Biology subjects

Kostarelos, K.

Publications and source records attributed to Kostarelos, K..

18 recordsLinked to original sources

bioRxiv2026

Plasma membrane-associated graphene oxide as aplatform for modulating signalling through cell-surfacereceptors: an integrin-focused proof-of-concept study

Graphene oxide (GO) has primarily been investigated as a carrier for intracellular delivery of therapeutic molecules. In previous work, we identified a cell type-dependent interaction pattern in which GO remained predominantly associated with the plasma membrane of cancer cells but was internalised by non-cancerous epithelial cells. Here, we explored whether plasma membrane-associated GO can be used as a platform to present bioactive ligands and influence cell-surface receptor signalling in cancer cells. To test this hypothesis, we targeted integrin receptors at the plasma membrane in glioblastoma cell models using an RGD-containing peptide non-covalently complexed with GO. We assessed GO-peptide interactions, cellular interactions/uptake, motility, and focal adhesion signalling readouts. Peptide association was quantified using a 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay, and GO was characterised by atomic force microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and colloidal measurements. Immediately after complexation, ~70% of RGD was associated with GO. Peptide association increased the nitrogen signal and shifted the principal GO XRD peak while retaining nanosheet morphology. Biological responses were examined in U87 and U251 glioblastoma cells with different integrin-positive fractions, and in non-cancerous BEAS-2B bronchial epithelial cells. Confocal microscopy showed that GO and GO:RGD remained predominantly localised on the plasma membrane in U87 and U251 cells, whereas greater intracellular localisation was observed in BEAS-2B cells. Importantly, GO:RGD significantly reduced key indicators of cell motility: cell velocity in U87 and U251 cells, with trajectory and mean-square-displacement analyses supporting restricted cellular movement. Free RGD had no significant effect, while GO alone produced a smaller reduction in motility only in U251 cells. No treatment significantly altered BEAS-2B motility. Flow cytometry also showed a reduced pFAK-associated signal in GO:RGD-treated U87 cells. These findings establish a proof of concept that the cell-line-dependent plasma membrane localisation of GO can be exploited as a membrane-associated nano-bio interface for cell-surface-active ligands, opening the way for the development of GO-based platforms that modulate receptor-mediated signalling and cell behaviour.

cell biology↗

bioRxiv2025

An artefact-resilient wide bandwidth bidirectional graphene neural interface

The ability to simultaneously record and modulate neural activity is critical for next-generation bidirectional neural interfaces aiming to enable adaptive neuromodulation therapies for neurological disorders. Active graphene transistor technologies are particularly promising for neural recordings, as they extend the ability to monitor brain activity at very low frequencies and support multiplexed operation for high-density neural interfaces. However, their limited charge injection capacity makes them unsuitable for stimulation. In this work, we present a bidirectional neural interface that combines nanoporous reduced graphene oxide (rGO) microelectrodes for high charge injection focal stimulation and graphene solution-gated field-effect transistors (gSGFETs) for brain activity monitoring, exploiting the advantages of both technologies in one single device. Using scalable cleanroom microfabrication techniques, we monolithically integrate these two graphene-based technologies into fully flexible probes. We evaluate the performance of the hybrid devices both in saline and in vivo, with a particular focus on transistor performance during stimulation. Our results demonstrate that the recording capability of this bidirectional neural interface, including the monitoring of infraslow and local field potential activity, is not compromised during stimulation. This work highlights the potential of this hybrid neural interface for both basic neurophysiological and clinical translation use.

neuroscience↗

bioRxiv2025

Graphene Micro-transistor Arrays Reveal Perfusion-Dependent Electrophysiological and Haemodynamic Signatures of Cortical Spreading Depolarizations in Ischaemic Stroke

