Biology subjects

Bhatia, D. D.

Publications and source records attributed to Bhatia, D. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Hypoxia Induced Modulation of Cellular and in vivo uptake of DNA nanocage implications in therapeutics

DNA tetrahedra (DNA Td) are promising nanocarriers for drug delivery, but how hypoxia affects their cellular internalisation remains poorly understood. We synthesised and characterised Cy5-labelled DNA Td and established chemical hypoxia; in HeLa, MDA-MB-231, and MCF-7 cells. Hypoxia was confirmed by HIF-1; nuclear translocation. Confocal microscopy revealed significantly reduced DNA Td uptake under hypoxia, whereas transferrin and cholera toxin B uptake increased, indicating cargo-selective regulation. Temperature-arrest experiments confirmed reduced energy-dependent internalisation. Pharmacological profiling showed a shift from clathrin-mediated and galectin/glycan-dependent pathways toward lipid raft/cholesterol-dependent uptake. Hypoxia increased plasma membrane electronegativity, suggesting a biophysical barrier to DNA Td uptake. Importantly, DOTMA complexation restored uptake to normoxic levels, identifying electrostatic repulsion as a key determinant. In zebrafish larvae, hypoxia significantly enhanced whole-larva DNA Td accumulation. These findings highlight surface charge engineering as a strategy for improving DNA nanostructure delivery under hypoxic conditions.

bioinformatics↗

bioRxiv2026

Programmable DNA Nanocages Enhance Levodopa Delivery for Neuroprotection in a Zebrafish Model of Parkinson's Disease

Parkinsons Disease (PD) is the second-most prevalent neurodegenerative disease, often characterized by neural motor dysfunction, oxidative stress, and dopamine receptor malfunction leading to improper dopamine levels in the system. DNA tetrahedron nanostructures are a promising drug delivery agent due to their biocompatibility and properties of controlled and sustained release. In this study we evaluated the potential of using TD-mediated Levodopa delivery for a MPTP induced Parkinsons Disease model in Zebrafish larvae. The induction of Parkinsonism led to morphological behaviour changes like the presence of tremors, erratic swimming behaviour, latency, and reduced locomotor activity, even elevated reactive oxygen species (ROS) and apoptosis was observed. These effects and symptoms were alleviated when the larvae were treated using TD:Levodopa conjugates, particularly at the 1:100 ratio. At the molecular level, genes like TH, DAT, SOX2, PARKIN and apoptotic genes like BCL2 and caspases showed alteration in expression in the Parkinsonism model and post treatment was induced. This highlights the potential of using DNA nanocages as a novel drug delivery agent as therapeutic strategy for Parkinsons disease. TOCDopamine loaded DNA nanocages with the capacity to overcome biological barriers for release of dopamine with neuroprotection activity in Parkinsons disease model of zebrafish. O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/742731v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1d2b94eorg.highwire.dtl.DTLVardef@183bc1aorg.highwire.dtl.DTLVardef@1b254ddorg.highwire.dtl.DTLVardef@e1b589_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Smartphone-Validated Portable Paper-Based Device Integrated with an Electropolymerized Molecularly Imprinted Polymer for Serotonin Detection in Serum Samples

