br Cell viability was determined using the
Cell viability was determined using the CellTiter 96 aqueous one solution cell proliferation assay kit MTS (Promega, Madison, WI, USA). Lung adenocarcinoma (NCI-H1437 from ATCC) MPP+Iodide were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS-CORNING) and 1% antibiotic-antimycotic (10,000 units penicillin, 10 mg streptomycin, and 25 mg/mL amphotericin B per mL (SIGMA)) in a 37 °C, 5% CO2 humidified incubator. Cells were grown to 75–85% confluence, detached with 0.25% trypsin-0.1% EDTA, and used for assay protocols.
NCI-H1437 cells were seeded by triplicate in a 96-well plate (5 × 103 cells/mL) and incubated under standard growth conditions for 24 h followed by the addition of CDDP in various concentrations (5–200 μg/mL), anionic core and cationic core nanogels, and drug loaded nanogels, were dispersed in PBS. In all assays, untreated cells were used as negative control (C-) and 5% DMSO as positive dead control (C+), cells were incubated for 24 and 48 h. After exposure, 20 μL of (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt (MTS)) were added, and the amount of formazan converted by viable cells was determined by measuring the absorbance at 490 nm on a 96-well microplate reader EPOCH (BioTek, Winooski, VT, USA).
The results were normalized to untreated cells (100%) to obtain the percentage of cell viability and are expressed as the average ± SEM (standard error of the mean) of triplicates. Results were examined statistically by an unpaired Student’s t test. All statistical analyses were performed using the GraphPad prism program, version 5.0. A value of *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statisti-cally significant.
2.6. In-vitro cellular uptake
The cellular uptake of nanogels was investigated by flow cytometry. A total of 1.5 × 105 NCI-H1437 cells were treated for 0.5 h at 37 °C with 50 μg/mL of fluorescent ACNF or CCNF nanogels, both empty and also with CDDP loaded nanogels. Experiments were performed in tri-plicate. After treatment, cells were washed three times with PBS (0.01 M) and the fluorescence was analyzed by an Applied biosystems Attune Acoustic Focusing Cytometer (Life Technologies, Carlsbad, CA). Cellular uptake of particles was considered as percentage of total events positive in BL1-A channel after singlets gating exclusion.
2.7. Fluorescence microscopy
The cellular uptake of nanogels was also visualized using fluores-cence microscopy. NCI-H1437 cells were cultured by duplicate in a 96-well plate (5 × 103 cells/mL) using RPMI-1640 medium supplemented with 10% FBS at 37 °C and 5% CO2 by using a humidified incubator for 24 h. Afterwards, ACNF and CCNF nanogels (50 μg/mL) were added into each well and incubated at 37 °C for 0.5 h. Then 100 μL of cold RPMI-1640 culture medium was added to stop the cellular uptake. NCl-H1437 cells were centrifuged at 400 × g for 5 min and the supernatant was removed. Next, the cells were treated for 5 min with Hoechst 33258 on PBS (1:1000) to stain the cell nuclei, washed once with PBS, and centrifuged at 400 × g for 5 min. Cell images were obtained in an in-verted microscope EVOS FLoid Cell Imaging Station (Life Technologies, Carlsbad, CA) at 20× magnification. Further image edition was achieved by using the ImageJ software.
3. Results and discussion
3.1. Design and characteristics of PEGylated nanogels
As previously mentioned, nanoparticle size and surface charge are two characteristics extremely important in the nanocarrier design for cellular uptake process, therefore the physicochemical properties, such as copolymer composition, Dh, PDI, ζ potential, and yield are presented in Table 1 for the synthesized nanogels. Although the reactivity ratios reported for DEAEMA: PEGMA solution polymerization are close to the unit , a strong composition drift in the CCN synthesis is estimated by 1H-RMN (Figure S1), this could be explained by the eﬀect of com-partmentalization exhibited in the SFEP polymerization, leading to an augmentation in the rate of polymerization of PEGMA , and also by a high PEG-chain steric hindrance, giving rise to a low diﬀusion of the DEAEMA molecules throughout the PEG-shell . A high content of PEG was obtained for both cationic nanogels (a dense shell), this is important to hold an eﬀective “Trojan Horse” eﬀect during the trans-portation in the bloodstream and the internalization process, since polymers of DEAEMA are toxic . The prepared CCNs showed sizes
below 111 nm with unimodal and very narrow distributions (PDI < 0.05), see Figure S2 in Supplementary material; which is ideal for drug delivery applications . Additionally, the positive surface charge of the nanoparticles increased with the higher content of DEAEMA. Thermal characteristics were also studied, (Supplementary material, Figure S3). More insights into the characteristics of this type of core-shell nanogels were reported earlier .