Cell viability imaging in tumor spheroids via DNA binding of a ruthenium(II) light-switch complex.

The famous ''light-switch'' ruthenium complex [Ru(bpy)2(dppz)](PF6)2 (1) has been long known for its DNA binding properties in vitro. However, the biological utility of this compound has been hampered by its poor cellular uptake in living cells. Here we report a bioimaging application of 1 as cell viability probe in both 2D cells monolayer and 3D multi-cellular tumor spheroids of various human cancer cell lines (U87, HepG2, A549). When compared to propidium iodide, a routinely used cell viability probe, 1 was found to enhance the staining of dead cells in particular in tumor spheroids. 1 has high photostability, longer Stokes shift, and displays lower cytotoxicity compared to propidium iodide, which is a known carcinogenic. Finally, 1 was also found to displace the classical DNA binding dye Hoechst in dead cells, which makes it a promising dye for time-dependent imaging of dead cells in cell cultures, including multi-cellular tumor spheroids.

Photosubstitution in a trisheteroleptic ruthenium complex inhibits conjunctival melanoma growth in a zebrafish orthotopic xenograft model.

In vivo data are rare but essential for establishing the clinical potential of ruthenium-based photoactivated chemotherapy (PACT) compounds, a new family of phototherapeutic drugs that are activated via ligand photosubstitution. Here a novel trisheteroleptic ruthenium complex [Ru(dpp)(bpy)(mtmp)](PF6)2 ([2](PF6)2, dpp = 4,7-diphenyl-1,10-phenanthroline, bpy = 2,2'-bipyridine, mtmp = 2-methylthiomethylpyridine) was synthesized and its light-activated anticancer properties were validated in cancer cell monolayers, 3D tumor spheroids, and in embryonic zebrafish cancer models. Upon green light irradiation, the non-toxic mtmp ligand is selectively cleaved off, thereby releasing a phototoxic ruthenium-based photoproduct capable notably of binding to nuclear DNA and triggering DNA damage and apoptosis within 24-48 h. In vitro, fifteen minutes of green light irradiation (21 mW cm-2, 19 J cm-2, 520 nm) were sufficient to generate high phototherapeutic indexes (PI) for this compound in a range of cancer cell lines including lung (A549), prostate (PC3Pro4), conjunctival melanoma (CRMM1, CRMM2, CM2005.1) and uveal melanoma (OMM1, OMM2.5, Mel270) cancer cell lines. The therapeutic potential of [2](PF6)2 was further evaluated in zebrafish embryo ectopic (PC3Pro4) or orthotopic (CRMM1, CRMM2) tumour models. The ectopic model consisted of red fluorescent PC3Pro4-mCherry cells injected intravenously (IV) into zebrafish, that formed perivascular metastatic lesions at the posterior ventral end of caudal hematopoietic tissue (CHT). By contrast, in the orthotopic model, CRMM1- and CRMM2-mCherry cells were injected behind the eye where they developed primary lesions. The maximally-tolerated dose (MTD) of [2](PF6)2 was first determined for three different modes of compound administration: (i) incubating the fish in prodrug-containing water (WA); (ii) injecting the prodrug intravenously (IV) into the fish; or (iii) injecting the prodrug retro-orbitally (RO) into the fish. To test the anticancer efficiency of [2](PF6)2, the embryos were treated 24 h after engraftment at the MTD. Optimally, four consecutive PACT treatments were performed on engrafted embryos using 60 min drug-to-light intervals and 90 min green light irradiation (21 mW cm-2, 114 J cm-2, 520 nm). Most importantly, this PACT protocol was not toxic to the zebrafish. In the ectopic prostate tumour models, where [2](PF6)2 showed the highest photoindex in vitro (PI > 31), the PACT treatment did not significantly diminish the growth of primary lesions, while in both conjunctival melanoma orthotopic tumour models, where [2](PF6)2 showed more modest photoindexes (PI ∼ 9), retro-orbitally administered PACT treatment significantly inhibited growth of the engrafted tumors. Overall, this study represents the first demonstration in zebrafish cancer models of the clinical potential of ruthenium-based PACT, here against conjunctival melanoma.

Mimicking Photosystem I with a Transmembrane Light Harvester and Energy Transfer-Induced Photoreduction in Phospholipid Bilayers.

