Riboflavin-targeted nanomedicines for cancer imaging and drug delivery

The development of nanomaterials and their physicochemical, in vitro, and in vivo evaluation in preclinical settings have a great impact on the improvement of cancer diagnosis and treatment. Nanomaterials can be used as contrast agents and/or drug delivery systems, and their efficiency in cancer therapy may be improved by their conjugation to a targeting ligand, which specifically binds to cellular receptors overexpressed in the tumor lesion. Riboflavin Transporters (RFT) and Riboflavin Carrier Protein (RCP) are overexpressed by many tumor cells and cells of the malignant stroma. This is why this thesis involves two studies focused on the preclinical evaluation of riboflavin (vitamin B2) as a tumor targeting ligand for nanomaterials. In the first study, flavin mononucleotide (a riboflavin derivative) was grafted on the surface of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, used as contrast agents for magnetic resonance imaging (MRI). The riboflavin-targeted USPIOs were further stabilized with different small biomolecules that influenced the size and functionality of the magnetic nanoparticles. The nanoparticles’ uptake by endothelial and numerous cancer cells was evaluated photometricaly and with MRI. The results showed that the surface modification of the contrast agents had a significant influence on the cellular internalization: riboflavin faciliated a receptor-mediated endocytosis of the magnetic nanoparticles by cancer cells, and the amount of targeting ligand on the particles’ surface regulated the degree of cellular uptake. In the second study, the targeting efficiency of riboflavin-functionalized polymers was evaluated in vivo - in mice bearing prostate and epidermoid tumors - using fluorescent molecular and computed tomography. The polymers had different molecular weights and hydrodynamic sizes (10 kDa, DH~6 nm and 40 kDa, DH~14 nm) and, therefore, displayed different pharmacokinetic profiles in terms of plasma residence and volume of distribution. The results led to the hypothesis that targeted drug delivery systems should have the size of diagnostic antibodies (DH~12 nm) in order to display an ideal balance between EPR-based accumulation, sufficient tumor penetration, active binding, and cellular internalization. Riboflavin targeting with drug carriers exhibiting rapid compartmental exchange and fast tissue clearance significantly improved tumor accumulation and cellular uptake in the cancer lesion. On the other hand, riboflavin targeting with long circulating drug carriers improved only their cellular internalization in the malignant lesion and hardly increased their tumor accumulation. In addition, the results showed that already small differences in probe size could influence drug carrier uptake in targeted cell populations in tumors

Amphiphilic Phospholipid-Based Riboflavin Derivatives for Tumor Targeting Nanomedicines

Riboflavin (RF) is an essential vitamin for cellular metabolism. Recent studies have shown that RF is internalized through RF transporters, which are highly overexpressed by prostate and breast cancer cells, as well as by angiogenic endothelium. Here, we present an optimized synthesis protocol for preparing tailor-made amphiphilic phospholipid-based RF derivatives using phosphoramidite chemistry. The prepared RF amphiphile-RfdiC14-can be inserted into liposome formulations for targeted drug delivery. The obtained liposomes had a hydrodynamic size of 115 ± 5 nm with narrow size distribution (PDI 0.06) and a zeta potential of -52 ± 3 mV. In vitro uptake studies showed that RfdiC14-containing liposomes were strongly internalized in HUVEC, PC3, and A431 cells, in a specific and transporter-mediated manner. To assess the RF targeting potential in vivo, an amphiphile containing PEG spacer between RF and a lipid was prepared-DSPE-PEG-RF. The latter was successfully incorporated into long-circulating near-infrared-labeled liposomes (141 ± 1 nm in diameter, PDI 0.07, zeta potential of -33 ± 1 mV). The longitudinal μCT/FMT biodistribution studies in PC3 xenograft bearing mice demonstrated similar pharmacokinetics profile of DSPE-PEG-RF-functionalized liposomes compared to control. The subsequent histological evaluation of resected tumors revealed higher degree of tumor retention as well as colocalization of targeted liposomes with endothelial cells emphasizing the targeting potential of RF amphiphiles and their utility for the lipid-containing drug delivery systems

Synthesis, characterization, and relaxation studies of Gd‐DO3A conjugate of chlorambucil as a potential theranostic agent