Accurate and decentralized quantification of serotonin, also known as 5-hydroxytryptamine (5-HT), in biological fluids is critically important for the diagnosis, prognosis, and therapeutic monitoring of neurological and psychiatric disorders. However, conventional analytical methods generally rely on centralized laboratory infrastructure, skilled personnel, and labor-intensive sample processing, which restrict their applicability in rapid near-patient and point-of-care settings. Herein, we report a portable molecularly imprinted polymer (MIP)-based electrochemical sensing platform for selective and on-site detection of serotonin using screen-printed carbon electrodes (SPCEs). The biomimetic recognition interface was fabricated through direct electropolymerization of a polydopamine recognition layer in the presence of serotonin as the template molecule, followed by template extraction to generate complementary recognition cavities for selective rebinding. The sensor fabrication parameters, including monomer concentration, electropolymerization cycles, template-to-monomer stoichiometry, and electrolyte pH, were systematically optimized to achieve improved sensitivity, selectivity, and signal stability. Under optimized conditions, the MIP/SPCE sensor exhibited a broad linear response from 10 pM -10 {micro}M in phosphate buffer, with a correlation coefficient of R2 = 0.974 and an ultralow limit of detection of 0.16 pM. The analytical applicability of the platform was further validated in spiked artificial serum, where the sensor achieved an LOD of 0.12 pM, satisfactory recovery values of 88.66-96.02%, and acceptable precision with RSD values [≤] 8.43% (n=3), confirming its reliability in a complex biological matrix. The developed sensor demonstrated excellent selectivity toward serotonin against physiologically relevant interferents, maintaining signal retention between 99% and 101%. In addition, the platform showed high operational repeatability with an RSD of 0.45%, good inter-electrode reproducibility with an RSD of 6.3%, and long-term storage stability, retaining 90-110% of its initial response over 28 days. Importantly, cross-platform validation using a smartphone-coupled potentiostat demonstrated strong analytical agreement with laboratory-grade instrumentation, as evidenced by R2 = 0.9967 and a slope of 1.023. These findings establish the proposed MIP/SPCE platform as a simple, low-cost, portable, and smartphone-compatible electrochemical device for field-deployable serotonin monitoring in clinically relevant samples.

bioengineering↗

bioRxiv2026

Rationally Anchored Geometry-Controlled DNA Tetrahedral Nanostructures for Attomolar Impedimetric IL-6 Detection

Interleukin-6 (IL-6) is a pleiotropic cytokine whose aberrant elevation drives life-threatening conditions, including sepsis, cytokine storm, and autoimmune disorders, yet existing clinical detection methods demand centralized laboratory infrastructure and multi-hour assay times incompatible with rapid point-of-care decision-making. Here, we report an impedimetric aptasensor built on a programmable tetrahedral DNA nanostructure (TDN) interface anchored to a disposable gold screen-printed electrode (Au-SPE) for the ultrasensitive, label-free detection of IL-6. By systematically varying the number of thiolated base vertices from zero to three, we establish a clear and previously unreported structure-function relationship between multipodal anchoring geometry and charge-transfer resistance modulation: tripodal thiolation yields the most rigid, upright, and electrochemically responsive interface, producing the steepest analytical signal gain upon IL-6 binding at the apex-localised aptamer. Under optimised conditions (pH 7.0, 0.05 {micro}M TDN, MCH passivation), the aptasensor exhibits a linear dynamic range of 0.0001-0.001 pg/mL, a limit of detection of 55 ag/mL , and a sensitivity of 1.55x107 {Omega} (pg mL-1)-1. Selectivity evaluation against seven physiologically relevant interferents such as TNF-, BSA, glucose, urea, ascorbic acid, glycine, and cysteine confirms negligible cross-reactivity, with relative responses ranging from 0.57% to 14.35% of the IL-6 signal. Spike-recovery experiments in human serum yield recoveries of 74.0-87.6% (%RSD < 4.5%), and the sensor retains functional activity for at least 21 days under refrigerated storage. This work demonstrates that thiolated vertex number is a critical and tunable design parameter for TDN-based biosensors, offering a modular, disposable platform for sub-femtogram cytokine detection with direct applicability to early sepsis diagnosis and inflammatory disease monitoring.

bioengineering↗

bioRxiv2026

Biofunctional 2D Graphitic Carbon Nitride-Hydrogel Heterointerfaces for Electrochemical Detection of Interleukin-6 toward Septic Cardiomyopathy Diagnostics in Clinical Biofluids