Invited for the cover of this issue are Andrea Pannwitz, Sylvestre Bonnet and co-workers at Leiden University and Johns Hopkins University. The image depicts an observer watching over a lipid bilayer "landscape" and a sky full of luminescent giant vesicles. Read the full text of the article at 10.1002/chem.202003391.

Flavin-Conjugated Nanobombs: Key Structural Requirements Governing Their Self-Assemblies' Morphologies.

In contrast to artificial molecules, natural photosensitizers have the benefit of excellent toxicity profiles and of life-compatible activating energy ranges. Flavins are such photosensitizers that were selected by nature in a plethora of light-triggered biochemical reactions. Flavin-rich nanoparticles could thus emerge as promising tools in photodynamic therapies and in active-targeting drug delivery. Self-assembled flavin-conjugated phospholipids improve the pharmacokinetics of natural flavins and, in the case of controlled morphologies, reduce photobleaching phenomena. The current article presents a proof of concept for the design of riboflavin-rich nanoparticles of tunable morphology from multilamellar patches to vesicular self-assemblies. Coarse-grained simulations of the self-assembling process revealed the key interactions governing the obtained nanomaterials and successfully guided the synthesis of new flavin-conjugates of predictable self-assembly. The obtained flavin-based liposomes had a 65 nm hydrodynamic diameter, were stable, and showed potential photosensitizer activity.

Mimicking Photosystem I with a Transmembrane Light Harvester and Energy Transfer-Induced Photoreduction in Phospholipid Bilayers.

Photosystem I (PS I) is a transmembrane protein that assembles perpendicular to the membrane, and performs light harvesting, energy transfer, and electron transfer to a final, water-soluble electron acceptor. We present here a supramolecular model of it formed by a bicationic oligofluorene 12+ bound to the bisanionic photoredox catalyst eosin Y (EY2- ) in phospholipid bilayers. According to confocal microscopy, molecular modeling, and time dependent density functional theory calculations, 12+ prefers to align perpendicularly to the lipid bilayer. In presence of EY2- , a strong complex is formed (Ka =2.1±0.1×106 m-1 ), which upon excitation of 12+ leads to efficient energy transfer to EY2- . Follow-up electron transfer from the excited state of EY2- to the water-soluble electron donor EDTA was shown via UV-Vis absorption spectroscopy. Overall, controlled self-assembly and photochemistry within the membrane provides an unprecedented yet simple synthetic functional mimic of PS I.

Biotin-decorated all-HPMA polymeric micelles for paclitaxel delivery.

To avoid poly(ethylene glycol)-related issues of nanomedicines such as accelerated blood clearance, fully N-2-hydroxypropyl methacrylamide (HPMAm)-based polymeric micelles decorated with biotin for drug delivery were designed. To this end, a biotin-functionalized chain transfer agent (CTA), 4-cyano-4-[(dodecylsulfanylthiocarbonyl)-sulfanyl]pentanoic acid (biotin-CDTPA), was synthesized for reversible addition-fragmentation chain-transfer (RAFT) polymerization. Amphiphilic poly(N-2-hydroxypropyl methacrylamide)-block-poly(N-2-benzoyloxypropyl methacrylamide) (p(HPMAm)-b-p(HPMAm-Bz)) with molecular weights ranging from 8 to 24 kDa were synthesized using CDTPA or biotin-CDTPA as CTA and 2,2'-azobis(2-methylpropionitrile) as initiator. The copolymers self-assembled in aqueous media into micelles with sizes of 40-90 nm which positively correlated to the chain length of the hydrophobic block in the polymers, whereas the critical micelle concentrations decreased with increasing hydrophobic block length. The polymer with a molecular weight of 22.1 kDa was used to prepare paclitaxel-loaded micelles which had sizes between 61 and 70 nm, and a maximum loading capacity of around 10 wt%. A549 lung cancer cells overexpressing the biotin receptor, internalized the biotin-decorated micelles more efficiently than non-targeted micelles, while very low internalization of both types of micelles by HEK293 human embryonic kidney cells lacking the biotin receptor was observed. As a consequence, the paclitaxel-loaded micelles with biotin decoration exhibited stronger cytotoxicity in A549 cells than non-targeted micelles. Overall, a synthetic pathway to obtain actively targeted poly(ethylene glycol)-free micelles fully based on a poly(HPMAm) backbone was established. These polymeric micelles are promising systems for the delivery of hydrophobic anticancer drugs.

