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Engineered nanogels are of high value for a targeted and controlled transport of compounds due to the ability to change their chemical properties by external stimuli. As it has been indicated that nanogels possess a high ability to penetrate the stratum corneum, it cannot be excluded that nanogels interact with dermal dendritic cells, especially in diseased skin. In this study the potential crosstalk of the thermore-sponsive nanogels (tNGs) with the dendritic cells of the skin was investigated with the aim to determine the immunotoxicological properties of the nanogels. The investigated tNGs were made of dendritic polyglycerol (dPG) and poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)), as polymer conferring thermoresponsive properties. Although the tNGs were taken up, they displayed neither cytotoxic and genotoxic effects nor any induction of reactive oxygen species in the tested cells. Interestingly, specific uptake mechanisms of the tNGs by the dendritic cells were depending on the nanogels cloud point temperature (Tcp), which determines the phase transition of the nanoparticle. The study points to caveolae-mediated endocytosis as being the major tNGs uptake mechanism at 37 degrees C, which is above the Tcp of the tNGs. Remarkably, an additional uptake mechanism, beside caveolae-mediated endocytosis, was observed at 29 degrees C, which is the Tcp of the tNGs. At this temperature, which is characterized by two different states of the tNGs, macropinocytosis was involved as well. In summary, our study highlights the impact of thermoresponsivity on the cellular uptake mechanisms which has to be taken into account if the tNGs are used as a drug delivery system.
pH-sensitive nanoparticles have a great potential for dermal and transfollicular drug delivery. In this study, pH-sensitive, dexamethasone-loaded Eudragit (R) L 100, Eudragit (R) L 100-55, Eudragit (R) S 100, HPMCP-50, HPMCP-55 and cellulose acetate phthalate nanoparticles were prepared by nanoprecipitation and characterized. The pH-dependent swelling, erosion, dissolution and drug release kinetics were investigated in vitro using dynamic light scattering and Franz diffusion cells, respectively. Their toxicity potential was assessed by the ROS and MTT assays. 100-700 nm nanoparticles with high drug loading and entrapment efficiency were obtained. The nanoparticles bear no toxicity potential. Cellulose phthalates nanoparticles were more sensitive to pH than acrylates nanoparticles. They dissolved in 10 mM pH 7.5 buffer and released > 80% of the drug within 7 h. The acrylate nanoparticles dissolved in 40 mM pH 7.5 buffer and released 65-70% of the drug within 7 h. The nanoparticles remained intact in 10 and 40 mM pH 6.0 buffers (HPMCP nanoparticles dissolved in 40 mM pH 6.0 buffer) and released slowly. The nanoparticles properties could be modulated by blending the different polymers. In conclusion, various pH-sensitive nanoparticles that could release differently on the skin surface and dissolve and release in the hair follicles were obtained.
Detection of cancer precursors contributes to cancer prevention, for example, in the case of colorectal cancer. To record more patients early, ultrasensitive methods are required for the purpose of noninvasive precursor detection in body fluids. Our aim was to develop a method for enrichment and detection of known as well as unknown driver mutations in the Adenomatous polyposis coli (APC) gene. By coupled wild-type blocking (WTB) PCR and high-resolution melting (HRM), referred to as WTB-HRM, a minimum detection limit of 0.01% mutant in excess wild-type was achieved according to as little as 1 pg mutated DNA in the assay. The technique was applied to 80 tissue samples from patients with colorectal cancer (n = 17), adenomas (n = 50), serrated lesions (n = 8), and normal mucosa (n = 5). Any kind of known and unknown APC mutations (deletions, insertions, and base exchanges) being situated inside the mutation cluster region was distinguishable from wild-type DNA. Furthermore, by WTB-HRM, nearly twice as many carcinomas and 1.5 times more precursor lesions were identified to be mutated in APC, as compared with direct sequencing. By analyzing 31 associated stool DNA specimens all but one of the APC mutations could be recovered. Transferability of the WTB-HRM method to other genes was proven using the example of KRAS mutation analysis. In summary, WTB-HRM is a new approach for ultrasensitive detection of cancer-initiating mutations. In this sense, it appears especially applicable for noninvasive detection of colon cancer precursors in body fluids with excess wild-type DNA like stool. Cancer Prev Res; 6(9); 898-907. (C) 2013 AACR.
Epigenetic silencing of tumor suppressor genes is involved in early transforming events and has a high impact on colorectal carcinogenesis. Likewise, colon cancers that derive from chronically inflamed bowel diseases frequently exhibit epigenetic changes. But there is little data about epigenetic aberrations causing colorectal cancer in chronically inflamed tissue. The aim of the present study was to evaluate the aberrant gain of methylation in the gene promoters of VIM, TFPI2 and ITGA4 as putative early markers in the development from inflamed tissue via precancerous lesions toward colorectal cancer.