Cortical spreading depolarizations (CSDs) are large-scale disruptions of neuronal homeostasis that contribute to secondary injury in ischaemic stroke. In healthy cortex, CSDs induce vasodilation to meet metabolic demand, whereas in ischaemic tissue, they can provoke vasoconstriction and sustained hypoperfusion, exacerbating damage. We present a multimodal neurotechnology platform integrating flexible, transparent graphene solution-gated field-effect transistor (gSGFET) arrays with laser speckle contrast imaging (LSCI), enabling simultaneous DC-coupled electrophysiological recordings and cerebral blood flow imaging with high spatiotemporal fidelity. gSGFETs enable stable, distortion-free recording of infraslow potentials essential for resolving CSD waveforms in vivo. In two murine stroke models, we show that CSD waveform duration and morphology scale with local perfusion, delineating electrophysiological subtypes predictive of tissue viability and haemodynamic response. In metabolically compromised cortex, CSDs exhibit double peaks or negative ultraslow components that result in vasoconstriction, contrasting with narrow, monophasic waveforms eliciting vasodilation in healthy regions. Systemic ketamine shortens CSD duration and converts vasoconstriction to vasodilation in perfusion-deficit tissue, revealing a mechanism for its neuroprotective action. This platform enables high-fidelity investigation of brain-blood flow interactions in vivo.

neuroscience↗

bioRxiv2025

Targeting therapeutic nanoparticles to the glioblastoma resection margin by harnessing post-operative spatiotemporal blood-brain barrier disruption

Resection surgery is the first-line therapy for glioblastoma (GBM) that is performed in >70% of patients, typically within days of suspected diagnosis. Current protocols for follow-on chemoradiotherapy have shown only modest efficacy in eliminating residual disease, leading to inevitable tumour recurrence. There remains a need for new approaches to swiftly and effectively treat post-operative residual disease to prevent the rapid early progression of recurrent GBM. Using syngeneic preclinical models of glioblastoma resection, we identified a spatially and temporally restricted window of blood brain barrier (BBB) disruption localised to the resection margin, during the immediate (15 min) and early (48-72h) postoperative periods. Intravenous administration of fluorescently labelled, clinically-used liposome nanoparticles during these periods demonstrated that selective accumulation at the postoperative resection margin, while largely being excluded from areas of the brain with an intact BBB, could be achieved. Confocal analysis confirmed the presence of extravasated nanoparticles within the margin parenchyma which largely interacted with microglial populations closely associated with residual tumour cells. Exploiting this, we performed intravenous administration of doxorubicin-loaded liposomes (DOX-Lipo) coinciding with the peak of postoperative BBB disruption and demonstrated both enhanced chemotherapy delivery and consequently complete inhibition of tumour recurrence from a single administration. Overall, this work underscores the importance of timing concomitant chemotherapy to the post-operative timeframe and demonstrates that clinically-used liposomal nanomedicines could be readily repurposed for early post-operative therapy in aggressive brain tumours.

pharmacology and toxicology↗

bioRxiv2024

Interplay between material properties and cellular effects drives distinct pattern of interaction of graphene oxide with cancer and non-cancer cells

Understanding how graphene oxide (GO) interacts with cells is crucial for its safe and efficient biomedical applications. Despite extensive research, a systematic investigation using a panel of cell lines, thoroughly characterized label-free nanomaterials, and complementary analytical techniques is lacking. Here, we examined the uptake of thin GO sheets with distinct lateral dimensions in 13 cell lines: 8 cancer (HeLa, A549, PC3, DU-145, LNCaP, SW-480, SH-SY5Y, U87-MG) and 5 non-cancer (BEAS-2B, NIH/3T3, PNT-2, HaCaT, 293T), using confocal microscopy, transmission electron microscopy, and flow cytometry. Our results reveal a striking difference in GO uptake: non-cancer cells internalized GO efficiently, while in cancer cells, GO predominantly interacted with the plasma membrane, showing minimal to no internalization. Comparison to other nanomaterials (polystyrene beads and graphene flakes) confirmed that cancer cells internalize materials similarly to non-cancer cells, indicating GO-specific interactions. We identified that GO thickness plays a pivotal role in this differential uptake. Additionally, GO disrupted the actin cytoskeleton, impairing migration in cancer but not in non-cancer cells. We propose that thin GO sheets act as a cue upon interaction with the plasma membrane of cancer cell lines, subsequently inducing actin filaments disruption leading to impaired endocytosis, migration activity, and reduced capacity of cancer cells towards GO uptake.