Systematic evaluation of design features enables efficient selection of Π electron-stabilized polymeric micelles.

Polymeric micelles (PM) based on poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) loaded with paclitaxel (PTX-PM) have shown promising results in overcoming the suboptimal efficacy/toxicity profile of paclitaxel. To get insight into the stability of PTX-PM formulations upon storage and to optimize their in vivo tumor-targeted drug delivery properties, we set out to identify a lead PTX-PM formulation with the optimal polymer composition. To this end, PM based on four different mPEG5k-b-p(HPMA-Bz) block copolymers with varying molecular weight of the hydrophobic block (17-3 kDa) were loaded with different amounts of PTX. The hydrodynamic diameter was 52 ± 1 nm for PM prepared using polymers with longer hydrophobic blocks (mPEG5k-b-p(HPMA-Bz)17k and mPEG5k-b-p(HPMA-Bz)10k) and 39 ± 1 nm for PM composed of polymers with shorter hydrophobic blocks (mPEG5k-b-p(HPMA-Bz)5k and mPEG5k-b-p(HPMA-Bz)3k). The best storage stability and the slowest PTX release was observed for PM with larger hydrophobic blocks. On the other hand, smaller sized PM of shorter mPEG5k-b-p(HPMA-Bz)5k showed a better tumor penetration in 3D spheroids. Considering better drug retention capacity of the mPEG5k-b-p(HPMA-Bz)17k and smaller size of the mPEG5k-b-p(HPMA-Bz)5k as two desirable design features, we argue that PM based on these two polymers are the lead candidates for further in vivo studies.

Photo-Uncaging of a Microtubule-Targeted Rigidin Analogue in Hypoxic Cancer Cells and in a Xenograft Mouse Model.

Marine alkaloid rigidins are cytotoxic compounds known to kill cancer cells at nanomolar concentrations by targeting the microtubule network. Here, a rigidin analogue containing a thioether group was "caged" by coordination of its thioether group to a photosensitive ruthenium complex. In the dark, the coordinated ruthenium fragment prevented the rigidin analogue from inhibiting tubulin polymerization and reduced its toxicity in 2D cancer cell line monolayers, 3D lung cancer tumor spheroids (A549), and a lung cancer tumor xenograft (A549) in nude mice. Photochemical activation of the prodrug upon green light irradiation led to the photosubstitution of the thioether ligand by water, thereby releasing the free rigidin analogue capable of inhibiting the polymerization of tubulin. In cancer cells, such photorelease was accompanied by a drastic reduction of cell growth, not only when the cells were grown in normoxia (21% O2) but also remarkably in hypoxic conditions (1% O2). In vivo, low toxicity was observed at a dose of 1 mg·kg-1 when the compound was injected intraperitoneally, and light activation of the compound in the tumor led to 30% tumor volume reduction, which represents the first demonstration of the safety and efficacy of ruthenium-based photoactivated chemotherapy compounds in a tumor xenograft.

Selective Cytotoxicity to HER2 Positive Breast Cancer Cells by Saporin-Loaded Nanobody-Targeted Polymeric Nanoparticles in Combination with Photochemical Internalization.

In cancer treatment, polymeric nanoparticles (NPs) can serve as a vehicle for the delivery of cytotoxic proteins that have intracellular targets but that lack well-defined mechanisms for cellular internalization, such as saporin. In this work, we have prepared PEGylated poly(lactic acid- co-glycolic acid- co-hydroxymethyl glycolic acid) (PLGHMGA) NPs for the selective delivery of saporin in the cytosol of HER2 positive cancer cells. This selective uptake was achieved by decorating the surface of the NPs with the 11A4 nanobody that is specific for the HER2 receptor. Confocal microscopy observations showed rapid and extensive uptake of the targeted NPs (11A4-NPs) by HER2 positive cells (SkBr3) but not by HER2 negative cells (MDA-MB-231). This selective uptake was blocked upon preincubation of the cells with an excess of nanobody. Nontargeted NPs (Cys-NPs) were not taken up by either type of cells. Importantly, a dose-dependent cytotoxic effect was only observed on SkBr3 cells when these were treated with saporin-loaded 11A4-NPs in combination with photochemical internalization (PCI), a technique that uses a photosensitizer and local light exposure to facilitate endosomal escape of entrapped nanocarriers and biomolecules. The combined use of saporin-loaded 11A4-NPs and PCI strongly inhibited cell proliferation and decreased cell viability through induction of apoptosis. Also the cytotoxic effect could be reduced by an excess of nanobody, reinforcing the selectivity of this system. These results suggest that the combination of the targeting nanobody on the NPs with PCI are effective means to achieve selective uptake and cytotoxicity of saporin-loaded NPs.

