Author Contributions
Conceptualization, C.P. and O.T.; methodology, L.C. and F.F.; validation, L.C., F.F. and F.D.F.; formal analysis, L.C., F.F. and F.D.F.; investigation, F.D.F.; resources, C.P. and O.T.; data curation, C.P., L.C., F.F. and O.T.; writing—original draft preparation, L.C. and F.D.F.; writing—review and editing, C.P. and O.T.; visualization, C.P., L.C. and F.F.; supervision, C.P. All authors have read and agreed to the published version of the manuscript.
Figure 1.
Size distribution by intensity of CS/hsDNA complexes prepared with CS of different molecular weights and at different N/P values (from N/P 5 to N/P20 by using 50 and 150KDa chitosan).
Figure 1.
Size distribution by intensity of CS/hsDNA complexes prepared with CS of different molecular weights and at different N/P values (from N/P 5 to N/P20 by using 50 and 150KDa chitosan).
Figure 2.
SEM micrograph of CS/hsDNA particles (CS molecular weight = 50 kDa, N/P ratio = 10). Scale bar: 2 µm.
Figure 2.
SEM micrograph of CS/hsDNA particles (CS molecular weight = 50 kDa, N/P ratio = 10). Scale bar: 2 µm.
Figure 3.
Two percent Agarose gel electrophoresis: (STD) DNA standards of different molecular weight, (A) CS/hsDNA microparticles, (B) CS/hsDNA in the presence of DNAse I, (C) CS/hsDNA in the presence of inactivated DNAse I.
Figure 3.
Two percent Agarose gel electrophoresis: (STD) DNA standards of different molecular weight, (A) CS/hsDNA microparticles, (B) CS/hsDNA in the presence of DNAse I, (C) CS/hsDNA in the presence of inactivated DNAse I.
Figure 4.
In vitro release kinetics of hsDNA from CS/hsDNA complexes. N/P ratio = 10, CS MW = 50 kDa.
Figure 4.
In vitro release kinetics of hsDNA from CS/hsDNA complexes. N/P ratio = 10, CS MW = 50 kDa.
Figure 5.
1H-NMR spectra of CS (A) and CS-FITC conjugate (B) in D2O acidified with DCl. The signals relating to CS (black line in box (A)) and CS FITC (blu line in box (B)) and the integrals (black lines in box (B)) relating to the diagnostic signals of the two species are reported.
Figure 5.
1H-NMR spectra of CS (A) and CS-FITC conjugate (B) in D2O acidified with DCl. The signals relating to CS (black line in box (A)) and CS FITC (blu line in box (B)) and the integrals (black lines in box (B)) relating to the diagnostic signals of the two species are reported.
Figure 6.
Size distribution by intensity of CS/siRNA (a), PEI/siRNA (b) and Escort IVTM/siRNA (c) complexes.
Figure 6.
Size distribution by intensity of CS/siRNA (a), PEI/siRNA (b) and Escort IVTM/siRNA (c) complexes.
Figure 7.
Cell viability of C8166 cells after treatment with CS/hsDNA complexes at concentrations of 100, 20 × 1010 µg/mL for 24, 48 and 72 h.
Figure 7.
Cell viability of C8166 cells after treatment with CS/hsDNA complexes at concentrations of 100, 20 × 1010 µg/mL for 24, 48 and 72 h.
Figure 8.
Optical microscope image of C8166 cells treated with different concentrations of CS/hsDNA complexes (CS MW = 50 KDa, N/P = 10): 250, 50, 10 and 0 µg/mL.
Figure 8.
Optical microscope image of C8166 cells treated with different concentrations of CS/hsDNA complexes (CS MW = 50 KDa, N/P = 10): 250, 50, 10 and 0 µg/mL.
Figure 9.
Fluorescence-activated cell sorting of C8166 treated with 0, 100, 20 and 10 µg/mL of FITC-CS/hsDNA particles for 4 h of incubation at RT (CS MW = 50 KDa, N/P = 10).
Figure 9.
Fluorescence-activated cell sorting of C8166 treated with 0, 100, 20 and 10 µg/mL of FITC-CS/hsDNA particles for 4 h of incubation at RT (CS MW = 50 KDa, N/P = 10).
