Trimethyl chitosan coated palladium nanoparticles as a photothermal agent and its in vitro evaluation in 2D and 3D model of breast cancer cells.

The potential of palladium has been scantily explored in biomedical applications. In the present study, palladium nanoparticles (PdNPs) were synthesized and were successfully coated with trimethyl-chitosan (TMC) to improve their biocompatibility. Coating with TMC improved the nanoparticle accumulation in MDAMB231 breast cancer cells, compared to nanoparticles coated with native chitosan. The TMC coated palladium nanoparticles (TMC/PdNPs) exhibited good biocompatibility and physiological stability, as compared to the plain(uncoated) PdNPs. TMC coated PdNPs resulted in photothermal therapeutic effect, when irradiated with a near-infrared (NIR) laser having the wavelength of 808-nm. The TMC/PdNPs resulted in good cytotoxic effect upon laser treatment in both, 2D monolayers and 3D spheroids of MDAMB231 cells, the latter mimicking the tumor microenvironment. These results clearly indicated that TMC/PdNPs acted as ideal photothermal agents for anti-cancer therapy in combination with a non-invasive near-infrared laser.

Establishment and characterization of lung co-culture spheroids for paclitaxel loaded Eudragit® RL 100 nanoparticle evaluation.

3D cell cultures are regarded as a better and more relevant approach for screening drugs and therapeutics, particularly due to their likeness with the in vivo conditions. Spheroids offer an intermediate platform between in vitro and in vivo models, for conducting tumor-based investigations. In this study, a simple setup was developed for consistent generation of lung co-culture spheroids, which were developed using the cancer cell lines A549, NCI H460, and fibroblast cells WI-38. The potential of these spheroids for evaluating the toxicity of Eudragit® RL 100 nanoparticles (ENP) was explored. Monodisperse ENP, having the size range of 140-200 nm was prepared using the nanoprecipitation method. These were loaded with the poorly water-soluble anticancer drug paclitaxel. The evaluation of toxicity and uptake of drug-loaded ENP revealed that 2D monolayers were more sensitive to treatment than 3D spheroids. Within spheroids, co-cultures were more resistant to the treatment than monocultures. Overall, our findings demonstrated that the lung co-culture spheroids were a suitable model for accelerating the efficacy and toxicity-related investigations of novel drug delivery systems.

In Vivo Studies of 3D Starch-Gelatin Scaffolds for Full-Thickness Wound Healing.

In this study, we have combined the wound-healing properties of two biodegradable polymers, viz., starch and gelatin, and have reinforced their mechanical strength through cross-linking. Further, scaffolds of this polymer combination were used to support an organotypic culture of human skin for wound healing. Human dermal fibroblasts (HDFs) and human epidermal keratinocytes (HEKs) were isolated and were seeded on the scaffolds on days 1 and 7, respectively. The scaffold was then air-lifted to develop a stratified epidermal layer. Hematoxylin and eosin (H&E) staining and immunohistochemical analysis ascertained that the histology of the skin organotypic culture was similar to that of the human skin. For in vivo animal investigations, the scaffolds were transplanted in a full-thickness wound mouse model, as a one-step procedure, wherein the artificial skin substitute showed the presence of well-defined epidermis and formation of stratum basale by day 14. By combining the inherent properties of both the materials, we have synthesized a cost-effective porous scaffold with good mechanical strength and excellent biocompatibility that can be easily adapted for commercial use. The aforementioned scaffold may integrate with the surrounding tissue, accelerate wound closure, and promote tissue reorganization and remodeling.

Protective nature of low molecular weight chitosan in a chitosan-Amphotericin B nanocomplex - A physicochemical study.

