Biodegradable silica nanoparticles for efficient linear DNA gene delivery

Figures & data

Scheme 1. p-DNA to l-DNA transformation. (a) PCR components and primer sequence design. (b) Diagram of the experimental procedure. (c) Agarose gel characterization of the synthesized l-DNA.

Table 1. Optimization of DNA@SiO₂ particle synthesis.

Entry	Synthesis					Characterization
	NH3 /M	TEOS/ M	H2O/M	DNA		Size (TEM)	Z-Size (DLS)	PDI (DLS)
				Shape	[DNA] / M (µg DNA/mg SiO2)
type a	1.06	0.24	3.32	plasmid	1.4 × 10^-5 (4.5 µg)	678 ± 51	–	–
type b	1.22	0.24	9.99	plasmid	1.4 × 10^-5 (4.5 µg)	388 ± 36	–	–
type c	0.34	0.24	14.0	plasmid	1.4 × 10^-5 (4.5 µg)	186 ± 15	–	–
p-DNA#1	1.22	0.24	9.99	plasmid	1.6 × 10^-6 (0.5 µg)	468 ± 28	601 ± 28	0.18 ± 0.08
p-DNA#2	1.22	0.24	9.99	plasmid	4.8 × 10^-6 (1.5 µg)	274 ± 19	358 ± 18	0.2 ± 0.1
p-DNA#3	1.22	0.24	9.99	plasmid	9.6 × 10^-6 (3.0 µg)	262 ± 16	324 ± 3	0.26 ± 0.02
p-DNA#4	1.22	0.24	9.99	plasmid	1.4 × 10^-5 (4.5 µg)	393 ± 22	413 ± 67	0.23 ± 0.06
p-DNA#5	1.22	0.24	9.99	plasmid	2.9 × 10^-5 (9 µg)	*	1874 ± 25	0.99 ± 0.05
p-DNA#6	1.22	0.24	9.99	plasmid	4.8 × 10^-5 (15 µg)	*	3770 ± 17	1.5 ± 0.2
l-DNA#1	1.22	0.24	9.99	linear	1.6 × 10^-6 (0.5 µg)	384 ± 40	325 ± 12	0.41 ± 0.02
l-DNA#2	1.22	0.24	9.99	linear	4.8 × 10^-6 (1.5 µg)	311 ± 18	314 ± 28	0.35 ± 0.03
l-DNA#3	1.22	0.24	9.99	linear	9.6 × 10^-6 (3.0 µg)	284 ± 20	236 ± 60	0.36 ± 0.06
l-DNA#4	1.22	0.24	9.99	linear	1.4 × 10^-5 (4.5 µg)	260 ± 22	250 ± 28	0.33 ± 0.03
l-DNA#5	1.22	0.24	9.99	linear	2.9 × 10^-5 (9 µg)	*	1034 ± 30	0.7 ± 0.3

Notes: Effect of different concentrations of the Stöber mixture (a-c). Effects of different amounts of DNA (plasmid and linear) on particle size and dispersity. Figures S1-S11 show the TEM and DLS results of characterization. Asterisk in places where (*) TEM sizes could not be estimated due to aggregates (see Figures S5–S6, Figures S11).

TEM: transmission electron microscopy; DLS: dynamic light scattering; PDI: polydispersity index; p-DNA: plasmid DNA; l-DNA: linear DNA.

Figure 1. p-DNA@SiO₂ particles. (a) TEM images of p-DNA#4 particles with 4.5 µg of plasmid H2B:YFP DNA. (b) Particle diameter (mean ± SD) measured by TEM within a scatter dot plot comparing p-DNA#1-6 (n = 100). Values for p-DNA#5 and p-DNA#6 are not included due to the presence of aggregates (see Figures S8–S9). (*)(**) p-DNA#5-6 not included due to the presence of aggregates (see Figures S5–S6). (c) DLS characterization of p-DNA#1-6 bar chart of Z-size (mean ± SD, left Y axis, n = 3) and dot plot in red showing the polydispersity index (PDI) (mean ± SD, right Y axis, n = 3). (d) Flow cytometry quantification of the transfection efficiency (mean fluorescence intensity) of the p-DNA#4 DNA@SiO₂ particles and a representative confocal microscopy image of HEK 293 cells expressing the recombinant H2B:YFP protein 72 h after transfection. Data are shown as the mean ± SD of 3 experimental replicas (n = 10,000 cells/replica, t-test, *p < 0.05, and ***p < 0.001).

Figure 2. l-DNA@SiO₂ particles. (a) TEM images of particles l-DNA#4 with 4.5 µg of linear H2B:YFP DNA. (b) Particle diameter (mean ± SD) measured by TEM within a scatter dot plot comparing l-DNA#1-5 (n = 100). (*) l-DNA#5 is not included due to the presence of aggregates (see Figure S11). (c) l-DNA#1-6 bar chart of Z-size (mean ± SD, left Y axis, n = 3) and dot plot in red showing polydispersity index (PDI) (mean ± SD, right Y axis, n = 3). (d) Flow cytometry quantification of the transfection efficiency (mean fluorescence intensity) of the l-DNA#4 DNA@SiO₂ particles and a representative confocal microscopy image of HEK 293 cells expressing the recombinant H2B:YFP protein 72 h after transfection. Data are shown as the mean ± SD of 3 experimental replicas (n = 10,000 cells/replica, t-test, *p < 0.05, and ***p < 0.001).

Figure 3. (a) Quantification of the transfection efficiency (mean fluorescence intensity) using p-DNA#4, l-DNA#2, and l-DNA#4 particles. Data are shown as the mean ± SD of 3 experimental replicas (n = 10,000 cells/replica, t-test, **p < 0.01, ***p < 0.001, and ****p < 0.0001). Protein expression profile from 24 h to 14 days. (b) Mechanistic proposal of differences in gene transfection efficiency for p-DNA#4, l-DNA#2, and l-DNA#4.

Figure 4. Effect of particle concentration on transfection efficiency and cell viability. (a) Flow cytometry quantification of transfected H2B:YFP protein by using l-DNA#4 particles (mean ± SD, n = 3) carrying different DNA@SiO₂ concentrations (µg/ml IMDM). (b) MTT assay showing % of viable cells 24 h and 72 h after l-DNA#4 treatment with the same particles (mean ± SD, n = 6).

Figure 5. Quantification of H2B:YFP protein expression (mean fluorescence intensity) using Lipofectamine^TM 2000 and DNA@SiO₂ system with p-DNA and l-DNA in HEK 293 cells. Data are shown as the mean ± SD of 3 experimental replicas (n = 10,000 cells/replica, t-test, *p < 0.05, and ***p < 0.001).

Data availability statement

The data presented in this study are available on request from the corresponding author.