Liposome-targeted recombinant human acid sphingomyelinase: Production, formulation, and in vitro evaluation.

Niemann-Pick disease type B is a hereditary rare condition caused by deficiency of the acid sphingomyelinase (ASM) that is needed for lysosomal hydrolysis of sphingomyelin to ceramide and phosphocholine. This deficiency leads to a massive accumulation of sphingomyelin in cells throughout the body, predominantly in the liver, spleen and lungs. Currently, there is no effective treatment available. Olipudase alfa (recombinant human acid sphingomyelinase; rhASM) is an investigational drug that has shown promising results. However, dose-dependent toxicity was observed in mice upon the intravenous administration of rhASM, potentially due to the systemic release of ceramide upon the extracellular degradation of sphingomyelin by rhASM. Using a nanocarrier to deliver the rhASM to cells could improve the therapeutic window by shielding the rhASM to prevent the off-target degradation of sphingomyelin. For this aim, we recombinantly expressed hASM in human cells and loaded it into different liposomal formulations at a drug-to-lipid ratio of 4% (w/w). Among four formulations, the liposomal rhASM formulation with the composition DPPC:DOPS:BMP:CHOL:DiD (59:20:10:10:1 mol%) was selected because of its superiority concerning the encapsulation efficiency of rhASM (21%) and cellular uptake by fibroblasts and macrophages. The selected liposomal rhASM formulation significantly reduced the accumulated lyso-sphingomyelin in NPD-B fibroblasts by 71%, part of this effect was stimulated by the used lipids, compared to 55% when using the free rhASM enzyme. More importantly, the undesired extracellular degradation of sphingomyelin was reduced when using the selected liposomal rhASM by 61% relative to the free rhASM. The presented in vitro data indicate that the liposomal rhASM is effective and may provide a safer intervention than free rhASM.

Thermosensitive liposomes for triggered release of cytotoxic proteins.

Lysolipid-containing thermosensitive liposomes (LTSL) are clinically-relevant drug nanocarriers which have been used to deliver small molecule cytostatics to tumors in combination with local hyperthermia (42 °C) to trigger local drug release. The objective of this study was to investigate the feasibility of LTSL for encapsulation and triggered release of macromolecular drugs such as plant-derived cytotoxins. As therapeutic protein we used Mistletoe lectin-1 (ML1) - a ribosome-inactivating protein with potent cytotoxic activity in tumor cells. Model macromolecules (dextrans, albumin) and ML1 were encapsulated in small unilamellar LTSL with varying lipid compositions by the thin film hydration method and extrusion. LTSLs showed molecular weight dependent heat-triggered release of the loaded cargo. The most promising composition, ML1 formulated in LTSL composed of 86:10:4 %mol DPPC:MSPC:DSPE-PEG2000, was further studied for bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging showed uptake of released ML1 after mild hyperthermia at 42 °C, subsequently leading to potent cytotoxicity by LTSL-ML1. Our study shows that LTSL in combination with localized hyperthermia hold promise as local tumor delivery strategy for macromolecular cytotoxins.

Influence of PEGylation of Vitamin-K-Loaded Mixed Micelles on the Uptake by and Transport through Caco-2 Cells.

The aim of the study is to investigate the uptake by and transport through Caco-2 cells of two mixed micelle formulations (based on egg phosphatidylcholine and glycocholic acid) of vitamin K, i.e., with and without DSPE-PEG2000. The uptake of vitamin K and fluorescently labeled mixed micelles with and without PEG coating showed similar kinetics and their uptake ratio remained constant over time. Together with the fact that an inhibitor of scavenger receptor B1 (BLT-1) decreased cellular uptake of vitamin K by ∼80% compared to the uptake in the absence of this inhibitor, we conclude that both types of micelles loaded with vitamin K can be taken up intactly by Caco-2 cells via this scavenger receptor. The amount of vitamin K in chylomicrons fraction from Caco-2 cell monolayers further indicates that mixed micelles (with or without PEGylation) are likely packed into chylomicrons after internalization by Caco-2 cells. Uptake of vitamin K from PEGylated mixed micelles increased four- to five-fold at simulated gastrointestinal conditions. In conclusion, PEGylated mixed micelles are stable upon exposure to simulated gastric conditions, and as a result, they do show overall a higher cellular uptake efficiency of vitamin K as compared to mixed micelles without PEG coating.

