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Nanoparticle-mediated photoporation has arisen as a universal intracellular delivery tool; however, the direct interaction of nanoparticles and cells hampers its clinical translation. Here, we report a uniform contactless intracellular delivery that transfects a large number of cells within a minute and avoids direct contact of nanoparticles and cells, thereby improving the cell viability. Our platform consists of an array of polydimethylsiloxane (PDMS) mixed reduced graphene oxide (rGO) nanoflakes on pyramidal microtips, uniformly distributed at the apex of the tip. The extraordinary optoelectronic properties of rGO were combined with micro-pyramidal cavities to entrap light in micro-cavities and efficiently convert it into heat through multiple reflections and absorptions. As a result, ultralow infra-red laser pulse irradiation could create cavitation bubbles followed by cell membrane deformation and biomolecular delivery. Using this delivery platform, we have achieved the delivery of small to large cargo (668 Da to 465 kDa) in various mammalian cells, including hard-to-transfect H9C2 cardiomyocytes. The best results were achieved for enzyme (465 kDa) delivery with a transfection efficiency and cell viability of 95% and 98%, respectively, in SiHa cells. The highly efficient cargo delivery tool demonstrated a safe and effective approach for cell therapy and diagnostics.
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Supervivencia Celular , Dimetilpolisiloxanos , Grafito , Grafito/química , Dimetilpolisiloxanos/química , Humanos , Transfección , Animales , Dispositivos Laboratorio en un Chip , Línea Celular , LuzRESUMEN
Factor XIIIa (FXIIIa) is a cysteine transglutaminase that catalyzes the last step in the coagulation process. An anion-binding site inhibition of FXIIIa is a paradigm-shifting strategy that may offer key advantages of controlled inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We previously reported a flavonoid trimer-based allosteric inhibitor of FXIIIa with moderate potency and selectivity. To further advance this approach, we evaluated a series of 27 variably sulfonated heparin mimetics against human FXIIIa. Only 13 molecules exhibited inhibitory activity at the highest concentration tested with IC50 values of 2-286 µM. Specifically, inhibitor 16 demonstrated an IC50 value of 2.4 ± 0.5 µM in a bisubstrate, fluorescence-based trans-glutamination assay. It also demonstrated a significant selectivity over other clotting factors including thrombin, factor Xa, and factor XIa as well as other cysteine enzymes including papain and tissue transglutaminase 2. Inhibitor 16 did not affect the viability of three human cell lines at a concentration that is 5-fold its FXIIIa-IC50. The molecule had a very weak effect on the activated partial thromboplastin time of human plasma at a concentration of >700 µM, further supporting its functional selectivity. Importantly, molecule 16 inhibited FXIIIa-mediated polymerization of fibrin(ogen) in a concentration-dependent manner as shown by the gel electrophoresis experiment. Michaelis-Menten kinetics revealed that the molecule competes with the Gln-donor protein substrate, i.e., dimethylcasein, but not with the Lys-donor small substrate, i.e., dansylcadaverine. Molecular modeling studies revealed that this type of molecule likely binds to an anion-binding site comprising the basic amino acids of Lys54, Lys61, Lys73, Lys156, and Arg244 among others. Overall, our work puts forward a new anion-binding site, selective, nontoxic, sulfonated heparin mimetic FXIIIa inhibitor 16 for further development as an effective and safer anticoagulant.
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Cyanobacteria have emerged as a microbial cell factory to produce a variety of bioproducts, including peptides and proteins. Cyanobacteria stand out among other organisms due to their photoautotrophic metabolism and ability to produce a wide range of metabolites. As photoautotrophic hosts can produce industrial compounds and proteins by using minimal resources such as sunlight, atmospheric carbon dioxide, and fewer nutrients, cyanobacteria are cost-effective industrial hosts. Therefore, the use of protein engineering tools for rational protein design, and the desired modification of enzyme activity has become a desirable undertaking in cyanobacterial biology. Protein engineering can improve their biological functions as well as the stability of their intracellular proteins. This review aims to highlight the success of protein engineering in the direction of cyanobacterial biotechnology and outlines the emerging technologies, current challenges, and prospects of protein engineering in cyanobacterial biotechnology.
