Nanomedicine Approaches to Negotiate Local Biobarriers for Topical Drug Delivery

AbstractTopical treatments have been widely adopted to address a broad range of conditions across multiple sites thanks to their convenience, versatility, and effectiveness. While bypassing systemic biobarriers and avoiding systemic side effects by delivering directly to the target tissue, topical treatments still face significant local biobarriers that limit their efficacy. The toolset available for nanodelivery systems and their inherent multifunctionality can contribute to simultaneously address otherwise intractable challenges related to barrier function evasion, drug solubility, bioavailability, pharmacokinetics, smart and sustained release, quantitative co‐delivery, and local targeting which are key to successful topical treatments. This review summarizes the outstanding challenges associated with the topical treatments of key diseases of the skin, mucosae, eyes, and ears, and highlights how nanodelivery systems are being developed to address them effectively

Preparation of human primary macrophages to study the polarization from monocyte-derived macrophages to pro- or anti-inflammatory macrophages at biomaterial interface in vitro.

BACKGROUND/PURPOSE Testing of dental materials when in contact with innate immune cells has been so far hindered by the lack of proper in vitro models. Human primary monocyte-derived macrophages (MDMs) would be an excellent option to this aim. However, the inability to detach them from the tissue culture plates contrast the possibility to culture them on biomaterials. The goal of the present work is to present and validate an innovative protocol to obtain MDMs from peripheral blood monocytes, and to reseed them in contact with biomaterials without altering their viability and phenotype. MATERIALS AND METHODS We differentiated MDMs on ultra-low attachment tissue culture plastics and recovered them with specific detachment solution in order to be reseeded on a secondary substrate. Therefore, using biological assays (RT-PCR, Western blot, and immunofluorescence) we compared their phenotype to MDMs differentiated on standard culture plates. RESULTS Transferred MDMs keep their differentiated M0 resting state, as well as the ability to be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages. CONCLUSION These data provide the dental material research community the unprecedented possibility to investigate the immunomodulatory properties of biomaterials for dental application

Human mesenchymal stem cells from chorionic villi and amniotic fluid are not susceptible to transformation after extensive in vitro expansion.

Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering. Increasing evidence suggests that MSCs isolated from fetal tissues are more plastic and grow faster than adult MSCs. In this study, we characterized human mesenchymal progenitor cells from chorionic villi (CV) and amniotic fluid (AF) isolated during the first and second trimesters, respectively, and compared them with adult bone marrow-derived MSCs (BM). We evaluated 10 CV, 10 AF, and 6 BM samples expanded until the MSCs reached senescence. We used discarded cells from prenatal analyses for all the experiments. To evaluate the replicative stability of these cells, we studied the telomerase activity, hTERT gene transcription, and telomere length in these cells. Spontaneous chromosomal alterations were excluded by cytogenetic analysis. We studied the expression of c-myc and p53, tumor-associated genes, at different passage in culture and the capacity of these cells to grow in an anchorage-independent manner by using soft agar assay. We isolated homogeneous populations of spindle-shaped CV, AF, and BM cells expressing mesenchymal immunophenotypic markers throughout the period of expansion. CV cells achieved 14 ± 0.9 logs of expansion in 118 days and AF cells achieved 21 ± 0.9 logs in 118 days, while BM cells achieved 11 × 0.4 logs in 84 days. Despite their high proliferation capacity, fetal MSCs showed no telomerase activity, no hTERT and c-myc transcriptions, and maintained long, stable telomeres. A constant expression level of p53 and a normal karyotype were preserved throughout long-term expansion, suggesting the safety of fetal MSCs. In conclusion, our results indicate that fetal MSCs could be an alternative, more accessible resource for cell therapy and regenerative medicine

Nanomedicine Approaches to Negotiate Local Biobarriers for Topical Drug Delivery

Topical treatments have been widely adopted to address a broad range of conditions across multiple sites thanks to their convenience, versatility, and effectiveness. While bypassing systemic biobarriers and avoiding systemic side effects by delivering directly to the target tissue, topical treatments still face significant local biobarriers that limit their efficacy. The toolset available for nanodelivery systems and their inherent multifunctionality can contribute to simultaneously address otherwise intractable challenges related to barrier function evasion, drug solubility, bioavailability, pharmacokinetics, smart and sustained release, quantitative co-delivery, and local targeting which are key to successful topical treatments. This review summarizes the outstanding challenges associated with the topical treatments of key diseases of the skin, mucosae, eyes, and ears, and highlights how nanodelivery systems are being developed to address them effectively