Interleukin-6 (IL-6) is a key pro-inflammatory cytokine closely associated with sepsis progression and septic cardiomyopathy (SCM), a severe clinical condition characterized by acute cardiac dysfunction and high mortality in critically ill patients. Rapid and sensitive monitoring of IL-6 in clinical biofluids is therefore crucial for early diagnosis, disease prognosis, and timely therapeutic intervention in emergency healthcare settings. Herein, we report a biofunctional, label-free electrochemical aptasensor based on a graphitic carbon nitride-incorporated chitosan hydrogel-modified gold screen-printed electrode (MCH/Apt-IL-6/g-C3N4@CS/Au-SPE) for ultrasensitive detection of IL-6 in clinical biofluids. The electroactive g-C3N4@CS hydrogel heterointerface was engineered via electrostatic interactions between the negatively charged surface functionalities of two-dimensional graphitic carbon nitride (g-C3N4) and the protonated amino groups (-NH3+) of chitosan (CS), yielding a porous, conductive, and biocompatible sensing matrix with enhanced aptamer immobilization and accelerated electron-transfer kinetics. Biocompatibility evaluation using MTT assay and confocal fluorescence imaging demonstrated that the hydrogel maintained excellent cellular compatibility at 5 mg/mL, preserving normal cytoskeletal organization, mitochondrial integrity, and nuclear morphology, while higher concentrations induced cellular stress responses. Under optimized experimental conditions, the developed aptasensor exhibited outstanding analytical performance with an ultrawide linear detection range from 1 fg/mL to 10 ng/mL, a high sensitivity of 2.162 A/[log10(ng/mL)] cm-2, a low detection limit of 0.460 pg/mL, and excellent linearity (R2 = 0.979). In addition, the sensor demonstrated remarkable selectivity toward common biological interferents, including ascorbic acid, cysteine, glucose, glycine, and urea, together with excellent reproducibility (RSD = 1.139%). Validation studies performed in spiked human serum samples further confirmed the reliability and practical applicability of the proposed sensing platform for rapid clinical analysis. Owing to its label-free detection strategy, disposable electrode format, high sensitivity, and favorable biocompatibility, the developed g-C3N4-hydrogel heterointerface-based aptasensor represents a promising next-generation platform for early septic cardiomyopathy diagnostics, inflammatory biomarker monitoring, and point-of-care electrochemical biosensing applications.

bioengineering↗

bioRxiv2026

Influence of Physicochemical Parameters on the in vitro Stability of DNA Tetrahedral Nanostructures

Tetrahedral DNA nanostructures (TDNs) are promising nanocarriers due to their structural precision, biocompatibility, and efficient cellular uptake. However, their stability under physiological conditions remains a key challenge. In this study, TDNs were synthesized via a one-pot thermal annealing method and characterized using native PAGE, dynamic light scattering (DLS), and zeta potential analysis, confirming uniform size ([~]13 nm) and negative surface charge. Their stability was systematically evaluated across different biological media (DMEM complete, serum-free DMEM, and E3), temperatures (4 {degrees}C, 25 {degrees}C, and 37 {degrees}C), and pH conditions (4.0, 7.0, and 8.5) over 24 h. Results revealed rapid degradation in serum-containing medium, increased instability at higher temperatures, and reduced stability under acidic conditions, while serum-free, lower-temperature, and neutral to mildly basic environments enhanced structural integrity. These findings highlight the strong environmental dependence of TDN stability and provide insights for optimizing their design for biomedical applications.

bioengineering↗

bioRxiv2026

Green Synthesis of Fluorescent Carbon Quantum Dots from Bearberry Extract via Hydrothermal and Microwave-Assisted Routes: Comparative Physicochemical Characterisation, Antioxidant Activity, and Biocompatibility Evaluation

Producing photoluminescent nanomaterials with controllable surface chemistry and predictable biological activity remains one of the outstanding problems in green nanoscience. The present study shows that, even when the same bearberry (Arctostaphylos uva-ursi) extract precursor is used, the mode of energy delivery during synthesis plays a determining role in shaping the surface composition, photophysical properties, and biological activity of the resulting carbon quantum dots (CQDs). Hydrothermal processing at 160 {degrees}C for 6 h yielded CQDs with an average particle size of 7.13 nm. Surface characterisation indicated abundant hydroxyl- and carbonyl-containing functionalities, while XPS analysis showed a comparatively higher proportion of graphitic sp2 carbon (43.06%). These structural features were accompanied by strong DPPH free-radical scavenging activity. Microwave-assisted synthesis, by contrast, yields 9.65 nm particles carrying a substantially greater surface carboxylate content (O-C=O: 19.06%), a higher fluorescence quantum yield, and enhanced intracellular uptake statistically significant in retinal epithelial cells at 200 {micro}g/mL (p < 0.001) and showing concentration-dependent accumulation in zebrafish larvae from 100 {micro}g/mL onwards (p < 0.05). XPS C 1s deconvolution, interpreted alongside FTIR difference spectroscopy, points to incomplete decarboxylation under microwave conditions as the primary mechanistic origin of these divergent properties. Cytocompatibility was uncompromised for both formulations across the full concentration range tested (10-250 {micro}g/mL) in RPE-1 and HeLa cells, with no statistically significant loss of viability at any concentration. Taken together, these results define a synthesis-route-encoded structure-property relationship that permits rational selection between an antioxidant-optimised and an imaging-optimised CQD formulation from the same green precursor feedstock.

