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.

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.

Water-powered self-propelled magnetic nanobot for rapid and highly efficient capture of circulating tumor cells.

Nanosized robots with self-propelling and navigating capabilities have become an exciting field of research, attributable to their autonomous motion and specific biomolecular interaction ability for bio-analysis and diagnosis. Here, we report magnesium (Mg)-Fe3O4-based Magneto-Fluorescent Nanorobot ("MFN") that can self-propel in blood without any other additives and can selectively and rapidly isolate cancer cells. The nanobots viz; Mg-Fe3O4-GSH-G4-Cy5-Tf and Mg-Fe3O4-GSH-G4-Cy5-Ab have been designed and synthesized by simple surface modifications and conjugation chemistry to assemble multiple components viz; (i) EpCAM antibody/transferrin, (ii) cyanine 5 NHS (Cy5) dye, (iii) fourth generation (G4) dendrimers for multiple conjugation and (iv) glutathione (GSH) by chemical conjugation onto one side of Mg nanoparticle. The nanobots propelled efficiently not only in simulated biological media, but also in blood samples. With continuous motion upon exposure to water and the presence of Fe3O4 shell on Mg nanoparticle for magnetic guidance, the nanobot offers major improvements in sensitivity, efficiency and speed by greatly enhancing capture of cancer cells. The nanobots showed excellent cancer cell capture efficiency of almost 100% both in serum and whole blood, especially with MCF7 breast cancer cells.

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.

Self-Propelling Targeted Magneto-Nanobots for Deep Tumor Penetration and pH-Responsive Intracellular Drug Delivery.

Self-propelling magnetic nanorobots capable of intrinsic-navigation in biological fluids with enhanced pharmacokinetics and deeper tissue penetration implicates promising strategy in targeted cancer therapy. Here, multi-component magnetic nanobot designed by chemically conjugating magnetic Fe3O4 nanoparticles (NPs), anti-epithelial cell adhesion molecule antibody (anti-EpCAM mAb) to multi-walled carbon nanotubes (CNT) loaded with an anticancer drug, doxorubicin hydrochloride (DOX) is reported. Autonomous propulsion of the nanobots and their external magnetic guidance is enabled by enriching Fe3O4 NPs with dual catalytic-magnetic functionality. The nanobots propel at high velocities even in complex biological fluids. In addition, the nanobots preferably release DOX in the intracellular lysosomal compartment of human colorectal carcinoma (HCT116) cells by the opening of Fe3O4 NP gate. Further, nanobot reduce ex vivo HCT116 tumor spheroids more efficiently than free DOX. The multicomponent nanobot's design represents a more pronounced method in targeting tumors with self-assisted anticancer drug delivery for 'far-reaching' sites in treating cancers.

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.

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.

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.

Designing Multicomponent Nanosystems for Rapid Detection of Circulating Tumor Cells.

Detection of circulating tumor cells (CTCs) in the blood circulation holds immense promise as it predicts the overall probability of patient survival. Therefore, CTC-based technologies are gaining prominence as a "liquid biopsy" for cancer diagnostics and prognostics. Here, we describe the design and synthesis of two distinct multicomponent magnetic nanosystems for rapid capture and detection of CTCs. The multifunctional Magneto-Dendrimeric Nano System (MDNS) composed of an anchoring dendrimer that is conjugated to multiple agents such as near infrared (NIR) fluorescent cyanine 5 NHS (Cy5), glutathione (GSH), transferrin (Tf), and iron oxide (Fe3O4) magnetic nanoparticle (MNP) for simultaneous tumor cell-specific affinity, multimodal high resolution confocal imaging, and cell isolation. The second nanosystem is a self-propelled microrocket that is composed of carbon nanotube (CNT), chemically conjugated with targeting ligand such as transferrin on the outer surface and Fe3O4 nanoparticles in the inner surface. The multicomponent nanosystems described here are highly efficient in targeting and isolating cancer cells thus benefiting early diagnosis and therapy of cancer.

Budding trends in integrated pest management using advanced micro- and nano-materials: Challenges and perspectives.

