Quality by design empowered preparation of itraconazole albumin nanoparticles for prostate cancer.

The current advent explores the potential of itraconazole (ITR) in prostate cancer (PCa), by its incorporation into albumin nanoparticles (NP). ITR as a repurposed moiety has displayed tremendous potential in various cancers. However, poor aqueous solubility poses hurdles towards its clinical translation. Amorphisation of ITR was observed post-incorporation within NP matrix which could prevent its precipitation in aqueous media. ITR NP was developed using quality by design and multivariate analysis and evaluated for cellular uptake, cell proliferation inhibition and the mechanism of PCa cell inhibition. Time and concentration-dependent serum stability and hemolytic potential revealed safety of ITR NP. Morphological changes and nuclear staining studies revealed the efficacy of ITR and ITR NP in promoting growth inhibition of PC-3 cells. Superior qualitative and quantitative uptake, reactive oxygen species (ROS) and mitochondrial impairment for ITR NP in comparison with ITR and control group was observed. Cell cycle study revealed remarkable G2/M phase inhibition in PC-3 cells. ITR NP demonstrated superior anticancer potential in 3D tumoroids mimicking the micro-metastatic lesions compared to control and ITR. Hence, ITR NP can be a favorable alternative therapeutic alternative in PCa.

Chitosan revokes controlled-cortical impact generated neurological aberrations in circadian disrupted mice via TLR4-NLRP3 axis.

The severity of inevitable neurological deficits and long-term psychiatric disorders in the aftermath of traumatic brain injury is influenced by pre-injury biological factors. Herein, we investigated the therapeutic effect of chitosan lactate on neurological and psychiatric aberrations inflicted by circadian disruption (CD) and controlled-cortical impact (CCI) injury in mice. Firstly, CD was developed in mice by altering sporadic day-night cycles for 2 weeks. Then, CCI surgery was performed using a stereotaxic ImpactOne device. Mice subjected to CCI displayed a significant disruption of motor coordination at 1-, 3- and 5-days post-injury (DPI) in the rotarod test. These animals showed anxiety- and depression-like behaviors in the elevated plus maze and forced-swim test at 14 and 15 DPI, respectively. Notably, mice subjected to CD + CCI exhibited severe cognitive impairment in Y-maze and novel object recognition tasks. The compromised neurological, psychiatric, and cognitive functions were mitigated in chitosan-treated mice (1 and 3 mg/mL). Immunohistochemistry and real-time PCR assay results revealed the magnified responses of prima facie biomarkers like glial-fibrillary acidic protein and ionized calcium-binding adaptor molecule 1 in the pericontusional brain region of the CD + CCI group, indicating aggravated inflammation. We also noted the depleted levels of brain-derived neurotrophic factor and augmented expression of toll-like receptor 4 (TLR4)-leucine-rich-containing family pyrin domain-containing 3 (NLRP3) signaling [apoptosis-associated-speck-like protein (ASC), caspase-1, and interleukin 1-ß] in the pericontusional area of CD + CCI group. CCI-induced changes in the astrocyte-glia and aggravated immune responses were ameliorated in chitosan-treated mice. These results suggest that the neuroprotective effect of chitosan in CCI-induced brain injury may be mediated by inhibition of the TLR4-NLRP3 axis.

Microfluidics-Based Blood Typing Devices: An In-Depth Overview.

Identification of correct blood types holds paramount importance in understanding the pathophysiological parameters of patients, therapeutic interventions, and blood transfusion. Considering the wide applications of blood typing, the requirement of centralized laboratory facilities is not well suited on many occasions. In this context, there has been a significant development of such blood typing devices on different microfluidic platforms. The advantages of these microfluidic devices offer easy, rapid test protocols, which could potentially be adapted in resource-limited settings and thereby can truly lead to the decentralization of testing facilities. The advantages of pump-free liquid transport (i.e., low power consumption) and biodegradability of paper substrates (e.g., reduction in medical wastes) make it a more preferred platform in comparison to other microfluidic devices. However, these devices are often coupled with some inherent challenges, which limit their potential to be used on a mass commercial scale. In this context, our Review offers a succinct summary of the recent development, especially to understand the importance of underlying facets for long-term sustainability. Our Review also delineates the role of integration with digital technologies to minimize errors in interpreting the readouts.