GSK-3 inhibition reverts mesenchymal transition in primary human corneal endothelial cells

Human corneal endothelial cells are organized in a tight mosaic of hexagonal cells and serve a critical function in maintaining corneal hydration and clear vision. Regeneration of the corneal endothelial tissue is hampered by its poor proliferative capacity, which is partially retrieved in vitro, albeit only for a limited number of passages before the cells undergo mesenchymal transition (EnMT). Although different culture conditions have been proposed in order to delay this process and prolong the number of cell passages, EnMT has still not been fully understood and successfully counteracted. In this perspective, we identified herein a single GSK-3 inhibitor, CHIR99021, able to revert and avoid EnMT in primary human corneal endothelial cells (HCEnCs) from old donors until late passages in vitro (P8), as shown from cell morphology analysis (circularity). In accordance, CHIR99021 reduced expression of α-SMA, an EnMT marker, while restored endothelial markers such as ZO-1, Na+/K+ ATPase and N-cadherin, without increasing cell proliferation. A further analysis on RNA expression confirmed that CHIR99021 induced downregulation of EnMT markers (α-SMA and CD44), upregulation of the proliferation repressor p21 and revealed novel insights into the β-catenin and TGFβ pathways intersections in HCEnCs. The use of CHIR99021 sheds light on the mechanisms involved in EnMT, providing a substantial advantage in maintaining primary HCEnCs in culture until late passages, while preserving the correct morphology and phenotype. Altogether, these results bring crucial advancements towards the improvement of the corneal endothelial cells based therapy

A fine-tuned β-catenin regulation during proliferation of corneal endothelial cells revealed using proteomics analysis

: Corneal endothelial (CE) dysfunction is the main indication for corneal transplantation, an invasive procedure with several limitations. Developing novel strategies to re-activate CE regenerative capacity is, therefore, of fundamental importance. This goal has proved to be challenging as corneal endothelial cells (CEnC) are blocked in the G0/G1 phase of the cell cycle in vivo and, albeit retaining proliferative capacity in vitro, this is further hindered by endothelial-to-mesenchymal transition. Herein we investigated the mechanisms regulating CEnC proliferation in vitro. Comparing the proteome of non-proliferating (in vivo-G0/G1) and proliferating (in vitro-G2/M) rabbit CEnC (rCEnC), 77 proteins, out of 3,328 identified, were differentially expressed in the two groups (p < 0.005). Literature and Gene Ontology analysis revealed β-catenin and transforming growth factor (TGF-β) pathways to be correlated with the identified proteins. Treatment of rCEnC with a β-catenin activator and inhibitor showed that β-catenin activation was necessary during rCEnC proliferation, but not sufficient for its induction. Furthermore, both pro-proliferative activity of basic fibroblast growth factor and anti-proliferative effects of TGF-β were regulated through β-catenin. Overall, these results provide novel insights into the molecular basis underlying the proliferation process that CEnC re-activate in vitro, consolidating the role of β-catenin and TGF-β

Preparation of human primary macrophages to study the polarization from monocyte-derived macrophages to pro- or anti-inflammatory macrophages at biomaterial interface in vitro

Background/purpose: Testing of dental materials when in contact with innate immune cells has been so far hindered by the lack of proper in vitro models. Human primary monocyte-derived macrophages (MDMs) would be an excellent option to this aim. However, the inability to detach them from the tissue culture plates contrast the possibility to culture them on biomaterials. The goal of the present work is to present and validate an innovative protocol to obtain MDMs from peripheral blood monocytes, and to reseed them in contact with biomaterials without altering their viability and phenotype. Materials and methods: We differentiated MDMs on ultra-low attachment tissue culture plastics and recovered them with specific detachment solution in order to be reseeded on a secondary substrate. Therefore, using biological assays (RT-PCR, Western blot, and immunofluorescence) we compared their phenotype to MDMs differentiated on standard culture plates. Results: Transferred MDMs keep their differentiated M0 resting state, as well as the ability to be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages. Conclusion: These data provide the dental material research community the unprecedented possibility to investigate the immunomodulatory properties of biomaterials for dental application