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Cianobacterias , Cianobacterias/genética , Ingeniería de Proteínas , Biotecnología , Dióxido de Carbono/metabolismoRESUMEN
Photothermal nanoparticle-sensitised photoporation is an emerging approach, which is considered an efficient tool for the intracellular delivery of biomolecules. Nevertheless, using this method to achieve high transfection efficiency generally compromises cell viability and uneven distribution of nanoparticles results in non-uniform delivery. Here, we show that high aspect ratio gold nano-burflowers, synthesised in a microfluidic device, facilitate highly efficient small to very-large cargo delivery uniformly using infrared light pulses without sacrificing cell viability. By precisely controlling the flow rates of shaping reagent and reducing agent, high-density (24 numbers) sharply branched spikes (â¼80 nm tip-to-tip length) of higher aspect ratios (â¼6.5) with a small core diameter (â¼45 nm) were synthesised. As produced gold burflower-shape nanoparticles are biocompatible, colloidally stable (large surface zeta potential value), and uniform in morphology with a higher plasmonic peak (max. 890 nm). Theoretical analysis revealed that spikes on the nanoparticles generate a higher electromagnetic field enhancement upon interaction with light pulses. It induces plasmonic nanobubbles in the vicinity of the cells, followed by pore formation on the membrane leading to diverse biomolecular delivery into cells. Our platform has been successfully implemented for uniform delivery of small to very large biomolecules, including siRNA (20-24 bp), plasmid DNA expressing green fluorescent protein (6.2 kbp), Cas-9 plasmid (9.3 kbp), and ß-galactosidase enzyme (465 kDa) into diverse mammalian cells with high transfection efficiency and cell viability. For very large biomolecules such as enzymes, the best results were achieved as â¼100% transfection efficiency and â¼100% cell viability in SiHa cells. Together, our findings demonstrate that the spiky gold nano-burflower shape nanoparticles manufactured in a microfluidic system exhibited excellent plasmonic behaviour and could serve as an effective tool in manipulating cell physiology.
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Nanopartículas del Metal , Nanoestructuras , Animales , Oro , Transfección , Línea Celular Tumoral , MamíferosRESUMEN
Cell patterning is a powerful technique for the precise control and arrangement of cells, enabling detailed single-cell analysis with broad applications in therapeutics, diagnostics, and regenerative medicine. This study presents a novel and efficient technique that enables massively parallel high throughput cell patterning and precise delivery of small to large biomolecules into patterned cells. The innovative cell patterning device proposed in this study is a standalone, ultrathin 3D SU-8 micro-stencil membrane, with a thickness of 10 µm. It features an array of micro-holes ranging from 40 µm to 80 µm, spaced apart by 50 µm to 150 µm. By culturing cells on top of this SU-8 membrane, the technique achieves highly efficient cell patterns varying from single-cell to cell clusters on a Petri dish. Utilizing this technique, we have achieved a remarkable reproducible patterning efficiency for mouse fibroblast L929 (80.5%), human cervical SiHa (81%), and human neuroblastoma IMR32 (89.6%) with less than 1% defects in undesired areas. Single-cell patterning efficiency was observed to be highest at 75.8% for L929 cells. Additionally, we have demonstrated massively parallel high throughput uniform transfection of large biomolecules into live patterned cells by employing an array of titanium micro-rings (10 µm outer diameter, 3 µm inner diameter) activated through infrared light pulses. Successful delivery of a wide range of small to very large biomolecules, including propidium iodide (PI) dye (668.4 Da), dextran (3 kDa), siRNA (13.3 kDa), and ß-galactosidase enzyme (465 kDa), was accomplished in cell patterns for various cancer cells. Notably, our platform achieved exceptional delivery efficiencies of 97% for small molecules like PI dye and 84% for the enzyme, with corresponding high cell viability of 100% and 90%, respectively. Furthermore, the compact and reusable SU-8-based membrane device facilitates highly efficient cell patterning, transfection, and cell viability, making it a promising tool for diagnostics and therapeutic applications.
