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The steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera. It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found that WFA dose-dependently (within the concentration range of 0.3-1.25 μM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhesion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome analysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, suggesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and impedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologies. Ministry of Education (MOE) National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This research was supported, in part, by the Singapore Ministry of Education (MOE) under its MOE Academic Research Fund (AcRF) Tier 2 Grant (MOE2017-T2-2-004), MOEAcRF Tier 1 Grant (2020-T1-001-062) and the National Research Foundation Singapore under its Open Fund–Large Collaborative Grant (OFLCG18May-0028) and administered by the Singapore Ministry of Health’s National Medical Research Council (NMRC). W. V. B. acknowledges funding support from the Foundation Against Cancer (Grant number-7872, Belgium), Hercules Foundation (Grant numberAUHA/13/012, Belgium), and Research Foundation - Flanders (FWO, grant numbers G059713N/G079614N, Belgium).

David Hutchinson,1,2 Judith Müller,3 Joseph E McCarthy,4 Yurii K Gun’ko,4,5 Navin Kumar Verma,6 Xuezhi Bi,7 Luisana Di Cristo,8 Laura Kickham,8 Dania Movia,8 Adriele Prina-Mello,5,8 Yuri Volkov5,8,9 1Royal Cornwall Hospital NHS Trust, Treliske, 2University of Exeter Medical School Cornwall, UK; 3University of Basel, Basel, Switzerland; 4School of Chemistry, 5Advanced Materials for BioEngineering Research Centre (AMBER), Trinity College Dublin, Dublin, Ireland; 6Lee Kong Chian School of Medicine, Nanyang Technological University, 7Bioprocessing Technology Institute, A*STAR Graduate Academy, Singapore; 8Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Dublin, Ireland; 9International Laboratory of Magnetically Controlled Nanosystems for Theranostics of Oncological and Cardiovascular Diseases, ITMO University, St. Petersburg, Russia Objective: The objective of the study was to determine whether the cadmium-derived materials induce intracellular protein citrullination. Methods: Human A549 lung epithelial cells were exposed to cadmium in soluble and nanoparticulate forms represented by cadmium chloride (CdCl2) and cadmium oxide (CdO), respectively, and their combinations with ultrafine carbon black (ufCB) produced by high temperature combustion, imitating cigarette burning. Protein citrullination in cell lysates was analyzed by Western immunoblotting and verified by immunofluorescent confocal microscopy. Target citrullinated proteins were identified by proteomic analysis. Results: CdO, ufCB and its combination with CdCl2 and CdO after high temperature combustion induced protein citrullination in cultured human lung epithelial cells, as detected by immunoblotting with anti-citrullinated protein antibody. Cytokeratins of type II (1, 2, 5, 6A, 6B and 77) and type I (9, 10) were identified as major intracellular citrullination targets. Immunofluorescent staining confirmed the localization of citrullinated proteins both in the cytoplasm and cell nuclei. Conclusion: Cadmium oxide nanoparticle exposure facilitated post-translational citrullination of proteins. Keywords: cadmium, COPD, nanoparticles, cytokeratins, citrullination, autoimmunity, proteomics

