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    2020-08-30

    
    of 30 μg/mL of CDDP and 48 h of treatment, (this CDDP concentration corresponds to its IC50 for 48 h exposure). Results reveal that the cell viability was reduced up to 50%, the same as by using free CDDP. Furthermore, when CCN1 and CCN2 nanogels were put in contact with the 33069-62-4 in a concentration representing an equivalent dosage of 90 μg/ mL of CDDP, a steep decrease in the cell survival was observed below to 25% for both CCNs, being comparable to the positive control.
    These results suggest that the nanogels managed to internalize to the cancer cells, which is possible due to the nanogels positive surface charge in contact with the negatively charged cell membrane surface via their electrostatic interaction, allowing the formation of endosomes, generating an acidic environment (pH 5.0). This cause the swelling of the nanogel, and consequently, the amine groups of the nanogel are being positively ionized, resulting in the CDDP being rapidly released in the cell interior, generating apoptosis.
    As previously stated, a higher amount on CDDP could be released from the cationic systems, as high as 60% at pH 5.0 (Fig. 2d). In this aspect, the investigation of nanoparticles with positive surface charge have shown that these could promote a higher internalization rate,-increase the cellular uptake, and exhibit perinuclear localization inside the cells [54].
    3.4. In-vitro cellular uptake
    Flow cytometry was used to study the cellular uptake of nanogels. Fluorescent labeled anionic and cationic nanogels were synthesized following the methodology reported here, but replacing the EGDMA by a fluorescent crosslinker: FDA, resulting in nanogels with green fluor-escence. These nanogels denoted as CCNF and ACNF were loaded also with CDDP and were used both empty and CDDP loaded, in studies of cellular uptake. In NCI-H1437 cells, the internalization rate of the CDDP loaded CCNF nanogel increased notably as compared with the empty cationic nanogel, nevertheless both cationic nanogels were in-ternalized. On the other hand, both empty and drug loaded ACNF na-nogels showed no internalization in NCLH-1437 cells (Fig. 5a). The percentages of cells internalized with empty and drug loaded cationic nanogels were 15.5% and 37.8% after 30 min of contact, respectively (Fig. 5b). Due to the previous mentioned, the toxicity effect in vitro to the cell-line appears to be a synergetic effect of both the drug (CDDP) and the cationic nanogel cores.
    Fluorescence microscopy was also used to visualize the inter-nalization of nanogels into NCI-H1437 cells at 0.5 h. Cell nuclei were counterstained with Hoechst 33258 in blue and anionic or cationic nanogels were visualized in green.
    Presumptively, the obtained images by this technique, supports the internalization of cationic nanogels, since these nanogels were observed in the interior of the cells. Otherwise, anionic nanogels were preferably located outside of the cells and they were accumulated in the cell membrane (Fig. 5c).
    4. Conclusions
    Surfactant free emulsion polymerization using PEGMA as poly-merizable stabilizer allowed for the synthesis of both cationic and an-ionic nanogels with Dh between 90–140 nm and PDI below 0.2. The cationic and anionic nanogels were loaded efficiently with cisplatin, showing DLE (close to 90 wt%) and DLC (approx. 30 wt%) due to the coordination of the drug with the cationic nanogel and the chelate formation with the anionic nanogel.
    The anionic nanogels showed an acceleration of cisplatin release at a pH of 6.8 (similar to tumor microenvironment pH) while the anionic nanogels only showed an accelerated release of cisplatin at pH = 5 (endosomal pH), those behaviors are related to the specific nanogel functional groups-cisplatin interactions.
    The cell compatibility of empty anionic and cationic nanogels was demonstrated in different concentrations (30–400 μg/mL). The cell viability of lung cancer cells depends on the dose and time of exposure to the cisplatin-loaded nanogels. Whereas the cisplatin-loaded anionic nanogels did not showed an affectation on cell viability with respect to the studied time (48 h), the cisplatin-loaded cationic nanogels exhibited a significant effect, showing cytotoxicity when the dose was increased and the exposure time was changed from 24 to 48 h.
    The internalization of nanogels into human lung cancer cells was verified by fluorescence microscopy and flow cytometry, in which 37.8% of the cells revealed that drug loaded CCNs were internalized after 30 min of contact. Otherwise, ACNs presented accumulation in the cell membrane, but no internalization was observed.
    The PEGMA-PDEAEMA cationic nanogels represent an interesting and promising option to transport CDDP into the human lung cancer cell line NCI-H1437.