Probing the Intracellular Delivery of Nanoparticles into Hard-to-Transfect Cells

Abstract

Hard-to-transfect cells are cells that are known to present special difficulties in intracellular delivery of exogenous entities. However, the special transport behaviors underlying the special delivery problem in these cells have so far not been examined carefully. Here, we combine single-particle motion analysis, cell biology studies, and mathematical modeling to investigate nanoparticle transport in bone marrow-derived mesenchymal stem cells (BMSCs), a technologically important type of hard-to-transfect cells. Tat peptide-conjugated quantum dots (QDs-Tat) were used as the model nanoparticles. Two different yet complementary single-particle methods, namely, pair-correlation function and single-particle tracking, were conducted on the same cell samples and on the same viewing stage of a confocal microscope. Our results reveal significant differences in each individual step of transport of QDs-Tat in BMSCs vs a commonly used model cell line, HeLa cells. Single-particle motion analysis demonstrates that vesicle escape and cytoplasmic diffusion are dramatically more difficult in BMSCs than in HeLa cells. Cell biology studies show that BMSCs use different biological pathways for the cellular uptake, vesicular transport, and exocytosis of QDs-Tat than HeLa cells. A reaction−diffusion−advection model is employed to mathematically integrate the individual steps of cellular transport and can be used to predict and design nanoparticle delivery in BMSCs. This work provides dissective, quantitative, and mechanistic understandings of nanoparticle transport in BMSCs. The investigative methods described in this work can help to guide the tailored design of nanoparticle-based delivery in specific types and subtypes of hard-to-transfect cells.

Mechanism-Driven Technology Development for Solving the Intracellular Delivery Problem of Hard-To-Transfect Cells

“…The results in our recent report indicate that a key transport barrier for nanoparticles in BMSCs is vesicle trapping . Compared with HeLa cells, escape from vesicles is found to be much more difficult in BMSCs for a model type of exogenous entities, Tat peptide-conjugated quantum dots (QDs-Tat) .…”

“…Pair-correlation function analysis (pCF) determined the transit time of uptake of single QDs-PDS1 in BMSCs to be 120 ms on average (Figure 3e), which is much shorter than that for QDs-Tat in BMSCs (420 ms). 14 Bulk measurement of the uptake of QDs-PDS1 in BMSCs indicates that the QD amount in cells reached a plateau at 12 h of incubation, before decreasing slightly in the following 36 h (Figure 3f). The small decrease between 12 and 36 h was probably due to exocytosis of QDs-PDS1.…”

“…The results in our recent report indicate that a key transport barrier for nanoparticles in BMSCs is vesicle trapping . Compared with HeLa cells, escape from vesicles is found to be much more difficult in BMSCs for a model type of exogenous entities, Tat peptide-conjugated quantum dots (QDs-Tat) . Here, we screen a variety of vesicle trapping–reducing methods reported in the literature, including vesicle-disrupting agents, − autophagy-inhibiting agents, ,, membrane fluidity enhancers, , and nanoparticle coating with direct cell-membrane-penetrating potential, − on BMSCs.…”

“…Compared with HeLa cells, escape from vesicles is found to be much more difficult in BMSCs for a model type of exogenous entities, Tat peptide-conjugated quantum dots (QDs-Tat) . Here, we screen a variety of vesicle trapping–reducing methods reported in the literature, including vesicle-disrupting agents, − autophagy-inhibiting agents, ,, membrane fluidity enhancers, , and nanoparticle coating with direct cell-membrane-penetrating potential, − on BMSCs. Based on the best method identified from the screening, we further develop gene delivery technology to enhance transfection efficiency in BMSCs.…”

“…As in our previous report, we employed QDs as the model exogenous entities and QDs-Tat as the benchmark of intracellular delivery performance of QDs. QDs were selected because of their excellent ability to emit intense and stable fluorescence, permitting convenient and reliable imaging and tracking .…”