Wound care management presents one of the substantial and tenacious challenges to the healthcare systems worldwide. Microbial colonization and subsequent biofilm formation after injury have garnered much attention, as there is an appreciable correlation between biofilms formation and delayed healing in chronic wounds. Nanotechnology has emerged as a potential platform for the management of treating acute and chronic wounds. This study presents the utility of electrospun nanofiber mats containing a natural extract (Gymnema sylvestre) that averts biofilm formation but supports human dermal fibroblasts (hDFs) attachment. The scaffolds exhibited good wettability, enhanced mechanical properties and contact mediated inhibition of Gram-positive and Gram-negative bacteria. MTS viability assay and confocal imaging further confirmed that the natural extract loaded mats remained non-cytotoxic for hDFs. Overall these findings evidenced the suitability of the Gymnema sylvestre (GS) functionalized electrospun poly-ε-caprolactone (PCL) nanofibers, as an effective wound dressing with broad spectrum anti-bacterial properties. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University National Medical Research Council (NMRC) National Research Foundation (NRF) The authors gratefully acknowledge the financial support from UGC-DAE Consortium for Scientific Research (CSR-KN/CRS-70/2015- 16/811) Kalpakkam, Tamilnadu, India, Translational and Clinical Research Flagship Program of the Singapore National Research Foundation (NMRC/TCR/008-SERI/2013), administered by the National Medical Research Council of the Singapore Ministry of Health. This work was supported by Co-operative Basic Research Grant from the Singapore National Medical Research Council (NMRC/CBRG/0048/ 2013) and SNEC Ophthalmic Technologies Incubator Program grant (Project no. R1181/83/2014) awarded to RL. NKV acknowledges funding support from Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Start-Up Grant (L0412290), Strategic Academic Initiative Grant (SAI-L0494003), the Ministry of Education - Singapore under its Singapore Ministry of Education Academic (AcRF) Research Fund Tier I (2015-T1-001-082) and Industry Alignment Fund Pre-Positioning Grant (IAF-PP H17/01/a0/0K9).

Designing biocompatible nanofibrous mats capable of preventing microbial colonization from resident and nosocomial bacteria for an extended period remains an unmet clinical need. In the present work, we designed antibiotic free durable antimicrobial nanofiber mats by taking advantage of synergistic interactions between polydopamine(pDA) and metal ions with varying degree of antimicrobial properties (Ag+, Mg2+, Ca2+, and Zn2+). Microscopic analysis showed successful pDA-mediated cross-linking of the gelatin nanofibers, which further improved by the inclusion of Ag+, Mg2+, and Ca2+ ions as supported by mechanical and thermal studies. Spectroscopic results reinforce the presence of strong interactions between pDA and metal ions in the composite nanofibers, leading to generation of robust polymeric nanofibers. We further showed that strong pDA–Ag interactions attenuated the cell cytotoxicity and anticell proliferative properties of silver ions for immortalized keratinocytes and primary human dermal fibroblasts. pDA–Ca2+/Zn2+ interactions rendered the composite structure sterile against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium strains, whereas the silver ion-incorporated composite mats displayed broad spectrum antibacterial activity against both Gram-positive/-negative bacteria and yeast strains. We showed that the strong pDA–Ag interactions help retaining long-term antimicrobial activity of the mats for at least 40 days while attenuating mammalian cell cytotoxicity of silver ions for skin cells. Overall, the results suggest the potential of pDA–metal ion interactions for engineering sterile nanofibrous mats and expanding the antibiotic armamentarium against drug-resistant pathogens.

Efficient intracellular nucleic acid delivery into mammalian cells remains a long-standing challenge owing to poor cell permeability and uptake of naked nucleic acids across the cell membrane and limited cargo stability. Conventional delivery methods have several drawbacks, such as cytotoxicity, limited cell-type applicability, low efficiency, hindrances that limit the potential of oligonucleotide delivery in functional genomics, therapeutics and diverse research applications. Thus, new approaches that are robust, safe, effective and valid across multiple cell types are much needed. Here, we demonstrate that GGL27, a TFPI-1-derived novel cationic host defence peptide, facilitates the delivery of nucleic acid cargo into the cytosol of a range of mammalian cells. The GGL27 peptide is non-cytotoxic and is internalized in a broad range of mammalian cell-types, including transformed cell lines and primary cells. GGL27 spontaneously forms complexes with nucleic acids of variable sizes, protects them from nuclease degradation, and delivers cargo effectively. Together, our observations demonstrate the versatile cell-penetrating property of GGL27, providing an excellent template for developing a simple, non-toxic peptide-based cytosolic delivery tool for wide use in biomedical research. Nanyang Technological University Skin Research Institute of Singapore (SRIS) This work was supported by the Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Postdoctoral Fellowship 2014 (L049201020), to RS. Authors acknowledge funding support, in part, by Start-Up Grants to N.K.V. (L0412290) and A.S., provided by Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, and the Skin Innovation Grant (SIG18015) to N.K.V. by the Skin Research Institute of Singapore.

