Mannose receptor targeted bioadhesive chitosan nanoparticles of clofazimine for effective therapy of tuberculosis.

Drug-resistant tuberculosis (TB) is one of the most lethal diseases, and it is imperative to exploit an advanced drug formulation for its effective treatment. This work aims to develop a mannose receptor-targeted bioadhesive chitosan nanoparticles for effective drug-resistant tuberculosis treatment. The clofazimine loaded chitosan nanoparticles were formulated; their size, charge, polydispersity (PDI), surface morphology, entrapment efficiency (EE) and in-vitro release pattern were established. Also, cellular uptake study on C2C12 cell lines and anti-mycobacterial activity against H37Rv (a standard strain of Mycobacterium tuberculosis) were evaluated. The particle sizes of formulated chitosan nanoparticles were in the range of 132-184 nm and EE was also found to be between 73 and 95%. The functionalization of bioadhesive chitosan nanoparticles with mannose was confirmed by infrared spectroscopy (FTIR). The uptake studies on the C2C12 cell lines showed that mannosylated nanoparticles were more efficiently internalized when compared to non-targeted nanoparticles. Further, luciferase reporter phage (LRP) assay against H37Rv strain showed that clofazimine nanoparticles were found to be 49.5 times superior in terms of inhibition and anti-mycobacterial activity than free clofazimine. This excellent activity might be attributed to enhanced drug delivery with a promising bioadhesion property of chitosan-based nanoparticles.

Low density culture of mammalian primary neurons in compartmentalized microfluidic devices.

This paper demonstrates the fabrication of a compartmentalized microfluidic device with docking sites to position a single neuron or a cluster of 5-6 neurons along with varying length of microgrooves and the optimization process for culturing primary mammalian neurons at low densities. The principle of centrifugation was employed to situate cells in desired locations followed by the application of a fluid flow to remove the extra or unwanted cells lying in the vicinity of the located neurons. The neuronal cell density was optimized by seeding 103 cells and 104 cells/microfluidic device. The speed of centrifugation was optimized as 1500 rpm for 1 min and a cell density of greater than or equal to 104 cells/microfluidic device was found to be suitable for loading maximum number of docking sites. The outcomes of the simulated experiments was found to be in compliance with the experimemtal verifications. Furthermore, the cells cultured within the microfluidic device were assessed for immunocytochemical staining and the axonal growth was quantified with the help of an Axofluidic software. Although, several in vitro microfluidic platforms have been developed that facilitate the investigations where communication between neurons or between neurons and other cell types is concerned, none of the partitioned devices so far has reported the presence of docking sites along with an array of grooves of varying lengths. These physically connected but fluidically isolated compartmentalized microfluidic devices may serve us in analysing the activity of a low density of neurons and the influence of axonal length in setting up a communication with other cell type.This platform is useful to gain insights into the processes of synapse formation, axonal guidance, cell-cell interaction, to name a few.

Printability assessment of psyllium husk (isabgol)/gelatin blends using rheological and mechanical properties.

The primary goal of this study is to highlight the rheological and mechanical properties of a new blend composed of naturally-derived hydrogel materials- psyllium husk (PH) and gelatin (G) for its potential use in three-dimensional (3D) printing technology. The mixtures were prepared at various weight ratios of 100PH, 75PH + 25G and 50PH + 50G. A suitable selection of the printable ink was made based on the preliminary screening steps of manual filament drop test and layer stacking by 3D printing. Printing of the common features such as hexagon and square grids helped evaluating shape fidelity of the chosen ink. Although 50PH + 50G blend was found meeting most of the criteria for an ideal 3D printable ink, rheological and mechanical characterizations have been performed for all the ratios of polymeric blends. This study documents the correlation between various factors of rheology that should be taken into account while categorizing any biomaterial as a printable ink. Yield stress was measured as 18.59 ± 4.21 Pa, 268.74 ± 13.56 Pa and 109.16 ± 9.85 Pa for 50PH + 50G, 75PH + 25G and 100PH, respectively. Similarly, consistency index (K) and flow index (n) were calculated using the power law equation and found as 49.303 ± 4.17, 530.59 ± 10.92, 291.82 ± 10.53 and 0.275 ± 0.04, 0.05 ± 0.005, 0.284 ± 0.04 for 50PH + 50G, 75PH + 25G and 100PH, respectively. The loss modulus (G″) was observed dominating over storage modulus (G') for 50PH + 50G, that depicts its liquid-like property; whereas storage modulus (G') was found dominating in case of 75PH + 25G and 100PH, indicating their solid-like characteristics. In addition, the loss tangent value (tan δ) of 50PH + 50G was observed exceeding unity (1.05), supporting its plastic behavior, unlike 75PH + 25G (0.5) and 100PH (0.33) whose loss tangent values were estimated less than unity revealing their elastic behavior. Also, 50PH + 50G was found to have the highest mechanical strength amongst the three blends with a Young's modulus of 9.170 ± 0.0881 kPa.

