Ariana Hudita

Impact of multicellular tumor spheroids as an in vivo-like tumor model on anticancer drug response

The incidence of colorectal cancer is higher in men than in women, amounting to 15% of cancer-related diseases as a whole. As such, undesirable effects, arising from the administration of current chemotherapeutic agents (the FOLFIRI/FOLFOX combinations), which are exerted on the remaining non-cancerous tissues and/or cells, have contributed to the occurrence of resistance to multiple drugs, thus markedly reducing their efficacy. However, the delivery of chemotherapeutic agents may be improved and their action may be more selectively targeted to diseased tissues/cells by means of developing biotechnologies and nano-techniques. Thus, the current focus is on creating biological tissue and related tumor models, by means of three-dimensional (3D) spheres, in an attempt to bridge the gap between results obtained in the pre-clinical phase and promising outcomes obtained in clinical trials. For this purpose, the characterization and use of so-called ‘multicellular tumor spheroids’, may prove to be invaluable. In this study, we focus on describing the efficacy of a model 3D system as compared to the traditional 2D tumor spheres in determining drug response, highlighting a potentially greater effect of the drugs following the encapsulation of respective liposomes. The results obtained demonstrate the successful preparation of a suspension of liposomes loaded with folinic acid, oxaliplatin and 5-fluorouracil (5-FU), and loaded with meso-tetra (4-sulfonatophenyl) porphyrin. Following its use on HT-29 colorectal cancer cells, an important comparative reduction was noted in the viability of the HT-29 cells, demonstrating the efficacy of multicellular tumor spheroids carrying liposomes loaded with therapeutic drugs. These findings indicate that the method of drug encapsulation in liposomes may improve the treatment efficacy of chemotherapeutic agents.

Poly(HydroxyButyrate-co-HydroxyValerate) (PHBHV) Nanocarriers for Silymarin Release as Adjuvant Therapy in Colo-rectal Cancer

The aim of this study was to address one of the major challenges of the actual era of nanomedicine namely, the bioavailability of poorly water soluble drugs such as Silymarin. We developed new, biodegradable, and biocompatible nanosized shuttles for Silymarin targeted delivery in colon-cancer cells. The design of these 100 nm sized carrier nanoparticles was based on natural polymers and their biological properties such as cellular uptake potential, cytotoxicity and 3D penetrability were tested using a colon cancer cell line (HT-29) as the in vitro culture model. Comparative scanning electron microscopy (SEM) and atomic force microscopy (AFM) measurements demonstrated that the Silymarin loaded Poly(3-HydroxyButyrate-co-3-HydroxyValerate) (PHBHV) nanocarriers significantly decreased HT-29 cells viability after 6 and 24 h of treatment. Moreover, in vivo-like toxicity studies on multicellular tumor spheroids showed that the Silymarin loaded PHBHV nanocarriers are able to penetrate 3D micro tumors and significantly reduce their size.

Colon Cancer Cells Gene Expression Signature As Response to 5- Fluorouracil, Oxaliplatin, and Folinic Acid Treatment

5-FU cytotoxicity mechanism has been assigned both to the miss-incorporation of fluoronucleotides into RNA and DNA and to the inhibition of thymidylate synthase. 5-FU is one of the most widely used chemotherapeutic drugs, although it has severe side effects that may vary between patients. Pharmacogenetic studies related to 5-FU have been traditionally focused on the rate-limiting catabolic enzyme, dihydropyrimidine dehydrogenase that breaks 80–85% of 5-FU into its inactive metabolite. Choosing the right dosing scheme and chemotherapy strategy for each individual patient remains challenging for personalized chemotherapy management. In the general effort toward reduction of colorectal cancer mortality, in vitro screening studies play a very important role. To accelerate translation research, increasing interest has been focused on using in vivo-like models such as three-dimensional spheroids. The development of higher throughput assays to quantify phenotypic changes in spheroids is an active research area. Consequently, in this study we used the microarray technology to reveal the HT-29 colorectal adenocarcinoma cells gene expression signature as response to 5-FU/OXP/FA treatment in a state of the art 3D culture system. We report here an increased reactive oxygen species production under treatment, correlated with a decrease in cell viability and proliferation potential. With respect to the HT-29 cells gene expression under the treatment with 5-FU/OXP/FA, we found 15.247 genes that were significantly differentially expressed (p < 0.05) with a fold change higher that two-fold. Among these, 7136 genes were upregulated and 8111 genes were downregulated under experimental conditions as compared to untreated cells. The most relevant and statistic significant (p < 0.01) pathways in the experiment are associated with the genes that displayed significant differential expression and are related to intracellular signaling, oxidative stress, apoptosis, and cancer.

Silk-Based Hydrogels for Biomedical Applications

Among the naturally occurring fibers, silk occupies a special position due to its properties. Silk fibroins, the unique proteins of silkworm fibers, are highmolecular-weight block copolymers consisting of a heavy (~370 kDa) and a light (~26 kDa) chain with varying amphiphilicity linked by a single disulphide bond. Bombyx mori silk is the most characterized silkworm silk. Researchers have investigated fibroin as one of the promising resources of biotechnology and biomedical materials due to its other unique properties including excellent biocompatibility, favorable oxygen permeability, and outstanding biodegradability, and the degradation product can be readily absorbed by the body with minimal inflammatory reaction. Silk hydrogels have been thoroughly studied for potential biotechnological applications due to their mechanical properties, biocompatibility, controllable degradation rates, and self-assembly into β-sheet networks. Hydrogels made from silk proteins have shown a potential in overcoming limitations of hydrogels prepared from conventional polymers. This chapter offers overview of the recent developments in silk protein-based hydrogels, both of fibroin and sericin proteins. It describes the approaches for obtaining silk hydrogels and ideas to improve the existing properties or to incorporate new features in the hydrogels by making composites. Characterization tools and modern bioapplications of the silk hydrogels for tissue engineering and controlled release are also reviewed. A special focus is given to silk fibroin composite hydrogels for bone tissue engineering applications.

In Vitro Effects of Cetylated Fatty Acids Mixture from Celadrin on Chondrogenesis and Inflammation with Impact on Osteoarthritis

Objective Cetylated fatty acids are a group of naturally occurring fats of plant and/or animal origin. Cetyl myristoleate, in particular, was initially involved in osteoarthritis related research as its therapeutic administration prevented experimentally induced arthritis in Swiss Albino mice. In this context, the aim of our study was to investigate the possible mechanisms of Celadrin cetylated fatty acids action at the cellular level inflammation related pain relief and chondrogenesis. Design For this, we tested the effects of the cetylated fatty acids mixture from Celadrin on an in vitro scaffold-free 3-dimensional mesenchymal stem cells culture model of chondrogenesis. Furthermore, we treated stimulated mouse macrophage cells with the cetylated fatty acids mixture to investigate the expression profile of secreted inflammatory cytokines. Results The cetylated fatty acids mixture from Celadrin significantly decreased the production of IL-6, MCP-1, and TNF, key regulators of the inflammatory process, in stimulated RAW264.7 mouse macrophage cells. The treatment with cetylated fatty acids mixture initiated and propagated the process of chondrogenesis as demonstrated by the increased expression and deposition of chondrogenic markers by the differentiating mesenchymal cells. Conclusion The cetylated fatty acids mixture from Celadrin reduces inflammation in vitro by significantly decreasing the expression of IL-6, MCP-1, and TNF in stimulated RAW264.7 mouse macrophage cells. These compounds facilitate the chondrogenic differentiation process of human adipose-derived stem cells by stimulating the expression of chondrogenic markers under chondrogenic induction conditions.