Formulation and In Vitro Characterization of Composite Biodegradable Magnetic Nanoparticles for Magnetically Guided Cell Delivery

Abstract

Purpose Cells modified with magnetically responsive nanoparticles (MNP) can provide the basis for novel targeted therapeutic strategies. However, improvements are required in the MNP design and cell treatment protocols to provide adequate magnetic properties in balance with acceptable cell viability and function. This study focused on select variables controlling the uptake and cell compatibility of biodegradable polymer-based MNP in cultured endothelial cells. Methods Fluorescent-labeled MNP were formed using magnetite and polylactide as structural components. Their magnetically driven sedimentation and uptake were studied fluorimetrically relative to cell viability in comparison to non-magnetic control conditions. The utility of surface-activated MNP forming affinity complexes with replication-deficient adenovirus (Ad) for transduction achieved concomitantly with magnetic cell loading was examined using the green fluorescent protein reporter. Results A high-gradient magnetic field was essential for sedimentation and cell binding of albumin-stabilized MNP, the latter being rate-limiting in the MNP loading process. Cell loading up to 160 pg iron oxide per cell was achievable with cell viability >90%. Magnetically driven uptake of MNP-Ad complexes can provide high levels of transgene expression potentially useful for a combined cell/gene therapy. Conclusions Magnetically responsive endothelial cells for targeted delivery applications can be obtained rapidly and efficiently using composite biodegradable MNP.

Real-time analysis of composite magnetic nanoparticle disassembly in vascular cells and biomimetic media

“…At the same time, it may be predicted that local delivery of antirestenotic drugs inhibiting the proliferation of the neointimal cells can reverse the accelerated MNP processing and slow the degradation of the cell-associated MNP fraction. Similar predictions may be made regarding the fate of MNPs used to generate magnetically responsive endothelial cells for targeted delivery and enhanced reendothelialization of injured arteries (15,17). MNPs loaded into magnetically guided cells may exhibit faster disassembly at earlier time points, but their processing likely will occur at a slower rate when the cells reach the contact-inhibited state.…”

“…Thus, although our experiments did not reveal a significant delay in the onset of MNP disintegration, a detectable lag may be anticipated for NPs formed from a polymer with a larger initial molecular weight or a higher degree of crystallinity (6,8). Composite PLA-based magnetic particles were chosen as a model biodegradable NP formulation for evaluation in the present studies because of their rapid and quantitative, magnetically controlled cell uptake (17), facilitating and synchronizing measurements of the internalized MNP disassembly in cultured cells, and also because of their direct relevance to magnetically targeted cell and drug delivery to sites of arterial injury, which is the therapeutic focus of our recent research (14,15,17). Particle-forming polymer was monitored in our experiments as the primary component determining structural integrity of the particles.…”

“…Notably, endocytosed MNPs caused no changes in cell morphology or GFP fluorescence compared with untreated cells and exhibited the characteristic perinuclear localization in the cell interior ( Fig. 2 B and C) (17). The evolution of N FRET as a function of the modeled MNP degradation status was accompanied by changes in the donor and acceptor emission intensities shown in Fig.…”

“…Uniformly sized PLA-based MNPs were formulated by using a modification of the emulsification-solvent evaporation method (17). In brief, an ethanolic solution of ferric chloride hexahydrate and ferrous chloride tetrahydrate (170 and 62.5 mg, respectively, in 2.5 mL) was added to an equivalent amount of sodium hydroxide dissolved in deionized water (5 mL).…”

“…Fluorescence intensity and spectra of individually labeled and colabeled MNPs were taken after dilution at the indicated ratios in the respective media. For MNP fluorescence measurements in the cell interior, quantitative magnetically driven internalization was achieved over 24 h, as described previously (17), by positioning plates with confluent cells on a high-gradient field magnetic separator (LifeSep 96F; Dexter Magnetic Technologies). Emission spectra were taken using λ ex = 540 and 590 nm, corresponding to the optimal excitation wavelengths for the donor and acceptor fluorophores determined in preliminary experiments using MNP [ As part of the validation of the approach, emission spectra of MNP[don/ acc] before and after digestion in an organic solvent (acetonitrile applied at a 100:1 volume ratio to MNPs in suspension) were taken using λ ex = 540 nm to confirm the complete reversal of the fluorescence pattern upon dissociation of the FRET pair-forming MNP components.…”