cell biology↗

bioRxiv2024

Flexible graphene-based neurotechnology for high-precision deep brain mapping and neuromodulation in Parkinsonian rats

Deep brain stimulation (DBS) is a neuroelectronic therapy for the treatment of a broad range of neurological disorders, including Parkinsons disease. Current DBS technologies face important limitations that impact their efficacy, such as large electrode size, invasiveness, and lack of adaptive therapy based on biomarker monitoring. The use of novel electrode materials is expected to contribute to overcome these limitations. In a previous study, we reported that nanoporous reduced graphene oxide (rGO) is a promising electrode material due to its high charge injection capacity and low impedance. Here, we investigate the potential benefits of using the rGO technology in DBS. To this end, we implant a flexible high-density array of rGO microelectrodes in the subthalamic nucleus (STN) of healthy and hemi-parkinsonian rats to investigate specific electrophysiological Parkinsonian biomarkers and to assess the effect of microscale stimulation. We demonstrate that these microelectrodes record action potentials with high signal-to-noise ratios (SNR > 6), allowing the precise localization of deep brain structures like the STN, and the tracking of multiunit-based biomarkers such as STN bursts. The bidirectional capability to deliver high-density focal stimulation and to record high-fidelity signals unlocks the visualization of the local neuromodulation of the multiunit biomarker. These findings demonstrate the potential of bidirectional high-resolution neural interfaces to investigate the mechanisms around DBS in preclinical models and suggest new avenues for the use of adaptive closed-loop operation based on electrophysiological biomarkers monitoring.

bioengineering↗

bioRxiv2023

Engineering the glioblastoma microenvironment using TLR7/8 agonist-complexed graphene oxide nanosheets

The glioblastoma (GBM) microenvironment is characterised as immunologically cold, with immunosuppressive components that compromise the efficacy of current immunotherapies. Tumour associated macrophages and microglia (TAMMs) that are activated towards an immunosuppressive, pro-tumoral state have been identified as major contributing factors to the coldness of GBM, while further promoting tumour progression and resistance to therapy. Based on this understanding, strategies such as macrophage reprogramming have been explored but have so far been limited by poor delivery and retention of reprogramming agents to the target cell populations within the GBM microenvironment. Consequently, clinical efficacy of such approaches has thus far shown limited success. Two-dimensional, graphene oxide (GO) nanosheets have been demonstrated to spread readily throughout the entire tumour microenvironment following a single intratumoral injection, interacting primarily with TAMMs. The current study aimed to investigate whether the immunosuppressive character of TAMMs in GBM can be ameliorated using GO sheets as a vector system to selectively deliver a TLR7/8 agonist (Resiquimod, R848), into these populations. GO enhanced the activity of R848 and induced the expression of M1-like markers on bone marrow derived macrophages in vitro. Using multi-parameter flow cytometry and histological analysis in a syngeneic, orthotopic mouse model of GBM, we observed that a single intratumoral injection of GO:R848 complex significantly elevated the proportion of macrophages and microglia expressing MHCII, TNF and CD86 (associated with a pro-inflammatory, anti-tumoral state), while downregulating their expression of the M2 markers ARG1 and YM1 (associated with an anti-inflammatory, pro-tumoral state). This local complex administration inhibited tumour progression and significantly reduced tumour burden. These data illustrate that immunomodulatory GO nanosheets can effectively alter the immune landscape of GBM and modulate the wider GBM microenvironment. ToC Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/558196v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@199bdb0org.highwire.dtl.DTLVardef@7473c3org.highwire.dtl.DTLVardef@15c14c6org.highwire.dtl.DTLVardef@57fdeb_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Engineering of a graphene oxide-based two-dimensional platform for immune activation and modulation