Initial screening of different cancer cell lines by using methylation-specific PCR revealed a putative colon cancer-specific methylation pattern. Additionally, a demethylation assay was performed to investigate the methylation-dependent gene silencing of ITGA4. The candidate markers were analyzed in colonic tissue specimens from patients with colorectal cancer (n = 15), adenomas (n = 76), serrated lesions (n = 13), chronic inflammation (n = 10) and normal mucosal samples (n = 9).
A high methylation frequency of VIM (55.6 %) was observed in normal colon tissue, whereas ITGA4 and TFPI2 were completely unmethylated in controls. A significant gain of methylation frequency with progression of disease as well as an age-dependent effect was detectable for TFPI2. ITGA4 methylation frequency was high in precancerous and cancerous tissues as well as in inflammatory bowel diseases (IBD).
The already established methylation marker VIM does not permit a specific and sensitive discrimination of healthy and neoplastic tissue. The methylation markers ITGA4 and TFPI2 seem to be suitable risk markers for inflammation-associated colon cancer.
Epigenetic histone modulation contributes to improvements in inflammatory bowel disease via EBI3
(2020)
Ulcerative colitis (UC) is characterized by relapsing-remitting inflammatory episodes paralleled by varying cytokine levels, suggesting that switching epigenetic processes might be involved. However, the epigenetic impact on cytokine levels in colitis is mostly unexplored. The heterodimeric interleukin (IL)-12 cytokine family have various functions in both pro- and anti-inflammatory processes. The family member IL-35 (EBI3/IL-12p35) was recently reported to play an anti-inflammatory role in UC. Therefore, we aimed to investigate a possible epigenetic regulation of the IL-35 subunits in vitro and in vivo, and to examine the epigenetic targeting of EBI3 expression as a therapeutic option for UC. Exposure to either the pro-inflammatory TNF alpha or to histone deacetylase inhibitors (HDACi) significantly increased EBI3 expression in Human Colon Epithelial Cells (HCEC) generated from healthy tissue. When applied in combination, a drastic upregulation of EBI3 expression occurred, suggesting a synergistic mechanism. Consequently, IL-35 was increased as well. In vivo, the intestines of HDACi-treated wild-type mice exhibited reduced pathological signs of colitis compared to non-treated colitic mice. However, the improvement by HDACi treatment was completely lost in Ebi3-deficient mice (Ebi3(-/-)). In fact, HDACi appeared to exacerbate the disease phenotype in Ebi3(-/-). In conclusion, our results reveal that under inflammatory conditions, EBI3 is upregulated by the epigenetic mechanism of histone acetylation. The in vivo data show that the deficiency of EBI3 plays a key role in colitis manifestation. Concordantly, our data suggest that conditions promoting histone acetylation, such as upon HDACi application, improve colitis by a mechanism involving the local formation of the anti-inflammatory cytokine IL-35.
Drug loaded dendritic core-multishell (CMS) nanocarriers are of especial interest for the treatment of skin diseases, owing to their striking dermal delivery efficiencies following topical applications. CMS nanocarriers are composed of a polyglycerol core, connected by amide-bonds to an inner alkyl shell and an outer methoxy poly(ethylene glycol) shell. Since topically applied nanocarriers are subjected to biodegradation, the application of conventional amide-based CMS nanocarriers (10-A-18-350) has been limited by the potential production of toxic polyglycerol amines. To circumvent this issue, three tailored ester-based CMS nanocarriers (10-E-12-350, 10-E-15-350, 10-E-18-350) of varying inner alkyl chain length were synthesized and comprehensively characterized in terms of particle size, drug loading, biodegradation and dermal drug delivery efficiency. Dexamethasone (DXM), a potent drug widely used for the treatment of inflammatory skin diseases, was chosen as a therapeutically relevant test compound for the present study. Ester-and amide-based CMS nanocarriers delivered DXM more efficiently into human skin than a commercially available DXM cream. Subsequent in vitro and in vivo toxicity studies identified CMS (10-E-15-350) as the most biocompatible carrier system. The anti-inflammatory potency of DXM-loaded CMS (10-E-15-350) nanocarriers was assessed in TNF alpha supplemented skin models, where a significant reduction of the pro-inflammatory cytokine IL-8 was seen, with markedly greater efficacy than commercial DXM cream. In summary, we report the rational design and characterization of tailored, biodegradable, ester-based CMS nanocarriers, and their subsequent stepwise screening for biocompatibility, dermal delivery efficiency and therapeutic efficacy in a top-down approach yielding the best carrier system for topical applications. (C) 2016 Elsevier B.V. All rights reserved.