Long-term culture of patient-derived mammary organoids in non-biogenic electrospun scaffolds for identifying metalloprotein and motor protein activities in aging and senescence.

We recently identified TMEM230 as a master regulator of the endomembrane system of cells. TMEM230 expression is necessary for promoting motor protein dependent intracellular trafficking of metalloproteins for cellular energy production in mitochondria. TMEM230 is also required for transport and secretion of metalloproteinases for autophagy and phagosome dependent clearance of misfolded proteins, defective RNAs and damaged cells, activities that decline with aging. This suggests that aberrant levels of TMEM230 may contribute to aging and regain of proper levels may have therapeutic applications. The components of the endomembrane system include the Golgi complex, other membrane bound organelles, and secreted vesicles and factors. Secreted cellular components modulate immune response and tissue regeneration in aging. Upregulation of intracellular packaging, trafficking and secretion of endosome components while necessary for tissue homeostasis and normal wound healing, also promote secretion of pro-inflammatory and pro-senescence factors. We recently determined that TMEM230 is co-regulated with trafficked cargo of the endomembrane system, including lysosome factors such as RNASET2. Normal tissue regeneration (in aging), repair (following injury) and aberrant destructive tissue remodeling (in cancer or autoimmunity) likely are regulated by TMEM230 activities of the endomembrane system, mitochondria and autophagosomes. The role of TMEM230 in aging is supported by its ability to regulate the pro-inflammatory secretome and senescence-associated secretory phenotype in tissue cells of patients with advanced age and chronic disease. Identifying secreted factors regulated by TMEM230 in young patients and patients of advanced age will facilitate identification of aging associated targets that aberrantly promote, inhibit or reverse aging. Ex situ culture of patient derived cells for identifying secreted factors in tissue regeneration and aging provides opportunities in developing therapeutic and personalized medicine strategies. Identification and validation of human secreted factors in tissue regeneration requires long-term stabile scaffold culture conditions that are different from those previously reported for cell lines used as cell models for aging. We describe a 3 dimensional (3D) platform utilizing non-biogenic and non-labile poly ε-caprolactone scaffolds that supports maintenance of long-term continuous cultures of human stem cells, in vitro generated 3D organoids and patient derived tissue. Combined with animal component free culture media, non-biogenic scaffolds are suitable for proteomic and glycobiological analyses to identify human factors in aging. Applications of electrospun nanofiber technologies in 3D cell culture allow for ex situ screening and the development of patient personalized therapeutic strategies and predicting their effectiveness in mitigating or promoting aging.

Self-associating cellulose-graft-poly(ε-caprolactone) to design nanoparticles for drug release.

The growing interest in the use of polysaccharides nanoparticles for biomedical applications is related to the recent progresses on the synthesis of cellulose-based polymers with the specific functionalities. In particular, cellulose graft copolymers are emerging as amphiphilic materials suitable to fabricate smart nanoparticles for drug delivery applications. In this work, a cellulose-graft-poly(ε-caprolactone) (cell-g-PCL) was synthetized and characterized by FTIR, TGA and DSC in order to validate the synthesis process. We demonstrated that fast evaporation processes promoted cell-g-PCL self-assembly to form nanomicellar structures with hydrodynamic radius ranged from 30 to 60 nm as confirmed by TEM analysis. Moreover, the application of controlled electrostatic forces on solvent evaporation - namely electrospraying - allowed generating round-like nanoscaled particles, as confirmed by SEM supported via image analysis. We demonstrated also that sodium diclofenac (DS) drastically influenced the mechanism of particle formation, favoring the deposition of erythrocyte-like particles with highly concave surfaces, not penalizing the encapsulation efficiency of nanoparticles (>80%). The release profile showed a fast delivery of DS - about 60% during the first 24 h - followed by a sustained release - about 20% during the next 6 days - strictly related to the peculiar weak interactions among amphiphilic polymer segments and water molecules, thus suggesting a successful use of electrosprayed cell-g-PCL nanoparticles for therapeutic treatments in nanomedicine.