Nanoscale-based tools for immunomodulation are expected to offer a rich battery of options for more targeted and safer approaches to achieve clinically effective manipulation of the local and systemic immune environment. In this study, we aimed to design nanoscale constructs based on graphene oxide (GO) nanosheets as platform carriers for the TLR7/8 agonist Resiquimod (R848). The non-covalent complexation of R848 molecules on the GO surface resulted in stable complexes by preserving their biological activity. The physicochemical properties, molecular quantification, as well as the overall performance of the complex were systematically investigated. We hypothesized the formation of GO:drug nano-constructs with strong colloidal stability over time, due to the strong {pi}-{pi} interactions between the R848 molecules and the GO surface, and identified that R848 loading efficiency consistently ranged around 75% (of starting molecules), quantified by HPLC and UV-Vis. The 2D morphology of the thin nanosheets was retained after complexation, determined by various (AFM and SEM) microscopic techniques. Based on the surface physicochemical characterization of the complexes by Raman, FTIR, XPS, and XRD, the formation of non-covalent interactions among the GO surface and the R848 molecules was confirmed. Most importantly, GO:R848 complexes did not compromise the biological activity of R848, and effectively activated macrophages in vitro. Collectively, this study demonstrates that thin GO sheets can act as platforms for the non-covalent association with small TLR7/8 agonist molecules, forming stable and highly reproducible complexes, that could be exploited as effective immunomodulatory agents.

bioengineering↗

bioRxiv2023

Functioning human lung organoids model pulmonary tissue response from carbon nanomaterial exposures

Human lung organoids (HLOs) are increasingly used to model development and infectious diseases, however their ability to recapitulate functional pulmonary tissue response to nanomaterial (NM) exposures has yet to be demonstrated. Here, we established a lung organoid exposure model that utilises microinjection to present NMs into the lumen of organoids. Our model assures efficient, reproducible and controllable exposure of the apical pulmonary epithelium, emulating real-life human exposure scenario. By comparing the impact of two well studied carbon-based NMs, graphene oxide sheets (GO) and multi-walled carbon nanotubes (MWCNT), we validated lung organoids as tools for predicting pulmonary NM-driven responses. In agreement with established in vivo data, we demonstrate that MWCNT, but not GO, elicit adverse effects on lung organoids, leading to a pro-fibrotic phenotype. Our findings reveal the capacity and suitability of HLOs for hazard assessment of NMs, aligned with the much sought-out 3Rs (animal research replacement, reduction, refinement) framework.

pharmacology and toxicology↗

bioRxiv2022

Graphene-based thin film microelectrode technology for in vivo high resolution neural recording and stimulation

Neuroprosthetic technology aims to restore nervous system functionality in cases of severe damage or degeneration by recording and stimulating the electrical activity of the neural tissue. One of the key factors determining the quality of the neuroprostheses is the electrode material used to establish electrical communication with the neural tissue, which is subject to strict electrical, electrochemical, and mechanical specifications as well as biological and microfabrication compatibility requirements. This work presents a nanoporous graphene-based thin film technology and its engineering to form flexible neural implants. Bench measurements show that the developed microelectrodes offer low impedance and high charge injection capacity throughout millions of pulses. In vivo electrode performance was assessed in rodents both from brain surface and intracortically showing high-fidelity recording performance, while stimulation performance was assessed with an intrafascicular implant that demonstrated low current thresholds and high selectivity for activating subsets of axons within the sciatic nerve. Furthermore, the tissue biocompatibility of the devices was validated by chronic epicortical and intraneural implantation. Overall, this works describes a novel graphene-based thin film microelectrode technology and demonstrates its potential for high-precision neural interfacing in both recording and stimulation applications.

bioengineering↗

bioRxiv2021

Full bandwidth electrophysiology of seizures and epileptiform activity enabled by flexible graphene micro-transistor depth neural probes

