Showing papers by "Tuhin Subhra Santra published in 2021"

A Review of Single-Cell Adhesion Force Kinetics and Applications.

Abstract: Cells exert, sense, and respond to the different physical forces through diverse mechanisms and translating them into biochemical signals. The adhesion of cells is crucial in various developmental functions, such as to maintain tissue morphogenesis and homeostasis and activate critical signaling pathways regulating survival, migration, gene expression, and differentiation. More importantly, any mutations of adhesion receptors can lead to developmental disorders and diseases. Thus, it is essential to understand the regulation of cell adhesion during development and its contribution to various conditions with the help of quantitative methods. The techniques involved in offering different functionalities such as surface imaging to detect forces present at the cell-matrix and deliver quantitative parameters will help characterize the changes for various diseases. Here, we have briefly reviewed single-cell mechanical properties for mechanotransduction studies using standard and recently developed techniques. This is used to functionalize from the measurement of cellular deformability to the quantification of the interaction forces generated by a cell and exerted on its surroundings at single-cell with attachment and detachment events. The adhesive force measurement for single-cell microorganisms and single-molecules is emphasized as well. This focused review should be useful in laying out experiments which would bring the method to a broader range of research in the future.

Pulsed laser assisted high-throughput intracellular delivery in hanging drop based three dimensional cancer spheroids.

Abstract: Targeted intracellular delivery of biomolecules and therapeutic cargo enables the controlled manipulation of cellular processes. Laser-based optoporation has emerged as a versatile, non-invasive technique that employs light-based transient physical disruption of the cell membrane and achieves high transfection efficiency with low cell damage. Testing of the delivery efficiency of optoporation-based techniques has been conducted on single cells in monolayers, but its applicability in three-dimensional (3D) cell clusters/spheroids has not been explored. Cancer cells grown as 3D tumor spheroids are widely used in anti-cancer drug screening and can be potentially employed for testing delivery efficiency. Towards this goal, we demonstrated the optoporation-based high-throughput intracellular delivery of a model fluorescent cargo (propidium iodide, PI) within 3D SiHa human cervical cancer spheroids. To enable this technique, nano-spiked core–shell gold-coated polystyrene nanoparticles (ns-AuNPs) with a high surface-to-volume ratio were fabricated. ns-AuNPs exhibited high electric field enhancement and highly localized heating at an excitation wavelength of 680 nm. ns-AuNPs were co-incubated with cancer cells within hanging droplets to enable the rapid aggregation and assembly of spheroids. Nanosecond pulsed-laser excitation at the optimized values of laser fluence (45 mJ cm−2), pulse frequency (10 Hz), laser exposure time (30 s), and ns-AuNP concentration (5 × 1010 particles per ml) resulted in the successful delivery of PI dye into cancer cells. This technique ensured high delivery efficiency (89.6 ± 2.8%) while maintaining high cellular viability (97.4 ± 0.4%), thereby validating the applicability of this technique for intracellular delivery. The optoporation-based strategy can enable high-throughput single cell manipulation, is scalable towards larger 3D tissue constructs, and may provide translational benefits for the delivery of anti-cancer therapeutics to tumors.

Metallic nanoparticles for biomedical applications

Abstract: Metallic nanoparticles have found various biomedical applications due to their intrinsic physicochemical properties. As the size decreases, the high surface area of particles gives rise to distinctive features, which are entirely different from that of a macro-sized structure. Several methods are involved in synthesizing metallic nanoparticles, and in general, it can be categorized into either bottom-up or top-down approaches. The top-down method consists of cutting down the bulk materials into nano-sized particles through physical, chemical, or mechanical treatments, whereas, in a bottom-up approach, nanoparticles are formed by joining individual atoms or molecules. The top-down approach produces metallic nanoparticles in naked form, which can further agglomerate and hence not suitable for biomedical applications. The bottom-up approach involves solid-state, liquid state, gas phase, biological, microfluidic-technology based, and other methods. Chemical reduction in the bottom-up approach is the most common method of metallic nanoparticle synthesis, which is flexible, simpler, inexpensive, and produces particles in homogenous form. Recently biological method of nanoparticle synthesis has become popular due to its toxic-free nature, inexpensiveness, sustainability, and eco-friendly. In this chapter, we describe the top-down and bottom-up approach and current trends in the synthesis of metallic nanoparticles for biomedical purposes. Further, it explains how the parameters can be tuned to get metallic nanoparticles with the desired shape, size, morphology, composition and crystallinity.

