Current Advancements and Future Road Map to Develop ASSURED Microfluidic Biosensors for Infectious and Non-Infectious Diseases.

Better diagnostics are always essential for the treatment and prevention of a disease. Existing technologies for detecting infectious and non-infectious diseases are mostly tedious, expensive, and do not meet the World Health Organization's (WHO) ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end user) criteria. Hence, more accurate, sensitive, and faster diagnostic technologies that meet the ASSURED criteria are highly required for timely and evidenced-based treatment. Presently, the diagnostics industry is finding interest in microfluidics-based biosensors, as this integration comprises all qualities, such as reduction in the size of the equipment, rapid turnaround time, possibility of parallel multiple analysis or multiplexing, etc. Microfluidics deal with the manipulation/analysis of fluid within micrometer-sized channels. Biosensors comprise biomolecules immobilized on a physicochemical transducer for the detection of a specific analyte. In this review article, we provide an outline of the history of microfluidics, current practices in the selection of materials in microfluidics, and how and where microfluidics-based biosensors have been used for the diagnosis of infectious and non-infectious diseases. Our inclination in this review article is toward the employment of microfluidics-based biosensors for the improvement of already existing/traditional methods in order to reduce efforts without compromising the accuracy of the diagnostic test. This article also suggests the possible improvements required in microfluidic chip-based biosensors in order to meet the ASSURED criteria.

Nanoparticle-based strategies to target HIV-infected cells.

Antiretroviral drugs employed for the treatment of human immunodeficiency virus (HIV) infections have remained largely ineffective due to their poor bioavailability, numerous adverse effects, modest uptake in infected cells, undesirable drug-drug interactions, the necessity for long-term drug therapy, and lack of access to tissues and reservoirs. Nanotechnology-based interventions could serve to overcome several of these disadvantages and thereby improve the therapeutic efficacy of antiretrovirals while reducing the morbidity and mortality due to the disease. However, attempts to use nanocarriers for the delivery of anti-retroviral drugs have started gaining momentum only in the past decade. This review explores in-depth the various nanocarriers that have been employed for the treatment of HIV infections highlighting their merits and possible demerits.

A paper microfluidic device based colorimetric sensor for the detection and discrimination of elapid versus viper envenomation.

Snake bites are a neglected tropical disease, causing mortality and severe damage to various vital organs like the nervous system, kidneys and heart. There is increasing interest in designing new antivenom treatments that are more specific to particular groups (either taxonomic or regional) of species, given the increasing evidence that current polyvalent Indian antivenom is ineffective in many situations. Under these circumstances, being able to detect the species, or a group of species, responsible for the envenomation becomes important. Unfortunately, no such diagnostic tool is available in the Indian market. Such a tool will need to be rapid, sensitive and affordable. To address this need, we have combined the power of nanotechnology and paper microfluidics and herein report a device that has the ability to detect and differentiate viper venom from elapid and scorpion venom. In principle, this assay is based on the release of the dye from the stimuli-responsive glutaraldehyde cross-linked methylene blue-loaded gelatin (GMG) nanoparticles in the presence of snake venom metalloproteases and serine proteases. The developed equipment-free assay can detect and discriminate viper venom from that of elapids and scorpions. The low-end detection limit of the sensor is ∼3.0 ng for the saw-scaled viper Echis carinatus, while the same for Russell's viper Daboia russelii is ∼6.0 ng. The performance of the sensor remains unaltered for different batches of GMG nanoparticles. Altogether, this finding establishes the role of nanotechnology and paper microfluidics in the rapid and accurate detection of viper venom.

Nanodiamonds synthesis using sustainable concentrated solar thermal energy: applications in bioimaging and phototherapy.

There is a renewed interest in nanodiamonds and their applications in biology and medicine, especially in bioimaging and photothermal therapy. This is due to their small size, chemical inertness and unique photo-properties such as bright and robust fluorescence, resistant to photobleaching and photothermal response under near infrared (NIR) irradiation. However, the biggest challenge limiting the wide-spread use of nanodiamonds is the high-energy consuming, dangerous and sophisticated synthetic methods currently adopted by industry named higher temperature high pressure approach, and detonation method. Despite over a decade of research towards the development of new synthetic approaches, most of the methods developed to date require sophisticated instrumentations and have high energy demand. To circumvent the reliance on high energy demanding sophisticated experimental setups, here we present a simple synthetic approach using solar energy as a sustainable sole energy source. Using low-grade coal as carbon precursor, we used high power magnifying glasses to concentrate and focus sunlight to induce synthesis of nanodiamonds. The synthesized nanodiamonds exhibit similar physicochemical and photo-properties as nanodiamonds synthesized using other synthetic approaches.In vitrostudies using macrophage Raw 264.7 cells demonstrated rapid uptake and bright fluorescence of the synthesized nanodiamonds with superior biocompatibility (≥95% cell viability). The synthesized nanodiamonds also exhibited dose dependent photothermal response under NIR irradiation.

