Evaluation of Riboflavin Transporters as Targets for Drug Delivery and Theranostics

Abstract

The retention and cellular internalization of drug delivery systems and theranostics for cancer therapy can be improved by targeting molecules. Since an increased uptake of riboflavin was reported for various cancers, riboflavin and its derivatives may be promising binding moieties to trigger internalization via the riboflavin transporters (RFVT) 1, 2, and 3. Riboflavin is a vitamin with pivotal role in energy metabolism and indispensable for cellular growth. In previous preclinical studies on mice, we showed the target-specific accumulation of riboflavin-functionalized nanocarriers in cancer cells. Although the uptake mechanism of riboflavin has been studied for over a decade, little is known about the riboflavin transporters and their expression on cancer cells, tumor stroma, and healthy tissues. Furthermore, evidence is lacking concerning the representativeness of the preclinical findings to the situation in humans. In this study, we investigated the expression pattern of riboflavin transporters in human squamous cell carcinoma (SCC), melanoma and luminal A breast cancer samples, as well as in healthy skin, breast, aorta, and kidney tissues. Low constitutive expression levels of RFVT1–3 were found on all healthy tissues, while RFVT2 and 3 were significantly overexpressed in melanoma, RFVT1 and 3 in luminal A breast cancer and RFVT1–3 in SCC. Correspondingly, the SCC cell line A431 was highly positive for all RFVTs, thus qualifying as suitable in vitro model. In contrast, activated endothelial cells (HUVEC) only presented with a strong expression of RFVT2, and HK2 kidney cells only with a low constitutive expression of RFVT1–3. Functional in vitro studies on A431 and HK2 cells using confocal microscopy showed that riboflavin uptake is mostly ATP dependent and primarily driven by endocytosis. Furthermore, riboflavin is partially trafficked to the mitochondria. Riboflavin uptake and trafficking was significantly higher in A431 than in healthy kidney cells. Thus, this manuscript supports the hypothesis that addressing the riboflavin internalization pathway may be highly valuable for tumor targeted drug delivery.

Riboflavin-Targeted Drug Delivery

“…Interestingly, FMN USPIO were internalized by tumor cells, endothelial cells, and macrophages. Although the uptake in macrophages and endothelial cells-to a certain degree-can be attributed to unspecific phagocytosis, enhanced uptake can also be the consequence of the upregulation of SLC52 transporters due to the enhanced metabolic activity of these cells [14,50]. In summary, FMN USPIO showed tumor specific accumulation in cancer cells and the tumor stroma, i.e., the tumor associated endothelial cells and macrophages.…”

“…In vivo, low levels of all three RF transporters have been found in most healthy tissues, whilst tumors seem to overexpress RFVTs: all three transporters were significantly overexpressed in squamous cell carcinoma (SCC), melanoma, and luminal A breast cancer. RFVT3, in particular, was shown to be 187-fold more expressed in SCC compared to healthy tissue [50]. High amounts of RFVT3 were also found in esophageal squamous cell carcinoma, squamous cell carcinoma, and glioma [51][52][53].…”

“…The substrate size is very important, as it has been shown that upon conjugation to larger molecules, the substrate can lose the ability to pass through the membrane. In vitro, high RFVT expression was observed in human epidermoid carcinoma (A431), human renal proximal tube epithelial cells (HK-2), and human umbilical vein endothelial cells (HUVEC) [50]. In vivo, low levels of all three RF transporters have been found in most healthy tissues, whilst tumors seem to overexpress RFVTs: all three transporters were significantly overexpressed in squamous cell carcinoma (SCC), melanoma, and luminal A breast cancer.…”

Molecular engineering of the last-generation CNTs in smart cancer therapy by grafting PEG–PLGA–riboflavin

“…Moreover, the derivatives of RF have been applied as tumor targeting ligands in vitro and in vivo, which has shown the enhanced tumor-specic accumulation of riboavintargeted nanotubes carrying paclitaxel in MCF-7 cells12 as well as riboavin-targeted liposomes in murine A431 and PC3 tumor xenogras. 12 The success in the practical implementation of anticancer nanomedicine is closely related to the drug delivery system. The major objective of delivery systems is to enhance the specicity of drug delivery to target tissues, minimize the adverse effects, prolong its half-life, protect the drug's molecule, and increase its biocompatibility.…”

Influence of Riboflavin Targeting on Tumor Accumulation and Internalization of Peptostar Based Drug Delivery Systems

“…9 RFVTs and the RF carrier protein have been shown to be highly expressed in breast, 10−14 prostate, 15 liver, 16 esophageal, 17,18 and brain 19 cancers, as well as in squamous cell carcinoma. 14 This makes RF a promising ligand for active targeting of drugs and drug delivery systems. 20 Like poly(ethylene glycol) (PEG), polysarcosine (pSar) is a protein-resistant material that can introduce stealth-like properties to a surface at high grafting densities.…”