Magnetically-controlled release from hydrogel-supported vesicle assemblies

Abstract

Magnetic nanoparticle–vesicle assemblies embedded within a hydrogel extravesicular matrix have been shown to release their contents in response to a remote magnetic trigger.

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The replication of cellular adhesion using phospholipidvesicles^1,2 implies that adhesion between vesicles in bulk should produce biomaterials that structurally mimic tissue.^1a,1b,2b However, current assembly techniques produce vesicle aggregates that are neither robust nor patterned, in contrast to tissue which contains arrays of adhering cells structurally reinforced by an extracellular matrix (ECM). Similarly, vesicles can be structurally reinforced by filling them with hydrogel (a cytoskeleton mimic), or surrounding them by a hydrogel matrix (an ECM mimic).³ The latter approach produces vesicle gels, biomaterials composed of individual vesiclesencased within a hydrogel,^4–6 which have shown great potential as drug delivery platforms.⁷ Nonetheless to elaborate vesicle gels into tissue-mimetic structures, cell–cell adhesion must be replicated by linking the vesicles together, and the resulting vesicle assemblies must be patterned within the hydrogel. Adding a mechanism for the controlled release of vesicle contents will then generate tissue-mimetic biomaterials that can chemically communicate with cells.

Herein we report a new type of tissue-mimetic material, magnetically-sensitive vesicle gels. These vesicle gels contain vesicles crosslinked by Fe₃O₄ magnetic nanoparticles, and these vesicle assemblies allow the materials to: (a) host membrane-bound enzymes/glycolipids; (b) be patterned by external magnetic fields; and (c) respond to alternating magnetic fields by releasing stored drugs or bio(macro)molecules. These magnetic vesicle gels are smart materials with potential applications in cell culture or drug delivery; the transparency of tissue to magnetic fields allows a remote magnetic signal to induce spatially controlled release of biologics without otherwise affecting the surrounding tissue.

The key compounds for the creation of our vesicle gels are 1, which give a histidine coating to 10 nm diameter Fe₃O₄ magnetic nanoparticles ([1-MNP]), and adhesive lipid Cu(2), which enables 800 nm phospholipidvesicles to adhere to these coated nanoparticles (Fig. 1a). Simply mixing vesicles with 5% mol/mol Cu(2) in their membranes with [1-MNP] provides assemblies of vesicles crosslinked by [1-MNP] (Fig. 1b,c); energy dispersive X-ray analysis showed co-localisation of Cu(2) and [1-MNP].^2b The perfluoroalkyl membrane anchor in Cu(2) is key as it causes phase separation of Cu(2) in liquid- and solid-ordered membranes,^2c strengthening the interaction with [1-MNP] at neutral pH.⁸ In particular, the solid-ordered membranes of thermally-sensitive vesicles at 20 °C ([Cu(2)-TSV]; 5% Cu(2), 9.5% dimyristoyl phosphatidylcholine, 85.5% mol/mol dipalmitoyl phosphatidylcholine) gave phase-separated Cu(2) (ESI† ), and this phase separation allowed thermally-sensitive magnetic nanoparticle–vesicle assemblies to be created at pH 7.4.

Fig. 1 (a) Histidine ligand 1 and adhesive lipid Cu(2). (b) Reversible crosslinking of Cu(2)-doped vesicles by coated Fe₃O₄nanoparticles ([1-MNP]) gives magnetic nanoparticle–vesicle assemblies, which are then fixed within a hydrogel matrix. (c) Representative cryo-ESEM micrograph of a magnetic nanoparticle–vesicle assembly embedded in alginate gel . (d) Patterning procedure: (i) [1-MNP] and [Cu(2)-TSV] are left to aggregate for 1 h (MOPS buffer, pH 7.4, 20 °C); (ii) the assemblies are magnetically sedimented using a 5 kG magnet, then the concentrate transferred into 1% wt/vol sodium alginate and magnetically positioned; (iii) infusion of CaCl₂ gives a self-supporting hydrogel. (e) An inverted cell culture well containing a magnetic vesiclegel patterned with regions of immobilised magnetic nanoparticle–vesicle assemblies; these regions appear orange due to encapsulated 5/6-CF.

To find a suitable extravesicular matrix, several gel -forming mixtures were screened, although those requiring large changes in temperature, pH or ionic strength to gel were excluded as these processes risked compromising vesicle integrity. In contrast to literature reports,^3d,5 alginate solutions were found to gel the surrounding matrix without the fibrils penetrating and disrupting the vesicles. The retention of 5/6-carboxyfluorescein (5/6-CF) encapsulated within vesicle assemblies in the gel was excellent, with only 3% leakage after 1 h, compared to 82% leakage after 0.5 h from vesicle-free alginate gel (ESI† ). The magnetic character of the vesicle assemblies made the patterning of vesicle gels straightforward. An external NdFeB magnet (5 kG) was first used to magnetically concentrate the nanoparticle–vesicle assemblies (Fig. 1d). The supernatant solution was removed and the sediment carefully transferred into 1% wt/vol sodium alginate solution. The magnetic vesicle assemblies could then be positioned in the alginate solution e.g. three small 5 kG magnets (3 mm diameter) placed under the cell culture well generated the pattern shown in Fig. 1e (ESI† ). The resulting mixture was cured by the infusion of CaCl₂ solution (0.1 M) through a polycarbonate filter (200 nm pore size), immobilising the magnetic nanoparticle–vesicle assemblies and fixing any magnetic patterning. The resulting vesicle gels were characterised by freeze-fracture cryo-scanning electron microscopy (cryo-SEM), which showed vesicles embedded within a matrix of alginate fibres (Fig. 1c). In accordance with the retention of 5/6-CF, there was no evidence of alginate fibres penetrating into the vesicles, with fibrous mats forming at vesicle assembly-hydrogel interfaces (ESI† ).^9b