Mapping the entire frequency bandwidth of neuronal oscillations in the brain is of paramount importance for understanding physiological and pathological states. The ability to record simultaneously infraslow activity (<0.1 Hz) and higher frequencies (0.1-600 Hz) using the same recording electrode would particularly benefit epilepsy research. However, commonly used metal microelectrode technology is not well suited for recording infraslow activity. Here we use flexible graphene depth neural probes (gDNP), consisting of a linear array of graphene microtransistors, to concurrently record infraslow and high frequency neuronal activity in awake rodents. We show that gDNPs can reliably record and map with high spatial resolution seizures, post-ictal spreading depolarisation, and high frequency epileptic activity through cortical laminae to the CA1 layer of the hippocampus in a mouse model of chemically-induced seizures. We demonstrate functionality of chronically implanted devices over 10 weeks by recording with high fidelity spontaneous spike-wave discharges and associated infraslow activity in a rat model of absence epilepsy. Altogether, our work highlights the suitability of this technology for in vivo electrophysiology research, in particular, to examine the contributions of infraslow activity to seizure initiation and termination.

neuroscience↗

bioRxiv2019

Dynamic interactions and intracellular fate of label-free GO within mammalian cells: role of lateral sheet size

Graphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is essential for successful clinical translation. In this study, we exploit intrinsic fluorescent properties of thin GO sheets to establish the relationship between lateral dimensions of the material, its cellular uptake mechanisms and intracellular fate over time. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO) were thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was initially interrogated using a range of pharmaceutical inhibitors and validated using polystyrene beads of different diameters (0.1 and 1 m). Subsequently, RNA-Seq was used to follow the changes in the uptake mechanism used to internalize s-GO flakes over time. Regardless of lateral dimensions, both types of GO were found to interact with the plasma membrane and to be internalized by a panel of cell lines studied. However, s-GO was internalized mainly via macropinocytosis while us-GO was mainly internalized via clathrin- and caveolae-mediated endocytosis. Importantly, we report the shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of s-GO at 24 h, mediated by upregulation of mTORC1/2 pathway. Finally, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results offer an insight into the mechanism of interaction of GO with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems.

cell biology↗

bioRxiv2019

Transient Reprogramming of Neonatal Cardiomyocytes to a Proliferative Dedifferentiated State

Zebrafish and urodele amphibians are capable of extraordinary myocardial regeneration thanks to the ability of their cardiomyocytes to undergo transient dedifferentiation and proliferation. Somatic cells can be temporarily reprogrammed to a proliferative, dedifferentiated state through transient expression of Oct3/4, Sox2, Klf4 and c-Myc (OSKM) transcription factors. Here, we utilized an OSKM-encoding non-integrating vector to induce transient reprogramming of mammalian cardiomyocytes in vitro. Reprogramming factor expression in neonatal rat cardiomyocytes triggered rapid cell dedifferentiation characterized by downregulation of cardiomyocyte specific gene and protein expression, sarcomere dis-assembly and loss of autorhythmic contractile activity. Concomitantly, a significant increase in cell cycle related gene expression and Ki67 positive cells was observed, indicating that dedifferentiated cardiomyocytes possess an enhanced proliferative capacity. A small proportion of cardiomyocytes progressed through mesenchymal to epithelial transition, further indicating the initiation of cell reprogramming. However, complete reprogramming to a pluripotent-like state was not achieved for the duration of the study (20 days), both in standard and embryonic stem cell culture media conditions. The transient nature of this partial reprogramming response was confirmed as cardiomyocyte-specific cell morphology, gene expression and contractile activity were recovered by day 15 after viral transduction. Further investigations into the complete downstream biological effects of ectopic OSKM expression in cardiomyocytes and the fate of these reprogrammed cells are warranted. Our results to date suggest that transient reprogramming could be a feasible strategy to recapitulate regenerative mechanisms of lower vertebrates and inform direct gene therapy approaches to cardiac regenerative medicine.