Influence of surface charge of graphene quantum dots on their uptake and clearance in melanoma cells.

Graphene quantum dots (GQDs) continue to draw interest in biomedical applications. However, their efficacy gets compromised due to their rapid clearance from the body. On one hand, rapid clearance is desired and considered advantageous in terms of their cytocompatibility, but on the other hand, it is a major limitation for their prolonged use as imaging and therapeutic probes. The uptake and clearance of GQDs have been described in vivo, however, their clearance in vitro is still not understood, one of the main reasons being that their uptake and clearance are a cell type-dependent phenomena. Studies on other types of quantum dots revealed the importance of surface charge in their uptake and retention in different cell types. However, the role of surface chemistry in GQD uptake and clearance has not been described previously. Here, we studied the influence of surface charge on GQDs (anionic and cationic) on their uptake and clearance in melanoma cells. Both cationic and anionic GQDs were synthesized using a hydrothermal method to have a relatively consistent size with an aim to study the role of surface charge in their uptake and clearance in isolation by avoiding size-dependent uptake bias. Both GQDs exhibited excellent biocompatibility with cell viability over 90% even at a high concentration of 200 µg mL-1. Using confocal microscopy and flow cytometry, we observed significantly faster and higher uptake of cationic GQDs compared to anionic GQDs. Consequently, relatively rapid clearance was observed in cells treated with anionic GQDs compared to those treated with cationic GQDs, highlighting the role of surface charge on GQDs in their uptake and clearance. Raman analysis of the cleared exocytosed GQDs revealed no sign of biodegradation of either type.

Stealth anti-CD4 conjugated immunoliposomes with dual antiretroviral drugs--modern Trojan horses to combat HIV.

Highly active antiretroviral therapy (HAART) is the currently employed therapeutic intervention against AIDS where a drug combination is used to reduce the viral load. The present work envisages the development of a stealth anti-CD4 conjugated immunoliposomes containing two anti-retroviral drugs (nevirapine and saquinavir) that can selectively home into HIV infected cells through the CD4 receptor. The nanocarrier was characterized using transmission electron microscopy, FTIR, differential scanning calorimetry, particle size and zeta potential. The cell uptake was also evaluated qualitatively using confocal microscopy and quantitatively by flow cytometry. The drug to lipid composition was optimized for maximum encapsulation of the two drugs. Both drugs were found to localize in different regions of the liposome. The release of the reverse transcriptase inhibitor was dominant during the early phases of the release while in the later phases, the protease inhibitor is the major constituent released. The drugs delivered via anti-CD4 conjugated immunoliposomes inhibited viral proliferation at a significantly lower concentration as compared to free drugs. In vitro studies of nevirapine to saquinavir combination at a ratio of 6.2:5 and a concentration as low as 5 ng/mL efficiently blocked viral proliferation suggesting that co-delivery of anti-retroviral drugs holds a greater promise for efficient management of HIV-1 infection.

Targeting strategies for delivery of anti-HIV drugs.

Human Immunodeficiency Virus (HIV) infection remains a significant cause of mortality globally. Though antiretroviral therapy has significantly reduced AIDS-related morbidity and mortality, there are several drawbacks in the current therapy, including toxicity, drug-drug interactions, development of drug resistance, necessity for long-term drug therapy, poor bio-availability and lack of access to tissues and reservoirs. To circumvent these problems, recent anti-HIV therapeutic research has focused on improving drug delivery systems through drug delivery targeted specifically to host cells infected with HIV or could potentially get infected with HIV. In this regard, several surface molecules of both viral and host cell origin have been described in recent years, that would enable targeted drug delivery in HIV infection. In the present review, we provide a comprehensive overview of the need for novel drug delivery systems, and the successes and challenges in the identification of novel viral and host-cell molecules for the targeted drug delivery of anti-HIV drugs. Such targeted anti-retroviral drug delivery approaches could pave the way for effective treatment and eradication of HIV from the body.