bioengineering↗

bioRxiv2026

Hamelia patens-Derived Red-Emitting Carbon Quantum Dots: Surface-State Luminescence, Antioxidant Potency, and In Vitro Bioimaging

Red-emitting carbon quantum dots (HP-CQDs) were synthesised for the first time from aqueous leaf extracts of Hamelia patens through single-step, reagent-free microwave-assisted carbonisation (750 W). The resulting nanoparticles displayed a narrow hydrodynamic size distribution centred at 3.9 nm, consistent with atomic force microscopy measurements showing a maximum height of 2.81 nm. Under 400 nm excitation, the CQDs exhibited a characteristic red emission maximum at 675 nm, representing a rare example of long-wavelength-emitting green CQDs derived from plant biomass. UV-Vis absorption bands at 224 and 256 nm were assigned to {pi}-{pi}* transitions of aromatic carbon domains and n-{pi}* transitions associated with carbonyl-containing surface groups, respectively. X-ray photoelectron spectroscopy (XPS) indicated a carbon-rich composition (C: 67.24%, O: 31.25%, N: 1.52%) with prominent C-O (42.67%) and C-C/C=C (42.64%) contributions. ATR-FTIR further confirmed the retention of hydroxyl, ether, and aliphatic functionalities following carbonisation. The excitation-wavelength-independent emission peak position implicates discrete surface molecular states rather than a heterogeneous distribution of emitters. HP-CQDs exhibit potent DPPH radical scavenging activity (ICOO = 141.8 {micro}g mLO{superscript 1}), comparable to ascorbic acid (ICOO = 114.8 {micro}g mLO{superscript 1}), and maintain >95% cell viability in both HeLa and RPE-1 cells up to 250 {micro}g mLO{superscript 1}. Confocal microscopy demonstrates concentration-dependent cytoplasmic accumulation and selective perinuclear localization at 300 {micro}g mLO{superscript 1}. In vivo biodistribution in zebrafish larvae confirms systemic uptake with statistically significant fluorescence enhancement at 500 {micro}g mLO{superscript 1} (p < 0.01), establishing HP-CQDs as biocompatible red-fluorescent probes with dual imaging-antioxidant functionality. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/724069v2_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@17be668org.highwire.dtl.DTLVardef@153d874org.highwire.dtl.DTLVardef@1a7e6edorg.highwire.dtl.DTLVardef@29f13a_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Curcumin-Magnesium complex loaded DNA hydrogels: concentration dependent swelling kinetics and selective cytotoxicity via Oxidative Stress induced apoptosis

Curcumin is a naturally occurring polyphenol that demonstrates considerable anti-cancer activity, however the aqueous insolubility, rapid metabolism and relatively low bioavailability are limiting to its clinical application. As such, a curcumin-magnesium (Cur-Mg) coordination complex was synthesized and subsequently encapsulated within DNA hydrogels (Cur-Mg-Hgel). The Cur-Mg complex was fully characterized using UV-Vis spectroscopy, FTIR and X-ray diffraction (XRD). UV-Vis, FTIR and XRD all support the formation of a coordination complex and suggest a decreased level of crystallinity compared to free curcumin. DNA hydrogels were formed and characterized using atomic force microscopy, rheology and swelling kinetic studies. In vitro cytotoxicity studies utilizing an MTT assay demonstrate dose dependent inhibition of HeLa cell proliferation and a slightly better retention of RPE-1 viability at low concentrations (suggesting some difference in sensitivity) though significant cell death is seen at higher concentrations and both cells. Intracellular production of ROS was measured using the DCFH-DA assay and is seen to increase when HeLa cells are treated with Cur-Mg-Hgel in comparison to un-treated controls. Annexin V/PI staining demonstrates primarily late or early apoptotic activity with minimal necrosis following treatment with Cur-Mg-Hgel. The evidence presented strongly supports the notion that Cur-Mg-Hgel is a ROS-modulating, pro-apoptotic Hydrogel suitable for cancer treatment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/724072v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@d23f8corg.highwire.dtl.DTLVardef@14a30f6org.highwire.dtl.DTLVardef@1d485e9org.highwire.dtl.DTLVardef@1a3adf7_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Membrane sialylation orchestrates cellular gateways: A spatiotemporal analysis of cellular transport using DNA nanocages via membrane charge modulation