CG-NAP, also known as AKAP450, is an anchoring/adaptor protein that streamlines signal transduction in various cell types by localizing signaling proteins and enzymes with their substrates. Great efforts are being devoted to elucidating functional roles of this protein and associated macromolecular signaling complex. Increasing understanding of pathways involved in regulating T lymphocytes suggests that CG-NAP can facilitate dynamic interactions between kinases and their substrates and thus fine-tune T cell motility and effector functions. As a result, new binding partners of CG-NAP are continually being uncovered. Here, we review recent advances in CG-NAP research, focusing on its interactions with kinases in T cells with an emphasis on the possible role of this anchoring protein as a target for therapeutic intervention in immune-mediated diseases. Published version

Chetna Dhand,1,2,* Yamini Balakrishnan,3,* Seow Theng Ong,4,* Neeraj Dwivedi,5 Jayarama R Venugopal,6 Sriram Harini,1 Chak Ming Leung,3 Kenny Zhi Wei Low,7 Xian Jun Loh,7 Roger W Beuerman,1,2 Seeram Ramakrishna,8 Navin Kumar Verma,1,4 Rajamani Lakshminarayanan1,2 1Anti-Infectives Research Group, Singapore Eye Research Institute, The Academia, Discovery Tower, Singapore; 2Ophthalmology and Visual Sciences Academic Clinical Program, Duke-NUS Graduate Medical School, Singapore; 3Department of Bioengineering, National University of Singapore, Singapore; 4Dermatology and Skin Biology, Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore; 5Department of Electrical and Computer Engineering, National University of Singapore, Singapore; 6Faculty of Industrial Sciences & Technology, Universiti Malaysia Pahang, Gambang, Malaysia; 7Department of Mechanical Engineering, Faculty of Engineering, Center for Nanofibers and Nanotechnology, National University of Singapore, Singapore; 8Soft Materials Department, Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research, Singapore *These authors contributed equally tothiswork Introduction: In search for cross-linkers with multifunctional characteristics, the present work investigated the utility of quaternaryammonium organosilane (QOS) as a potential cross-linker for electrospun collagen nanofibers. We hypothesized that the quaternary ammonium ions improve the electrospinnability by reducing the surface tension and confer antimicrobial properties, while the formation of siloxane after alkaline hydrolysis could cross-link collagen and stimulate cell proliferation. Materials and methods: QOScollagen nanofibers were electrospun by incorporating various concentrations of QOS (0.1%–10% w/w) and were cross-linked in situ after exposure to ammonium carbonate. The QOS cross-linked scaffolds were characterized and their biological properties were evaluated in terms of their biocompatibility, cellular adhesion and metabolic activity for primary human dermal fibroblasts and human fetal osteoblasts. Results and discussion: The study revealed that 1) QOS cross-linking increased the flexibility of otherwise rigid collagen nanofibers and improved the thermal stability; 2) QOS cross-linked mats displayed potent antibacterial activity and 3) the biocompatibility of the composite mats depended on the amount of QOS present in dope solution – at low QOS concentrations (0.1% w/w), the mats promoted mammalian cell proliferation and growth, whereas at higher QOS concentrations, cytotoxic effect was observed. Conclusion: This study demonstrates that QOS cross-linked mats possess anti-infective properties and confer niches for cellular growth and proliferation, thus offering a useful approach, which is important for hard and soft tissue engineering and regenerative medicine. Keywords: anti-infective wound dressing, cyto-compatible nanofibre, electrospinning, cost-effective cross-linker, tissue regeneration, antimicrobial

The cyclic depsipeptide, teixobactin, kills a number of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium tuberculosis without detectable resistance. To date, teixobactin is the only molecule in its class that has shown in vivo antibacterial efficacy. In this work, we designed and synthesized 10 new in vivo ready teixobactin analogues. These analogues showed highly potent antibacterial activities against Staphylococcus aureus, MRSA, and vancomycin-resistant enterococci (VRE) in vitro. One analogue, d-Arg4-Leu10-teixobactin, 2, was found to be noncytotoxic in vitro and in vivo. Moreover, topical instillation of peptide 2 in a mouse model of S. aureus keratitis decreased the bacterial bioburden (>99.0% reduction) and corneal edema significantly as compared to untreated mouse corneas. Collectively, our results have established the high therapeutic potential of a teixobactin analogue in attenuating bacterial infections and associated severities in vivo. MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Accepted version

