Effect of molecular weight of polyethyleneimine on loading of CpG oligodeoxynucleotides onto flake-shell silica nanoparticles for enhanced TLR9-mediated induction of interferon-α

Figures & data

Figure 1 Characterization of flake-shell SiO₂ nanoparticles. (A) Field-emission scanning electron microscopy image. (B) Scanning transmission electron microscopic image. (C) Nitrogen adsorption-desorption isotherms.

Table 1 Surface charge and maximum loading capacity of CpG ODNs on SiO₂ nanoparticles coated with PEIs of different number-average molecular weights

	Zeta potential (mV)		Maximum loading capacity for CpG ODNs (μg/mg nanoparticles)
	Before loading of CpG ODNs	After loading of CpG ODNs
Smooth-surfaced SiO2 nanoparticles coated with 3% PEI and
Mn = 600	6.9 ± 1.8	−23.3 ± 3.7	17.9 ± 2.4
Mn = 1800	10.3 ± 1.2	−26.1 ± 4.9	31.5 ± 2.5
Mn = 10,000	17.8 ± 1.3	−27.4 ± 2.3	49.3 ± 1.3
Flake-shell SiO2 nanoparticles coated with 3% PEIs with
Mn = 600	5.4 ± 0.7	−24.1 ± 3.4	97.7 ± 11.5
Mn = 1800	8.4 ± 1.0	−27.1 ± 4.4	180.7 ± 13.0
Mn = 10,000	11.9 ± 1.7	−31.2 ± 5.7	321.0 ± 12.0

Abbreviations: Mn, number-average molecular weight; SiO₂, mesoporous silica; ODN, oligodeoxynucleotides; PEI, polyethyleneimine.

Figure 2 Characterization of flake-shell SiO₂ nanoparticles coated with PEI-600, PEI-1800, and PEI-10,000. (A) Scanning transmission electron microscopic images of flake-shell SiO₂ nanoparticles before and after coating with PEI. (B) Particle size distribution of flake-shell SiO₂ nanoparticles measured using dynamic light scattering. The polydispersity index was 0.26 for the SiO₂ nanoparticles without PEI. The polydispersity index was 0.36, 0.37, and 0.13 for PEI-600, PEI-1800, and PEI-10,000, respectively. (C) Thermogravimetric analysis of flake-shell SiO₂ nanoparticles coated with PEI. (D) N₂ adsorption-desorption isotherms of flake-shell SiO₂ nanoparticles after coating with PEI.

Abbreviation: PEI, polyethyleneimine.

Figure 3 Cytotoxicity of PEI-coated SiO₂ nanoparticles. Relative cell viability of smooth-surfaced (A) and flake-shell (B) SiO₂ nanoparticles coated with PEI of Mns 600, 1800, and 10,000. Peripheral blood mononuclear cells were exposed to PEI-coated SiO₂ nanoparticles at various concentrations for 48 hours.

Abbreviations: PEI, polyethyleneimine, NPs, nanoparticles; Mn, number-average molecular weight.

Figure 4 Loading capacity of CpG ODN2006×3-PD on SiO₂ nanoparticles. Loading capacity of CpG ODN2006×3-PD on smooth-surfaced SiO₂ nanoparticles (A) and flake-shell SiO₂ nanoparticles (B) coated with polyethyleneimine of Mns 600, 1800, and 10,000. CpG ODN2006×3-PD solutions (46 μL) of various concentrations were incubated with 40 μg of SiO₂ nanoparticles coated with polyethyleneimine.

Abbreviations: PEI, polyethyleneimine; NPs, nanoparticles; Mn, number-average molecular weight; ODN, oligodeoxynucleotides.

Figure 5 IFN-α induction by CpG ODNs in peripheral blood mononuclear cells. (A) IFN-α induction by free CpG ODN2006×3-PD and CpG ODN2216. Free class A CpG ODN2216 induced IFN-α in a dose-dependent manner, but free CpG ODN2006×3-PD did not induce IFN-α. (B) IFN-α induction by CpG ODN2006×3-PD loaded on SiO₂ nanoparticles. Naked smooth-surfaced and flake-shell SiO₂ nanoparticles did not induce IFN-α, but CpG ODN2006×3-PD loaded on smooth-surfaced and flake-shell SiO₂ nanoparticles coated with PEI induced IFN-α. The SiO₂ nanoparticles loaded with CpG ODN2006×3-PD were applied to peripheral blood mononuclear cells at a concentration of 50 μg/mL. The concentrations of CpG ODN2006×3-PD on smooth-surfaced SiO₂ nanoparticles coated with PEI of Mns 600, 1800, and 10,000 were estimated to be 33, 62, and 98 pmol/mL medium, respectively, from the loading capacities. Similarly, the concentrations of CpG ODN2006×3-PD on flake-shell SiO₂ nanoparticles coated with PEI of Mns 600, 1800, and 10,000 were estimated to be 196, 364, and 650 pmol/mL medium, respectively.

Abbreviations: PEI, polyethyleneimine, NPs, nanoparticles; Mn, number-average molecular weight; ODN, oligodeoxynucleotides; IFN-α, interferon alpha.

Figure 6 IFN-α induction by the same density of CpG ODN2006×3-PD on flake-shell SiO2 nanoparticles coated with PEI of Mns 600, 1800, and 10,000. The loading amount of CpG ODN2006×3-PD was about 100 μg/mg nanoparticles (200 pmol/mL medium), which is similar to the maximum loading capacity of flake-shell SiO₂ nanoparticles coated with PEI of Mn 600. The flake-shell SiO₂ nanoparticles loaded with CpG ODN2006×3-PD were applied to peripheral blood mononuclear cells at a concentration of 50 μg nanoparticles/mL. The SiO₂ nanoparticles coated with PEI of Mns 1800 and 10,000 showed a significantly lower level of IFN-α induction despite having the same density of CpG ODN2006×3-PD as the SiO₂ nanoparticles coated with PEI of Mn 600.

Abbreviations: PEI, polyethyleneimine, NPs, nanoparticles; Mn, number-average molecular weight; ODN, oligodeoxynucleotides; IFN-α, interferon alpha.

Figure 7 Intracellular localization of CpG ODN2006×3-PD delivered by flake-shell SiO₂ nanoparticles coated with PEI of Mns 600, 1800, and 10,000. FITC-labeled CpG ODN2006×3-PD was loaded on the SiO₂ nanoparticles through PEI.

Notes: The loading amount of FITC-labeled CpG ODN2006×3-PD was approximately 100 μg/mg nanoparticles, and the SiO₂ nanoparticles were applied to the cells at a concentration of 50 μg/mL. Each image was obtained from a cross-section of cells using confocal laser fluorescence microscopy. Bar = 10 μm.

Abbreviations: FITC, fluorescein isothiocyanate; PEI, polyethyleneimine, NPs, nanoparticles; Mn, number-average molecular weight; ODN, oligodeoxynucleotides.