Fabrication of TiO2 microspikes for highly efficient intracellular delivery by pulse laser-assisted photoporation.

Abstract: 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.

Nanomaterials: An Introduction

Abstract: Nanotechnology offers a significant advantage in science, engineering, medicine, medical surgery, foods, packing, clothes, robotics, and computing from the beginning of the twenty-first century. As the potential scientific discovery always contains some good and bad effects on human civilization and the environment, nanotechnology is not an exception. The major drawbacks include economic disruption along with imposing threats to security, privacy, health, and environment. The introduction of the chapter discusses the historical background of nanotechnology. Later it also discusses the advancement of nanotechnology to date with its benefits. Major drawbacks of nanotechnology arise in human health due to the enormous involvement in medicine, food, agriculture, etc. This chapter also deals with environmental nano pollution and its effect on society, highlighting the social-economic disruption due to the rapid use of nanotechnology. Nano pollution affects not only human beings but also other living beings like microorganisms, animals and plants, which are briefly reviewed. This chapter also demonstrates the safety and security of nanotechnological developments, current policy and regulation status, challenges, and future trends. Finally, it is concluded, while nanotechnology offers more efficient power sources, faster and modern computers and technologies, life-saving medical treatments, but due to some negative impacts, it bounds us to think twice before any further advanced technological applications.

Microfluidic Based Physical Approaches towards Single-Cell Intracellular Delivery and Analysis

Abstract: The ability to deliver foreign molecules into a single living cell with high transfection efficiency and high cell viability is of great interest in cell biology for applications in therapeutic development, diagnostics, and drug delivery towards personalized medicine. Various physical delivery methods have long demonstrated the ability to deliver cargo molecules directly to the cytoplasm or nucleus and the mechanisms underlying most of the approaches have been extensively investigated. However, most of these techniques are bulk approaches that are cell-specific and have low throughput delivery. In comparison to bulk measurements, single-cell measurement technologies can provide a better understanding of the interactions among molecules, organelles, cells, and the microenvironment, which can aid in the development of therapeutics and diagnostic tools. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great interest. In recent years, single-cell technologies have become increasingly robust and accessible, although limitations exist. This review article aims to cover various microfluidic-based physical methods for single-cell intracellular delivery such as electroporation, mechanoporation, microinjection, sonoporation, optoporation, magnetoporation, and thermoporation and their analysis. The mechanisms of various physical methods, their applications, limitations, and prospects are also elaborated.

Editorial for the Special Issue on Micro/Nanofluidic Devices for Single Cell Analysis, Volume II.

Abstract: The functional, genetic, or compositional heterogeneity of healthy and diseased tissues promotes significant challenges to drug discovery and development [...]

