Can ultrasound enable efficient intracellular uptake of molecules? A retrospective literature review and analysis

Abstract

Most applications of therapeutic ultrasound (US) for intracellular delivery of drugs, proteins, DNA/RNA and other compounds would benefit from efficient uptake of these molecules into large numbers of cells without killing cells in the process. In this study we tested the hypothesis that efficient intracellular uptake of molecules can be achieved with high cell viability after US exposure in vitro. A search of the literature for studies with quantitative data on uptake and viability yielded 26 published papers containing 898 experimental data points. Analysis of these studies showed that just 7.7% of the data points corresponded to relatively efficient uptake (>50% of cells exhibiting uptake). Closer examination of the data showed that use of Definity US contrast agent (as opposed to Optison) and elevated sonication temperature at 37°C (as opposed to room temperature) were associated with high uptake, which we further validated through independent experiments carried out in this study. Although these factors contributed to high uptake, almost all data with efficient uptake were from studies that had not accounted for lysed cells when determining cell viability. Based on retrospective analysis of the data, we showed that not accounting for lysed cells can dramatically increase the calculated uptake efficiency. We further argue that if all the data considered in this study were re-analyzed to account for lysed cells, there would be essentially no data with efficient uptake. We therefore conclude that the literature does not support the hypothesis that efficient intracellular uptake of molecules can be achieved with high cell viability after US exposure in vitro, which poses a challenge to future applications of US that require efficient intracellular delivery.

Fig. 1

Intracellular uptake efficiency vs. cell viability after US exposure. Data points were obtained from literature: (a) + (Tata et al. 1997); × (Miller et al. 1999); (Cochran and Prausnitz 2001); |, , , (Guzman et al. 2001, 2002, 2003); (Keyhani et al. 2001); ◁ (Larina et al. 2005); ✫ (Mehier-Humbert et al. 2005); *, (Hallow et al. 2006, 2007); – (Hutcheson et al. 2010); ◁ (Karshafian et al. 2010); ∎ (Li et al. 2008); ◯ (van Wamel et al. 2002); ◆ (Forbes et al. 2011); ◀ (Sundaram et al. 2003); ● (Miller and Dou 2009); ▶ (Hassan et al. 2010); ◻, ▽, (Karshafian et al. 2007, 2005); ★ (Lai et al. 2006); ▽ (Karshafian et al. 2009); ▲ (Han et al. 2007); ◇ (Kinoshita and Hynynen 2005); △ (Karshafian et al. 2004). Each data point represents the average of n ≥3 replicates collected at various experimental conditions as described in the original papers and summarized in Table 1. The solid line is where uptake efficiency equals cell viability. The dashed line is where the uptake efficiency equals 50%.

Fig. 2

Intracellular uptake efficiency vs. cell viability after US exposure. Data points were obtained from literature, as described in Fig. 1 and Table 1. The solid line is where uptake efficiency equals cell viability. The dashed line is where the uptake efficiency equals 50%. The graphs exhibit the same data presented to distinguish between experiments using (a) US energy density less than (●) and higher than (◇)100 J/cm²; (b) mechanical index less than (●) and higher than (◇) 0.7; (c) megahertz US (●) and kilohertz US (◇); (d) large molecules (●, MW >1 kDa, e.g., dextran, BSA and DNA) and small molecules (◇, MW <1 kDa, e.g., calcein); (e) KHCT cells (●), prostate cancer cells (◇), CHO cells (B), AoSMC cells (□), rat mammary cells (△), ex vivo artery (×) and other cell lines (+); (f) 37°C (●) and room temperature (◇); (g) Definity (●), Optison (◇) and other contrast agents (×); and (h) analysis not accounting (●) and accounting (◇) for cell debris to calculate cell viability.

Fig. 3

Identification of US exposure characteristics associated with efficient intracellular uptake. (a) The fraction of data points in the efficient uptake group found among the data points having the given exposure characteristics. (b) The fraction of data points with given US exposure characteristics found among the data points in the efficient uptake group (i.e., uptake efficiency .50%). These graphs were generated based on the data shown in Figure 2.

Fig. 4

The effect of temperature and US contrast agent on intracellular uptake efficiency. DU145 prostate cancer cells were exposed to US as described in Materials and Methods at 37°C or 23°C with 0.1 vol% Definity or 2 vol% Optison (*p , 0.05, n 5 3 replicates, data points show average 6 standard deviation).

Fig. 5

The effect of accounting for lysed cells when determining cell viability and uptake efficiency. (a) The uptake efficiency vs. cell viability after US exposure determined by accounting for cells lysed during US exposure (>, data obtained directly from Hallow et al. (2006)) and without accounting for lysed cells (C, reanalysis of data from Hallow et al. (2006)). Each data point represents the average of n $3 replicates. The solid line is where uptake efficiency equals cell viability. The dashed line is where uptake efficiency equals 50%. The differences between (b) intracellular uptake and (●) cell viability calculated with and without accounting for lysed cells. Differences were determined by subtracting the values calculated accounting for lysed cells from values calculated without accounting for lysed cells.