The following sections describe the results obtained for cell viability using nanogels without the fluorescent tag. The cell toxicity was tested for nanogels made using EBA and MBA as a cross-linker, followed by the nanogels with a comonomer (AA and AEA) with different degrees of cross-linker (MBA).
O N O
O
HO O
HN
O HO
reduction
non-fluorescent (indigo)
fluroescent (Em: 590nm) (pink)
(17) (18)
3.3.2.2.1 EBA-based nanogels
The first nanogels synthesised in this project were prepared using EBA as a cross-linker. The nanogels with the lowest degree of cross-linker were selected for testing in cells because of their lower LCST value, closer to skin temperature (as described in Section 2.5.1). The composition of the selected preparations is described in the table below.
NIPAM EBA DF74 80% 20%
DF75 90% 10%
DF76 95% 5%
Table 3.3 – Composition EBA-based nanogels.
In the early stages of this thesis, DF74 to DF76 were tested for their toxicity in keratinocytes K17 for 24 and 48hours at concentrations of 1, 5 and 10 mg/mL.
The results are presented below.
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1mg/mL 5mg/mL 10mg/mL
Viable cells
Concentration of nanogel DF74
24h 48h
Figure 3.2 – Cell viability results respect a control of untreated K17 cells for nanogels DF74, DF75 and DF76 incubated for 24 and 48 hours. Error bars represent standard deviation (n=3).
In all cases, nanogels DF74-DF76 show an excellent cell viability (almost 100% for all the samples and concentrations), demonstrating that these materials are non-toxic when incubated with the cells for 48h. The simple structure of the material, similar to that of a protein (with peptide bonds) and its high solubility in water make this material an excellent candidate as a drug delivery system.
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1mg/mL 5mg/mL 10mg/mL
Viable cells
Concentration of nanogel DF75
24h 48h
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1mg/mL 5mg/mL 10mg/mL
Viable cells
Concentration of nanogel DF76
24h 48h
3.3.2.2.2 MBA-based nanogels
The next set of nanoparticles to be analysed for cell cytotoxicity in K17 were those prepared using MBA as a cross-linker. The composition can be found in the table below.
NIPAM MBA
DF98 80% 20%
DF99 90% 10%
DF100 95% 5%
Table 3.4 – Composition MBA-based nanogels.
As with EBA, the results showed a low toxicity for the cells. Having observed the excellent performance of the EBA counterparts, these materials were tested directly for 48 hours. The results are presented in the graph below.
Figure 3.3 - Cell viability results respect a control of untreated K17 cells for nanogels DF98, DF99 and DF100 incubated for 48hours.
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120%
2.5mg/mL 5mg/mL 7.5mg/mL 10mg/mL
Viable Cell after 48h
Concentration of nanogels
DF98 DF99 DF100
The MBA nanogels show excellent cell viability. The statistical significance of changes in cell viability was calculated using a t-test (p<0.05) and showed that the results below 100% were not different from a control. These results are similar to those obtained for EBA. The key finding is that the concentration of nanogels used in these experiments is very high, up to 10mg/ml and well above the values frequently reported in the literature.
NIPAM microgels cross-linked with MBA have been reported by Mozumdar et al. to withstand concentrations up 0.2 mg/mL in HeLa cells and Nguyen et al. reported values of up to 1 mg/mL in fibroblast.9,10
3.3.2.2.3 MBA-based, charged non-fluorescent nanogels
As described in Chapter II, one of the main interests was to evaluate the effect of different surface charges on the skin permeability properties. This is why several nanogels were prepared with comonomers AEA and AA (introducing positive and negative charge, respectively) at different cross-linking concentrations. The composition of these materials is shown in the table below.
NIPAM MBA AA AEA Charge
DF172 75% 20% 5% -
Negative
DF173 85% 10% 5% -
DF174 90% 5% 5% -
DF180 75% 20% - 5%
Positive
DF181 85% 10% - 5%
DF182 90% 5% - 5%
Table 3.5 – Negatively (AA) and positively (AEA) charged MBA-based NIPAM nanogels composition.
These six different preparations were tested in K17 keratinocyte cells for 48 hours to assess their cytotoxicity. The results are presented in the following two graphs; the first one for DF172-DF174 (negatively-charged nanogels) and the second one for DF180-DF182 (positively-charged).
Figure 3.4 - Cell viability results respect a control of untreated K17 cells for negatively-charged nanogels D172, DF173 and DF174 incubated for 48hours.
The incorporation of AA does not seem to affect the cell viability of the nanogels, except for high concentrations of nanogel DF172. Only the concentrations of 5 and 10 mg/mL for DF172 are statistically different from a control. For nanogels DF173 and DF174, although a decrease in cell viability can be observed for the highest concentrations, a test-t showed that the values are not significantly different from a control. Excluding the highest concentrations of the preparation DF172, the nanogels showed excellent cell viability, comparable to the non-charged nanogels. The viability rate was also higher than that previously reported in the literature for similar negatively-charged nanogels, where up to 0.2 µg/mL was successfully tested.11,12
The results of the cell viability test for positively-charged nanogels are shown
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120%
10mg/mL 5mg/mL 2mg/mL 1mg/mL
Viable cell after 48h
Concentration of nanogel
DF172 (20%MBA) DF173 (10% MBA) DF174 (5% MBA)
Figure 3.5 - Cell viability results respect a control of untreated K17 cells for positively-charged nanogels D180, DF181 and DF182 incubated for 48hours.
As can be seen above, the positively-charged nanogels DF181 and DF182 showed increasing cell toxicity with decreasing MBA concentration and consequently a lower LCST. DF182 kills or prevents the normal growth of the cells present in the test. DF181 had a cell viability of around 70%. This can only be explained by a toxic effect of the nanogels.
In these experiments, for the first time, the presence of aggregates (polymer thermoresponding) was observed under the microscope for samples DF181 and DF182. The nanogels were forming spherical precipitates that were more abundant in wells containing higher concentrations of the nanogels. The lower the LCST of the nanogels, the higher was the quantity of aggregates in each well.