The development of new organometallic compounds as anticancer agents is currently an active area of research. Here, we report the design, synthesis and characterization of a panel of 10 new ferrocenyl–phosphine derivatives (FD1–FD10) and the analysis of their anti-proliferative activities in hematolymphoid cells representing non-Hodgkin cutaneous T-cell lymphoma (CTCL). The gold-coordinated ferrocenyl–phosphine complex FD10 exhibited a significant and dose-dependent cytotoxicity in 4 different CTCL cell lines – HuT78, HH, MJ and MyLa. FD10 concentrations causing 50% cell growth inhibition (IC50) of HuT78, HH, MJ and MyLa cells at 24 h were recorded to be 5.55 ± 0.20, 7.80 ± 0.09, 3.16 ± 0.10 and 6.46 ± 0.24 μM respectively. Further mechanistic studies showed that FD10 induced apoptosis in CTCL cells by an intrinsic pathway mediated via the activation of caspase-3 and poly(ADP-ribose)polymerase. It suppressed the expression and activity of STAT3 oncoprotein in CTCL cells. FD10 caused robust G0/G1 phase cell cycle arrest and reduced the expression levels of Akt S473 phosphorylation and c-Myc, both are key cell cycle regulator proteins. Taken together, this study highlights anticancer properties of the ferrocenyl–phosphine gold organometallic complex FD10 and suggests that further development of this novel class of molecule may contribute to new drug discovery for certain hematolymphoid malignancies. MOE (Min. of Education, S’pore) Published version

Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48 nm), which shortened during reticulocyte maturation (average spectrin length of 41 nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes. ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Accepted version

Post-transcriptional gene silencing holds great promise in discovery research for addressing intricate biological questions and as therapeutics. While various gene silencing approaches, such as siRNA and CRISPR-Cas9 techniques, are available, these cannot be effectively applied to “hard-to-transfect” primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called “GapmeR”, is an emerging new class of gene silencing molecule. Here, we show that GapmeR internalizes into human primary T-cells through macropinocytosis. Internalized GapmeR molecules can associate with SNX5-positive macropinosomes in T-cells, as detected by super-resolution microscopy. Utilizing the intrinsic self-internalizing capability of GapmeR, we demonstrate significant and specific depletion (>70%) of the expression of 5 different endogenous proteins with varying molecular weights (18 kDa Stathmin, 80 kDa PKCε, 180 kDa CD11a, 220 kDa Talin1 and 450 kDa CG-NAP/AKAP450) in human primary and cultured T-cells. Further functional analysis confirms CG-NAP and Stathmin as regulators of T-cell motility. Thus, in addition to screening, identifying or verifying critical roles of various proteins in T-cell functioning, this study provides novel opportunities to silence individual or multiple genes in a subset of purified human primary T-cells that would be exploited as future therapeutics.

In this study, we report that the integrin LFA-1 cross-linking with its ligand ICAM-1 in human PBMCs or CD4+ T cells promotes Th1 polarization by upregulating IFN-γ secretion and T-bet expression. LFA-1 stimulation in PBMCs, CD4+ T cells, or the T cell line HuT78 activates the Notch pathway by nuclear translocation of cleaved Notch1 intracellular domain (NICD) and upregulation of target molecules Hey1 and Hes1. Blocking LFA-1 by a neutralizing Ab or specific inhibition of Notch1 by a γ-secretase inhibitor substantially inhibits LFA-1/ICAM-1–mediated activation of Notch signaling. We further demonstrate that the Notch pathway activation is dependent on LFA-1/ICAM-1–induced inactivation of glycogen synthase kinase 3β (GSK3β), which is mediated via Akt and ERK. Furthermore, in silico analysis in combination with coimmunoprecipitation assays show an interaction between NICD and GSK3β. Thus, there exists a molecular cross-talk between LFA-1 and Notch1 through the Akt/ERK–GSK3β signaling axis that ultimately enhances T cell differentiation toward Th1. Although clinical use of LFA-1 antagonists is limited by toxicity related to immunosuppression, these findings support the concept that Notch inhibitors could be attractive for prevention or treatment of Th1-related immunologic disorders and have implications at the level of local inflammatory responses.

