Kinetic Analysis of the Uptake and Release of Fluorescein by Metal-Organic Framework Nanoparticles.

Metal-organic framework nanoparticles (MOF NPs) are promising guest-host materials with applications in separation, storage, catalysis, and drug delivery. However, on- and off-loading of guest molecules by porous MOF nanostructures are still poorly understood. Here we study uptake and release of fluorescein by two representative MOF NPs, MIL-100(Fe) and MIL-101(Cr). Suspensions of these MOF NPs exhibit well-defined size distributions and crystallinity, as verified by electron microscopy, dynamic light scattering, and X-ray diffraction. Using absorbance spectroscopy the equilibrium dissociation constants and maximum numbers of adsorbed fluorescein molecules per NP were determined. Time-resolved fluorescence studies reveal that rates of release and loading are pH dependent. The kinetics observed are compared to theoretical estimates that account for bulk diffusion into NPs, and retarded internal diffusion and adsorption rates. Our study shows that, rather than being simple volumetric carriers, MOF-NPs are dominated by internal surface properties. The findings will help to optimize payload levels and develop release strategies that exploit varying pH for drug delivery.

Impact of plasma protein binding on cargo release by thermosensitive liposomes probed by fluorescence correlation spectroscopy.

Thermosensitive liposomes (TSLs) whose phase-transition temperature (Tm) lies slightly above body temperature are ideal candidates for controlled drug release via local hyperthermia. Recent studies, however, have revealed disruptive shifts in the release temperature Tr in mouse plasma, which are attributed to undefined interactions with blood proteins. Here, we study the effects of four major plasma proteins - serum albumin (SA), transferrin (Tf), apolipoprotein A1 (ApoA1) and fibrinogen (Fib) - on the temperature-dependent release of fluorescein di-ß-D-galactopyranoside (FDG) from TSLs. The amount of fluorescein released was quantified by fluorescence correlation spectroscopy (FCS) after hydrolysis of FDG with ß-galactosidase (ß-Gal). This approach is more sensitive and thus superior to previous release assays, as it is impervious to the confounding effects of Triton on conventional fluorescence measurements. The assay determines the molar release ratio, i.e. the number of molecules released per liposome. We show that shifts in the Tr of release do not reflect protein affinities for the liposomes derived from adsorption isotherms. We confirm a remarkable shift in induced release towards lower temperatures in the presence of mouse plasma. In contrast, exposure to rat or human plasma, or fetal bovine serum (FBS), has no effect on the release profile.

Multifunctional Nanoparticles by Coordinative Self-Assembly of His-Tagged Units with Metal-Organic Frameworks.

Self-assembly of individual units into multicomponent complexes is a powerful approach for the generation of functional superstructures. We present the coordinative interaction of oligohistidine-tags (His-tags) with metal-organic framework nanoparticles (MOF NPs). By this novel concept, different molecular units can be anchored on the outer surface of MOF NPs in a self-assembly process generating multifunctional nanosystems. The article focuses on two main objectives: first, the detailed investigation of the assembly process and fundamental establishment of the novel functionalization concept; and second, its subsequent use for the development of biomacromolecule (e.g., peptides and proteins) delivery vehicles. Three exemplary MOF structures, MIL-88A, HKUST-1, and Zr-fum, based on different metal components, were selected for the external binding of various His-tagged synthetic peptides and recombinant or chemically H6-modified proteins. Evidence for simultaneous assembly of different functional units with Zr-fum MOF NPs as well as their successful transport into living cells illustrate the promising potential of the self-assembly approach for the generation of multifunctional NPs and future biological applications. Taking the high number of possible MOF NPs and different functional units into account, the reported functionalization approach opens great flexibility for the targeted synthesis of multifunctional NPs for specific purposes.

Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells.

PURPOSE: Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. METHODS: Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. RESULTS: The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. CONCLUSION: Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.

pH-Reversible Cationic RNase A Conjugates for Enhanced Cellular Delivery and Tumor Cell Killing.

Intracellularly-acting therapeutic proteins are considered promising alternatives for the treatment of various diseases. Major limitations of their application are low efficiency of intracellular delivery and possible reduction of protein activity during derivatization. Herein, we report pH-sensitive covalent modification of proteins with a histidine-rich cationic oligomer (689) for efficient intracellular transduction and traceless release of functional proteins. Enhanced Green fluorescent protein (EGFP), as model for the visualization of protein transduction, and RNase A, as therapeutic protein with antitumoral effect, were modified with the pH-sensitive bifunctional AzMMMan linker and varying amounts of cationic oligomer. The modification degree showed impact on the internalization and cellular distribution of EGFP as well as the biological effect of RNase A conjugates, which mediated considerable toxicity against cancer cells at optimal ratio. The presented conjugates demonstrate their qualification to achieve efficient intracellular delivery and controlled release without protein inactivation and potential prospective applications in protein-based therapies.

MOF nanoparticles coated by lipid bilayers and their uptake by cancer cells.

We report the synthesis of MOF@lipid nanoparticles as a versatile and powerful novel class of nanocarriers based on metal-organic frameworks (MOFs). We show that the MOF@lipid system can effectively store dye molecules inside the porous scaffold of the MOF while the lipid bilayer prevents their premature release. Efficient uptake of the MOF@lipid nanoparticles by cancer cells makes these nanocarriers promising for drug delivery and diagnostic purposes.