Multifaceted roles of pollen in the management of cancer.

Oral drug delivery of microparticles demonstrates shortcomings like aggregation, decreased loading capacity and batch-to-batch variation, which limits its scale-up. Later, porous structures gained attention because of their large surface-to-volume ratio, high loading capacity and ability to carry biomacromolecules, which undergo degradation in GIT. But there are pitfalls like non-uniform particle size distribution, the impact of porogen properties, and harsh chemicals. To circumvent these drawbacks, natural carriers like pollen are explored in drug delivery, which withstands harsh environments. This property helps to subdue the acid-sensitive drug in GIT. It shows uniform particle size distribution within the species. On the other side, they contain phytoconstituents like flavonoids and polysaccharides, which possess various pharmacological applications. Therefore, pollen has the capability as a carrier system and therapeutic agent. This review focuses on pollen's microstructure, composition and utility in cancer management. The extraction strategies, characterisation techniques and chemical structure of sporopollenin exine capsule, its use in the oral delivery of antineoplastic drugs, and emerging cancer treatments like photothermal therapy, immunotherapy and microrobots have been highlighted. We have mentioned a note on the anticancer activity of pollen extract. Further, we have summarised the regulatory perspective, bottlenecks and way forward associated with pollen.

Structural characterization and in silico toxicity prediction of degradation impurities of roxadustat.

Roxadustat is the first drug approved for anemia due to chronic kidney disease. Drug degradation profile is very crucial for assessing the quality and safety of the drug substances and their formulations. Forced degradation studies are conducted for quick prediction of drug degradation products. Forced degradation of roxadustat was carried out as per ICH guidelines, and nine degradation products (DPs) were observed. These DPs (DP-1 to DP-9) were separated using the reverse phase HPLC gradient method with an XBridge column (250 mm × 4.6 mm, 5 µm). The mobile phase consisted of 0.1% formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 1.0 ml/min. The chemical structures of all the DPs were proposed by using LC-Q-TOF/MS. DP-4 and DP-5, the two major degradation impurities, were isolated, and NMR was used to confirm their chemical structures. Based on our experiments, the roxadustat was found stable to thermal degradation in solid state and oxidative conditions. However, it was unstable in acidic, basic, and photolytic conditions. A very remarkable observation was made about DP-4 impurity. DP-4 was generated as a common degradation impurity in alkaline hydrolysis, neutral hydrolysis as well as photolysis conditions. DP-4 has a similar molecular mass to roxadustat but is structurally different. DP-4 is chemically, (1a-methyl-6-oxo-3-phenoxy-1,1a,6,6a-tetrahydroindeno [1,2-b] aziridine-6a-carbonyl) glycine. In silico toxicity study was conducted using Dereck software to gain the best knowledge of the drug and its degradation products towards carcinogenicity, mutagenicity, teratogenicity, and skin sensitivity. A further study using molecular docking confirmed the potential interaction of DPs with proteins responsible for toxicity. DP-4 shows a toxicity alert due to the presence of aziridine moiety.

A critical review of Roxadustat formulations, solid state studies, and analytical methodology.

This review aims to collate information about the analytical methodologies, bioanalytical methodologies, pharmaceutical formulations, solid-state studies, and the current and future market scenario for a relatively new class of drugs, Roxadustat. Roxadustat is a hypoxia-inducible factor propyl hydroxylase inhibitor that significantly increases blood hemoglobin via the action of transcriptional activator HIF. As the molecule has a promising role in stimulating erythropoiesis, it is considered an ideal therapeutic agent for patients with anemia. In the current review, an attempt has been made to compile the pharmacological, pharmacokinetic, and pharmacodynamic characteristics of Roxadustat and systematically present product development data. This drug has several polymorphs of cocrystal, co-former, and salt, which have been explained in detail in the current work. The comprehensive review summarizes all the chromatographic methods and is presented in table form. This review has extensively covered Liquid chromatography-tandem mass spectrometry methods used to analyze Roxadustat in the biological matrix. The literature needs more data on forced degradation study, impurity profiling, gas chromatography, analytical methods for assay, dissolution, and different formulation aspects of Roxadustat.

