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  • br In the case of the cationic nanogels a nanogel


    In the case of the cationic nanogels, a nanogel containing 100% of PDEAEMA resulted to be cytotoxic at any studied concentration (100–400 μg/mL), see Figure S11 in the Supplementary material. This behavior is not surprising and it Cenicriviroc is related with its surface charge (+19.1 mV), evidently positive (Figure S2, in Supplementary material). The majority of nanocarriers with positive surface charge correlates with higher cellular uptake and greater cytotoxicity in the cells, prob-ably by causing more pronounced disruption of plasma-membrane in-tegrity, stronger mitochondrial and lysosomal damage, and a higher number of autophagosomes [52]. As shown in Figure S12c and d, PE-Gylated cationic nanogels containing 15 and 20 wt% of PDEAEMA (CCN1 and CCN2, respectively) demonstrated that those systems did 
    not induced significant toxicities to the cell line NCI-H1437 in the studied concentrations (30–400 μg/mL). Despite the cationic surface charge, nanogels possessed excellent biocompatibility, which can be attributed to the PEG shielding effect. A recent work reported that PEGylated PDEAEMA nanogels with similar surface charges and na-noparticle structure did not exhibited significant toxicities against colon cancer, lung cancer and fibroblasts cell lines, further confirming the biocompatibility properties of the PEG/PDEAEMA nanogels of certain PDEAEMA content [42].
    The cytotoxic effect of cisplatin to the human lung cancer cell line NCI-H1437 was studied in a dose and time-dependent manner (Figure S13a, in the Supplementary material). The cell viability decreased sharply from 100% to 60% at low CDDP concentrations (5–50 μg/mL) after 24 h exposure time, obtaining values of median lethal dose (IC50) = 50–55 μg/mL. Increasing the exposure time to 48 h (Figure S13b) resulted in a steeper decrease in the cell viability at lower CDDP concentrations, for instance from 76 to 25% with the concentrations of 10 and 30 μg/mL, respectively. This behavior has been reported pre-viously. For instance, during the evaluation of free CDDP in the lung cancer cell line A549, it was observed that at 24 h, 308 μg/mL of the drug was needed to reach the IC50, while at 48 h, the mean lethal dose decreased drastically to 2.54 μg/mL [53]. Also, this effect has been observed in other cell lines for example in gastric cancer [27], breast cancer [50], uterine/cervical cancer [49], and prostate cancer [47,48]. Therefore, this confirms that the cell survival is dependent on the dose and time of the exposure.
    The cytotoxicity studies of CDDP-loaded anionic and cationic na-nogels against human lung cancer cell line NCI-H1437 are shown in Fig. 4. The CDDP concentration was calculated considering the DLC of each nanogel. In the case of the anionic nanogel ACN1 loaded with CDDP (˜30% w/w) three different CDDP concentrations (17, 33 and 50 μg/mL) were tested. Results showed a high cell survival of 80% after 24 h with the highest concentration tested (Fig. 4a), while after 48 h it decreased slightly to 75% (Fig. 4b). This behavior can be related to the slow release rate of CDDP from the nanogels at pH 7.4 (˜ 20% after 50 h), discussed before in the drug release section. The low cytotoxicity of the CDDP loaded ACN1 could be also attributable to the low affinity
    Fig. 5. Cell internalization studies using fluorescein O,O′-diacrylate (FDA) cross-linked anionic and cationic nanogels (ACNF and CCNF respectively, 50 μg/mL for 0.5 h of incubation): a) Representative histograms of FDA fluorescence in NCI-H1437 cells in contact with empty and drug-loaded nanogels; b) Percentage of cells positive for FDA-empty or FDA-drug loaded anionic and cationic nanogels. Data represents mean ± SD of triplicates, significances have been determined by one-way ANOVA (***p < 0.001 vs untreated cells); c) Fluorescence microscopy images of NCI-H1437 cells exposed to ACNF and CCNF nanogels.
    of the anionic nanogel to be internalized into the cell, and therefore the release of the drug could take place in the periphery of the cells and the drug could accessto cytoplasm by diffusional transport. In a literature report on CDDP loaded in pH-thermal dual responsive anionic nanogels, the difference in cytotoxicity between the free CDDP and the nanogel encapsulated CDDP was time exposure dependent [50]. Studies on three cancer cell lines, MCF-7, Hela and A549 cell lines, showed that the IC50 changed drastically after testing for 24, 48 and 96 h of study, due to the slow release rate of CDDP from the nanogels [50].
    In the case of the cationic nanogels, CCN1 and CCN2 nanogels concentration were the equivalent of 55 μg/mL CDDP, observing a cell survival close to 100% after 24 h exposure (Fig. 4c). This is intimately related to the slow release rate of CDDP from the nanogels at pH 7.4 (∼ 10% after 50 h), resulting in a high cell viability. Fig. 4d shows the results for CCN1 and CCN2 nanogels loaded with an equivalent dosage