Having shown we could pattern these magnetic vesicle assemblies in a gel , the next objective was to release the vesicle contents in response to an external stimulus. We started by demonstrating thermally-triggered release from vesicles.¹⁰vesicle compositions have been developed with triggering temperatures in the physiologically useful 31 to 40 °C range,⁹ and the [Cu(2)-TSV] composition used was designed to trigger at 37 °C (ESI† ). Indeed, [1-MNP]–[Cu(2)-TSV] vesicle assemblies encapsulating 5/6-CF (0.5 mM) gave rapid dye release after warming to 40 °C (Fig. 2a). Similarly, heating vesicle gels containing [1-MNP]–[Cu(2)-TSV] to 40 °C also gave rapid 5/6-CF release (Fig. 2a,b), with >50% of the 5/6-CF released within 900 s (100% release achieved after 24 h incubation with Triton X-100). This procedure was repeated with encapsulated fluorescein-labeled dextran (4 kDa FITC-dextran, 20 mM); it should be difficult for molecules of this size to traverse even weakened bilayers and diffuse through the alginate matrix. However, 20% of the FITC-dextran was released in a small burst phase (the first 600 s at 40 °C), with slower release (to 35%) after 1.5 h. This release of a 4 kDa biomacromolecule suggests glycosaminoglycans or proteins could also be candidates for triggered release from these magnetic vesicle gels.

Fig. 2 (a) Contents release from [1-MNP]–[Cu(2)-TSV] assemblies after warming to 40 °C; (○) 5/6-CF release from vesicle assemblies in suspension; (●) 5/6-CF release from a section of magnetic vesiclegel ; (◆) FITC-dextran release from a section of magnetic vesiclegel . Errors are ±0.03. (b) Sections of magnetic vesiclegel in buffer at 20 °C (left, with orange encapsulated 5/6-CF), and after 90 min at 40 °C (right, showing green fluorescence from released 5/6-CF).

The next step was to replace thermal release of encapsulated materials by a remote trigger that will not adversely affect surrounding healthy cells or tissue.¹¹ Magnetically-triggered release is an ideal alternative as tissue is transparent to alternating magnetic fields (AMFs), but AMFs will rapidly heat magnetite nanoparticles (Fig. 3a).¹² Exposure of the (10 ± 2) nm diameter Fe₃O₄ superparamagnetic nanoparticles in our magnetic vesicle gels to a ∼400 kHz AMF should only heat the volume adjacent to the nanoparticles, melting and permeabilizing the [Cu(2)-TSV] in the gel without heating the bulk (Fig. 3a).¹³ Gratifyingly, exposure of a 0.125 cm³ block of magnetically-responsive vesiclegel with encapsulated 5/6-CF (in 2 mL MOPS buffer) to a 392 kHz alternating magnetic field resulted in rapid release of 5/6-CF, with a faster rate (100% release after 600 s) than direct thermal triggering (Fig. 3b). The surrounding solution was not heated (only ∼2 °C increase after 600 s), showing that the thermal energy was transferred directly to the vesicle assemblies in the gel ; indeed an alginate gel which contained thermally-sensitive vesicles that were not crosslinked with Fe₃O₄nanoparticles did not release 5/6-CF upon exposure to an AMF (ESI† ).

Fig. 3 (a) Scheme depicting the magnetically-triggered release of 5/6-CF from Fe₃O₄nanoparticle–vesicle assemblies embedded near the surface of a vesiclegel section. (b) (●) Rate of 5/6-CF release from a 0.125 cm³ section of magnetic vesiclegel in buffer (2 mL), after exposure to a 392 kHz alternating magnetic field (AMF). (○) Corresponding change in the temperature of the buffer during exposure to the 392 kHz AMF.

The combination of magnetic patterning and magnetically-induced release will allow these materials to act as smart interfaces for the conversion of electrical signals into chemical messengers, leading to potential applications as smart scaffolds for stem cells or remotely triggered in vivodrug delivery systems. Research into using bioadhesive interactions to crosslink magnetic nanoparticles with vesicles and tagging cell recognition sites onto the extravesicular matrix is ongoing.

We thank Dr P. Hill for cryo-ESEM microscopy. This work was supported by the BBSRC (KPL) and the Leverhulme Trust (RJM).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Synthesis and characterisation of magnetically-responsive vesicle gels (differential scanning calorimetry, fluorescence microscopy, cryo-ESEM). See DOI: 10.1039/b901472a

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