Intracellular delivery of colloids: Past and future contributions from microinjection.

The manipulation of single cells and whole tissues has been possible since the early 70's, when semi-automatic injectors were developed. Since then, microinjection has been used to introduce an ever-expanding range of colloids of up to 1000 nm in size into living cells. Besides injecting nucleic acids to study transfection mechanisms, numerous cellular pathways have been unraveled through the introduction of recombinant proteins and blocking antibodies. The injection of nanoparticles has also become popular in recent years to investigate toxicity mechanisms and intracellular transport, and to conceive semi-synthetic cells containing artificial organelles. This article reviews colloidal systems such as proteins, nucleic acids and nanoparticles that have been injected into cells for different research aims, and discusses the scientific advances achieved through them. The colloids' intracellular processing and ultimate fate are also examined from a drug delivery perspective with an emphasis on the differences observed for endocytosed versus microinjected material.

Microinjection for the ex Vivo Modification of Cells with Artificial Organelles.

Microinjection is extensively used across fields to deliver material intracellularly. Here we address the fundamental aspects of introducing exogenous organelles into cells to endow them with artificial functions. Nanocarriers encapsulating biologically active cargo or extreme intraluminal pH were injected directly into the cytosol of cells, where they bypassed subcellular processing pathways and remained intact for several days. Nanocarriers' size was found to dictate their intracellular distribution pattern upon injection, with larger vesicles adopting polarized agglomerated distributions and smaller colloids spreading evenly in the cytosol. This in turn determined the symmetry or asymmetry of their dilution following cell division, ultimately affecting the intracellular dose at a cell population level. As an example of microinjection's applicability, a cell type relevant for cell-based therapies (dendritic cells) was injected with vesicles, and its migratory properties were studied in a co-culture system mimicking lymphatic capillaries.

Presumed LRP1-targeting transport peptide delivers ß-secretase inhibitor to neurons in vitro with limited efficiency.

Interfering with the activity of ß-secretase to reduce the production of Aß peptides is a conceivable therapeutic strategy for Alzheimer's disease. However, the development of efficient yet safe inhibitors is hampered by secondary effects, usually linked to the indiscriminate inhibition of other substrates' processing by the targeted enzyme. Based on the spatial compartmentalization of the cleavage of the amyloid precursor protein by ß-secretase, we hypothesized that by exploiting the endocytosis receptor low-density lipoprotein receptor-related protein it would be possible to direct an otherwise cell-impermeable inhibitor to the endosomes of neurons, boosting the drug's efficacy and importantly, sparing the off-target effects. We used the transport peptide Angiopep to build an endocytosis-competent conjugate and found that although the peptide facilitated the inhibitor's internalization into neurons and delivered it to the endosomes, the delivery was not efficient enough to potently reduce ß-secretase activity at the cellular level. This is likely connected to the finding that in the cell lines we used, Angiopep's internalization was not mediated by its presumed receptor to a significant extent. Additionally, Angiopep exploited different internalization mechanisms when applied alone or when conjugated to the inhibitor, highlighting the impact that drug conjugation can have on transport peptides.

Suppression of nanoparticle cytotoxicity approaching in vivo serum concentrations: limitations of in vitro testing for nanosafety.

Nanomaterials challenge paradigms of in vitro testing because unlike molecular species, biomolecules in the dispersion medium modulate their interactions with cells. Exposing cells to nanoparticles known to cause cell death, we observed cytotoxicity suppression by increasing the amount of serum in the dispersion medium towards in vivo-relevant conditions.

Low dose of amino-modified nanoparticles induces cell cycle arrest.

The interaction of nanoscaled materials with biological systems is currently the focus of a fast-growing area of investigation. Though many nanoparticles interact with cells without acute toxic responses, amino-modified polystyrene nanoparticles are known to induce cell death. We have found that by lowering their dose, cell death remains low for several days while, interestingly, cell cycle progression is arrested. In this scenario, nanoparticle uptake, which we have recently shown to be affected by cell cycle progression, develops differently over time due to the absence of cell division. This suggests that the same nanoparticles can trigger different pathways depending on exposure conditions and the dose accumulated.

Role of cell cycle on the cellular uptake and dilution of nanoparticles in a cell population.

Nanoparticles are considered a primary vehicle for targeted therapies because they can pass biological barriers and enter and distribute within cells by energy-dependent pathways. So far, most studies have shown that nanoparticle properties, such as size and surface, can influence how cells internalize nanoparticles. Here, we show that uptake of nanoparticles by cells is also influenced by their cell cycle phase. Although cells in different phases of the cell cycle were found to internalize nanoparticles at similar rates, after 24 h the concentration of nanoparticles in the cells could be ranked according to the different phases: G2/M > S > G0/G1. Nanoparticles that are internalized by cells are not exported from cells but are split between daughter cells when the parent cell divides. Our results suggest that future studies on nanoparticle uptake should consider the cell cycle, because, in a cell population, the dose of internalized nanoparticles in each cell varies as the cell advances through the cell cycle.