Tailored Polymersomes for Enhanced Oral Drug Delivery: pH-Sensitive Systems for Intestinal Delivery of Immunosuppressants.

Ensuring precise drug release at target sites is crucial for effective treatment. Here, pH-responsive nanoparticles for oral administration of mycophenolate mofetil, an alternative therapy for patients with inflammatory bowel disease unresponsive to conventional treatments is developed. However, its oral administration presents challenges due to its low solubility in the small intestine and high solubility and absorption in the stomach. Therefore, this aim is to design a drug delivery system capable of maintaining drug solubility compared to the free drug while delaying absorption from the stomach to the intestine. Successful synthesis and assembly of a block copolymer incorporating a pH-responsive functional group is achieved. Dynamic light scattering indicated a significant change in hydrodynamic size when the pH exceeded 6.5, confirming successful incorporation of the pH-responsive group. Encapsulation and controlled release of mycophenolate mofetil are efficiently demonstrated, with 90% release observed at intestinal pH. In vitro cell culture studies confirmed biocompatibility, showing no toxicity or adverse effects on Caco-2 cells. In vivo oral rat studies indicated reduced drug absorption in the stomach and enhanced absorption in the small intestine with the developed formulation. This research presents a promising drug delivery system with potential applications in the treatment of inflammatory bowel disease.

Sialic Acid-Imprinted Fluorescent Core-Shell Particles for Selective Labeling of Cell Surface Glycans.

The expression of cell surface glycans terminating with sialic acid (SA) residues has been found to correlate with various disease states there among cancer. We here report a novel strategy for specific fluorescence labeling of such motifs. This is based on sialic acid-imprinted core-shell nanoparticles equipped with nitrobenzoxadiazole (NBD) fluorescent reporter groups allowing environmentally sensitive fluorescence detection at convenient excitation and emission wavelengths. Imprinting was achieved exploiting a hybrid approach combining reversible boronate ester formation between p-vinylphenylboronic acid and SA, the introduction of cationic amine functionalities, and the use of an NBD-appended urea-monomer as a binary hydrogen-bond donor targeting the SA carboxylic acid and OH functionalities. The monomers were grafted from 200 nm RAFT-modified silica core particles using ethylene glycol dimethacrylate (EGDMA) as cross-linker resulting in a shell thickness of ca. 10 nm. The particles displayed strong affinity for SA in methanol/water mixtures (K = 6.6 × 10(5) M(-1) in 2% water, 5.9 × 10(3) M(-1) in 98% water, B(max) ≈ 10 µmol g(-1)), whereas binding of the competitor glucuronic acid (GA) and other monosaccharides was considerably weaker (K (GA) = 1.8 × 10(3) M(-1) in 98% water). In cell imaging experiments, the particles selectively stained different cell lines in correlation with the SA expression level. This was further verified by enzymatic cleavage of SA and by staining using a FITC labeled SA selective lectin.

A new animal product free defined medium for 2D and 3D culturing of normal and cancer cells to study cell proliferation and migration as well as dose response to chemical treatment.

Cell culturing methods are increasingly used to reduce and replace the use of live animals in biomedical research and chemical toxicity testing. Although live animals are avoided when using cell culturing methods, they often contain animal-derived components of which one of the most commonly used is foetal bovine serum (FBS). FBS is added to cell culture media among other supplements to support cell attachment/spreading and cell proliferation. The safety, batch-to-batch variation, and ethical problems with FBS are acknowledged and therefore world-wide efforts are ongoing to produce FBS free media. Here, we present the composition of a new defined medium with only human proteins either recombinant or derived from human tissues. This defined medium supports long-term culturing/routine culturing of normal cells and of cancer cells, and can be used for freezing and thawing of cells, i.e. for cell banking. Here, we show for our defined medium, growth curves and dose response curves of cells grown in two and three dimensions, and applications such as cell migration. Cell morphology was studied in real time by phase contrast and phase holographic microscopy time-lapse imaging. The cell lines used are human cancer-associated fibroblasts, keratinocytes, breast cancer JIMT-1 and MDA-MB-231 cells, colon cancer CaCo-2 cells, and pancreatic cancer MiaPaCa-2 cells as well as the mouse L929 cell line. In conclusion, we present the composition of a defined medium without animal-derived products which can be used for routine culturing and in experimental settings for normal cells and for cancer cells, i.e. our defined medium provides a leap towards a universal animal product free cell culture medium.