cell biology↗

bioRxiv2019

Selective Liposomal Transport Through Blood Brain Barrier Disruption in Ischaemic Stroke Reveals Two Distinct Therapeutic Opportunities

The development of new therapies for stroke continues to face repeated translational failures. Brain endothelial cells form paracellular and transcellular barriers to many blood-borne therapies and the development of efficient delivery strategies is highly warranted. Here, in a mouse model of stroke, we show selective recruitment of clinically used liposomes into the ischaemic brain that correlates with biphasic blood brain barrier (BBB) breakdown. Intravenous administration of liposomes into mice exposed to transient middle cerebral artery occlusion took place at early (0.5h and 4h) and delayed (24h and 48h) timepoints, covering different phases of BBB disruption after stroke. Using a combination of in vivo real-time imaging and histological analysis we show that selective liposomal brain accumulation coincides with biphasic enhancement in transcellular transport followed by a delayed impairment to the paracellular barrier. This process precedes neurological damage in the acute phase and maintains long-term liposomal co-localisation within the neurovascular unit, which could have great potential for neuroprotection. Levels of liposomal uptake by glial cells are similarly selectively enhanced in the ischaemic region late after experimental stroke (2-3 days), highlighting their potential for blocking delayed inflammatory responses or shifting the polarization of microglia/macrophages towards brain repair.\n\nThese findings demonstrate the capability of liposomes to maximise selective translocation into the brain after stroke and identify for the first time two windows for therapeutic manipulation. This emphasizes the benefits of selective drug delivery for efficient tailoring of new stroke treatments.

neuroscience↗

bioRxiv2018

Graphene Oxide as 2D Platform for Complexation and Intracellular Delivery of siRNA

The development of efficient and safe nucleic acid delivery vectors remains an unmet need holding back translation of gene therapy approaches into bedside. Graphene oxide (GO) could help bypass such bottleneck thanks to its large surface area, versatile chemistry and biocompatibility, which could overall enhance transfection efficiency while abolishing some of the limitations linked to the use of viral vectors. Here, we aimed to assess the capacity of bare GO, without any further surface modification, to complex a short double-stranded nucleic acid of biological relevance (siRNA) and mediate its intracellular delivery. GO formed stable complexes with siRNA at 10:1, 20:1 and 50:1 GO:siRNA mass ratios. Complexation was further corroborated by atomistic molecular dynamics simulations. GO:siRNA complexes were promptly internalized in a primary mouse cell culture, as early as 4 h after exposure. At this time point, intracellular siRNA levels were comparable to those provided by a lipid-based transfection reagent that achieved significant gene silencing. Time-lapse tracking of internalized GO and siRNA evidenced a sharp decrease of intracellular siRNA from 4 to 12 h, while GO was sequestered in large vesicles, which may explain the lack of biological effect (i.e. gene silencing) achieved by GO:siRNA complexes. This study underlines the potential of non-surface modified GO flakes to act as 2D siRNA delivery platforms, without the need for cationic functionalization, but warrants further vector optimization to allow effective release of the nucleic acid and achieve efficient gene silencing.

bioengineering↗

bioRxiv2018

Exposure to graphene oxide sheets alters the expression of reference genes used for real-time RT-qPCR normalization

Backgroundstudies that unravel the interactions between thin, 2D graphene oxide (GO) sheets and the biological milieu, including cells and tissues, are multiplying quickly as the biomedical applications of those and other 2D materials continue to be explored. Many of such studies rely on real-time RT-qPCR as a powerful, yet relatively simple technique to determine gene expression. However, a systematic investigation of potential GO-induced changes in the expression of reference genes, crucial for appropriate normalization of qPCR data that ensures reliability of the results, is still lacking. In this study, we aimed to cover this gap by investigating the stability of the expression of ten (10) candidate reference genes upon exposure to increasing, but subtoxic, concentrations of GO, with two established algorithms (Bestkeeper and NormFinder). The study was performed in a human cancer cell line (MCF7) and in mouse, non-cancerous primary cells (mouse embryonic fibroblasts, MEFs), to assess different behaviors between cell types.\n\nResultsBestkeeper and NormFinder algorithms evidenced significant deviations in the expression of various reference genes. Ribosomal proteins scored among the most significantly dysregulated targets in both cell types. Expression of ACTB and GAPDH, the most frequent calibrators in real-time RT-qPCR studies, was also affected, although differences existed between cell lines.\n\nConclusionsthis study illustrates the need to validate reference genes for appropriate real-time RT-qPCR normalization, according to specific experimental conditions, when GO-cell interactions occur.

