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Riboflavin transporters (RFTs) and the riboflavin carrier protein (RCP) are highly upregulated in many tumor cells, tumor stem cells, and tumor neovasculature, which makes them attractive targets for nanomedicines. Addressing cells in different tumor compartments requires drug carriers, which are not only able to accumulate via the EPR effect but also to extravasate, target specific cell populations, and get internalized by cells. Reasoning that antibodies are among the most efficient targeting systems developed by nature, we consider their size (-10-15 nm) to be ideal for balancing passive and active tumor targeting. Therefore, small, short-circulating (10 kDa, -7 nm, t1/2 - 1 h) and larger, longer-circulating (40 kDa, -13 nm, t1/2 - 13 h) riboflavin-targeted branched PEG polymers were synthesized, and their biodistribution and target site accumulation were evaluated in mice bearing angiogenic squamous cell carcinoma (A431) and desmoplastic prostate cancer (PC3) xenografts. The tumor accumulation of the 10 kDa PEG was characterized by rapid intercompartmental exchange and significantly improved upon active targeting with riboflavin (RF). The 40 kDa PEG accumulated in tumors four times more efficiently than the small polymer, but its accumulation did not profit from active RF-targeting. However, RF-targeting enhanced the cellular internalization in both tumor models and for both polymer sizes. Interestingly, the nanocarriers' cell-uptake in tumors was not directly correlated with the extent of accumulation. For example, in both tumor models the small RF-PEG accumulated much less strongly than the large passively targeted PEG but showed significantly higher intracellular amounts 24 h after iv administration. Additionally, the size of the polymer determined its preferential uptake by different tumor cell compartments: the 10 kDa RF-PEGs most efficiently targeted cancer cells, whereas the highest uptake of the 40 kDa RF-PEGs was observed in tumor-associated macrophages. These findings imply that drug carriers with sizes in the range of therapeutic antibodies show balanced properties with respect to passive accumulation, tissue penetration, and active targeting. Besides highlighting the potential of RF-mediated (cancer) cell targeting, we show that strong tumor accumulation does not automatically mean high cellular uptake and that the nanocarriers' size plays a critical role in cell- and compartment-specific drug targeting.

Theranostics 7(6), 1499-1510 (2017). doi:10.7150/thno.17263, Published by Ivyspring, Wyoming, NSW

Nataliia Beztsinna,1,* Yoanna Tsvetkova,2,* Jithin Jose,3 Boutayna Rhourri-Frih,1 Wa’el Al Rawashdeh,2 Twan Lammers,2 Fabian Kiessling,2 Isabelle Bestel1 1Institute of Chemistry & Biology of Membranes & Nanoobjects (CBMN), UMR 5248, University of Bordeaux, Pessac, France; 2Institute for Experimental Molecular Imaging, RWTH Aachen University Clinic, Aachen, Germany; 3Fujifilm VisualSonics BV, Amsterdam, the Netherlands *These authors contributed equally to this work Abstract: Photoacoustic imaging is an emerging method in the molecular imaging field, providing high spatiotemporal resolution and sufficient imaging depths for many clinical applications. Therefore, the aim of this study was to use photoacoustic imaging as a tool to evaluate a riboflavin (RF)-based targeted nanoplatform. RF is internalized by the cells through a specific pathway, and its derivatives were recently shown as promising tumor-targeting vectors for the drug delivery systems. Here, the RF amphiphile synthesized from a PEGylated phospholipid was successfully inserted into a long-circulating liposome formulation labeled with the clinically approved photoacoustic contrast agent – indocyanine green (ICG). The obtained liposomes had a diameter of 124 nm (polydispersity index =0.17) and had a negative zeta potential of –26 mV. Studies in biological phantoms indicated a stable and concentration-dependent photoacoustic signal (Vevo® LAZR) of the ICG-containing RF-functionalized liposomes. In A431 cells, ahigh uptake of RF-functionalized liposomes was found and could be blocked competitively. First, studies in mice revealed ~3 times higher photoacoustic signal in subcutaneous A431 tumor xenografts (P