disease virus F gene encapsulated in LDH@SiO₂-NPs.

Figure 5 Fourier transform infrared spectroscopy spectrum (A) and XRD curve (B) for each of the LDH and pFDNA-LDH@SiO₂-NPs.

Notes: The XRD patterns of the pFDNA-LDH@SiO₂-NPs exhibited typical diffractions of LDH including (003) and (006), indicative of the presence of LDH crystallite. The characteristic peak at 20° of pFDNA-LDH@SiO₂-NPs showed the existence of SiO₂.

Abbreviations: LDH, layered double hydroxide; NPs, nanoparticles; pFDNA-LDH@SiO₂-NPs, Newcastle disease virus F gene encapsulated in LDH@SiO₂-NPs; XRD, X-ray diffraction.

Figure 6 In vitro release profiles of DNA from pFDNA-LDH@SiO₂-NPs in phosphate-buffered saline (pH 7.4).

Notes: The experiment was repeated three times and measured in triplicate. Data are presented as the mean ± standard deviation (n=3).

Abbreviations: LDH, layered double hydroxide; NPs, nanoparticles; pFDNA-LDH@SiO₂-NPs, Newcastle disease virus F gene encapsulated in LDH@SiO₂-NPs.

Figure 7 In vitro expression of the pFDNA-LDH@SiO₂-NPs in 293 T cells assayed by indirect immunofluorescence (×40).

Notes: (A) Naked plasmid pVAX1-F(o) DNA group. (B) Transfected pFDNA-LDH@SiO₂-NPs group. (C) Blank LDH@SiO₂-NPs group. (D) 293 T cell group as the negative control.

Abbreviations: LDH, layered double hydroxide; NPs, nanoparticles; pFDNA-LDH@SiO₂-NPs, Newcastle disease virus F gene encapsulated in LDH@SiO₂-NPs.

Figure 8 Histopathological analyses of normal duodena, proventriculi, and myocardia.

Notes: (A) Tissues of duodena were immunized with pFDNA-LDH@SiO₂-NPs IM (a), pFDNA-LDH@SiO₂-NPs IN (b), and PBS IM (c). (B) Tissues of proventriculi were immunized with pFDNA-LDH@SiO₂-NPs IM (a), pFDNA-LDH@SiO₂-NPs IN (b), and PBS IM (c). (C) Tissues of myocardia were immunized with pFDNA-LDH@SiO₂-NPs IM (a), pFDNA-LDH@SiO₂-NPs IN (b), and PBS IM (c).

Abbreviations: IM, intramuscularly; IN, intranasally; LDH, layered double hydroxide; NPs, nanoparticles; PBS, phosphate-buffered saline; pFDNA-LDH@SiO₂-NPs, Newcastle disease virus F gene encapsulated in LDH@SiO₂-NPs.

Table 1 IgG antibody titers in serum samples of specific pathogen-free chickens immunized with PBS IM, LDH@SiO₂-NPs IM, naked plasmid pVAX1-F(o) DNA IM, pFDNA-LDH@ SiO₂-NPs IM, and pFDNA-LDH@SiO₂-NPs IN (unit: log 2), respectively

Group	Weeks post-immunization
	1	2	3	4	5	6	7	8
pFDNA-LDH@SiO2-NPs IN	3.333±0.577^a	4.667±0.577^a	6.000±0^a	7.667±0.577^a	8.667±0.577^a	8.667±0.577^a	7.667±1.155^a	7.333±0.577^a
pFDNA-LDH@SiO2-NPs IM	3.333±0.577^a	4.667±0.577^a	5.000±0^a	5.333 ±1.155^b	7.333±0.577^a	6.333±0.577^b	7.000±0^a	5.667±0.577^b
Naked plasmid pVAX1-F(o) DNA IM	3.000±1.000^a	4.667±0.577^a	4.667±0.577^a	5.667±0.577^b	3.000±1.000^b	2.667±1.155^c	3.000±1.000^b	3.000±1.000^c
Blank LDH@SiO2-NPs IM	2.000±1.000^b	1.667±0.577^b	2.000±1.000^b	1.667 ±1.155^c	1.667±1.155^c	2.000±0^d	2.000±1.000^b	1.667±0.577^d
PBS IM	1.333±0.577^b	1.333±0.577^b	1.667±0.577^b	1.000±0^c	1.333±0.577^c	1.333±0.577^d	1.667±1.155^b	1.000±0^d

Notes: Values are presented as mean ± standard deviation of five experiments in each group. Values within the same column with the different superscript letters are significantly different (P<0.05; Student’s t-test).

Abbreviations: IM, intramuscularly; IN, intranasally; LDH, layered double hydroxide; NPs, nanoparticles; PBS, phosphate-buffered saline; pFDNA-LDH@SiO₂-NPs, Newcastle disease virus F gene encapsulated in LDH@SiO₂-NPs.