Chitosan reduced in-situ synthesis of gold nanoparticles on paper towards fabricating highly sensitive, stable uniform SERS substrates for sensing applications.

Surface-Enhanced Raman Spectroscopy (SERS) is a powerful surface-sensitive technique for molecular analysis. Its use is limited due to high cost, non-flexible rigid substrates such as silicon, alumina or glass and less reproducibility due to non-uniform surface. Recently, paper-based SERS substrates, a low-cost and highly flexible alternative, received significant attention. We report here a rapid, inexpensive method for chitosan-reduced, in-situ synthesis of gold nanoparticles (GNPs) on paper devices towards direct utilization as SERS substrates. GNPs have been prepared by reducing chloroauric acid with chitosan as a reducing and capping reagent on the cellulose-based paper surface at 100 °C, under the saturated humidity condition (100 % humidity). GNPs thus obtained were uniformly distributed on the surface and had fairly uniform particle size with a diameter of 10 ± 2 nm. Substrate coverage of resulting GNPs directly depended on the precursor's ratio, temperature and reaction time. Techniques such as TEM, SEM, and FE-SEM were utilized to determine the shape, size, and distribution of GNPs on paper substrate. SERS substrate produced by this simple, rapid, reproducible and robust method of chitosan-reduced, in situ synthesis of GNPs, showed exceptional performance and long-term stability, with a detection limit of up to 1 pM concentration of test analyte, R6G. Present paper-based SERS substrates are cost-effective, reproducible, flexible, and suitable for field applications.

Tuning Mesoporous Silica Nanoparticles in Novel Avenues of Cancer Therapy.

The global menace of cancer has led to an increased death toll in recent years. The constant evolution of cancer therapeutics with novel delivery systems has paved the way for translation of innovative therapeutics from bench to bedside. This review explains the significance of mesoporous silica nanoparticles (MSNs) as delivery vehicles with particular emphasis on cancer therapy, including novel opportunities for biomimetic therapeutics and vaccine delivery. Parameters governing MSN synthesis, therapeutic agent loading characteristics, along with tuning of MSN toward cancer cell specificity have been explained. The advent of MSN in nanotheranostics and its potential in forming nanocomposites for imaging purposes have been illustrated. Additionally, various hurdles encountered during the bench to bedside translation have been explained along with potential avenues to circumvent them. This also opens up new horizons in drug delivery, which could be useful to researchers in the years to come.

Ready-to-use vertical flow paper device for instrument-free room temperature reverse transcription.

Paper-based nucleic acid detection and diagnosis are currently gaining much interest in point-of-care (POC) applications. The major steps involved in any nucleic acid amplification testing (NAAT) based diagnostics are nucleic acid isolation, reverse transcription (RT) (in the case of RNA), amplification and detection. RT is an important step in quantifying the viral load in case of disease diagnosis as well as quantifying gene expression levels in other molecular studies. cDNA synthesis is routinely carried out using a thermal cycler, with the process requiring temperatures between 40ºC to 65ºC. Here we report for the first time an instrument-free RT, performed at room temperature on cellulose-based paper devices. cDNA synthesis on paper was confirmed by RT-PCR and Sanger sequencing of the PCR products. Purified RNA from varied sources such as cell lysate, tissue and blood were used to test the methodology. Synthetic hepatitis C virus (HCV) RNA and human blood RNA were used as proof-of-concept to demonstrate the use of these devices in diagnostic applications. Further, ready-to-use paper-based reverse transcription (PRT) devices have been developed, wherein only the RNA sample is added on the device and the cDNA can be eluted after 30 min of incubation at room temperature. The devices were found to be stable for 30 days at - 20ºC storage. The cellulose-based PRT devices are simple, time saving and user-friendly for a complete instrument-free cDNA synthesis at room temperature.

