ABSTRACT
Nanoparticles (NPs) are currently being developed as vehicles for in vivo drug delivery. Two of the biggest barriers facing this therapy are the site-specific targeting and consequent cellular uptake of drug-loaded NPs(1). In vitro studies in 2D cell cultures have shown that an external magnetic field (MF) and functionalization with cell-penetrating peptides (CPPs) have the capacity to overcome these barriers. This study aimed to investigate if the potential of these techniques, which has been reported in 2D, can be successfully applied to cells growing in a 3D environment. As such, this study provides a more realistic assessment of how these techniques might perform in future clinical settings. The effect of a MF and/or penetratin attachment on the uptake of 100 and 200 nm fluorescent iron oxide magnetic NPs (mNPs) into a fibroblast-seeded 3D collagen gel was quantified by inductively coupled plasma mass spectrometry. The most suitable mNP species was further investigated by fluorescence microscopy, histology, confocal microscopy, and TEM. Results show that gel mNP uptake occurred on average twice as fast in the presence of a MF and up to three times faster with penetratin attachment. In addition, a MF increased the distance of mNP travel through the gel, while penetratin increased mNP cell localization. This work is one of the first to demonstrate that MFs and CPPs can be effectively translated for use in 3D systems and, if applied together, will make excellent partners to achieve therapeutic drug delivery in vivo.
Subject(s)
Carrier Proteins/chemistry , Cell Culture Techniques/methods , Drug Carriers/chemistry , Drug Carriers/metabolism , Ferric Compounds/chemistry , Magnetic Fields , Nanoparticles/chemistry , Biological Transport , Cell-Penetrating Peptides , Extracellular Matrix/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Mass Spectrometry , Microscopy , RheologyABSTRACT
The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an oncoprotein expressed in several EBV-associated malignancies. We have utilised mice expressing the Cao strain of LMP1 in epithelia to explore the consequences of expression in vivo, specifically the changes that occur prior to neoplasia, in the hyperplastic but degenerating tissue. Epidermal growth factor receptor (EGFR) ligands (transforming growth factor alpha (TGFalpha), heparin-binding EGF-like growth factor and epiregulin) are constitutively induced by LMP1, leading to EGFR phosphorylation but also down-regulation, degradation or turn-over, with the appearance of cleaved EGFR fragments. This is accompanied by down-regulation of Akt and activation of caspase-3 and p38 mitogen-activated protein kinase (MAPK). Surprisingly, removal of TGFalpha (using the null strain) does not ameliorate the LMP1-induced phenotype, but instead accelerates the deterioration. Consistent with this, EGFR is reduced less rapidly and MAPK/ERK kinase (MEK) and extracellular-signal-regulated kinase (ERK) are initially activated in the null background, suggesting that TGFalpha or excess of the ligands together act to divert phosphorylated EGFR into a cleavage pathway. In addition, LMP1 leads to the activation of c-Jun N-terminal kinase 2 (JNK2) followed by JNK1 in the effected tissue. Specific AP1 family members FosB, Fra-1 and JunB are constitutively induced and serum response factor, AP1 and nuclear factor kappaB (incorporating p65) are activated in the transgenic tissue compared with wild-type. This system allows the analysis of early events resulting from the expression of a viral oncogene with broad impact in the signalling milieu and the attempts at homeostasis in the responding tissue. It reveals what regulatory circuits are in place in a normal tissue, thus facilitating further prediction of causative events in carcinogenic progression.
