Designing 3D-nanosubstrates mimicking biological cell growth: pitfalls of using 2D substrates in the evaluation of anticancer efficiency.

Designing nano-substrates (NS) that support three-dimensional (3D) cell growth using physico-chemical interventions mimicking the cellular microenvironment is highly challenging. Here we report NS that assist 3D cell development (3D NS) using multi-components on a glass substrate (2D GS), which mimics the ex vivo tissue microenvironment and promotes 3D cell growth superior to conventional 2D cell culturing methodologies. 3D NS were chemically fabricated by linking the combination of advanced materials imparting different physico-chemical traits, for example, multiwalled carbon nanotubes (CNT), graphene (G), bovine serum albumin (BSA), and iron oxide magnetic nanoparticles (MNP). We compared cell-substrate interactions resulting in cellular morphological changes, influence on the cell circularity index (CI), nuclear-cytoplasmic ratios (N/C), and nuclear compression or derangements using human colorectal carcinoma cells (HCT116) and cervical cancer (HeLa) cells. We observed the increase in N/C, extended on the 3D NS micro-environment as indicative of cellular adaptation and the transformation. HCT116 and HeLa cells on 2D GS showed an N/C ratio <0.3, and 3D NS cultured cells exhibited a higher N/C ratio (>0.5). The most significant increase in the ratio, relative to arrested cell spreading, was observed with G-3D NS. Furthermore, 3D NS were evaluated for the cell viability differentiations using the anticancer drug doxorubicin (Dox). The drug-treated cells on 3D NS demonstrated far-displaced N/C ratios compared to 2D GS. In conclusion, 3D NS systems implicate an 'in vitro to in vivo' relevance for the outcome in cell biology, cell proliferation and migration, and in anticancer drug efficacy evaluation.

Correction: Designing 3D-nanosubstrates mimicking biological cell growth: pitfalls of using 2D substrates in the evaluation of anticancer efficiency.

Correction for 'Designing 3D-nanosubstrates mimicking biological cell growth: pitfalls of using 2D substrates in the evaluation of anticancer efficiency' by Ashwini Patil et al., Nanoscale, 2021, 13, 17473-17485, DOI: 10.1039/d1nr03816h.

Nanocarrier anticancer drug-conjugates cause higher cellular deformations: culpable for mischief.

Here we report nanocarrier-anticancer drug conjugates culpable for cellular deformations, critically evidenced through image-based analysis as a measure of karyoplasmic ratio (KR) and nuclear surface area (NSA). Multiwalled carbon nanotubes (MWCNTs) were coordinated additionally with Fe3O4 nanoparticles (NPs) to evaluate the symbiotic influence, and further conjugated to Dox for evaluating the cellular kinetics and for measuring cell deformations. Cellular entry kinetics of the CNT (CNT-Dox and CNT-Cys-Fe3O4-Dox) nanocarriers and their efficiency in nuclear localization were evaluated using cervical cancer (HeLa) cells. Of note, the Dox-bound nanocarriers showed significantly enhanced cell toxicity over the free form of the drug. CNT-Dox and CNT-Cys-Fe3O4-Dox influx occurred within 4 hours, while maximum cellular retention of Dox was observed for CNT-Dox at 24 h. However, the highest KR (∼0.51) was observed for CNT-Dox within 8 hours indicating similar cellular deformations using nanocarrier anticancer drug-conjugates to that of free Dox (KR ∼0.50) at 4 hours. In addition, we observed increased NSA at 4 h in Dox treatment whereas in the case of the Dox conjugated nanocarrier, increased NSA was noted at 8 h treatment. At 8 h exposure of HeLa cells with Dox conjugates, we observed that the cells fall into distinct regions of the morphospace with respect to KR and NSA. Conclusively, nano delivery systems considered for clinical and biomedical translations must take into account the possible negative influences imparting higher cellular deformations and secondary adverse effects over the free form of the drug.

A graphene-sandwiched DNA nano-system: regulation of intercalated doxorubicin for cellular localization.

