High field gradient targeting of magnetic nanoparticle-loaded endothelial cells to the surfaces of steel stents

Abstract

A cell delivery strategy was investigated that was hypothesized to enable magnetic targeting of endothelial cells to the steel surfaces of intraarterial stents because of the following mechanisms: (i) preloading cells with biodegradable polymeric superparamagnetic nanoparticles (MNPs), thereby rendering the cells magnetically responsive; and (ii) the induction of both magnetic field gradients around the wires of a steel stent and magnetic moments within MNPs because of a uniform external magnetic field, thereby targeting MNP-laden cells to the stent wires. In vitro studies demonstrated that MNP-loaded bovine aortic endothelial cells (BAECs) could be magnetically targeted to steel stent wires. In vivo MNP-loaded BAECs transduced with adenoviruses expressing luciferase (Luc) were targeted to stents deployed in rat carotid arteries in the presence of a uniform magnetic field with significantly greater Luc expression, detected by in vivo optical imaging, than nonmagnetic controls.

Fig. 1.

A schematic representation of a stented blood vessel showing that magnetically responsive cells are attracted to steel stent struts in a uniform, magnetic field because of a generated magnetic force (F⃗_mag) that directly depends on the strength of the total magnetic field (B⃗), high field magnetic gradients (∇B⃗_g) induced on the stent struts, and the magnetic moments (m⃗) induced on MNP-loaded cells by the uniform magnetic field B⃗₀. The total magnetic field (B⃗) is a sum of a gradient field (B⃗_g) of the stent and a uniform field (B⃗₀). The steel mesh structure of the stent is shown in both the open stented blood vessel fragment and as individual strut areas in Inset.

Fig. 2.

Characterization of MNP and MNP-cell loading. (a) Transmission EM of albumin-stabilized MNPs. Note the small size and the large number of individual oleic acid-coated magnetite grains distributed in the MNP polylactide–polymeric matrix. (b) Magnetization curve of MNPs, which exhibit superparamagnetic behavior showing no significant hysteresis and a remnant magnetization on the order of 0.5% of the saturation magnetization value. (c) Magnetization curves of 304-grade (left-sided y axis) and 316L-grade (right-sided y axis) stainless-steel stents. The 304-grade stainless-steel stent exhibits a near superparamagnetic behavior showing slight hysteresis and a remnant magnetization on the order of 7% of the saturation magnetization value. By comparison, the 316L-grade stent shows far less magnetic responsiveness. (d) MNP-cell loading studies. The kinetics of MNP uptake by BAECs in culture as a function of MNP dose, incubation time, and the presence or absence of a magnetic field of 500 G by using a fixed magnet applied directly to the underside of the cell culture plates. The MNP uptake was determined by fluorescence of internalized MNPs. (e) Micrographs of BAECs in culture with bright field and red fluorescent images qualitatively showing the relative amount of MNPs internalized within cells at different time points at the applied MNP dose of 9 μg per well. Green fluorescent micrographs show cell viability as assessed by Calcein green staining. (Magnification: ×100.) (f) Cell viability as a function of MNP dose and incubation time as determined by Alamar blue assays. (g) Magnetization curve of BAECs loaded with MNPs demonstrates superparamagnetic behavior showing no significant hysteresis and a remnant magnetization on the order of <1% of the saturation magnetization value. Cells not loaded with MNPs exhibit diamagnetic behavior.

Fig. 3.

Magnetic targeting of MNP-preloaded BAECs under flow conditions in vitro and in vivo. (a) In vitro capture kinetics of magnetically responsive BAECs onto a 304-grade stainless-steel stent in the presence of a uniform field of 1,000 G and a nonpulsatile flow rate of 30 ml/min. The initial capture rate was estimated to be 1% of cells per min. The data were obtained by measuring the fluorescence of MNPs. (b and c) Magnetically responsive BAECs captured in vitro onto a 304-grade stainless-steel stent as evidenced by the red fluorescence of MNPs (b) and Calcein green staining of live cells (c). (d) MNP-loaded BAECs captured in vivo onto a deployed 304-grade stainless-steel stent in the rat carotid artery. BAECs preloaded with fluorescent MNPs were transthoracically injected into the left ventricular cavity. Animals were exposed to a magnetic field of 1,000 G for 5 min, including the period of injection. The animals were killed 5 min after delivery, and the explanted stents were immediately examined by fluorescence microscopy. (e) Control rats underwent an identical procedure where no magnetic field was used. (Magnification: b–e, ×40.) (f) In vivo local magnetic cell delivery in a rat carotid stenting model under stop-flow conditions. A catheter was introduced via the external carotid into the common carotid artery and was positioned distal to a deployed stent. The cell suspension was delivered into isolated arterial segments for 15 s. (g) In vivo cell delivery under uninterrupted blood-flow conditions. A catheter was introduced via the external carotid into the common carotid and advanced beyond the stent to the aortic arch. The cells were injected at this site at the rate of 1 ml/min for 1 min. For both delivery protocols (f and g) in the magnetic group (Mag+), the injection was carried out with animals placed in a magnetic field of 1,000 G, and the field was maintained for a total of 5 min after delivery. In control rats (Mag− group), no magnetic field was applied. In both settings, BAECs were first transduced in culture with AdLuc and then loaded with MNPs. The animals were imaged 48 h after delivery by local perivascular administration of luciferin admixed in a pluronic gel. The signal emitted from the stented arterial segment due to the luciferase transgene expression was significantly higher in the animals that received cells in the presence of a magnetic field (Mag+ group).