Ellagic acid encapsulated chitosan nanoparticles as anti-hemorrhagic agent.

Ellagic acid, a naturally occurring polyphenol was encapsulated in chitosan particles prepared by ionotropic gelation and characterized for its physicochemical properties. A maximum encapsulation efficiency of 49% was achieved. The blood clotting time and clot retraction time were calculated for different concentrations of ellagic acid, chitosan and ellagic acid-encapsulated chitosan. A reduction of 34% in the clot time and 16.4% in the retraction time was observed in ellagic acid-encapsulated chitosan when compared with free ellagic acid at concentrations as low as 0.1mg/mL. The physical blend in comparison to free ellagic acid displayed a reduction of 13.8% and 4.6% in the clotting time and retraction time respectively under similar conditions. This suggests that the encapsulation of ellagic acid favors thrombosis due to synergistic action of chitosan and ellagic acid on same molecular targets. This study demonstrates the potential of ellagic acid-chitosan system as an effective anti-hemorrhagic system.

Evaluation of chitosan nanoformulations as potent anti-HIV therapeutic systems.

BACKGROUND: Antiretroviral Therapy (ART) is currently the major therapeutic intervention in the treatment of AIDS. ART, however, is severely limited due to poor availability, high cytotoxicity, and enhanced metabolism and clearance of the drug molecules by the renal system. The use of nanocarriers encapsulating the anti-retroviral drugs may provide a solution to the aforementioned problems. Importantly, the application of mildly immunogenic polymeric carrier confers the advantage of making the nanoparticles more visible to the immune system leading to their efficient uptake by the phagocytes. METHODS: The saquinavir-loaded chitosan nanoparticles were characterized by transmission electron microscopy and differential scanning calorimetry and analyzed for the encapsulation efficiency, swelling characteristics, particle size properties, and the zeta potential. Furthermore, cellular uptake of the chitosan nanocarriers was evaluated using confocal microscopy and Flow cytometry. The antiviral efficacy was quantified using viral infection of the target cells. RESULTS: Using novel chitosan carriers loaded with saquinavir, a protease inhibitor, we demonstrate a drug encapsulation efficiency of 75% and cell targeting efficiency greater than 92%. As compared to the soluble drug control, the saquinavir-loaded chitosan carriers caused superior control of the viral proliferation as measured by using two different viral strains, NL4-3 and Indie-C1, and two different target T-cells, Jurkat and CEM-CCR5. CONCLUSION: Chitosan nanoparticles loaded with saquinavir were characterized and they demonstrated superior drug loading potential with greater cell targeting efficiency leading to efficient control of the viral proliferation in target T-cells. GENERAL SIGNIFICANCE: Our data ascertain the potential of chitosan nanocarriers as novel vehicles for HIV-1 therapeutics.

Investigation on the stability of saquinavir loaded liposomes: implication on stealth, release characteristics and cytotoxicity.

Although anti-retroviral therapy is the most efficient disease management strategy for HIV-AIDS, its applications are limited by several factors including the low bioavailability and first pass metabolism of the drugs. Nanocarriers such as liposomes have been developed to circumvent some of these problems. We report here preparation of novel liposome formulations for efficient delivery of anti-retroviral drugs to mammalian cells in culture. The liposomes were prepared and surface was modified using poly (ethylene glycol). Encapsulation efficiency of the anti-retroviral drug saquinavir was found to be approximately 33% and also exhibited sustained release of the drug. Although PEGylated liposomes were more stable in protein-supplemented media, had better colloidal stability and exhibited lesser sonochemical stability due to lower cavitation threshold. The cell viability studies using Jurkat T-cells revealed that the PEGylated liposomes loaded with saquinavir were less cytotoxic as compared to the non-PEGylated liposomes or free drug confirming the potential of the liposomes as a sustained drug-release system. The drug delivery potential of the liposomes loaded with Alexa flour 647 was evaluated using Jurkat T-cells and flow cytometry showing uptake upto 74%. Collectively, our data demonstrate efficient targeting of mammalian cells using novel liposome formulations with insignificant levels of cytotoxicity.