Control of the sub-cellular localization of nanoparticles (NPs) with enhanced drug-loading capacity, employing graphene oxide (GO), iron oxide (Fe3O4) NPs and sandwiched deoxyribonucleic acid (DNA) bearing intercalated anticancer drug doxorubicin (DOX) has been investigated in this work. The nanosystems G-DNA-DOX-Fe3O4 and Fe3O4-DNA-DOX differentially influence serum protein binding and deliver DOX to lysosomal compartments of cervical cancer (HeLa) cells with enhanced retention. Stern-Volmer plots describing BSA adsorption on the nanosystems demonstrated the quenching constants, K sv for G-DNA-DOX-Fe3O4 and Fe3O4-DNA-DOX (0.025 mL µg-1 and 0.0103 mL µg-1 respectively). Nuclear DOX intensity, measured at 24 h, was ∼2.0 fold higher for Fe3O4-DNA-DOX in HeLa cells. Parallelly, the cytosol displayed ∼2.2 fold higher DOX intensity for Fe3O4-DNA-DOX compared to G-DNA-DOX-Fe3O4. Fe3O4-DNA-DOX was more efficacious in the cytotoxic effect than G-DNA-DOX-Fe3O4 (viability of treated cells: 33% and 49% respectively). The DNA:nanosystems demonstrated superior cytotoxicity compared to mole-equivalent free DOX administration. The results implicate DNA:DOX NPs in influencing the cellular uptake mechanism and were critically subject to cellular localization. Furthermore, cell morphology analysis evidenced maximum deformation attributed to free-DOX with 34% increased cell roundness, 63% decreased cell area and ∼1.9 times increased nuclear-to-cytoplasmic (N/C) ratio after 24 h. In the case of Fe3O4-DNA-DOX, the N/C ratio increased 1.2 times and a maximum ∼37% decrease in NSA was noted suggesting involvement of non-canonical cytotoxic pathways. In conclusion, the study makes a case for designing nanosystems with controlled and regulated sub-cellular localization to potentially exploit secondary cytotoxic pathways, in addition to optimized drug-loading for enhanced anticancer efficacy and reduced adverse effects.

Cellular regeneration and proliferation on polymeric 3D inverse-space substrates and the effect of doxorubicin.

Spatial arrangement for cells and the opportunity thereof have implications in cell regeneration and cell proliferation. 3D inverse space (3DIS) substrates with micron-sized pores are fabricated under controlled environmental conditions from polymers such as poly(lactic-co-glycolic) acid (PLGA), poly(lactic acid) (PLA) and poly(styrene) (PS). The characterization of 3DIS substrates by optical microscopy, scanning probe microscopy (SPM), etc. shows pores within 1-18 µm diameter and prominent surface roughness extending up to 3.9 nm in height over its base. Conversely, to compare two-dimensional (2D) versus 3DIS substrates, the crucial variables of cell height, cell spreading area and cell volume are compared using lung adenocarcinoma (A549) cells. The results indicate an average cell thickness of ∼6 µm on a glass substrate whereas cells on PLGA 3DIS were ∼12 µm in height, occasionally reaching 20 µm, with a 40% decreased cell spreading area. A549 cells cultured on polymer 3DIS substrates show a cell regeneration growth pattern, dependent on the available spatial volume. Furthermore, PLGA 3DIS cell culture systems with and without graded doxorubicin (DOX) pre-treatment result in potent cell inhibition and cell proliferation, respectively. Additionally, standard DOX administration to A549 cells in the PLGA 3DIS system revealed altered drug sensitivity. 3DIS demonstrates utility in facilitating cellular regeneration and mimicking cell proliferation in defined spatial arrangements.

Cell deformation and acquired drug resistance: elucidating the major influence of drug-nanocarrier delivery systems.

Cancer diagnosis and its stage-wise assessment are determined through invasive solid tissue biopsies. Conversely, cancer imaging is enriched through emission tomography and longitudinal high-resolution analysis for the early detection of cancer through altered cell morphology and cell-deformation. Similarly, in post multiple chemo-cycle exposures, the tumor regression and progression thereafter are not well understood. Here, we report chemo-cycles of doxorubicin (Dox) carrying nanoparticles (NPs) to be highly indicative of cell deformation and a progressive indicator of phenotypic expressions of acquired drug resistance (ADR). We designed graphene (G) based nanocarriers by chemically conjugating multiple components: (i) G; (ii) iron oxide (Fe3O4) NPs; and (iii) Dox through a cysteine (Cys) linker (G-Dox and G-Cys-Fe3O4-Dox). Although Dox underwent cell diffusion, the G-based nanocarriers followed a receptor-mediated endocytosis which created a profound impact on the cell membrane integrity. ADR owing to Dox and G-based nanocarriers was analyzed through a cytotoxicity assay, cell morphology deformation parameters and cellular uptake kinetic patterns. Interestingly, after the third chemo-cycle, G-Dox incubated cells showed the greatest decrease in the alteration of the nuclear surface area (NSA) of ∼28%, a ∼40% reduction of the cell surface area (CSA) and a ∼32% increase in the cell roundness (CRd). Our results suggested that the G-based nanocarriers induced the cell deformation process, subsequently resulting in ADR. Although the G-based nanocarriers initiated ADR, G-Dox was most cytotoxic to cancer cells and induced the maximum cell morphology deformation within our scope of study. This outcome implies caution is needed when using G-based nanocarriers and other multi-component nanosystems for Dox delivery as they lead to possible phenotypic expressions of drug resistance in cancer cells.