One of the most vital supports to sustain human life on the planet earth is the agriculture system that has been constantly challenged in terms of yield. Crop losses due to insect pest attack even after excessive use of chemical pesticides, are major concerns for humanity and environment protection. By the virtue of unique properties possessed by micro and nano-structures, their implementation in Agri-biotechnology is largely anticipated. Hence, traditional pest management strategies are now forestalling the potential of micro and nanotechnology as an effective and viable approach to alleviate problems pertaining to pest control. These technological innovations hold promise to contribute enhanced productivity by providing novel agrochemical agents and delivery systems. Application of these systems engages to achieve: i) control release of agrochemicals, ii) site-targeted delivery of active ingredients to manage specific pests, iii) reduced pesticide use, iv) detection of chemical residues, v) pesticide degradation, vi) nucleic acid delivery and vii) to mitigate post-harvest damage. Applications of micro and nano-technology are still marginal owing to the perception of low economic returns, stringent regulatory issues involving safety assessment and public awareness over their uses. In this review, we highlight the potential application of micro and nano-materials with a major focus on effective pest management strategies including safe handling of pesticides.

Self-propelled carbon nanotube based microrockets for rapid capture and isolation of circulating tumor cells.

Here, we report a non-invasive strategy for isolating cancer cells by autonomously propelled carbon nanotube (CNT) microrockets. H2O2-driven oxygen (O2) bubble-propelled microrockets were synthesized using CNT and Fe3O4 nanoparticles in the inner surface and covalently conjugating transferrin on the outer surface. Results show that self-propellant microrockets can specifically capture cancer cells.

Calcium phosphate nanocapsule crowned multiwalled carbon nanotubes for pH triggered intracellular anticancer drug release.

We report calcium phosphate (CaP) nanocapsule crowned multiwalled carbon nanotubes (CNT-GSH-G4-CaP) as a novel platform for intracellular delivery of an anticancer drug. As a proof-of-concept, CNT-GSH-G4-CaP demonstrates release of anticancer drug doxorubicin hydrochloride (DOX) within intracellular lysosomes from the interior cavity of CNT upon pH triggered CaP dissolution. Importantly, we found that the CNT with a CaP nanolid can efficiently prevent untimely drug release at physiological pH but promotes DOX release at increased acidic milieu as observed in subcellular compartments such as lysosomes (∼5.0). This "zero premature release" characteristic is of clinical significance in delivering cytotoxic drugs, by reducing systemic toxicity and thus beneficial for the effective anticancer treatment. We envision that this pH triggered CaP crowned CNT nanosystem would lead to a new generation of self-regulated platforms for intracellular delivery of a variety of anticancer drugs.

Biophysical interactions of polyamidoamine dendrimer coordinated Fe3O4 nanoparticles with insulin.

Advanced delivery systems, such as nano/micro carriers have not been studied significantly for their molecular interactions with serum proteins and other biologically relevant macromolecules. Here, we investigated the effect of surface chemistry of iron oxide (Fe3O4) nanoparticles on molecular interactions with human insulin by fluorescence, XRD and FTIR spectroscopy. Nanoparticles of Fe3O4 were chemically modified as Fe3O4-glutathione (GSH) and Fe3O4-GSH-polyamidoamine generation 4 (PAMAM G4) dendrimer. Our results demonstrate that, Fe3O4 and its conjugates such as Fe3O4-GSH, Fe3O4-GSH-G4 quenched insulin fluorescence, indicating strong interactions between insulin protein molecule and Fe3O4. The fluorescence quenching constants Ksv were obtained as 0.0367 x 10(3), 0.0303 x 10(3) and 0.0131 x 10(3) M and the binding constant K were found to be 27.095, 8.404 and 6.026 mM for Fe3O4, Fe3O4-GSH and Fe3O4-GSH-PAMAM G4, respectively. Both the Ksv and K (binding constant) values revealed that the interaction of Fe3O4 with insulin to be stronger over to dendrimer conjugates. In addition, the FTIR spectra suggested that the presence of nanoparticles results in secondary structure alteration in the insulin conformation. The study implies the critical evaluation of new delivery systems in establishing the biocompatibility, especially when delivered by systemic route.