Negatively charged DNA nanostructures, such as tetrahedral nanocages, are internalized by cells despite the electrostatic repulsion from the anionic cell membrane, and, paradoxically, cancer cells, which carry intrinsically higher negative charge due to overexpression of sialic acids on their cell surface, show markedly higher uptake than normal cells. This contradiction exposes a fundamental gap in our understanding of how these anionic nanostructures overcome this repulsion. Using chemical modulation of cell-surface sialylation in RPE1 cells to create three groups with altered sialylation levels, together with inhibitor-based dissection of endocytic pathways, we demonstrate that an increase in cell surface sialylation governs the uptake of DNA tetrahedra not through electrostatics but by structurally remodeling the cell membrane via rearrangement of the GM1 lipid raft microdomain, recruiting caveolae-mediated endocytosis as an additional pathway alongside clathrin-mediated endocytosis, thereby increasing the intake of the nanostructure. These findings reframe tumor hyper-sialylation as a determinant of the uptake of anionic nanostructures, such as DNA tetrahedra, and as a targetable parameter for rational optimization of DNA-based nanotherapeutics against cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/722926v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@10eede7org.highwire.dtl.DTLVardef@124dd56org.highwire.dtl.DTLVardef@13f5355org.highwire.dtl.DTLVardef@780ecf_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Inhibition of miR-1307 Reverses Resistance to Cisplatin in Drug-Resistant Oral Squamous Cell Carcinoma

BackgroundChemo-resistance remains a major clinical challenge in Oral Squamous Cell Carcinoma (OSCC), attributed to the intrinsically resistant cells. Although tumour-derived extracellular vesicles (EVs) have been implicated in cell-cell communication, their role in propagating chemo-resistance remains poorly defined. This study aims to identify salivary EV-associated miRNAs capable of predicting chemoresistance and to delineate the role of miR-1307-5p in modulating CSC-driven therapeutic refractoriness. MethodsSalivary EV-derived expression profile of miR-1307-5p was assessed by qPCR in chemo resistant OSCC patients and further validated in TCGA small RNA sequencing datasets. Expression was validated by qPCR and correlated with clinicopathological outcomes. Functional assays including cell-cycle analysis, apoptosis, migration/invasion, 3D spheroids, angiogenesis, and CAM assays were performed in miR-1307-5p inhibited CD44 CSC subpopulation compared to its vehicular control. Transcriptomic profiling cross-referencing with TCGA was conducted to identify potential novel targets of miR-1307-5p. Chemo-sensitisation was assessed by treating the knockdown chemo resistant cells with low dose cisplatin and validating it using in-vitro functional assays and orthotopic xenograft model. ResultsmiR-1307-5p was significantly elevated in salivary EVs of chemo resistant OSCC patients and correlated with poor overall survival (p = 0.03). The miRNA was markedly enriched in endogenously resistant CD44 CSCs. Silencing of miR-1307-5p induced G2/M arrest, triggered apoptosis, impaired invasion, and reduced angiogenesis both in-vitro and in ex-vivo assays. Transcriptomic profiling, TCGA validation, and integrative pathway analysis identified key oncogenic hubs which converge on PI3K-AKT, MAPK/ERK, and YAP signalling pathways governing EMT. Inhibition of miR-1307-5p restored cisplatin sensitivity in resistant CSCs, with low-dose cisplatin producing substantial tumour suppression in-vitro and in-vivo. Reduced CD44 expression in xenograft models confirmed CSC reprogramming. EVs from anti-miR-treated cells confer chemo sensitisation upon uptake by resistant CSCs. Xenograft models substantiated that EVs can initiate tumour formation and that EV-mediated delivery of anti-miR-1307-5p drives significant tumour regression. ConclusionThis study identifies salivary EV-derived miR-1307-5p as a clinically relevant biomarker of chemoresistance in OSCC and reveals its mechanistic role in sustaining CSC-driven therapeutic failure. Targeting miR-1307-5p offers a promising avenue for restoring cisplatin sensitivity and developing exosome-based therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/709730v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@19f88e0org.highwire.dtl.DTLVardef@d36b95org.highwire.dtl.DTLVardef@3c2579org.highwire.dtl.DTLVardef@c04ef5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