DO3A-based macrocycles serve as attractive templates from which clinically useful theranostic agents can be obtained after coupling with molecular targeted therapeutic drugs. In this study, we describe the chemical synthesis, relaxation, and cytotoxicity studies of a new DO3A conjugate of chlorambucil (CHL) as a magnetic resonance imaging (MRI) theranostic agent. A convenient route of synthesis is reported, which allowed conjugation of the macrocyclic ligand (DO3A) to the chemotherapeutic drug (CHL) via tyrosine for the preparation of an attractive chelate-drug ensemble (DO3A-TR-CHL). The structures of all intermediates and final compound have been determined by H-1, C-13 NMR, and MS. The efficacy of DO3A-TR-CHL as a non-ionic magnetic contrast agent was tested by performing relaxometric studies on its gadolinium complex. The complex exhibited relaxivities (7.11 mm(-1)/s) higher than that of currently used MR contrast agents and showed enhanced contrast in T-1-weighted images. MTT assays revealed that both DO3A-TR-CHL and Gd(III)-DO3A-TR-CHL conjugates exhibited dose-dependent toxicity and an enhanced antiproliferative activity against tumor (A549 and HeLa) cell lines compared to that of parent drug (CHL), thereby demonstrating their potential to be used as a magnetic resonance imaging theranostic for improved molecular imaging and therapy of human cancers

Photoacoustic imaging of tumor targeting with riboflavin-functionalized theranostic nanocarriers

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 (R) LAZR) of the ICG-containing RF-functionalized liposomes. In A431 cells, a high uptake of RF-functionalized liposomes was found and could be blocked competitively. First, studies in mice revealed similar to 3 times higher photoacoustic signal in subcutaneous A431 tumor xenografts (P<0.05) after injection of RF-functionalized liposomes compared to control particles. In this context, the application of a spectral unmixing protocol confirmed the initial quantitative data and improved the localization of liposomes in the tumor. In conclusion, the synthesized RF amphiphile leads to efficient liposomal tumor targeting and can be favorably detected by photoacoustic imaging with a perspective of theranostic applications

Photoacoustic imaging of tumor targeting with riboflavin-functionalized theranostic nanocarriers

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 (R) LAZR) of the ICG-containing RF-functionalized liposomes. In A431 cells, a high uptake of RF-functionalized liposomes was found and could be blocked competitively. First, studies in mice revealed similar to 3 times higher photoacoustic signal in subcutaneous A431 tumor xenografts (P<0.05) after injection of RF-functionalized liposomes compared to control particles. In this context, the application of a spectral unmixing protocol confirmed the initial quantitative data and improved the localization of liposomes in the tumor. In conclusion, the synthesized RF amphiphile leads to efficient liposomal tumor targeting and can be favorably detected by photoacoustic imaging with a perspective of theranostic applications

Noninvasive Assessment of Elimination and Retention using CT-FMT and Kinetic Whole-body Modeling

Fluorescence-mediated tomography (FMT) is a quantitative three-dimensional imaging technique for preclinical research applications. The combination with micro-computed tomography (μCT) enables improved reconstruction and analysis. The aim of this study is to assess the potential of μCT-FMT and kinetic modeling to determine elimination and retention of typical model drugs and drug delivery systems. We selected four fluorescent probes with different but well-known biodistribution and elimination routes: Indocyanine green (ICG), hydroxyapatite-binding OsteoSense (OS), biodegradable nanogels (NG) and microbubbles (MB). μCT-FMT scans were performed in twenty BALB/c nude mice (5 per group) at 0.25, 2, 4, 8, 24, 48 and 72 h after intravenous injection. Longitudinal organ curves were determined using interactive organ segmentation software and a pharmacokinetic whole-body model was implemented and applied to compute physiological parameters describing elimination and retention. ICG demonstrated high initial hepatic uptake which decreased rapidly while intestinal accumulation appeared for around 8 hours which is in line with the known direct uptake by hepatocytes followed by hepatobiliary elimination. Complete clearance from the body was observed at 48 h. NG showed similar but slower hepatobiliary elimination because these nanoparticles require degradation before elimination can take place. OS was strongly located in the bones in addition to high signal in the bladder at 0.25 h indicating fast renal excretion. MB showed longest retention in liver and spleen and low signal in the kidneys likely caused by renal elimination or retention of fragments. Furthermore, probe retention was found in liver (MB, NG and OS), spleen (MB) and kidneys (MB and NG) at 72 h which was confirmed by ex vivo data. The kinetic model enabled robust extraction of physiological parameters from the organ curves. In summary, μCT-FMT and kinetic modeling enable differentiation of hepatobiliary and renal elimination routes and allow for the noninvasive assessment of retention sites in relevant organs including liver, kidney, bone and spleen. © Ivyspring International Publisher

Balancing Passive and Active Targeting to Different Tumor Compartments Using Riboflavin-Functionalized Polymeric Nanocarriers

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, <i>t</i>_1/2 ∼ 1 h) and larger, longer-circulating (40 kDa, ∼13 nm, <i>t</i>_1/2 ∼ 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