Recent advances in bioprinting technologies for engineering different cartilage-based tissues.

Inadequate self-repair and regenerative efficiency of the cartilage tissues has motivated the researchers to devise advanced and effective strategies to resolve this issue. Introduction of bioprinting to tissue engineering has paved the way for fabricating complex biomimetic engineered constructs. In this context, the current review gears off with the discussion of standard and advanced 3D/4D printing technologies and their implications for the repair of different cartilage tissues, namely, articular, meniscal, nasoseptal, auricular, costal, and tracheal cartilage. The review is then directed towards highlighting the current stem cell opportunities. On a concluding note, associated critical issues and prospects for future developments, particularly in this sphere of personalized medicines have been discussed.

Recent advances in bioprinting technologies for engineering cardiac tissue.

Annually increasing incidence of cardiac-related disorders and cardiac tissue's minimal regenerative capacity have motivated the researchers to explore effective therapeutic strategies. In the recent years, bioprinting technologies have witnessed a great wave of enthusiasm and have undergone steady advancements over a short period, opening the possibilities for recreating engineered functional cardiac tissue models for regenerative and diagnostic applications. With this perspective, the current review delineates recent developments in the sphere of engineered cardiac tissue fabrication, using traditional and advanced bioprinting strategies. The review also highlights different printing ink formulations, available cellular opportunities, and aspects of personalized medicines in the context of cardiac tissue engineering and bioprinting. On a concluding note, current challenges and prospects for further advancements are also discussed.

Combined substrate micropatterning and FFT analysis reveals myotube size control and alignment by contact guidance.

Most important evaluating criteria for in vitro skeletal muscle models include the extent of differentiation and the degree of alignment in the tissue model. Substrate micropatterning is considered as an effective tool as it recreates in vivo like cellular microenvironment and helps in understanding the fundamental concepts and mechanisms underlying myogenesis. However, the influence of micropatterning based contact guidance cues over satellite cell alignment and myotube formation needs to be explored and studied further. In the present work, we demonstrate the regulation of myotube size control and alignment through the substrate micropatterning. For this purpose, primary myoblast cells (i.e., satellite cells) isolated from rat hind limb muscle were characterized and cultured for a period of 14 days on micropatterned glass substrates processed by the microchannnel flowed plasma process. Several characteristic parameters of muscle differentiation, including the fusion index, maturation index, and average width of the myotubes were quantified. The functional behavior of cultured myotubes exhibiting spontaneous contractions was assessed through kymograph to determine the twitch frequency. In addition, we evaluated the degree of alignment of myotubes on micropatterned substrates through examining orientation order parameter and two-dimensional fast Fourier transform analysis. Altogether, the outcomes reveal that the contact guidance cues arising due to micropatterning of the substrates could be a key regulator for controlling the size and degree of alignment of myotubes during the myogenesis process.

Fabrication and Cytocompatibility Evaluation of Psyllium Husk (Isabgol)/Gelatin Composite Scaffolds.

Psyllium husk or isabgol contains xylan backbone linked with arabinose, rhamnose, and galacturonic acid units (arabinoxylans). In this study, we demonstrate the fabrication and characterization of a macroporous three-dimensional (3D) composite scaffold by mixing psyllium husk powder (PH) and gelatin (G) in different ratios, viz.100 PH, 75/25 PH/G, and 50/50 PH/G (w/w), using an EDC-NHS coupling reaction followed by freeze-drying method. The reaction was performed in aqueous as well as in alcoholic media to determine the most appropriate solvent system for this purpose. The mechanical strength of the scaffold system was improved from 151 to 438 kPa. The fabricated scaffolds exhibited enhanced structural stability, remarkable swelling capacity, and escalated cell growth and proliferation. ATR-FTIR analysis showed the presence of amide and ester bonds indicating covalent crosslinking. SEM micrographs revealed the porous nature of the scaffolds with pores ranging from 30 to 150 µm, and further pore size distribution curve indicated that 75/25 PH/G (w/w%) EDC-NHS-alcohol scaffold exhibited the best fit to the Gaussian distribution. Swelling capacity of the 100 PH EDC-NHS-alcohol scaffolds was found to be nearly 40% from its original weight in 48 h. MTT assay using fibroblast cells revealed ~ 80% cellular proliferation by 6th day within the fabricated scaffolds in comparison to control. Graphical Abstract ᅟ.

