Superparamagnetic Nanoparticles as a Powerful Systems Biology. Characterization Tool in the Physiological Context

Recently, functionalized superparamagnetic iron oxide nanoparticles (SPIONs) have been utilized for protein separation and therapeutic delivery of DNA and drugs. The development of new methods and tools for the targeting and identification of specific biomolecular interactions within living systems is of great interest in the fields of systems biology, target and drug identification, drug delivery, and diagnostics. Magnetic separation of organelles and proteins from complex whole-cell lysates allows enrichment and elucidation of intracellular interaction partners for a specific immobilized protein or peptide on the surface of SPIONs

Advanced surface derivatization of superparamagnetic iron oxide nanoparticles in a fixed bed magnetic reactor for bio-application

There is an enormous interest in exploiting nanoparticles in various biomedical applications since their size scale is similar to that of biological molecules (e.g., proteins, DNA) and structures (e.g., viruses and bacteria). As the field continues to develop, quantitative and qualitative studies on particle-cell interaction, with respect to their size and surface, are required in order to advance nanotechnology for biomedical applications. This will be important for assessing nanoparticle toxicity (i.e. translocation into cells and interference with viability and cellular function), for advancing nanoparticles for imaging, drug delivery, and therapeutic applications (i.e. targeting specific cells, organs, or tumors), and for designing multifunctional nanoparticles. Due to the lack of systematic study to date, data are difficult to compare since the parameters and particles in each of the published studies differ substantially. However, the scientific community, in general, agrees that the size and colloidal behavior play a crucial role in cellular interaction/uptake, biodistribution, clearance and cytotoxicity. Surface functionalization of nanoparticles still remains a difficult task and represents not only a chemical challenge but constitutes a basic requirement for future scientific investigations. Alteration of surface charges and/or stabilization by the addition of bi- / multi-functional molecules, such as differently charged proteins or plasmids, frequently leads to particle flocculation and rapid sedimentation. The biological functionality, in such cases, is achieved by covalent binding of bio-active molecules on a preexisting single surface coating. A fixed bed magnetic reactor has been developed with a quadrupole repulsive arrangement of permanent magnets which allows for surface derivatization by magnetically immobilizing superparamagnetic iron oxide nanoparticles (SPIONs). The yield and quality of the resulting functionalized SPIONs was significantly improved with reduction in reaction times using solid phase synthesis strategy. In this way, pH changes across the isoelectric point, washing steps or even solvent exchanges could be easily tolerated thereby avoiding the problems of colloidal instability during the derivatization steps. It was shown that the surface functionalization of SPIONs using a magnetic fixed bed reactor was superior to the liquid phase reaction in terms of reaction yield, particle size distribution, colloidal stability and scalability. In particular, cell organelle targeting peptide derivatized on SPIONs surface was obtained from the reactor. The combination of functionalized SPIONs and their ability to be recovered using a magnetic column coupled with biomolecular mass spectrometry has allowed to explore a complex intracellular pathway using a peptide that is known to target HeLa cell organelle. Here the concept of biomolecular interaction network elucidation with an organelle-targeting peptide was demonstrated. Besides that, the colloidal stability and cellular uptake of polymer coated SPIONs were also studied. Preliminary results showed that minor modifications of the nanoparticle surface lead to an altered behavior in stability, uptake, and toxicity. Also, different charges on the particle surfaces were found responsible for differential uptake of particles in cell media. Colloidal stability and its influence on biological properties will provide a profound base for future discussions on toxicity and potential application of nanoparticles in the field of biomedicine

Influence of serum supplemented cell culture medium on colloidal stability of polymer coated iron oxide and polystyrene nanoparticles with impact on cell interactions in vitro

When nanoparticles interact with cells, the possible cellular responses to the particles depend on an array of parameters, in both particle and biological aspects. On the one hand, the physicochemical properties of the particles (e.g., material, size, shape, and surface charge) are known to play a key role in particle-cell interactions. On the other hand, it has been shown that prior to coming into contact with cells, nanoparticle interaction with the surrounding biological fluid may lead to a change of the initial particle properties. For example, the colloidal behavior of nanoparticles is strongly influenced by the density and viscosity of the surrounding media in both in vitro and in vivo systems

Protein Corona Composition of Superparamagnetic Iron Oxide Nanoparticles with Various Physico-Chemical Properties and Coatings

