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  • br CDDP drug release experiments from both PEGylated nanogels


    CDDP drug release experiments from both PEGylated nanogels were carried out in vitro. The percentage of CDDP released from the CCN1 and ACN1 under different conditions are shown in Fig. 2c and d, re-spectively. In a phosphate buffer saline (PBS) solution at pH 7.4, the free CDDP drug diffuses continuously from the dialysis bag to the medium reaching 92% in 6 h. Afterwards, the driving force for drug 
    release is decreasing as a function of CDDP concentration in the release medium (Fig. 2c and d); this is attributed to the fact that CDDP release tends toward equilibrium, indicating that the saturation concentration of CDDP in the release medium is achieved; this result agrees with previously reported CDDP release profiles in the literature [45].
    In contrast, when cisplatin is loaded in the nanogels, at physiolo-gical condition (pH 7.4) after 50 h, there was only around 25% and 10% of CDDP released from ACN1 and CCN1, respectively. Whereas at pH 6.8 (tumor extracellular pH) the nanogel ACN1 released 50% of CDDP, the nanogel CCN1 released only 10% of CDDP. ACN showed a faster release profile, which is attributed to the fact that the interaction of PSB 1115 groups with the drug are through the carboxylate groups, they are partially neutralized at a slight acidic pH, while the interactions with the amino groups of DEAEMA units are through the lone electron pair (Fig. 3). In addition to that, the water solubility of PDEAEMA is only minimal at pH 6.8 as reflected by the minimal nanogel expansion at that pH (Fig. 2a). At a more acidic pH of 5.0 (endosomes), a higher content of CDDP could be released for both systems, as high as 60%, being the ACN1 release slightly faster in the early stage (Fig. 2c). At this pH value the aqueous solubility of PDEAEMA is not a problem anymore, since these units are ionized, as reflected in their swelling and ζ-po-tential behavior (Fig. 2a and b). Ionization of amine groups in DEAEMA removes the availability of the lone electron pair for interactions with the drug (Fig. 3).
    3.3. In-vitro therapeutic efficacy
    Anticancer activity of free CDDP against human lung cancer cell line NCI-H1437 was examined using the cell proliferation MTS assay after incubation for 24 h and 48 h at 37 °C, at pH 7.4 and at 5% of CO2. Cell viability in the presence of the empty nanogels was also examined prior to the incorporation of CDDP at 24 h. Surprisingly, nanogels composed of only P2MBA (no PEGMA shell) were found to be nontoxic up to a concentration of 400 μg/mL. This indicates biocompatibility for the cell
    Fig. 3. Schematic illustration of the CDDP interactions via coordination to a ligand with ACN (left) and CCN (right).
    A. Gonzalez-Urias, et al.
    Fig. 4. Cytotoxic effect of CDDP loaded nano-gels on human lung cancer cell line NCI-H1437:
    a) ACN1 at different equivalent CDDP con-centrations after 24 h, b) ACN1 at different equivalent CDDP concentrations after 48 h, c) CCN1 and CCN2 at a 55 μg/mL CDDP con-centration after 24 h, d) CCN1 and CCN2 at different equivalent CDDP concentrations after 48 h. Cell viability (%) is expressed as function of untreated cells (C−). The results represent the average ± SEM of triplicates. Positive
    C−(unpaired Student’s t-test).
    line NCI-H1437, as shown in Figure S11 (Supplementary material). This type of nanogel was the largest tested (309 nm) with the highest ne-gative ζ potential (−29.9 mV, Figure S2 in the Supplementary mate-rial). According to the hypothesis named “wrapping time” of the membrane, nanocarriers with large size need stronger driving force and additional energy for a cellular internalization process, therefore, cel-lular uptake amounts decrease with the augmentation of particle size of nanogels. Additionally, the higher the surface charge, the lower the cellular uptake [21]. In the case of the PEGylated anionic nanogels, as expected the two nanogels evaluated (ACN1 and ACN2) did not reduce the cell viability at any studied concentration (30–300 μg/mL), it is evident that those nanogels were non-cytotoxic up to high concentra-tions (Figure S12 in Supplementary material). Different anionic poly-mers have been used in the design of nanocarriers for the supply of CDDP, for example: poly (lactic-co-glycolic acid) [46–48], poly (L-glutamic acid) [49], poly (hyaluronic acid) [27], poly (methacrylic acid) [26,50,51], poly (acrylic acid) [44], among others, which contain ionizable COOH groups in their chemical structure, generating the co-ordination or un-coordination of CDDP when the pH of the medium was changed. In addition, they have been shown to be non-toxic, when the nanocarriers are empty. In this study P2MBA is used, in which the acid group is a salicylic acid derivative; besides being biocompatible the aromatic ring imparts this polyacid partial hydrophobicity, resulting in a sharp volume pH-transition that can be used for an advantageous pH-sensitive drug release profile, as described above.