Effects of oligotryptophan end-tagging on the uptake of arginine-rich peptides into melanoma cells was investigated under various conditions and compared to that into non-malignant keratinocytes, fibroblasts, and erythrocytes, also monitoring resulting cell toxicity. In parallel, biophysical studies on peptide binding to, and destabilization of, model lipid membranes provided mechanistic insight into the origin of the selectivity between melanoma and non-malignant cells. Collectively, the results demonstrate that W-tagging represents a powerful way to increase selective peptide internalization in melanoma cells, resulting in toxicity against these, but not against the non-malignant cells. These effects were shown to be due to increased peptide adsorption to the outer membrane in melanoma cells, caused by the presence of anionic lipids such as phosphatidylserine and ganglioside GM1, and to peptide effects on mitochondria membranes and resulting apoptosis. In addition, the possibility of using W-tagged peptides for targeted uptake of nanoparticles/drug carriers in melanoma was demonstrated, as was the possibility to open up the outer membrane of melanoma cells in order to facilitate uptake of low Mw anticancer drugs, here demonstrated for doxorubicin.

A new drug concentration meter is developed. In vivo drug release can be monitored precisely via a self-indicating drug delivery system consisting of a new aggregation-induced emission thermoresponsive hydrogel. By taking the advantage of a self-indicating system, one can easily detect the depletion of drugs, and reinject to maintain a dosage in the optimal therapeutic window. ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) Accepted version

An international symposium for nanosafety was held recently at the Nanyang Technological University in Singapore. Topics relating to understanding nanomaterial properties, tools, and infrastructure required for predicting hazardous outcomes, measuring nanomaterial exposure levels, systems approach for risk assessment and public’s perception of nanotechnology were covered. The need for a multidisciplinary approach, across both natural and social sciences, for developing sustainable nanotechnology solutions was heavily emphasized. This commentary highlights the major issues discussed and the commitment of the nanosafety research community in Singapore to contribute collectively to realise the vision of sustainable nanotechnology.

Rab GTPases control membrane traffic and receptor-mediated endocytosis. Within this context, Rab5a plays an important role in the spatial regulation of intracellular transport and signal transduction processes. Here, we report a previously uncharacterized role for Rab5a in the regulation of T-cell motility. We show that Rab5a physically associates with protein kinase C ε (PKC ε ) in migrating T-cells. Following stimulation of T- cells through the integrin LFA-1 or the chemokine receptor CXCR4, Rab5a is phosphorylated on a N-terminal T7 site by PKC ε . Both Rab5a and PKC ε dynamically interact at the centrosomal region of migrating cells, and PKC ε -mediated phosphorylation on T7 regulates Rab5a trafficking to the cell leading edge. Further, we demonstrate that Rab5a T7 phosphorylation is functionally necessary for Rac1 activation, actin rearrangement and T- cell motility. We present a novel mechanism by which a PKC ε -Rab5a-Rac1 axis regulates cytoskeleton remodelling and T-cell migration, both of which are central for the adaptive immune response. Accepted version

Although recent decades have seen an improved cure rate for newly diagnosed paediatric acute lymphoplastic leukaemia (ALL), the treatment options for adult ALL, T-cell ALL (T-ALL) and relapsed disease remain poor. We have developed a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds and established their anticancer efficacy in a variety of human tumour cell types. Here, we demonstrate that PBOX-15 inhibits cell growth, and induces G2/M cell cycle arrest and apoptosis in both T-ALL and B-cell ALL (B-ALL) cells. In addition, prior to PBOX-15-induced apoptosis, PBOX-15 decreases ALL cell adhesion, spreading and migration. Concurrently, PBOX-15 differentially down-regulates β1-, β2- and α4-integrin expression in ALL cells and significantly decreases integrin-mediated cell attachment. PBOX-15 interferes with the lateral mobility and clustering of integrins in both B-ALL and T-ALL cells. These data suggest that PBOX-15 is not only effective in inducing apoptosis in ALL cells, but also has the potential to disrupt integrin-mediated adhesion of malignant lymphocytes, which represents a novel avenue for regulating leukaemic cell homing and migration.