Luminescent Gold Nanocluster-Decorated Polymeric Hybrid Particles with Assembly-Induced Emission.

Ultrasmall gold atom clusters (<2 nm in diameter) or gold nanoclusters exhibit emergent photonic properties (near-infrared absorption and emission) compared to larger plasmonic gold particles because of the significant quantization of their conduction band. Although single gold nanocluster properties and applications are being increasingly investigated, little is still known about their behavior and properties when assembled into suprastructures, and even fewer studies are investigating their use for biomedical applications. Here, a simple synthetic pathway combines gold nanoclusters with thermosensitive diblock copolymers of poly(ethylene glycol) (PEG) and poly( N-isopropylacrylamide) (PNIPAm) to form a new class of gold-polymer, micelle-forming, hybrid nanoparticle. The nanohybrids' design is uniquely centered on enabling the temperature-dependent self-assembly of gold nanoclusters into the hydrophobic cores of micelles. This nonbulk assembly not only preserves but also enhances the attractive near-infrared photonics of the gold nanoclusters by significantly increasing their native fluorescent signal. In parallel to the fundamental insights into gold nanocluster ordering and assembly, the gold-polymer nanohybrids also demonstrated great potential as fluorescent live-imaging probes in vitro. This innovative material design based on the temperature-dependent, self-assembly of gold nanoclusters within a polymeric micelle's core shows great promise toward bioassays, nanosensors, and nanomedicine.

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, 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.

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® 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 ~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.

Small nanosized poly(vinyl benzyl trimethylammonium chloride) based polyplexes for siRNA delivery.

The success of siRNA gene therapy requires the availability of safe and efficient delivery systems. In the present study, we investigated poly(vinyl benzyl trimethylammonium chloride) (PVTC) and its block copolymer with poly(oligo(ethyleneglycol) methacrylate) (POEGMA) as delivery vector for siRNA. Small polyplexes ranging from 8 to 25nm in diameter were formed in aqueous solution by spontaneous self-assembly of both the homopolymer and block copolymer with siRNA and the formed particles were stable at physiological ionic strength. It was shown that when human ovarian adenocarcinoma cells were transfected, siRNA polyplexes based on PVTC (40kDa) and PVTC-POEGMA-4 (PP4, 34kDa) efficiently induced luciferase gene silencing to the same extent as the formulation based on a commercial lipid (Lipofectamine®) (∼80%), and showed higher gene silencing than the linear polyethylenimine formulation linear polyethylenimine (∼35%). Importantly, the POEGMA block polymers displayed a significantly lower cytotoxicity as compared to L-pEI. siRNA polyplexes based on the block polymers displayed high cellular uptake resulting in ∼50% silencing of luciferase expression also in the presence of serum. These results demonstrate that PVTC-based polymers are promising siRNA delivery vectors.

Iron nanomedicines induce Toll-like receptor activation, cytokine production and complement activation.

Approximately a dozen of intravenous iron nanomedicines gained marketing authorization in the last two decades. These products are generally considered as safe, but have been associated with an increased risk for hypersensitivity-like reactions of which the underlying mechanisms are unknown. We hypothesized that iron nanomedicines can trigger the innate immune system. We hereto investigated the physico-chemical properties of ferric gluconate, iron sucrose, ferric carboxymaltose and iron isomaltoside 1000 and comparatively studied their interaction with Toll-like receptors, the complement system and peripheral blood mononuclear cells. Two out of four formulations appeared as aggregates by Scanning Transmission Electron Microscopy analysis and were actively taken up by HEK293T- and peripheral blood mononuclear cells in a cholesterol-dependent manner. These formulations triggered in vitro activation of intracellular Toll-like receptors 3, -7 and -9 in a dose- and serum-dependent manner. In parallel experiments, we determined that these compounds activated the complement system. Finally, we found that uptake of aggregation-prone iron nanomedicines by peripheral blood mononuclear cells in whole blood induced production of the proinflammatory cytokine IL-1ß, but not IL-6.

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.