A novel method for fabrication of paper-based microfluidic devices using BSA-ink.

This paper describes the fabrication of paper-based microfluidic devices using a novel, inexpensive ink composed of bovine serum albumin (BSA), utilizing BSA's thermal denaturation and aggregation to create a hydrophobic barrier on Whatman® Grade 4 filter paper. A 20% aqueous solution of BSA was inked onto the paper using a pen plotter at moderate speed (5 cm/s) with desired shape and size followed by heating at 80 °C to denature the BSA leading to hydrophobic barriers formation, whereas below 80 °C the barrier layer is prone to collapse. The minimum line gap and line width of ~1 mm and ~1.3 mm were achieved. Finally, a proof-of-concept glucose sensing was shown while addressing the issue of the coffee ring effect using the biopolymer NanoCheck-ATH® from ChitoLytic Inc. The glucose concentration limit of detection (LOD) as low as 0.2 mg/mL was estimated. The developed technique offers ease of fabrication, high reproducibility, cost-effectiveness, and is environmentally friendly.

Complex target SELEX-based identification of DNA aptamers against Bungarus caeruleus venom for the detection of envenomation using a paper-based device.

Complex target SELEX always have been an intriguing approach to the scientific community, as it offers the potential discovery of novel biomarkers. We herein successfully performed SELEX on Bungarus caeruleus venom to develop a panel of highly affine aptamers that specifically recognizes the B. caeruleus (common krait) venom and was able to discriminate the B. caeruleus venom from Cobra, Russell's, and Saw-scaled viper's venom. The aptamers generated against the crude venom also lead to the identification of the specific component of the venom, which is ß-Bungarotoxin, a toxin uniquely present in the B. caeruleus venom. The best performing aptamer candidates were used as a molecular recognition element in a paper-based device and were able to detect as low as 2 ng krait venom in human serum background. The developed aptamer-based paper device can be used for potential point-of-care venom detection applications due to its simplicity and affordability.

Cross-linked chitosan biofunctionalized paper-based microfluidic device towards long term stabilization of blood typing antibodies.

Long term stability of antibodies at room temperature is a major challenge in the commercialization of point-of-care devices for diagnostics. Since chitosan has been proven to be an excellent biofunctionalization material, the effects of four different biofunctionalization processes were studied to improve the room temperature stability of antibodies immobilized on chitosan modified paper-based microfluidic devices using blood typing antibodies as candidates. The devices used in this work have a flower-shaped design with 4 test zones at each corner. In three zones Anti-A, Anti-B, and Anti-D (Anti-Rh) antibodies are immobilized and the fouth zone represents the control (no antibodies) after biofunctionalization. The biofunctionalization of the paper devices was done with chitosan and chitosan cross-linked with sodium triphosphate pentabasic, glutaraldehyde, and sodium hydroxide. These devices were used for blood typing assays using real blood samples. A similar assay was also performed on unmodified (non-biofunctionalized) paper devices for comparison. Chitosan based biofunctionalized paper-devices showed better stability, up to 100 days as compared to 14 days on unmodified paper, at room temperature. Such biofunctionalized paper-based devices will be suitable for on-field and remote testing without any technical expertise and requirement for the cold chain.

Fabrication of cost-effective and efficient paper-based device for viscosity measurement.

Use of paper-based devices for affordable diagnostics is gaining interest due to unique advantages such as affordability, portability, easy disposability and inherent capillarity. As capillary transportation is an integral component of paper-based devices, low sample volume with faster measurement becomes an additional advantage. We have developed a simple, paper-based microfluidic device suitable for measuring the viscosity of Newtonian fluids as well as a few non-Newtonian fluids with sample volume as little as 12-20 µL. The results could be obtained much faster than the conventional methods. A comparative analysis of the results obtained with our paper-based viscometer and with that of the conventional Ostwald viscometer shows a correlation coefficient greater than 0.99. Apart from viscosity measurement, the paper-based devices were tested for protein denaturation and polymer molecular weight determination. Our results show that the paper-based viscometer could be a potential alternative for the conventional viscometers in the viscosity range from 0.9 cP up till ∼40 cP, with added benefits in terms of time, cost and low sample volume requirement.

