ABSTRACT
Recent innovations in single-cell technologies have opened up exciting possibilities for profiling the omics of individual cells. Minimally invasive analysis tools that probe and remove the contents of living cells enable cells to remain in their standard microenvironment with little impact on their viability. This negates the requirement of lysing cells to access their contents, an advancement from previous single-cell manipulation methods. These novel methods have the potential to be used for dynamic studies on single cells, with many already providing high intracellular spatial resolution. In this article, we highlight key technological advances that aim to remove the contents of living cells for downstream analysis. Recent applications of these techniques are reviewed, along with their current limitations. We also propose recommendations for expanding the scope of these technologies to achieve comprehensive single-cell tracking in the future, anticipating the discovery of subcellular mechanisms and novel therapeutic targets and treatments, ultimately transforming the fields of spatial transcriptomics and personalised medicine.
ABSTRACT
Bioinspired macromolecules can aid nucleation and crystallisation of minerals by mirroring processes observed in nature. Specifically, the iron oxide magnetite (Fe3O4) is produced in a dedicated liposome (called a magnetosome) within magnetic bacteria. This process is controlled by a suite of proteins embedded within the liposome membrane. In this study we look to synthetically mimic both the liposome and nucleation proteins embedded within it using preferential orientation polymer design. Amphiphilic block co-polymers self-assemble into vesicles (polymersomes) and have been used to successfully mimic liposomes. Carboxylic acid residue-rich motifs are common place in biomineralisation nucleating proteins and several magnetosome membrane specific (Mms) proteins (namely Mms6) have a specific carboxylic acid motifs that are found to bind both ferrous and ferric iron ions and nucleate the formation of magnetite. Here we use a combination of 2 diblock co-polymers: Both have the hydrophobic 2-hydroxypropyl methacrylate (PHPMA) block with either a poly(ethylene glycol) (PEG) block or a carboxylic acid terminated poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) block. These copolymers ((PEG113-PHPMA400) and (PMPC28-PHPMA400) respectively) self-assemble in situ to form polymersomes, with PEG113-PHPMA400 displaying favourably on the outer surface and PMPC28-PHPMA400 on the inner lumen, exposing numerous acidic iron binding carboxylates on the inner membrane. This is a polymersome mimic of a magnetosome (PMM28) containing interior nucleation sites. The resulting PMM28 were found to be 246 ± 137 nm in size. When the PMM28 were subjected to electroporation (5 pulses at 750 V) in an iron solution, iron ions were transported into the PMM28 polymersome core where magnetic iron-oxide was crystallised to fill the core; mimicking a magnetosome. Furthermore it has been shown that PMM28 magnetopolymersomes (PMM28Fe) exhibit a 6 °C temperature increase during in vitro magnetic hyperthermia yielding an intrinsic loss power (ILP) of 3.7 nHm2 kg-1. Such values are comparable to commercially available nanoparticles, but, offer the added potential for further tuning and functionalisation with respect to drug delivery.