Figure 9 IgA antibody levels in serum (A), tracheal fluid (B), bile (C), and Harderian glands (D) of specific pathogen-free chickens immunized with PBS IM, blank LDH@SiO₂-NPs IM, naked plasmid pVAX1-F(o) DNA IM, pFDNA-LDH@SiO₂-NPs IM, and pFDNA-LDH@SiO₂-NPs IN.

Notes: IgA antibody levels are presented as mean ± standard deviation of five experiments. Data with different small letters show significant difference (P<0.05).

Abbreviations: IM, intramuscularly; IN, intranasally; LDH, layered double hydroxide; NPs, nanoparticles; PBS, phosphate-buffered saline; pFDNA-LDH@SiO₂-NPs, Newcastle disease virus F gene encapsulated in the LDH@SiO₂-NPs.

Table 2 The stimulation index of T lymphocyte proliferation in specific pathogen-free chickens post-immunization

Group	Stimulation index
	7 days post-immunization	14 days post-immunization	21 days post-immunization	28 days post-immunization	35 days post-immunization	42 days post-immunization
pFDNA-LDH@SiO2-NPs IN	0.522±0.021^a	0.689±0.029^a	1.110±0.019^a	1.444±0.039^a	1.866±0.07^a	2.056±0.042^a
pFDNA-LDH@SiO2-NPs IM	0.517±0.003^a	0.690±0.014^a	1.104±0.028^a	1.234±0.071^b	1.745±0.096^b	1.803±0.028^b
Naked plasmid pVAX1-F(o) DNA IM	0.515±0.031^a	0.671±0.017^a	1.009±0.007^a	1.228±0.057^b	1.566±0.037^c	1.606±0.025^c
Blank LDH@SiO2-NPs IM	0.402±0.007^b	0.478±0.004^b	0.727±0.075^c	0.805±0.047^c	1.121±0.281^d	1.044±0.055^d
PBS IM	0.364±0.03^b	0.441±0.030^b	0.701±0.056^c	0.743±0.028^d	1.100±0.021^d	1.008±0.023^d

Notes: Values are presented as mean ± standard deviation of five experiments in each group. Values within the same column with the different superscript letters are significantly different (P<0.05; Student’s t-test).

Abbreviations: IM, intramuscularly; IN, intranasally; LDH, layered double hydroxide; NPs, nanoparticles; PBS, phosphate-buffered saline; pFDNA-LDH@SiO₂-NPs, Newcastle disease virus F gene encapsulated in LDH@SiO₂-NPs.

Table 3 Protection of the immunized specific pathogen-free chickens after being challenged with the highly virulent Newcastle disease virus strain F₄₈E₉

Treatment	Number of dead chickens/total number of chickens	Mortality (%)	Protection (%)
PBS IM	5/5	100	0
Blank LDH@SiO2-NPs IM	5/5	100	0
Naked plasmid pVAX1-F(o) DNA IM	3/5	60	40
pFDNA-LDH@SiO2-NPs IM	2/5	40	60
pFDNA-LDH@SiO2-NPs IN	0/5	0	100

Abbreviations: IM, intramuscularly; IN, intranasally; LDH, layered double hydroxide; NPs, nanoparticles; PBS, phosphate-buffered saline; pFDNA-LDH@SiO₂-NPs, Newcastle disease virus F gene encapsulated in LDH@SiO₂-NPs.

Figure 10 Histopathological analyses of the duodena and myocardia of chickens challenged with the highly virulent NDV strain F₄₈E₉.

Notes: (A) Tissues of the duodena from chickens immunized with pFDNA-LDH@SiO₂-NPs IN. (B) Tissues of the myocardia from chickens immunized with pFDNA-LDH@SiO₂-NPs IN. (C) Tissues of the duodena from chickens immunized with pFDNA-LDH@SiO₂-NPs IM, naked plasmid pVAX1-F(o) DNA IM, or PBS IM. (D) Tissues of the myocardia from chickens in groups immunized with the pFDNA-LDH@SiO₂-NPs IM, naked plasmid pVAX1-F(o) DNA IM, or PBS IM.

Abbreviations: IM, intramuscularly; IN, intranasally; LDH, layered double hydroxide; NDV, Newcastle disease virus; NPs, nanoparticles; PBS, phosphate-buffered saline; pFDNA-LDH@SiO₂-NPs, NDV F gene encapsulated in the LDH@SiO₂-NPs.

Figure S1 Preparation of pFDNA-LDH@SiO2-NPs.

Abbreviations: APTES, 3-aminopropyl-triethoxysilane; LDH, layered double hydroxide; NPs, nanoparticles; pFDNA-LDH@SiO₂-NPs, Newcastle disease virus F gene encapsulated in LDH@SiO₂-NPs; pFDNA-SiO₂-NPs, Newcastle disease virus F gene encapsulated in SiO₂-NPs; TEOS, tetraethyl orthosilicate.