International audienc

Magnetic, paramagnetic and/or superparamagnetic nanoparticles

The present invention relates to nanoparticles having a mean diameter of < 500nm and comprising, at their surface, a selected material. The nanoparticles are taken up by cells under physiological conditions and can be used to isolate interaction partners of the selected material within the cells. The present invention provides important advantages in that it opens up new ways of identifying cellular components and of delivering a substance of interest specifically to a selected cell compartment. The nanoparticles are also useful as a tool of diagnosis and for the constitution of chemical libraries

Fixed bed reactor for solid phase surface derivatization of superparamagnetic nanoparticles

The functionalization of nanoparticles is conditio sine qua non in studies of specific interaction with a biological target. Often, their biological functionality is achieved by covalent binding of bioactive molecules on a preexisting single surface coating. The yield and quality of the resulting coated and functionalized superparamagnetic iron oxide nanoparticles (SPIONs) can be significantly improved and reaction times reduced by using solid-phase synthesis strategies. In this study, a fixed bed reactor with a quadrupole repulsive arrangement of permanent magnets was assayed for SPION surface derivatization. The magnet array around the fixed bed reactor creates very high magnetic field gradients that enables the immobilization of SPIONs with a diameter as low as 9 nm. The functionalization on the surface of immobilized 25 nm 3-(aminopropyl)trimethoxysilane-coated SPIONs (APS-SPIONs) was performed using fluorescein-isothiocyanate directly, and by the SV40 large T-antigen nuclear localization signal peptide (PKKKRKVGC) conjugated to acryloylpoly(ethylene glycol)-N-hydroxysuccinimide, where the PEG reagent is conjugated first to create a functionalized nanoparticle and the peptide is added to the acryloyl group. We show that the yield of reactant grafted on the surface of the APS-coated SPIONs was higher in solid-phase within the fixed bed reactor compared to conventional liquid-phase chemistry. In summary, the functionalization of SPIONs using a magnetically fixed bed reactor was superior to the liquid-phase reaction in terms of the yield, reaction times required for derivatization, size distribution, and scalability

Improved dynamic response assessment for intra-articular injected iron oxide nanoparticles

The emerging importance of nanoparticle technology, including iron oxide nanoparticles for monitoring development, progression, and treatment of inflammatory diseases such as arthritis, drives development of imaging techniques. Studies require an imaging protocol that is sensitive and quantifiable for the detection of iron oxide over a wide range of concentrations. Conventional signal loss measurements of iron oxide nanoparticle containing tissues saturate at medium concentrations and show a nonlinear/nonproportional intensity to concentration profile due to the competing effects of T1 and T2 relaxation. A concentration calibration phantom and an in vivo study of intra-articular injection in a rat knee of known concentrations of iron oxide were assessed using the difference-ultrashort echo time sequence giving a positive, quantifiable, unambiguous iron signal and monotonic, increasing concentration response over a wide concentration range in the phantom with limited susceptibility artifacts and high contrast in vivo to all other tissues. This improved dynamic response to concentration opens possibilities for quantification due to its linear nature at physiologically relevant concentrations

MULTIFUNCTIONALIZED SPIONs FOR NUCLEAR TARGETING: CELL UPTAKE AND GENE EXPRESSION

Superparamagnetic iron oxide nanoparticles (SPIONs) are used in many biological applications, which necessitate intracellular targeting. Here, we investigate intracellular localization and gene expression in HeLa cells after treatment with functionalized SPIONs. Functional groups investigated included positive amino propyl silane (APS), polyethylene glycol and targeting peptides: nuclear targeting peptide (NTP) and/or cancer cell uptake promoting peptide (cRGD). Results revealed that the intracellular localization of SPIONs was strongly dependent on the surface chemistry. Nuclear targeted SPIONs functionalized with only NTP or both NTP and cRGD were mostly localized in perinuclear endosomes with a small fraction entering the nucleus. The biocompatibility of cells after treatment was also dependent on surface chemistry, where SPIONs functionalized with both NTP and cRGD exhibited a more significant reduction of cell proliferation compared to NTP or cRGD individually. Interestingly, gene expression after treatment with SPIONs was similar, regardless of the surface functionalization or intracellular localization. The results of this study showed that cellular uptake and intracellular localization predominantly depended on the surface chemistry, while gene expression exhibited a more generic response to SPION treatment