Chitosan as biomaterial in drug delivery and tissue engineering.

Chitin is one of the most abundant polysaccharide found on earth. The deacetylated form of chitin viz. chitosan has been reported for its various important pharmacological properties and its role in tissue engineering and regenerative medicine is also well documented. Chitosan based bone graft substitutes are biocompatible, biodegradable, osteoconductive, osteoinductive and structurally similar to bone, with excellent mechanical strength and cost effectiveness. Chitosan based hydrogels and wound healing bandages have also found a great market in the field of medicine. More recently, chitosan has gained popularity for its use as a matrix molecule for drug delivery and also finds an upcoming utility in the area of dentistry. The present article has tried to review the latest research on chitosan based tissue engineering constructs, drug delivery vehicles as well as dental care products. An attempt has also been made to discuss the various modifications of chitosan that enhance its use for a given set of applications which would pave a way for future applied research in the field of biomedical innovation and regenerative medicine.

Chitosan stabilized gold nanoparticle mediated self-assembled gliP nanobiosensor for diagnosis of Invasive Aspergillosis.

Diagnosis of Invasive Aspergillosis (IA) casused by Aspergillus fumigatus in miniaturized setting is challenging with great importance in human health. In this direction, we have designed a sensitive electrochemical nanobiosensor for diagnosis of IA through detecting the virulent glip target gene (glip-T) in a miniaturized experimetal setting. The sensor probe was fabricated using 1,6-Hexanedithiol and chitosan stabilized gold nanoparticle mediated self-assembly of glip probes (glip-P) on gold electrode. It was characterized by UV-visible spectroscopy, cyclic voltametry and electrochemical impedance spectroscopy. The ability of sensor to detect glip-T was analysed based on the hybridyzation reaction and the signal obtained using toluidine blue as indicator molecule. Analytical parameters were optimized in terms of glip-P concentration, temperature, reaction time, and concentration of toluidine blue. The biosensor showed the dynamic range between 1 × 10-14- 1 × 10-2 M with the detection limit of 0.32 ±â€¯0.01 × 10-14(RSD < 5.2%). The regeneration of biosensor was evaluated and the interference due to non-target oligonucleotide sequences was evaluated individualy as well as in mixed sample to validate the high selectivity of the designed sensor. The stability of the designed sensor was examined and practical applicability of biosensor was tested by detecting glip-T in real sample environment.

Modulation of stem cell differentiation by the influence of nanobiomaterials/carriers.

Stem cells, either neural [NSCs] or mesenchymal [MSCs], possess tremendous untapped potential for cell therapy. Unlike the NSCs, MSCs are multi-potent and they have high self-renewal capability and broad tissue distribution. Since they do not produce significant immune rejection on post-transplantation; they are better suited for cell-based therapies. However, several critical issues need to be addressed to maximize stem cell-derived therapeutic effects. The key factor affecting the therapeutic application of stem cells is exposure to hostile conditions in vivo such as oxidative stress, which results in considerably low survival rate of these cells at transplanted sites, thereby reducing the therapeutic efficiency. Such limitation has led scientists to design clinically relevant, innovative and multifaceted solutions including the use of nanobiomaterials. Use of cytocompatible nanobiomaterials holds great promise and has gained attention of researchers, worldwide. Various nanobiomaterials are being explored to increase the survival efficiency and direct differentiation of stem cells to generate tissue-specific cells for biomedical research and futuristic therapies. These materials have superior cytocompatability, mechanical, electrical, optical, catalytic and magnetic properties. Non-invasive visualization of the biological system has been developed using magnetic nanoparticles and magnetic resonance imaging [MRI] approaches. Apart from viral vectors, non-viral carriers such as DNA nano carriers, single stranded RNA nanoparticles, liposomes and carbon nanotubes/wires are being exploited for gene delivery into stem cells. This article reviews potential application of various biocompatible nanomaterials in stem cell research and development.