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The recent advancements of single-cell analysis have significantly enhanced the ability to understand cellular physiology when compared to bulk cellular analysis. Here a massively parallel single-cell patterning and very large biomolecular delivery is reported. Micro-pillar polydimethyl siloxane stamp with different diameters (40-100 µm with 1 cm × 1 cm patterning area) is fabricated and then imprint distinct proteins and finally pattern single-cell to small clusters of cells depending on the micro-pillar diameters. The maximum patterning efficiency is achieved 99.7% for SiHa, 96.75% for L929, and 98.6% for MG63 cells, for the 100 µm micro-pillar stamp. For intracellular delivery of biomolecules into the patterned cells, a titanium micro-dish device is aligned on top of the cells and exposed by infrared light pulses. The platform successfully delivers small to very large biomolecules such as PI dyes (668 Da), dextran 3000 Da, siRNA (20-24 bp), and large size enzymes (464 KDa) in SiHa, L929 and MG63 cells. The delivery efficiency for PI dye, Dextran 3000, siRNA, and enzyme for patterned cells are ≈95 ± 3%, 97 ± 1%, 96 ± 1% and 94 ± 3%, with cell viability of 98 ± 1%. Thus, the platform is compact, robust, easy for printing, and potentially applicable for single-cell therapy and diagnostics.
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Dextranos , Proteínas , Animales , Impresión , Análisis de la Célula Individual , ARN Interferente Pequeño , MamíferosRESUMEN
Uniform transfection of biomolecules into live cells with high delivery efficiency and cell viability is an immensely important area of biological research and has many biomedical applications. In the present study, we report highly efficient, uniform parallel intracellular delivery of small to very large biomolecules into diverse cell types using a titanium micro-ring (TMR) device activated by infrared (IR) light pulse. A TMR array device (2 cm × 2 cm) consists of a 10 µm outer diameter and 3 µm inner diameter for each micro-ring, and 10 µm interspacing between two micro-rings. Upon IR (1050 nm) pulse laser irradiation on the TMR device, photothermal cavitation bubbles are generated, disrupting the cell plasma membrane, and biomolecules are gently delivered into the cells by a simple diffusion process. This TMR device successfully delivered diverse types of small to very large biomolecules such as propidium iodide (PI; 668.4 Da) dye, dextran (3 kDa), small interfering RNA (13.3 kDa), enhanced green fluorescent protein expression plasmid DNA (6.2 kb), and ß-galactosidase enzyme (465 kDa) into human cervical (SiHa), mouse fibroblast (L929), and mouse neural crest-derived (N2a) cancer cells. For smaller molecules (PI dye), delivery efficiency and cell viability were achieved at â¼96% and â¼97%, respectively, with a laser fluence of 21 mJ cm-2 for 250 pulses. In contrast, â¼85% transfection efficiency and â¼90% cell viability were achieved for plasmid DNA with 45 mJ cm-2 laser fluence for 250 pulses in SiHa cells. Moreover, the intracellular delivery of ß-galactosidase enzyme was confirmed with confocal microscopy and flow cytometry analysis resulting in â¼83% co-staining of ß-galactosidase enzyme and calcein AM. Based on these efficient deliveries of diverse types of biomolecules in different cell types, the device has the potential for cellular diagnostic and therapeutic applications.
