Topical administration permits targeted, sustained delivery of therapeutics to human skin. Delivery to the skin, however, is typically limited to lipophilic molecules with molecular weight of < 500 Da, capable of crossing the stratum corneum. Nevertheless, there are indications protein delivery may be possible in barrier deficient skin, a condition found in several inflammatory skin diseases such as psoriasis, using novel nanocarrier systems.Methods: Water in water thermo-nanoprecipitation; dynamic light scattering; zeta potential measurement; nanoparticle tracking analysis; atomic force microscopy; cryogenic transmission electron microscopy; UV absorption; centrifugal separation membranes; bicinchoninic acid assay; circular dichroism; TNFα binding ELISA; inflammatory skin equivalent construction; human skin biopsies; immunohistochemistry; fluorescence microscopy; western blot; monocyte derived Langerhans cells; ELISAResults: Here, we report the novel synthesis of thermoresponsive nanogels (tNG) and the stable encapsulation of the anti-TNFα fusion protein etanercept (ETR) (~150 kDa) without alteration to its structure, as well as temperature triggered release from the tNGs. Novel tNG synthesis without the use of organic solvents was conducted, permitting in situ encapsulation of protein during assembly, something that holds great promise for easy manufacture and storage. Topical application of ETR loaded tNGs to inflammatory skin equivalents or tape striped human skin resulted in efficient ETR delivery throughout the SC and into the viable epidermis that correlated with clear anti-inflammatory effects. Notably, effective ETR delivery depended on temperature triggered release following topical application.Conclusion: Together these results indicate tNGs hold promise as a biocompatible and easy to manufacture vehicle for stable protein encapsulation and topical delivery into barrier-deficient skin.

(2017) Biocompatibility and characterization of polyglycerol-based thermoresponsive nanogels designed as novel drug-delivery systems and their intracellular localization in keratinocytes, Nanotoxicology, 11:2, 267-277, DOI: 10.1080/17435390.2017 ABSTRACTNovel nanogels that possess the capacity to change their physico-chemical properties in response to external stimuli are promising drug-delivery candidates for the treatment of severe skin diseases. As thermoresponsive nanogels (tNGs) are capable of enhancing penetration through biological barriers such as the stratum corneum and are taken up by keratinocytes of human skin, potential adverse consequences of their exposure must be elucidated. In this study, tNGs were synthesized from dendritic polyglycerol (dPG) and two thermoresponsive polymers. tNG_dPG_tPG are the combination of dPG with poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)) and tNG_dPG_pNIPAM the one with poly(N-isopropylacrylamide) (pNIPAM). Both thermoresponsive nanogels are able to incorporate high amounts of dexamethasone and tacrolimus, drugs used in the treatment of severe skin diseases. Cellular uptake, intracellular localization and the toxicological properties of the tNGs were comprehensively characterized in primary normal human keratinocytes (NHK) and in spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (HaCaT). Laser scanning confocal microscopy revealed fluorescently labeled tNGs entered into the cells and localized predominantly within lysosomal compartments. MTT assay, comet assay and carboxy-H2DCFDA assay, demonstrated neither cytotoxic or genotoxic effects, nor any induction of reactive oxygen species of the tNGs in keratinocytes. In addition, both tNGs were devoid of eye irritation potential as shown by bovine corneal opacity and permeability (BCOP) test and red blood cell (RBC) hemolysis assay. Therefore, our study provides evidence that tNGs are locally well tolerated and underlines their potential for cutaneous drug delivery. ARTICLE HISTORY

Epigenetic silencing of tumour suppressor genes is a key hallmark of colorectal carcinogenesis. Despite this, the therapeutic potential of epigenetic agents capable of reactivating these silenced genes remains relatively unexplored. Evidence has shown the dietary antioxidant vitamin C (ascorbate) acts as an inducer of the ten-eleven translocation (TET) dioxygenases, an enzyme family that catalyses a recently described mechanism of DNA demethylation linked to gene re-expression. In this study, we set out to determine whether vitamin C can enhance the known anti-neoplastic actions of the DNA-demethylating agents decitabine (DAC) and azacytidine (AZA) in colorectal cancer cells. Administration of vitamin C alone significantly enhanced global levels of 5-hydroxymethyl-2’-deoxycytidine (5-hmdC), without altering 5-methyl-2’-deoxycytidine (5-mdC), as would be expected upon the activation of TET dioxygenases. Concomitant treatment of vitamin C with either AZA or DAC resulted in an unexpectedly high increase of global 5-hmdC levels, one that administration of any these compounds alone could not achieve. Notably, this was also accompanied by increased expression of the tumour suppressor p21 (CDKN1A), and a significant increase in apoptotic cell induction. Our in vitro data leads us to hypothesize that the reactivation of genes in colorectal cancer cells by AZA or DAC can be improved when the 5-hmdC levels are simultaneously increased by the TET activator vitamin C. The dual administration of demethylating agents and vitamin C to colorectal cancer patients, a demographic in which vitamin C deficiencies are common, may improve responses to epigenetic therapies.

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