Figure 10.
Percentage of HIV-1 RNA reduction in C8166 cell line treated for 4 h pre (A) and post (B) single cycle HIV-1 infection, by PCR analysis. Both cell lines were treated with each nano delivery system (CS and control systems) loaded with both hsDNA and siRNA tat/rev at N/P = 10, with 20 µg/mL. * p = 0.0015; ** p = 0.0001; *** p < 0.0001 (Chi-square test).
Figure 10.
Percentage of HIV-1 RNA reduction in C8166 cell line treated for 4 h pre (A) and post (B) single cycle HIV-1 infection, by PCR analysis. Both cell lines were treated with each nano delivery system (CS and control systems) loaded with both hsDNA and siRNA tat/rev at N/P = 10, with 20 µg/mL. * p = 0.0015; ** p = 0.0001; *** p < 0.0001 (Chi-square test).
Table 1.
Weight ratios (w/w) between CS and nucleic acid used to synthetize polymeric particles with different N/P values.
Table 1.
Weight ratios (w/w) between CS and nucleic acid used to synthetize polymeric particles with different N/P values.
N/P | CS (mg) | HsDNA or siRNA (µg) |
---|
5 | 1 | 320 |
10 | 1 | 160 |
20 | 1 | 80 |
Table 2.
Weight ratios (w/w) between PEI and nucleic acid used to synthetize polymeric particles with different N/P values.
Table 2.
Weight ratios (w/w) between PEI and nucleic acid used to synthetize polymeric particles with different N/P values.
N/P | PEI (mg) | hsDNA or siRNA (µg) |
---|
10 | 214 | 160 |
20 | 214 | 80 |
Table 3.
Hydrodynamic diameter (nm) of CS/hsDNA complexes prepared with CS of different molecular weights and at different N/P values.
Table 3.
Hydrodynamic diameter (nm) of CS/hsDNA complexes prepared with CS of different molecular weights and at different N/P values.
MW (kDa) | N/P 5 | N/P 10 | N/P 20 |
---|
50 | 546.6 nm | 560.3 nm | 639.3 nm |
150 | 443.1 nm | 456.8 nm | 593.3 nm |
Table 4.
Z-potential (mV) of CS/hsDNA complexes prepared with CS of different molecular weights and at different N/P values.
Table 4.
Z-potential (mV) of CS/hsDNA complexes prepared with CS of different molecular weights and at different N/P values.
MW (kDa) | N/P 5 | N/P 10 | N/P 20 |
---|
50 | +16.0 | +35.0 | +48.0 |
150 | +10.0 | +12.0 | +16.0 |
Table 5.
Hydrodynamic diameter, PdI, Z-potential and entrapment efficiency (EE) of CS/siRNA tat/rev complexes prepared with CS 50 KDa and N/P = 10.
Table 5.
Hydrodynamic diameter, PdI, Z-potential and entrapment efficiency (EE) of CS/siRNA tat/rev complexes prepared with CS 50 KDa and N/P = 10.
Ø (nm) | PdI | Z-Potential (mV) | EE (%) |
---|
432.2 | 0.3 | +40.2 | 79.9 ± 4.8 |
Table 6.
Hydrodynamic diameter, PdI, Z-potential and entrapment efficiency (EE) of PEI/siRNA and Escort/siRNA.
Table 6.
Hydrodynamic diameter, PdI, Z-potential and entrapment efficiency (EE) of PEI/siRNA and Escort/siRNA.
| Ø (nm) | PdI | Z-Potential (mV) | EE (%) |
---|
PEI/siRNA | 88.7 | 0.2 | +38 | 93.0 ± 3.2 |
Escort/siRNA | 150.2 | 0.3 | 0 | 95.0 ± 2.8 |
Table 7.
Percentage of FITC+ cells treated with FITC-CS/hsDNA particles. C8166 cells were treated for 4 h before or after a single cycle of infection with HIV-1.
Table 7.
Percentage of FITC+ cells treated with FITC-CS/hsDNA particles. C8166 cells were treated for 4 h before or after a single cycle of infection with HIV-1.
| Pre-Infection | Post-Infection | Control |
---|
C8166 FITC+ | 46.1% | 63.3% | 75.5% |