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Rayos Infrarrojos , Rayos Láser , Humanos , Ratones , Animales , Membrana Celular/metabolismo , Transfección , Supervivencia Celular , beta-Galactosidasa/metabolismo , MamíferosRESUMEN
Objective: Cathepsin G (CatG) is a cationic serine protease with wide substrate specificity. CatG is reported to play a role in several inflammatory pathologies. Thus, we aimed at identifying a potent and allosteric inhibitor of CatG to be used as a platform in further drug development opportunities. Methods: Chromogenic substrate hydrolysis assays were used to evaluate the inhibition potency and selectivity of SPGG towards CatG. Salt-dependent studies, Michaelis-Menten kinetics and SDS-PAGE were exploited to decipher the mechanism of CatG inhibition by SPGG. Molecular modelling was also used to identify a plausible binding site. Key findings: SPGG displayed an inhibition potency of 57 nM against CatG, which was substantially selective over other proteases. SPGG protected fibronectin and laminin against CatG-mediated degradation. SPGG reduced VMAX of CatG hydrolysis of a chromogenic substrate without affecting KM, suggesting an allosteric mechanism. Resolution of energy contributions indicated that non-ionic interactions contribute ~91% of binding energy, suggesting a substantial possibility of specific recognition. Molecular modelling indicated that SPGG plausibly binds to an anion-binding sequence of 109SRRVRRNRN117. Conclusion: We present the discovery of SPGG as the first small molecule, potent, allosteric glycosaminoglycan mimetic inhibitor of CatG. SPGG is expected to open a major route to clinically relevant allosteric CatG anti-inflammatory agents.
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Physiological bioengineering of multilayered tissues requires an optimized geometric organization with comparable biomechanics. Currently, polymer-reinforced three-dimensional (3D) graphene foams (GFs) are gaining interest in tissue engineering due to their unique morphology, biocompatibility, and similarity to extracellular matrixes. However, the homogeneous reinforcement of single polymers throughout a GF matrix does not provide tissue-level organization. Therefore, a triple-layered structure is developed in a GF matrix to closely mimic native tissue structures of the periodontium of the teeth. The scaffold aims to overcome the issue of layer separation, which generally occurs in multilayered structures due to the poor integration of various layers. The 3D GF matrix was reinforced with a polycaprolactone (PCL), polyvinyl alcohol (PVA), and PCL-hydroxyapatite (HA) mixture, added sequentially, via spin coating, vacuum, and hot air drying. Later, PVA was dissolved to create a middle layer, mimicking the periodontal fibers, while the layers present on either side resembled cementum and alveolar bone, respectively. Scanning electron microscopy and micro-computed tomography revealed the structure of the scaffold with internal differential porosities. The nanoindentation and tensile testing demonstrated the closeness of mechanical properties to that of native tissues. The biocompatibility was assessed by the MTT assay with MG63 cells (human osteosarcoma cells) exhibiting high adhesion and proliferation rate inside the 3D architecture. Summing up, this scaffold has the potential for enhancing the regeneration of various multilayered tissues.
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The physical energy activated techniques for cellular delivery and analysis is one of the most rapidly expanding research areas for a variety of biological and biomedical discoveries. These methods, such as electroporation, optoporation, sonoporation, mechanoporation, magnetoporation, etc., have been widely used in delivering different biomolecules into a range of primary and patient-derived cell types. However, the techniques when used individually have had limitations in delivery and co-delivery of diverse biomolecules in various cell types. In recent years, a number of studies have been performed by combining the different membrane disruption techniques, either sequentially or simultaneously, in a single study. The studies, referred to as combinatorial, or hybrid techniques, have demonstrated enhanced transfection, such as efficient macromolecular and gene delivery and co-delivery, at lower delivery parameters and with high cell viability. Such studies can open up new and exciting avenues for understanding the subcellular structure and consequently facilitate the development of novel therapeutic strategies. This review consequently aims at summarising the different developments in hybrid therapeutic techniques. The different methods discussed include mechano-electroporation, electro-sonoporation, magneto-mechanoporation, magnetic nanoparticles enhanced electroporation, and magnetic hyperthermia studies. We discuss the clinical status of the different methods and conclude with a discussion on the future prospects of the combinatorial techniques for cellular therapy and diagnostics.