A novel 3D polycaprolactone high-throughput system for evaluation of toxicity in normoxia and hypoxia.

Two-dimensional (2D) culturing of cancer cells has been indispensable for the development of anti-cancer drugs. Drug development, however, is lengthy and costly with a high attrition rate, calling to mind that 2D culturing does not mimic the three-dimensional (3D) tumour microenvironment in vivo. Thus, began the development of 3D culture models for cancer research. We have constructed a 3D 96-well plate using electrospun fibres made of biocompatible polycaprolactone (PCL). Finely-cut PCL fibre pieces in water/ethanol solution was pipetted to the wells of hydrophobic 96-well plates. A fibrous network of approximately 200 µm thickness and high porosity was formed after crosslinking and drying. Human JIMT-1 breast cancer cells or fibroblasts were seeded into the network. Confocal microscopy shows that the cells grow throughout the fibre network. The toxicity of paclitaxel and an experimental salinomycin analogue was assessed and compared in 2D and 3D cultures incubated under conditions of normoxia and hypoxia often found in tumours. The toxicity of both compounds is lower when the cells are cultured in 3D compared to 2D in either normoxia or hypoxia. We conclude that our 96-well assay is a cost-efficient tool that may be used for high-throughput pre-clinical screening of potential anti-cancer compounds.

Identification of extracellular matrix proteins secreted by human dermal fibroblasts cultured in 3D electrospun scaffolds.

The appreciation that cell interactions in tissues is dependent on their three dimensional (3D) distribution has stimulated the development of 3D cell culture models. We constructed an artificial 3D tumour by culturing human breast cancer JIMT-1 cells and human dermal fibroblasts (HDFs) in a 3D network of electrospun polycaprolactone fibres. Here, we investigate ECM components produced by the cells in the artificial 3D tumour, which is an important step in validating the model. Immunostaining and confocal fluorescence microscopy show that the ECM proteins fibronectin, collagen I, and laminin are deposited throughout the entire 3D structure. Secreted soluble factors including matrix metalloproteinases (MMPs) and interleukine-6 (IL-6) were analysed in collected medium and were found to be mainly derived from the HDFs. Treatment with transforming growth factor-ß1 (TGF-ß1), a major cytokine found in a tumour, significantly alters the MMP activity and IL-6 concentration. In addition, TGF-ß1 treatment, changes the morphology of the HDFs to become more elongated and with increased linearized actin filaments compared to non-treated HDFs. Collectively, these novel findings suggest that the artificial 3D tumour displays a clear cell distribution and ECM deposition that resembles a tumour environment in vivo, suggesting an innovative biological model to study a human tumour.

Unique animal friendly 3D culturing of human cancer and normal cells.

Two-dimensional cell culturing has proven inadequate as a reliable preclinical tumour model due to many inherent limitations. Hence, novel three-dimensional (3D) cell culture models are needed, which in many aspects can mimic a native tumour with 3D extracellular matrix. Here, we present a 3D electrospun polycaprolactone (PCL) mesh mimicking the collagen network of tissue. The naturally hydrophobic PCL mesh was subjected to O2 plasma treatment to obtain hydrophilic fibres. Biocompatibility tests performed using L929 fibroblasts show that the 3D PCL fibre unit compartments were non-toxic. The human breast cancer cell lines MCF-7 and JIMT-1, the normal-like human breast cell line MCF-10A, and human adult fibroblast were cultured in PCL meshes and cell proliferation was monitored using the AlamarBlue® assay. Confocal microscopy and cryosectioning show that the cells penetrated deep into the fibre mesh within 1 week of cell culturing. The cancer cells form spheroids with the cells attaching mainly to each other and partly to the fibres. The human adult fibroblasts stretch out along the fibres while the MCF-10A cells stretch between fibres. Overall, we show that normal and cancer cells thrive in the 3D meshes cultured in fetal bovine free medium which eliminates the use of collagen as an extracellular matrix support.