Programmable DNA nanocages to modulate pollen tube growth via active uptake

Delivering biomolecules into pollen tubes that deliver sperm cells for plant fertilization remains technically challenging due to thick cell walls and rapid polarized growth, hindering reproductive engineering. DNA nanotechnology offers a promising alternative over current delivery methods due to their biocompatibility, programmable design, low cytotoxicity, and stimulus-responsive properties, yet their application in plants remains underexplored. Here, we provide the first demonstration of tetrahedral DNA nanostructures (TDNs) as nanocarriers for active, endocytosis-mediated uptake into Arabidopsis pollen tubes, enabling spermidine delivery that shortens pollen tube elongation through actin reorganization and ROS modulation. TDN-treated pollen tubes grew through the Arabidopsis stigma and style, underwent capacitation, and maintained attraction to ovules in a semi-in-vivo assay, preserving reproductive fitness. Furthermore, we demonstrate that functionalization of TDNs with nuclear localization signal peptide significantly enhances nuclear localization. Collectively, these findings establish DNA nanostructures as effective nanocarriers for targeted biomolecule delivery and precise pollen tube modulation, advancing crop reproductive engineering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/710033v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@a09c01org.highwire.dtl.DTLVardef@623943org.highwire.dtl.DTLVardef@9d954corg.highwire.dtl.DTLVardef@1b4c35b_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

bioengineering↗

bioRxiv2025

DNA Nanocage-Mediated Aspirin Delivery Mitigates Excitotoxicity and Promotes Neuroprotection in Ischemic Stroke-Induced Zebrafish

Oxidative stress, apoptosis, excitotoxicity, and compromised neuronal function are the hallmarks of ischaemic stroke, a major cause of neurological impairment. Because of their controlled-release properties and biocompatibility, DNA tetrahedron nanostructures (TD) offer a viable platform for targeted drug delivery. In this work, we used a zebrafish larval model of ischaemic stroke to assess the neuroprotective potential of TD-mediated aspirin administration and investigated the TD:Aspirin complexation modes using molecular dynamics (MD) simulations. In addition to morphological abnormalities, elevated reactive oxygen species (ROS), thrombus formation, intracellular calcium accumulation, and dysregulated expression of neuroprotective and apoptotic genes, stroke induction resulted in significant behavioural deficits, including decreased locomotor activity, increased latency, and erratic swimming. Treatment with TD:Aspirin, especially at a 1:100 ratio, markedly improved locomotor behaviour, restored production of BDNF, GDNF, MBP, -tubulin, GRIN2B, SLC8A, NOS1, and caspases, normalised heart rate, lowered ROS generation and apoptosis, and decreased thrombus and calcium deposition. TD:Aspirin showed better efficacy than free Aspirin and TD-only groups, perhaps as a result of improved stability, bioavailability, and sustained release. These results demonstrate the potential of drug delivery via DNA nanocages as a novel therapeutic approach for ischaemic stroke, offering neuroprotection, functional recovery, and neuronal homeostasis restoration. TOC GraphicDNA Nanocage-Mediated Aspirin Delivery Mitigates Excitotoxicity and Promotes Neuroprotection in Ischemic Stroke-Induced Zebrafish O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/696221v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@18dd327org.highwire.dtl.DTLVardef@21b8b4org.highwire.dtl.DTLVardef@1e53283org.highwire.dtl.DTLVardef@5771e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Programmable DNA-Silk fibroin hydrogel scaffold with temporal mechanical reinforcement for mesenchymal stem cells differentiation and accelerated wound healing in a full-thickness skin defect mice model