Structure effect of carbon nanovectors in regulation of cellular responses.

Carbon nanostructures such as multiwalled carbon nanotubes (CNT) and graphene (G) are potential candidates in a large number of biomedical applications. However, there is limited understanding and connection between the physicochemical properties of diverse carbon nanostructures and biological systems, particularly with regard to cellular responses. It is also crucial to understand how the structure and surface composition of carbon nanostructures affect the cellular internalization process. Here, through in vitro cellular entry kinetics and cytotoxicity studies using MCF-7 breast cancer cells and H460 human lung cancer cells, we show that the structure and surface composition of CNT and G conjugates with various molecules such as PAMAM dendrimers (G4) and G4-poly(ethylene glycol) (PEG) are directly related to their cellular internalization ability and toxicity. Interestingly, the cellular association of CNT and G nanoconjugates was observed to be structure and surface composition dependent. We found that CNT conjugates internalized more compared to G conjugates. Furthermore, G4 conjugated CNT internalized more compared to G4-PEG conjugated CNT, whereas, higher internalization was found for G4-PEG conjugated G than G4 conjugated G. We have also correlated the cytotoxicity and cellular uptake mechanisms of CNT, G, and their conjugates through zeta potential measurements, fluorescence quenching studies and by fluorescence-activated cell sorting. Altogether these studies suggest different biological activities of the carbon nanostructures, with the shape and surface composition playing a primary role.

Poly(ethylene glycol) versus dendrimer prodrug conjugates: influence of prodrug architecture in cellular uptake and transferrin mediated targeting.

Many polymer based drug delivery nanosystems are currently being explored for delivering cytotoxic agents to the tumors. However, very few strategies delineate the comparative carrier ability of nanosystems, in similar experimental settings. As a result, it remains unclear how to optimally design polymer based multicomponent prodrug systems for delivery applications. The present study is aimed to design polymeric prodrug conjugate carriers for the comparative cellular delivery ability of anticancer drug doxorubicin hydrochloride (DOX) using linear poly(ethylene glycol) (PEG), hyperbranched poly(amido amine) (PAMAM) G4 dendrimer, and PAMAM G4 dendrimer-PEG conjugate using MCF-7 cells. Furthermore, the cellular targetability and in vitro anticancer activity of DOX conjugates is evaluated using transferrin (Tf) as a targeting ligand. Interestingly, conjugation of DOX to PAMAM G4-OH dendrimer significantly influences the cytotoxicity of DOX leading to -4 fold decrease in the IC50 dose when compared to pegylated DOX. This study establishes the rational and comparative structural activity relationship of polymeric prodrug carriers for delivery of anticancer drugs. The schematic representation of design of prodrug conjugates with varied polymeric architecures is as shown below (Fig. 1).

Transferrin-mediated rapid targeting, isolation, and detection of circulating tumor cells by multifunctional magneto-dendritic nanosystem.

A multicomponent magneto-dendritic nanosystem (MDNS) is designed for rapid tumor cell targeting, isolation, and high-resolution imaging by a facile bioconjugation approach. The highly efficient and rapid-acting MDNS provides a convenient platform for simultaneous isolation and high-resolution imaging of tumor cells, potentially leading towards an early diagnosis of cancer.

Cellular imaging using biocompatible dendrimer-functionalized graphene oxide-based fluorescent probe anchored with magnetic nanoparticles.