molecular biology↗

bioRxiv2018

A Novel Scavenging Tool for Cancer Biomarker Discovery based on the Blood-Circulating Nanoparticle Protein Corona

The prominent discrepancy between the significant investment towards plasma biomarker discovery and the very low number of biomarkers currently in clinical use stresses the need for novel discovery technologies. The discovery of protein biomarkers present in human blood by proteomics is tremendously challenging, owing to the large dynamic concentration range of blood proteins. Here, we describe the use of blood-circulating lipid-based nanoparticles (NPs) as a scavenging tool to comprehensively analyse the blood circulation proteome. We aimed to exploit the spontaneous interaction of NPs with plasma proteins once injected in the bloodstream, known as protein corona and to facilitate the discovery of previously unreported biomarker molecules for cancer diagnostics. We employed two different tumor models, a subcutaneous melanoma model (B16-F10) and human lung carcinoma xenograft model (A549) and comprehensively compared by mass spectrometry the in vivo protein coronas formed onto clinically used liposomes, intravenously administered in healthy and tumor-bearing mice. The results obtained demonstrated the ability of blood-circulating liposomes to surface-capture and amplify low molecular weight (MW) and low abundant tumor specific proteins (intracellular products of tissue leakage) that could not be detected by plasma analysis, performed in comparison. Most strikingly, the NP (liposomal) corona formed in the xenograft model was found to consist of murine host response proteins, as well as human proteins released from the inoculated and growing human cancer cells. This study offers direct evidence that the in vivo NP protein corona could be deemed as a valuable tool of the blood proteome in experimental disease models to allow the discovery of potential biomarkers.\n\nToC Graphic\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC=\"FIGDIR/small/382192_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (29K):\norg.highwire.dtl.DTLVardef@130bde1org.highwire.dtl.DTLVardef@102058aorg.highwire.dtl.DTLVardef@1b9e61dorg.highwire.dtl.DTLVardef@1b29ba1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2017

Non-viral induction of transient cell reprogramming in skeletal muscle to enhance tissue regeneration

Somatic cells can be reprogrammed to pluripotency in vivo by overexpression of defined transcription factors. While their sustained expression triggers tumorigenesis, transient reprogramming induces pluripotency-like features and proliferation only temporarily, without teratoma formation. We sought to achieve transient reprogramming within mouse skeletal muscle with a localized injection of plasmid DNA (pDNA) and hypothesized that this would enhance regeneration after severe injury. Intramuscular administration of reprogramming pDNA rapidly upregulated pluripotency (Nanog, Ecat1, Rex1) and early myogenesis genes (Pax3) in the healthy gastrocnemius of various mouse strains. Mononucleated cells expressing such markers appeared promptly in clusters among myofibers, but proliferated only transiently and did not lead to the generation of teratomas. Nanog was also upregulated in the gastrocnemius when reprogramming factors were administered 7 days after laceration of its medial head. Enhanced tissue regeneration after reprogramming was manifested by the accelerated appearance of centro-nucleated myofibers and reduced fibrosis. These results suggest that in vivo transient reprogramming may constitute a novel strategy towards the acceleration of regeneration following muscle injury, based on the induction of transiently-proliferative, pluripotent-like cells in situ. Further research to achieve clinically meaningful functional regeneration is warranted.

developmental biology↗