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Electroporación , Técnicas de Transferencia de Gen , Humanos , Transfección , Electroporación/métodos , Terapia Genética/métodos , Supervivencia CelularRESUMEN
Cyanobacteria are known for their metabolic potential and carbon capture and sequestration capabilities. These cyanobacteria are not only an effective source for carbon minimization and resource mobilization into value-added products for biotechnological gains. The present review focuses on the detailed description of carbon capture mechanisms exerted by the various cyanobacterial strains, the role of important regulatory pathways, and their subsequent genes responsible for such mechanisms. Moreover, this review will also describe effectual mechanisms of central carbon metabolism like isoprene synthesis, ethylene production, MEP pathway, and the role of Glyoxylate shunt in the carbon sequestration mechanisms. This review also describes some interesting facets of using carbon assimilation mechanisms for valuable bio-products. The role of regulatory pathways and multi-omics approaches in cyanobacteria will not only be crucial towards improving carbon utilization but also will give new insights into utilizing cyanobacterial bioresource for carbon neutrality.
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Carbono , Cianobacterias , Carbono/metabolismo , Cianobacterias/metabolismo , BiotecnologíaRESUMEN
BACKGROUND: Ethacrynic acid (EA) is a loop diuretic that is approved orally and parenterally to manage edema-associated diseases. Nevertheless, it was earlier reported that it is also associated with bleeding upon its parenteral administration. In this report, we investigated the effects of EA on human factor XIIIa (FXIIIa) of the coagulation process using a variety of techniques. METHODS: A series of biochemical and computational methods have been used in this study. The potency and efficacy of human FXIIIa inhibition by EA was evaluated using a bisubstrate-based fluorescence trans-glutamination assay under near physiological conditions. To establish the physiological relevance of FXIIIa inhibition by EA, the effect on FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) - α2-antiplasmin complex was evaluated using SDS-PAGE experiments. The selectivity profile of EA against other coagulation proteins was assessed by evaluating EA's effect on human clotting times in the activated partial thromboplastin time (APTT) and the prothrombin time (PT) assays. We also used molecular modeling studies to put forward a putative binding mode for EA in the active site of FXIIIa. Results involving EA were the average of at least three experiments and the standard error ± 1 was provided. In determining the inhibition parameters, we used non-linear regression analysis. RESULTS: FXIIIa is a transglutaminase that works at the end of the coagulation process to form an insoluble, rigid, and cross-linked fibrin rich blood clot. In fact, inhibition of FXIIIa-mediated biological processes has been reported to result in a bleeding diathesis. Inhibition of FXIIIa by EA was investigated given the nucleophilic nature of the thiol-containing active site of the enzyme and the Michael acceptor-based electrophilicity of EA. In a bisubstrate-based fluorescence trans-glutamination assay, EA inhibited FXIIIa with a moderate potency (IC50 ~ 105 µM) and efficacy (∆Y ~ 66%). In SDS-PAGE experiments, EA appears to significantly inhibit the FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) - α2-antiplasmin complex which indicates that EA affects the physiological functions of FXIIIa. Interestingly, EA did not affect the clotting times of human plasma in the APTT and the PT assays at the highest concentration tested of 2.5 mM suggesting the lack of effects on the coagulation serine proteases and potentially the functional selectivity of EA with respect to the clotting process. Molecular modeling studies demonstrated that the Michael acceptor of EA forms a covalent bond with catalytic residue of Cys314 in the active site of FXIIIa. CONCLUSIONS: Overall, our studies indicate that EA inhibits the physiological function of human FXIIIa in vitro which may potentially contribute to the bleeding complications that were reported with the association of the parenteral administration of EA.