We describe a novel multicomponent graphene nanostructured system that is biocompatible, and has strong NIR optical absorbance and superparamagnetic properties. The fabrication of the multicomponent nanostructure system involves the covalent attachment of 3 components; Fe(3)O(4)(Fe) nanoparticles, PAMAM-G4-NH(2) (G4) dendrimer and Cy5 (Cy) on a graphene oxide (GO) surface to synthesize a biologically relevant multifunctional system. The resultant GO-G4-Fe-Cy nanosystem exhibits high dispersion in an aqueous medium, and is magnetically responsive and fluorescent. In vitro experiments provide a clear indication of successful uptake of the GO-G4-Fe-Cy nanosystem by MCF-7 breast cancer cells, and it is seen to behave as a bright and stable fluorescent marker. The study also reveals varied cellular distribution kinetics profile for the GO nanostructured system compared to free Cy. Furthermore, the newly developed GO nanostructured system is observed to be non-toxic to MDA-MB-231 cell growth, in striking contrast to free G4 dendrimer and GO-G4 conjugate. The GO-G4-Fe-Cy nanostructured system characterized by multifunctionality suggests the merits of graphene for cellular bioimaging and the delivery of bioactives.

ZnO, SiO2, and SrO doping in resorbable tricalcium phosphates: Influence on strength degradation, mechanical properties, and in vitro bone-cell material interactions.

To understand the combined effects of ZnO, SiO(2), and SrO doping on mechanical and biological properties of tricalcium phosphate (TCP) ceramics, dense ß-TCP compacts of different compositions (pure ß-TCP; 1.0 wt % SrO; 0.25 wt % ZnO; 1.0 wt % SrO + 0.5 wt % SiO(2); and 1.0 wt % SrO + 0.25 wt % ZnO) were prepared via dry pressing followed by sintering at 1250°C. X-ray diffraction of sintered compacts revealed that dopants retarded ß- to α-TCP phase transformation during sintering. Doping with SrO, SrO/SiO(2), and SrO/ZnO reduced compressive strength of the samples to 56% (173 ± 25 MPa), 57% (170 ± 15 MPa), and 47% (208 ± 72 MPa) of pure ß-TCP (396 ± 58 MPa), respectively. However, addition of ZnO resulted in only 7% (365 ± 69 MPa) strength degradation. The impact of dopants on long-term in vitro strength degradation was evaluated by soaking in simulated body fluid (SBF) for a period of 8 weeks. In all cases, excellent apatite growth was observed on doped ß-TCP samples. However, strength degradation rates were different depending on dopant chemistry and composition. Maximum degradation was observed in undoped and ZnO-doped ß-TCP samples, which degraded to 41% and 68% of the original strength before soaking in SBF. Finally, in vitro cell-materials interaction study using human fetal osteoblast cells demonstrated that addition of dopants improved cell attachment and proliferation. These results indicate that tailorable strength and strength degradation behavior can be achieved in ß-TCP via compositional modifications using small amount of dopants.

Poly(ethylene glycol)-Prodrug Conjugates: Concept, Design, and Applications.

Poly(ethylene glycol) (PEG) is the most widely used polymer in delivering anticancer drugs clinically. PEGylation (i.e., the covalent attachment of PEG) of peptides proteins, drugs, and bioactives is known to enhance the aqueous solubility of hydrophobic drugs, prolong circulation time, minimize nonspecific uptake, and achieve specific tumor targetability through the enhanced permeability and retention effect. Numerous PEG-based therapeutics have been developed, and several have received market approval. A vast amount of clinical experience has been gained which has helped to design PEG prodrug conjugates with improved therapeutic efficacy and reduced systemic toxicity. However, more efforts in designing PEG-based prodrug conjugates are anticipated. In light of this, the current paper highlights the synthetic advances in PEG prodrug conjugation methodologies with varied bioactive components of clinical relevance. In addition, this paper discusses FDA-approved PEGylated delivery systems, their intended clinical applications, and formulations under clinical trials.

Enhancing surface interactions with colon cancer cells on a transferrin-conjugated 3D nanostructured substrate.

A transferrin-conjugated PEG-Fe(3) O(4) nanostructured matrix is developed to explore cellular responses in terms of enhanced cell adhesion, specific interactions between ligands in the matrix and molecular receptors on the cell membrane, comparison of cell shapes on 2D and 3D surfaces, and effect of polymer architecture on cell adhesion. Integration of such advanced synthetic nanomaterials into a functionalized 3D matrix to control cell behavior on surfaces will have implications in nanomedicine.