In vivo evaluation of riboflavin receptor targeted fluorescent USPIO in mice with prostate cancer xenografts

Abstract

Riboflavin (Rf) receptors bind and translocate Rf and its phosphorylated forms (e.g. flavin mononucleotide, FMN) into cells where they mediate various cellular metabolic pathways. Previously, we showed that FMN-coated ultrasmall superparamagnetic iron oxide (FLUSPIO) nanoparticles are suitable for labeling metabolically active cancer and endothelial cells in vitro. In this study, we focused on the in vivo application of FLUSPIO using prostate cancer xenografts. Size, charge, and chemical composition of FLUSPIO were evaluated. We explored the in vitro specificity of FLUSPIO for its cellular receptors using magnetic resonance imaging (MRI) and Prussian blue staining. Competitive binding experiments were performed in vivo by injecting free FMN in excess. Bio-distribution of FLUSPIO was determined by estimating iron content in organs and tumors using a colorimetric assay. AFM analysis and zeta potential measurements revealed a particulate morphology approximately 20-40 nm in size and a negative zeta potential (-24.23 ± 0.15 mV) in water. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry data confirmed FMN present on the USPIO nanoparticle surface. FLUSPIO uptake in prostate cancer cells and human umbilical vein endothelial cells was significantly higher than that of control USPIO, while addition of excess of free FMN reduced accumulation. Similarly, in vivo MRI and histology showed specific FLUSPIO uptake by prostate cancer cells, tumor endothelial cells, and tumor-associated macrophages. Besides prominent tumor accumulation, FLUSPIO accumulated in the liver, spleen, lung, and skin. Hence, our data strengthen our hypothesis that targeting riboflavin receptors is an efficient approach to accumulate nanomedicines in tumors opening perspectives for the development of diagnostic and therapeutic systems.

One-pot synthesis of dextran-coated iron oxide nanoclusters for real-time regional lymph node mapping

“…Among the abovementioned indices, the ALT and AST indices are closely related to the liver function of animals, while CREA and BUN levels relate to kidney function. 18 The results revealed that there were no significant differences in any of the indices of the treated rat compared to the untreated rat within an observation period of 3 months, which proved that the DIONs might have no harmful impact on liver and kidney function ( Figure 5D). In order to further evaluate the biosafety, the testing of the blood iron content was performed.…”

“…They were washed with doubledistilled water 3 times and then observed by fluorescence microscopy (IX-70, Olympus Corporation, Tokyo, Japan). 18 Blue particles in the cell indicated success of staining. Then, we chose the DIONs with an appropriate concentration that had a satisfactory blue staining effect for the following comparison.…”

Photoacoustic imaging of tumor targeting with riboflavin-functionalized theranostic nanocarriers

“…[23][24][25][26] Thus, a major argument for using RF as a ligand for targeted drug delivery is that it targets both cancerous cells and the tumor stroma including the angiogenic endothelium. 19,27,28 This feature was demonstrated in several preclinical studies with a variety of used carriers -from iron oxide nanoparticles to polymers. 17,18,22,27 In our previous study, we validated the efficacy of cancer and endothelial cell targeting in vitro and in vivo by RF amphiphiles inserted into a liposome formulation.…”

“…In this context, riboflavin (RF)-based targeting represents a promising approach for cell labeling, 17,18 tumor contrast enhancement, 19 and cell-specific drug delivery. [20][21][22] Due to its involvement in cell growth and development, RF is of high demand in metabolically active cells such as various malignant (eg, breast, prostate cancer, and hepatocellular carcinoma) and rapidly growing endothelial cells.…”

“…The higher accumulation of RF-functionalized liposomes is assumed to be associated with endothelial cell binding. 19,27 Even though the addition of a targeting ligand on the surface of long-circulating liposomes might carry the risk of reducing nanomedicines' blood half-life or tumor extravasation, it is still a valuable alternative for the intracellular delivery of fragile and highly active biomolecules (eg, nucleic acids or proteins). 34 In this context, the endothelial cell targeting is a highly promising approach as it allows fast binding and internalization and does not require deep tumor extravasation.…”