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Antifibrinolíticos , Ácido Etacrínico , Factor XIIIa , Antifibrinolíticos/farmacología , Coagulación Sanguínea , Ácido Etacrínico/farmacología , Factor XIIIa/antagonistas & inhibidores , Fibrina/química , HumanosRESUMEN
The influence of nanowire (NW) surface states increases rapidly with the reduction of diameter and hence severely degrades the optoelectronic performance of narrow-diameter NWs. Surface passivation is therefore critical, but it is challenging to achieve long-term effective passivation without significantly affecting other qualities. Here, we demonstrate that an ultrathin InP passivation layer of 2-3 nm can effectively solve these challenges. For InAsP nanowires with small diameters of 30-40 nm, the ultrathin passivation layer reduces the surface recombination velocity by at least 70% and increases the charge carrier lifetime by a factor of 3. These improvements are maintained even after storing the samples in ambient atmosphere for over 3 years. This passivation also greatly improves the performance thermal tolerance of these thin NWs and extends their operating temperature from <150 K to room temperature. This study provides a new route toward high-performance room-temperature narrow-diameter NW devices with long-term stability.
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Single-neuron actions are the basis of brain function, as clinical sequelae, neuronal dysfunction or failure for most of the central nervous system (CNS) diseases and injuries can be identified via tracing single-neurons. The bulk analysis methods tend to miscue critical information by assessing the population-averaged outcomes. However, its primary requisite in neuroscience to analyze single-neurons and to understand dynamic interplay of neurons and their environment. Microfluidic systems enable precise control over nano-to femto-liter volumes via adjusting device geometry, surface characteristics, and flow-dynamics, thus facilitating a well-defined micro-environment with spatio-temporal control for single-neuron analysis. The microfluidic platform not only offers a comprehensive landscape to study brain cell diversity at the level of transcriptome, genome, and/or epigenome of individual cells but also has a substantial role in deciphering complex dynamics of brain development and brain-related disorders. In this review, we highlight recent advances of microfluidic devices for single-neuron analysis, i.e., single-neuron trapping, single-neuron dynamics, single-neuron proteomics, single-neuron transcriptomics, drug delivery at the single-neuron level, single axon guidance, and single-neuron differentiation. Moreover, we also emphasize limitations and future challenges of single-neuron analysis by focusing on key performances of throughput and multiparametric activity analysis on microfluidic platforms.
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Highly efficient intracellular delivery strategies are essential for developing therapeutic, diagnostic, biological, and various biomedical applications. The recent advancement of micro/nanotechnology has focused numerous researches towards developing microfluidic device-based strategies due to the associated high throughput delivery, cost-effectiveness, robustness, and biocompatible nature. The delivery strategies can be carrier-mediated or membrane disruption-based, where membrane disruption methods find popularity due to reduced toxicity, enhanced delivery efficiency, and cell viability. Among all of the membrane disruption techniques, the mechanoporation strategies are advantageous because of no external energy source required for membrane deformation, thereby achieving high delivery efficiencies and increased cell viability into different cell types with negligible toxicity. The past two decades have consequently seen a tremendous boost in mechanoporation-based research for intracellular delivery and cellular analysis. This article provides a brief review of the most recent developments on microfluidic-based mechanoporation strategies such as microinjection, nanoneedle arrays, cell-squeezing, and hydroporation techniques with their working principle, device fabrication, cellular delivery, and analysis. Moreover, a brief discussion of the different mechanoporation strategies integrated with other delivery methods has also been provided. Finally, the advantages, limitations, and future prospects of this technique are discussed compared to other intracellular delivery techniques.
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Microfluidic platforms gain popularity in biomedical research due to their attractive inherent features, especially in nanomaterials synthesis. This review critically evaluates the current state of the controlled synthesis of nanomaterials using microfluidic devices. We describe nanomaterials' screening in microfluidics, which is very relevant for automating the synthesis process for biomedical applications. We discuss the latest microfluidics trends to achieve noble metal, silica, biopolymer, quantum dots, iron oxide, carbon-based, rare-earth-based, and other nanomaterials with a specific size, composition, surface modification, and morphology required for particular biomedical application. Screening nanomaterials has become an essential tool to synthesize desired nanomaterials using more automated processes with high speed and repeatability, which can't be neglected in today's microfluidic technology. Moreover, we emphasize biomedical applications of nanomaterials, including imaging, targeting, therapy, and sensing. Before clinical use, nanomaterials have to be evaluated under physiological conditions, which is possible in the microfluidic system as it stimulates chemical gradients, fluid flows, and the ability to control microenvironment and partitioning multi-organs. In this review, we emphasize the clinical evaluation of nanomaterials using microfluidics which was not covered by any other reviews. In the future, the growth of new materials or modification in existing materials using microfluidics platforms and applications in a diversity of biomedical fields by utilizing all the features of microfluidic technology is expected.