Chitosan, a biopolymer of immense potential, has been well-explored over the past decade in the biomedical field. Despite its numerous biological properties like anti-microbial, anti-inflammatory, anti-diabetic etc., limited studies have been conducted on its ability to protect therapeutic molecules in nano-formulations. Amphotericin B (AMP) is one such therapeutic molecule which requires protection as it possesses inherent limitations of poor water-solubility, susceptibility to oxidation- and/or light-induced degradation etc. Although AMP formulations have been quite successful in treating chronic fungal infections, their high cost, prolonged nature of therapy and instability over longer durations has necessitated the development of alternative carriers. In the present study, a stable nanoparticulate formulation was successfully prepared using low molecular weight chitosan and the anti-fungal agent AMP and this was found to offer protection to the labile AMP. The developed nanocomplexes could prevent degradation of AMP up to six months, in solution form, unlike the native drug which degraded in <24 h. Antifungal studies of the developed nanocomplexes revealed a surface charge-dependent antifungal activity. Furthermore, the nanocomplexes did not affect the viability of mammalian cells and showed excellent intracellular uptake and retention, and hence suggested potential of the nanocomplexes in alleviating systemic fungal infections.

CHO Cells adapted to inorganic phosphate limitation show higher growth and higher pyruvate carboxylase flux in phosphate replete conditions.

Inorganic phosphate (Pi ) is an essential ion involved in diverse cellular processes including metabolism. Changes in cellular metabolism upon long term adaptation to Pi limitation have been reported in E. coli. Given the essential role of Pi , adaptation to Pi limitation may also result in metabolic changes in animal cells. In this study, we have adapted CHO cells producing recombinant IgG to limiting Pi conditions for 75 days. Not surprisingly, adapted cells showed better survival under Pi limitation. Here, we report the finding that such cells also showed better growth characteristics compared to control in batch culture replete with Pi (higher peak density and integral viable cell density), accompanied by a lower specific oxygen uptake rate and cytochrome oxidase activity towards the end of exponential phase. Surprisingly, the adapted cells grew to a lower peak density under glucose limitation. This suggests long term Pi limitation may lead to selection for an altered metabolism with higher dependence on glucose availability for biomass assimilation compared to control. Steady state U-13 C glucose labeling experiments suggest that adapted cells have a higher pyruvate carboxylase flux. Consistent with this observation, supplementation with aspartate abolished the peak density difference whereas supplementation with serine did not abolish the difference. This supports the hypothesis that cell growth in the adapted culture might be higher due to a higher pyruvate carboxylase flux. Decreased fitness under carbon limitation and mutations in the sucABCD operon has been previously reported in E. coli upon long term adaptation to Pi limitation, suggestive of a similarity in cellular response among such diverse species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:749-758, 2017.

In situ pH maintenance for mammalian cell cultures in shake flasks and tissue culture flasks.

pH in animal cell cultures decreases due to production of metabolites like lactate. pH control via measurement and base addition is not easily possible in small-scale culture formats like tissue-culture flasks and shake flasks. A hydrogel-based system is reported for in situ pH maintenance without pH measurement in such formats, and is demonstrated to maintain pH between 6.8 and 7.2 for a suspension CHO cell line in CD CHO medium and between 7.3 and 7.5 for adherent A549 cells in DMEM:F12 containing 10% FBS. This system for pH maintenance, along with our previous report of hydrogels for controlled nutrient delivery in shake flasks can allow shake flasks to better mimic bioreactor-based fed batch operation for initial screening during cell line and process development for recombinant protein production in mammalian cells.

Controlled release of nutrients to mammalian cells cultured in shake flasks.

Though cell culture-based protein production processes are rarely carried out under batch mode of operation, cell line and initial process development operations are usually carried out in batch mode due to simplicity of operation in widely used scale down platforms like shake flasks. Nutrient feeding, if performed, is achieved by bolus addition of concentrated feed solution at different intervals, which leads to large transient increases in nutrient concentrations. One negative consequence is increased waste metabolite production. We have developed a hydrogel-based nutrient delivery system for continuous feeding of nutrients in scale down models like shake flasks without the need for manual feed additions or any additional infrastructure. Continuous delivery also enables maintaining nutrient concentrations at low levels, if desired. The authors demonstrate the use of these systems for continuous feeding of glucose and protein hydrolysate to a suspension Chinese Hamster Ovary (CHO) culture in a shake flask. Glucose feeding achieved using the glucose-loaded hydrogel resulted in a 23% higher integral viable cell density and an 89% lower lactate concentration at the end of the culture when compared with a bolus-feed of glucose.