Background Nanomaterials such as SiO2 nanoparticles (SiO2NP) are finding increasing applications in the biomedical and biotechnological fields such as disease diagnostics, imaging, drug delivery, food, cosmetics and biosensors development. Thus, a mechanistic and systematic evaluation of the potential biological and toxic effects of SiO2NP becomes crucial in order to assess their complete safe applicability limits. Results In this study, human monocytic leukemia cell line THP-1 and human alveolar epithelial cell line A549 were exposed to a range of amorphous SiO2NP of various sizes and concentrations (0.01, 0.1 and 0.5 mg/ml). Key biological indicators of cellular functions including cell population density, cellular morphology, membrane permeability, lysosomal mass/pH and activation of transcription factor-2 (ATF-2) were evaluated utilizing quantitative high content screening (HCS) approach and biochemical techniques. Despite the use of extremely high nanoparticle concentrations, our findings showed a low degree of cytotoxicity within the panel of SiO2NP investigated. However, at these concentrations, we observed the onset of stress-related cellular response induced by SiO2NP. Interestingly, cells exposed to alumina-coated SiO2NP showed low level, and in some cases complete absence, of stress response and this was consistent up to the highest dose of 0.5 mg/ml. Conclusions The present study demonstrates and highlights the importance of subtle biological changes downstream of primary membrane and endocytosis-associated phenomena resulting from high dose SiO2NP exposure. Increased activation of transcription factors, such as ATF-2, was quantitatively assessed as a function of i) human cell line specific stress-response, ii) SiO2NP size and iii) concentration. Despite the low level of cytotoxicity detected for the amorphous SiO2NP investigated, these findings prompt an in-depth focus for future SiO2NP-cell/tissue investigations based on the combined analysis of more subtle signalling pathways associated with accumulation mechanisms, which is essential for establishing the bio-safety of existing and new nanomaterials.

Background Aerosolized therapeutics hold great potential for effective treatment of various diseases including lung cancer. In this context, there is an urgent need to develop novel nanocarriers suitable for drug delivery by nebulization. To address this need, we synthesized and characterized a biocompatible drug delivery vehicle following surface coating of Fe3O4 magnetic nanoparticles (MNPs) with a polymer poly(lactic-co-glycolic acid) (PLGA). The polymeric shell of these engineered nanoparticles was loaded with a potential anti-cancer drug quercetin and their suitability for targeting lung cancer cells via nebulization was evaluated. Results Average particle size of the developed MNPs and PLGA-MNPs as measured by electron microscopy was 9.6 and 53.2 nm, whereas their hydrodynamic swelling as determined using dynamic light scattering was 54.3 nm and 293.4 nm respectively. Utilizing a series of standardized biological tests incorporating a cell-based automated image acquisition and analysis procedure in combination with real-time impedance sensing, we confirmed that the developed MNP-based nanocarrier system was biocompatible, as no cytotoxicity was observed when up to 100 μg/ml PLGA-MNP was applied to the cultured human lung epithelial cells. Moreover, the PLGA-MNP preparation was well-tolerated in vivo in mice when applied intranasally as measured by glutathione and IL-6 secretion assays after 1, 4, or 7 days post-treatment. To imitate aerosol formation for drug delivery to the lungs, we applied quercitin loaded PLGA-MNPs to the human lung carcinoma cell line A549 following a single round of nebulization. The drug-loaded PLGA-MNPs significantly reduced the number of viable A549 cells, which was comparable when applied either by nebulization or by direct pipetting. Conclusion We have developed a magnetic core-shell nanoparticle-based nanocarrier system and evaluated the feasibility of its drug delivery capability via aerosol administration. This study has implications for targeted delivery of therapeutics and poorly soluble medicinal compounds via inhalation route.