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Nanoestructuras , Puntos Cuánticos , Biopolímeros , Dispositivos Laboratorio en un Chip , Microfluídica/métodos , Nanoestructuras/químicaRESUMEN
The anticoagulant activity of lignosulfonic acid sodium (LSAS), a non-saccharide heparin mimetic, was investigated in this study. LSAS is a relatively safe industrial byproduct with similar polyanionic characteristics to that of heparin. Human plasma clotting assays, fibrin polymerization testing, and enzyme inhibition assays were exploited to investigate the anticoagulant activity of LSAS. In normal human plasma, LSAS selectively doubled the activated partial thromboplastin time (APTT) at ~308 µg/mL. Equally, LSAS doubled APTT at ~275 µg/mL in antithrombin-deficient plasma. Yet, LSAS doubled APTT at a higher concentration of 429 µg/mL using factor XI-deficient plasma. LSAS did not affect FXIIIa-mediated fibrin polymerization at 1000 µg/mL. Enzyme assays revealed that LSAS inhibits factor XIa (FXIa) with an IC50 value of ~8 µg/mL. LSAS did not inhibit thrombin, factor IXa, factor Xa, factor XIIIa, chymotrypsin, or trypsin at the highest concentrations tested and demonstrated significant selectivity against factor XIIa and plasmin. In Michaelis-Menten kinetics, LSAS decreased the VMAX of FXIa hydrolysis of a tripeptide chromogenic substrate without significantly changing its KM indicating an allosteric inhibition mechanism. The inhibitor also disrupted the generation of FXIa-antithrombin complex, inhibited factor XIIa-mediated and thrombin-mediated activation of the zymogen factor XI to FXIa, and competed with heparin for binding to FXIa. Its action appears to be reversed by protamine sulfate. Structure-activity relationship studies demonstrated the advantageous selectivity and allosteric behavior of LSAS over the acetylated and desulfonated derivatives of LSAS. LSAS is a sulfonated heparin mimetic that demonstrates significant anticoagulant activity in human plasma. Overall, it appears that LSAS is a potent, selective, and allosteric inhibitor of FXIa with significant anticoagulant activity in human plasma. Altogether, this study introduces LSAS as a promising lead for further development as an anticoagulant.
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The introduction of foreign cargo into living cells with high delivery efficiency and cell viability is a challenge in cell biology and biomedical research. Here, we demonstrate a nanosecond pulse laser-activated photoporation for highly efficient intracellular delivery using titanium dioxide (TiO2) microspikes as a substratum. The TiO2 microspikes were formed on titanium (Ti) substrate using an electrochemical anodization process. Cells were cultured on top of the TiO2 microspikes as a monolayer, and the biomolecule was added. Due to pulse laser exposure of the TiO2 microspike-cell membrane interface, the microspikes heat up and induce cavitation bubbles, which rapidly grow, coalesce and collapse to induce explosion, resulting in very strong fluid flow at the cell membrane surface. Thus, the cell plasma membrane disrupts and creates transient nanopores, allowing delivery of biomolecules into cells by a simple diffusion process. By this technique, we successfully delivered propidium iodide (PI) dye in HeLa cells with high delivery efficiency (93%) and high cell viability (98%) using 7 mJ pulse energy at 650 nm wavelength. Thus, our TiO2 microspike-based platform is compact, easy to use, and potentially applicable for therapeutic and diagnostic purposes.