Cellular uptake and intracellular degradation of poly(alkyl cyanoacrylate) nanoparticles

Abstract

Background Poly(alkyl cyanoacrylate) (PACA) nanoparticles have shown promise as drug carriers both to solid tumors and across the blood–brain barrier. Efficient drug delivery requires both high cellular uptake of the nanoparticles and release of the drug from the nanoparticles. Release of hydrophobic drugs from PACA nanoparticles is primarily governed by nanoparticle degradation, and this process has been poorly studied at the cellular level. Here we use the hydrophobic model drug Nile Red 668 (NR668) to investigate intracellular degradation of PACA nanoparticles by measuring changes in NR668 fluorescence emission and lifetime, as the spectral properties of NR668 depend on the hydrophobicity of the dye environment. We also assess the potential of poly(butyl cyanoacrylate) (PBCA) and poly(octyl cyanoacrylate) (POCA) nanoparticles for intracellular drug delivery in the prostate cancer cell line PC3 and rat brain endothelial cell line RBE4 and the role of endocytosis pathways in PACA nanoparticle uptake in those cell lines.ResultsFluorescence lifetime imaging, emission spectra analysis and Förster resonance energy transfer indicated that the intracellular degradation was in line with the degradation found by direct methods such as gas chromatography and scanning electron microscopy, showing that PBCA has a faster degradation rate compared to POCA. The combined P(BCA/OCA) nanoparticles had an intermediate degradation rate. The uptake of POCA and PBCA nanoparticles was much higher in RBE4 than in PC3 cells. Endocytosis inhibition studies showed that both clathrin- and caveolin-mediated endocytosis were involved in PACA nanoparticle uptake, and that the former played a predominant role, particularly in PC3 cells.ConclusionsIn the present study, we used three different optical techniques to show that within a 24-hour period PBCA nanoparticles degraded significantly inside cells, releasing their payload into the cytosol, while POCA nanoparticles remained intact. This indicates that it is possible to tune the intracellular drug release rate by choosing appropriate monomers from the PACA family or by using hybrid PACA nanoparticles containing different monomers. In addition, we showed that the uptake of PACA nanoparticles depends not only on the monomer material, but also on the cell type, and that different cell lines can use different internalization pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0156-7) contains supplementary material, which is available to authorized users.

Labeling nanoparticles: Dye leakage and altered cellular uptake

“…Various approaches are reported in the literature to determine leakage of fluorescent dyes from NPs (12,18,20,(23)(24)(25)(26)(27)(28)(29). Several studies are based on in vitro dye/drug release in aqueous solution by separation methods (25).…”

“…Microscopy is another method used for studying uptake and distribution of NPs (18,22,37,43). However, microscopy should be employed with care in the assessment of dye leakage, as free hydrophobic dyes will bind to intracellular hydrophobic molecules resulting in both diffuse and spotted staining pattern (12,24,30,33), thereby making the dye hard to separate from the fluorescence of intact NPs. In a previous study, we have shown that PBCA-NPs with NR668 are taken up in PC3 cells by endocytosis, verified by the use of time consuming intracellular spectral microscopy and fluorescence-lifetime imaging microscopy (FLIM) (12).…”

“…In order to understand the behavior of NPs in vitro and in vivo it is necessary to trace them. This is commonly done by encapsulation of fluorescent dyes in the NPs, which allows their detection with optical techniques (6)(7)(8)(9)(10)(11)(12). Encapsulating dyes and drugs in NPs might change the properties both of the NPs and the encapsulated molecules.…”

“…The hydrophobic analogue NR668 was found to leak much less than NR from nanoemulsions (NEs) (18), and to be suitable for tracing the PBCA-NPs, as it was not released from the NPs until they were endocytosed and degraded (12). In search of alternative dyes for stable NP labeling and potential Förster resonance energy transfer (FRET) pairs, we performed the current study and applied a flow cytometric cell based assay to screen a variety of dye-NP combinations.…”

“…The other dyes were synthesized; NR668 designed to be more hydrophobic than NR, the oligothiophene p-HTAM has been used in a previous study of cellular uptake of NPs (12), and similarly to NR668, p-HTAH was designed to be more hydrophobic than p-HTAM. We demonstrate a large variability in NP-dye retention, and surprisingly found that encapsulation of different dyes significantly alters NP uptake in cells.…”