DNA-based hydrogels have attracted significant attention in biomedical applications due to their programmability, biocompatibility, and tunable functionalities. However, their clinical translation is hindered by high synthesis costs, limited mechanical strength, poor stability, and complex synthesis protocols. In this work, we present a novel DNA-silk fibroin hybrid hydrogel that overcomes these limitations by combining the unique biofunctionality of DNA with the superior mechanical and chemical properties of silk fibroin. Salmon-derived DNA, an abundant and low-cost source, was incorporated with silk fibroin to form hybrid networks via a simple, scalable process. Silk fibroin served as a robust scaffold, enhancing mechanical strength, controlling degradability, and improving structural stability, while the entangled DNA-silk network reduced rapid DNA degradation. The resulting hybrid hydrogel demonstrated temporal mechanical reinforcement and prolonged stability, with in vitro studies demonstrating that the developed hybrid hydrogel enhanced the chondrogenic and osteogenic differentiation of Infrapatellar Fat Pad Mesenchymal Stem Cells (IFP-MSCs). Additionally, hemostatic and in vivo studies demonstrated the potential of DNA-silk fibroin hydrogel as a promising biomaterial for rapid hemostasis and accelerated wound healing. Overall, the developed DNA-silk fibroin hybrid hydrogel offers strong potential for mesenchymal tissue engineering, hemostatic adjuvant and wound healing.

bioengineering↗

bioRxiv2025

Protocol for the Synthesis of Green Fluorescence Carbon Quantum Dots and Biological Application

Carbon quantum dots (CQDs) are materials that are made up of particles that are only a few n anometers wide. They have no dimensions and can change their fluorescence qualities. They are also verybiocompatible and have a lot of potential to change the chemistry of their surface. Their eco-friendly synthesis from precursors, like citric acid (CA) and ascorbic acid (AA), provides a sustainable approach to highly fluorescent nanomaterials with promising applications in bioimaging, drug delivery, and other biomedical fields. Green-emitting carbon quantum dots (GCQDs) were synthesized via a simple reflux approach using citric acid and ascorbic acid as biomolecular precursors in a solvent solution of ethanol-water (1:2). The obtained GCQDs solution was purified using a dialysis process. Besides this, pH maintenance, lyophilization, and further submission for detailed physicochemical evaluation and characterization are done. The spectroscopic analysis, including UV-visible spectroscopy, fluorescence spectroscopy, and FTIR, confirmed the presence of surface functional groups and strong fluorescence properties and quantum yield calculations of the synthesized GCQDs. Microscopic and structural analyses using XRD, AFM, and TEM revealed the nanoscale, predominantly spherical morphology of the synthesized GCQDs. In addition, in vitro biological evaluations such as MTT assay and cellular uptake analysis were undertaken to evaluate cytocompatibility and intracellular distribution. We study the endocytosis pathway of these GCQDs, with size variations ranging from 3 to 5 nm, in mouse tissue-derived primary cells, tissues, and zebrafish embryos. GCQDs were internalized into mouse kidney and liver primary cells through a clathrin-mediated pathway. The findings confirmed that the produced GCQDs exhibit good water solubility, favorable biocompatibility, and significant potential as candidates for biomedical imaging applications.

bioengineering↗

bioRxiv2025

Protocol for green synthesis of excellent fluorescent mango leaves-derived carbon for advanced theragnostic in biological applications

Carbon quantum dots (CQDs) are zero-dimensional fluorescence nanoparticles that are less than 10 nm in size. They have unique properties like tunable surface functionality, biocompatibility, fluorescence, and low toxicity. This study outlines a detailed protocol for synthesizing CQDs from mango leaf powder using a reflux method at 160 C and 400 rpm for a duration of two hours. The synthesized CQDs were comprehensively characterized using the following techniques, including UV-visible spectroscopy, fluorescent spectrophotometry, atomic force microscopy, dynamic light scattering and zeta potential measurement, Fourier transform infrared spectroscopy, and scanning electron microscopy. In vitro analysis was conducted through an MTT assay, revealing that the CQDs exhibit reduced toxicity in RPE1 cells compared to the control group. Additionally, a concentration-dependent cellular uptake assay was performed to further evaluate the biological performance of the CQDs. The findings suggest the potential of CQDs as safe and effective nanomaterials for various biological applications, including drug delivery and bioimaging.

