Magnetosomes for bioassays by merging fluorescent liposomes and magnetic nanoparticles: encapsulation and bilayer insertion strategies

Magnetized liposome (magnetosomes) labels can overcome diffusion limitations in bioassays through fast and easy magnetic attraction. Our aim therefore was to advance the understanding of factors influencing their synthesis focusing on encapsulation strategies and synthesis parameters. Magnetosome synthesis is governed by the surface chemistry and the size of the magnetic nanoparticles used. We therefore studied the two possible magnetic labelling strategies, which are the incorporation of small, hydrophobic magnetic nanoparticles (MNPs) into the bilayer core (b-liposomes) and the entrapment of larger hydrophilic MNPs into the liposomes’ inner cavity (i-liposomes). Furthermore, they were optimized and compared for application in a DNA bioassay. The major obstacles observed for each of these strategies were on the one hand the need for highly concentrated hydrophilic MNPs, which is limited by their colloidal stability and costs, and on the other hand the balancing of magnetic strength vs. size for the hydrophobic MNPs. In the end, both strategies yielded magnetosomes with good performance, which improved the limit of detection of a non-magnetic DNA hybridization assay by a factor of 3–8-fold. Here, i-liposomes with a magnetization yield of 5% could be further improved through a simple magnetic pre-concentration step and provided in the end an 8-fold improvement of the limit of detection compared with non-magnetic conditions. In the case of b-liposomes, Janus-like particles were generated during the synthesis and yielded a fraction of 15% magnetosomes directly. Surprisingly, further magnetic pre-concentration did not improve their bioassay performance. It is thus assumed that magnetosomes pull normal liposomes through the magnetic field towards the surface and the presence of more magnetosomes is not needed. The overall stability of magnetosomes during storage and magnetic action, their superior bioassay performance, and their adaptability towards size and surface chemistry of MNPs makes them highly valuable signal enhancers in bioanalysis and potential tools for bioseparations. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-02503-0) contains supplementary material, which is available to authorized users.

Fig. S1 TEM image of original b-liposomes with incorporated MNPs with hydrophobic surface coating. To reduce the distortion of the membrane, particles accumulate at one side instead of spreading across the whole bilayer, forming Janus shaped vesicles as also reported by other scientists previously [1].

Calculation of Particle Amount per Liposome and Costs for Particles
The theoretical number of particles that fit into the inner cavity per liposome is calculated according to The theoretical mass of particles that has to be inserted in the synthesis to achieve one particle per liposomes 1 / (ic for inner cavity and b for bilayer) was calculated according to Equation 4 and 5, respectively: With this amounts and masses, also the costs for each synthesis were calculated, either by simply multiplying the amount of particles for inner cavity encapsulation with the price of these particles, or -for the self-synthesized particles for bilayer insertion -taking into account the prices of the chemicals and the working time per synthesis multiplied with an average salary of a laboratory assistant in Germany. Of course, these calculations are just rough approximations and make no claim to completeness, but still they show the enormous discrepancy between both methods.

DNA Hybridization Assay Preliminary Studies
To validate the magnetic abilities of the synthesized magnetosomes, a DNA hybridization sandwich assay was performed in parallel with and without the presence of an external magnetic field (Fig. S3 left). In a first assay with original b-liposomes, LOD, LOQ and the maximum signal to noise ratio (max S/N) were 1.2 times better without than with magnet, respectively, although the sensitivity increased by 1.6 times. Therefore, optimization of this assay was necessary.
Another liposome system encapsulated hydrophilic magnetic particles inside the inner cavity of the liposomes, aside with the signal molecules. A first assay showed no significant change when conducting the assay with or without the presence of an external magnetic field (Fig. S3 right).

Liposome Stability
For examination of the long-term stability of i-and b-liposomes, the size distribution of liposomes was determined with dynamic light scattering directly after synthesis as well as after 9 and 11 months of storage (4 °C in the dark), respectively. Figure S4 shows the distribution of the hydrodynamic diameter for two different batches of b-liposomes and one batch of i-liposomes. No significant change in hydrodynamic diameter could be observed and the polydispersity index is still very low. Thus, and as no visible precipitation or lysis of the liposomes is observable, it can be assumed that magnetic liposomes are stable over at least 9 months of storage. For examination of the stability of liposomes under electromagnetic attraction, the size distribution of liposomes was determined with dynamic light scattering, then the cuvettes with liposome solution were placed next to a strong neodymium magnet for 60 min and after this time, the size distribution was measured again. As visible in Figure S5, no significant change in diameter and distribution was observable, as well as no visible precipitation or lysis. Thus, liposomes are assumed stable under the influence of an external magnetic field.