Foil assisted replica molding for fabrication of microfluidic devices and their application in vitro.

We present a simple, rapid, benchtop, Foil Assisted Rapid Molding (FARM) method for the fabrication of microfluidic devices. This novel technique involves the use of aluminium foil, pen and an X-Y plotter to create semi-circular or plano-concave, shallow microchannels. It is an easy do-it-yourself (DIY) technique for creating a microfluidic device in three simple steps: (1) create a channel design using the CAD software, (2) plot the patterns on aluminium foil and (3) use the reverse of the engraved foil as a mold to create microfluidic devices. In this report, we present a detailed study of the proposed method by varying a range of parameters such as foil thickness, tip material, and tip sizes and by investigating their effect on the creation of channels with varying geometry. Furthermore, we demonstrated the cytocompatibility of these devices in vitro.

Droplet-based microfluidics.

Droplet-based microfluidics or digital microfluidics is a subclass of microfluidic devices, wherein droplets are generated using active or passive methods. The active method for generation of droplets involves the use of an external factor such as an electric field for droplet generation. Two techniques that fall in this category are dielectrophoresis (DEP) and electrowetting on dielectric (EWOD). In passive methods, the droplet generation depends on the geometry and dimensions of the device. T-junction and flow focusing methods are examples of passive methods used for generation of droplets. In this chapter the methods used for droplet generation, mixing of contents of droplets, and the manipulation of droplets are described in brief. A review of the applications of digital microfluidics with emphasis on the last decade is presented.

Rapid and cost-effective fabrication of selectively permeable calcium-alginate microfluidic device using "modified" embedded template method.

In this paper, we have presented a non-lithographic embedded template method for rapid and cost-effective fabrication of a selectively permeable calcium-alginate (Ca-alginate) based microfluidic device with long serpentine delay channel. To demonstrate the versatility of the presented method, we have demonstrated two different strategies to fabricate serpentine long delay channels without using any sophisticated microfabrication techniques, in formal lab atmosphere. The procedure presented here, also, enables the preparation of a multilayered microfluidic device with channels of varying dimensions, in a single device without using any sophisticated micromachining instrumentation. In addition, we have also qualitatively studied the diffusion of small and large molecules from a Ca-alginate based microfluidic device and proposed a method to effectively control the out-flow of macro biomolecules from the crosslinked Ca-alginate matrix to create a selectively permeable matrix required for various biological and biomimetic applications, as mentioned in the Introduction section of this work.

Bromo-oxidation reaction in enzyme-entrapped alginate hollow microfibers.

In this article, the authors present the fabrication of an enzyme-entrapped alginate hollow fiber using a microfluidic device. Further use of enzyme-entrapped alginate hollow fibers as a biocatalytic microchemical reactor for chemical synthesis is also deliberated in this article. To ensure that there is no enzyme leaching from the fiber, fiber surfaces were coated with chitosan. To confine the mobility of reactants and products within the porous hollow fibers the entire fibers were embedded into a transparent polydimethylsiloxane (PDMS) matrix which also works as a support matrix. A vanadium-containing bromoperoxidase enzyme isolated from Corallina confusa was used as a model enzyme to demonstrate the use of these alginate hollow-fiber reactors in bromo-oxidation of phenol red to bromophenol blue at different dye flow rates. Stability of the entrapped enzyme at different temperatures and the effect of the chitosan coating on the reaction conversion were also studied. It was observed that molecules as big as 27 kDa can be retained in the matrix after coating with chitosan while molecules with molecular-weight of around 378 Da can still diffuse in and out of the matrix. The kinetic conversion rate in this microfluidic bioreactor was more than 41-fold faster when compared with the standard test-tube procedure.