bioengineering↗

bioRxiv2025

Preclinical evaluation of Targeted IL-1β Knockdown via CD44-Immunoliposomes: A Nano-therapy against the Inflammatory Microenvironment

Chronic inflammation, characterized by the infiltration of macrophages and the heightened release of pro-inflammatory cytokines, is the underlying cause of the pathogenesis of many critical diseases. Therapeutic interventions for controlling inflammation via gene knockdown of inflammatory mediators have emerged as a promising approach for regulating uncontrolled inflammation. This study explores the potential of siIL-1{beta}-anti-CD44-Liposomes (SIL) as a potent anti-inflammatory therapy against pro-inflammatory RAW264.7 macrophages via gene specific knockdown of IL-1{beta} mRNA through RNAi, and the subsequent down-regulation of the pro-inflammatory cytokine loop. The designed SIL exhibited a uniform size of 131.1 {+/-} 0.5 nm with a quasi-spherical morphology and sustained release of siIL-1{beta} within 24 hours. The reduction in pro-inflammatory cytokines like IL-1{beta}, TNF-, and IL-6 and inflammatory enzymes iNOS and COX-2; and the simultaneous increase in the anti-inflammatory cytokine IL-4, is indicative of the formulations therapeutic efficacy in reducing inflammation at a cellular level. The effects of SIL on the Macrophage-T cell crosstalk also uncovers the liposomes efficacy in reducing cytokine-mediated T cell effector functions. The nuanced effects of siIL-1{beta}-anti-CD44-Liposomes on in-vivo model of chronic inflammation underscore their potential for precise therapeutic interventions in inflammatory conditions, with multifaceted anti-inflammatory effects on tissue levels and cytokine levels. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/683015v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@f4d0f9org.highwire.dtl.DTLVardef@c3c24aorg.highwire.dtl.DTLVardef@8c115org.highwire.dtl.DTLVardef@bc863d_HPS_FORMAT_FIGEXP M_FIG Schematic representation of the study: CD44 receptors are elevated in LPS activated macrophages, which can be targeted by using anti-CD44 Liposomes, for RNA therapy. IL-1 knockdown via siRNA leads to lower inflammatory nature of macrophages, compromising its the antigen presentation and T-cell activation. This lowers the cytokine storm in inflammatory milieu and lower tissue damage can be achieved. Created with BioRender.com. C_FIG

pharmacology and toxicology↗

bioRxiv2025

Generation of uniform-sized spheroids using PDMS-based microwell devices as 3D cancer models for pharmacological profiling of drugs

Cancer researchers now consider spheroids a valuable in vitro model for cancer research and personalized medicine. They are used to studying cancer development, testing drug effectiveness, and potentially guiding treatment decisions for individual patients. Spheroids represent the simplest form of three-dimensional (3D) cellular arrangement and encapsulate the essential tumor microenvironment. These characteristics are crucial for studying processes such as tumor invasion, metastasis, angiogenesis, and cell cycle kinetics. In particular, spheroids excel in chemo-response assays where traditional monolayer cell cultures often fall short. A PDMS microwell device was developed to generate uniform-sized cancer spheroids. This device is user-friendly and capable of producing a large number of spheroids. The device measures 13 mm in diameter (1200 microwells per well if the device has microwells 400 m in size, and 300 microwells per well if the device has microwells 800 m in size). It advances 3D cultures by requiring only a small volume of cell culture supplements and is easy to manage. The hydrophobic nature of the PDMS device prevents cells from adhering to it, thereby promoting spheroid formation. Spheroids can be created on microwell devices, and subsequent experiments may either be conducted on the device or transferred to cell culture dishes for additional 3D biological assays. Seeding cells is notably easier compared to other 3D cell culture techniques, and the number of cells in each spheroid can be adjusted according to specific requirements. Overall, the PDMS-based microwell device offers a simple and efficient means to produce large quantities of uniform-sized spheroids for 3D cell culture studies, showcasing high throughput, short generation times, long-term effectiveness, and ease of handling.

bioengineering↗