Culturing melanocytes and fibroblasts within three-dimensional macroporous PDMS scaffolds: towards skin dressing material.

In the present study, we propose a platform for topical wound dressing material using a polydimethylsiloxane (PDMS) scaffold in order to enhance the skin healing process. In vitro co-culture assessment of epidermal-origin mouse B16-F10 melanocyte cells and mouse L929 fibroblast cells in three-dimensional polymeric scaffolds has been carried out towards developing bio-stable, interconnected, highly macroporous, PDMS based tissue-engineered scaffolds, using the salt leaching method. To determine a suitable ratio of salt to PDMS pre-polymer in the scaffold, two different samples with ratios 2:1 and 3:1 [w/w], were fabricated. Effective pore sizes of both scaffolds were observed to lie in the desirable range of 152-165 µm. In addition, scaffolds were pre-coated with collagen and investigated as a podium for culturing the chosen cells (fibroblast and melanocyte cells). Experimental results demonstrate not only a high proliferative potential of the skin tissue-specific cells within the fabricated PDMS based scaffolds but also confirm the presence of several other essential attributes such as high interconnectivity, optimum porosity, excellent mechanical strength, gaseous permeability, promising cell compatibility, water absorption capability and desired surface wettability. Therefore, scaffolds facilitate a high degree of cellular adhesion while providing a microenvironment necessary for optimal cellular infiltration and viability. Thus, the outcomes suggest that PDMS based macroporous scaffold can be used as a potential candidate for skin dressing material. In addition, the fabricated PDMS scaffolds may also be exploited for a plethora of other applications in tissue engineering and drug delivery.

Development of optically sensitive liver cells.

Optogenetics is a new and emerging field that involves techniques of optics and genetic engineering to influence cellular functionality. In this work, we have successfully incorporated a non-selective cationic channel channelrhodopsin-2 (ChR2) into human hepatocellular carcinoma (HepG2) cells. A plasmid construct AAV-CAG-ChR2-GFP was used for liposomal transfection into the cells. ChR2 is a light sensitive membrane channel that opens upon illumination with blue light. Plasmid DNA isolation from E. coli XL 10 gold bacteria by alkaline lysis method resulted in a DNA concentration of 1150 µg/mL. A significant difference (p < 0.05) was observed between the fluorescent intensities of transfected cells and the control. The percentage of transfected cells was estimated to be 41.26%. Overall, the study delivers an optimized methodology to produce the transfected HepG2 cells that can be controlled with the light stimulation. Although ChR2 has mostly been associated with excitable cells, we anticipate that its presence into HepG2 cells may also result changes in biological functionalities by modulating the concentration of cations inside the cell. Furthermore, the transfected HepG2 cells can be co-cultured with fibroblasts such as NIH 3T3 to form liver spheroids that can serve as models for toxicological and pharmacological studies.

Pharmacokinetics, biodistribution, in vitro cytotoxicity and biocompatibility of Vitamin E TPGS coated trans resveratrol liposomes.

The clinical application of trans resveratrol (RSV) in glioma treatment is largely limited because of its rapid metabolism, fast elimination from systemic circulation and low biological half life. Therefore, the objectives of this study were to enhance the circulation time, biological half life and passive brain targeting of RSV using d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) coated liposomes (RSV-TPGS-Lipo). In addition to basic liposomal characterizations, in vitro anticancer potential against C6 glioma cell lines and cellular internalization of liposomes were carried out by MTT assay and confocal laser scanning microscopy (CLSM), respectively. Pharmacokinetics and tissue distribution studies were also carried out after intravenous administration in Charles Foster rats. RSV-TPGS-Lipo 2 showed significantly higher cytotoxicity than RSV-Lipo (uncoated liposomes) and RSV. Both uncoated and TPGS coated liposomes showed excellent cellular uptake. RSV, RSV-Lipo and RSV-TPGS-Lipo 2 were found to be haemocompatible and safe after i.v. administration. Area under the curve (AUC) and plasma half life (t1/2) after i.v. administration of RSV-TPGS-Lipo 2 was found to be approximately 5.73 and 6.72 times higher than that of RSV-Lipo as well as 29.94 and 29.66 times higher than that of RSV, respectively. Thus, the outcome indicates that RSV-TPGS-Lipo 2 is a promising carrier for glioma treatment with improved pharmacokinetic parameters. Moreover, brain accumulation of RSV-Lipo and RSV-TPGS-Lipo 2 was found to be significantly higher than that of RSV (P<0.05). Results are suggesting that both RSV-Lipo and RSV-TPGS-Lipo 2 are the promising tools of RSV for the treatment of brain cancer.