Hydrogels: Biomaterials for Sustained and Localized Drug Delivery

Abstract: Hydrogels are three dimensional (3D) cross-linked polymer networks capable of holding a large volume of water. The hydrophilic polymeric system sometimes exists as a colloidal gel inside water, i.e., dispersion medium. Hydrogels aim to mimic the 3D microenvironment of cells with the advantage of surpassing adverse gastrointestinal effects on the drug, therefore increasing patient compliance. This polymer-based hydrogel formulation has tunable properties such as porosity, tensile strength, drug loading capacity, and release kinetics that contribute towards better biocompatible hydrogel design. The monomeric units in hydrogels bind through physical and chemical forces such as hydrophobic interaction, hydrogen bonding, UV crosslinking, and many others. Albeit hydrogel is known for its water holding capacity and high biocompatibility, the cytotoxicity of hydrogel depends on the polymer selection. Deformable and injectable hydrogels that can alter its physical state in room and body temperature are in the research pipeline to avoid surgery for implantation. Further, environmental stimuli-responsive hydrogels like pH, temperature-sensitive hydrogels are evolving as ‘Smart drug delivery’ systems. This distinctive property of tunable hydrogel design and formulation finds its application in sustained and localized drug delivery. This chapter discusses the different classifications of the hydrogel, along with its crosslinking chemistry involved. We also have summarised various forms of hydrogel from lab scale to industrial level. Finally, this chapter also covers the synthesis, functionalization, tailoring mechanism of the hydrogel matrix, followed by in vitro, ex vivo, and in vivo characterization and drug loading/delivery efficiency.

Nanomaterials: Versatile Drug Carriers for Nanomedicine

Abstract: Certain medicines and therapies have emerged for the treatment of different ailments. But in many cases, they have poor solubility, lower bioavailability, inability to cross the blood-brain barrier (BBB), and drug resistance. It becomes essential to establish standard treatment systems for overcoming such challenges. In this connection, the nanomaterial application in medicine and pharmaceuticals has rapidly gained interest with revolutionary prospects. The idea of nano-carriers first observed in a biological system consisting of nanoparticles is committed to locomotory function and protein cargos like importin, exportin. This observation led to the development of biomimetic nanomaterials for drug delivery. The synthesized nanomaterials exhibit useful properties such as large surface area, maximum bioavailability, reduced toxicity, high specificity along with enhanced permeability and retention (EPR) effect. These properties contribute to enhancing the efficacy of drugs having short half-lives, monitoring drugs for sustained release, enhancing the rate of dissolution of drugs, and reducing required dosage volume. Thus, increased therapeutic action and fewer side effects are to improve the quality of human life. Currently, many nano-carriers such as niosomes, dendrimers, fullerene, polymer-based nanoparticles, micelle, liposomes, hydrogels, metallic, mesoporous silica, quantum dots, etc. show potential for better drug delivery systems. These help in carrying entities like drug molecules, DNA/RNA, proteins, viruses, cell receptor sites, lipid bilayers, and variable antibody region for drug delivery in therapeutics. Such nano-therapeutics and diagnostics will unfold the secrets of human longevity and help reduce human illness, including cardiovascular disease, genetic disorder, immunodeficiency, cancer, and even viral infections. This chapter highlights recent advancements and applications of nano-carriers for drug delivery in medicine, especially wound healing therapeutics. It also discusses different approaches to enhance drug cargo capacity, improve cell delivery efficiency, to avoid host immune systems, and to achieve specific cell targeting.

Gold-Polystyrene Core-Shell Hybrid Nanoparticles Mediated Highly Efficient Intracellular Delivery Using Light Pulses

Abstract: Cellular transfection is a method by which exogenous biomolecules can be introduced into the cells. In the fields of molecular and cellular biology, cellular transfection is considered an essential tool, specifically for applications such as drug delivery, cellular therapy, and biomedical imaging. Photoporation based cellular transfection approach uses high-intensity light energy to create membrane pores, sometimes nanoparticles are incorporated to achieve the result at low intensity. In this work, hybrid nanoparticles, composed of gold and polystyrene are used to mediate the photoporation. Laser fluence, exposure time, wavelength, the concentration of nanoparticles, and concentration of exogenous molecules are optimized for the successful delivery of propidium iodide dye, quantum dots, and plasmid into CL1-0, AGS, and P-19 cells. The best results (delivery efficiency of 92% and cell viability of 98%) are achieved in delivering propidium iodide dye into CL1-0 cells.