Table S1 Scaling analysis results of for electroendocytosis (EED) and electroporation (EP) respectively. (a) EED exponent,n; (b) EP exponent, n; (c) Statistical analysis of the exponents.

tD(ms)
tT (s) / 0.2 / 0.5 / 1.0
10 / 2.280.15 / 2.290.20 / 2.310.21
30 / 2.860.47 / 2.210.06 / 2.090.59
60 / 1.880.29 / 1.780.08 / 1.980.32

(a)

tD(ms)
tT (s) / 0.2 / 0.5 / 1.0
10 / 9.330.88 / 6.910.40 / 8.060.79
30 / 6.850.75 / 4.470.34 / 5.050.45
60 / 5.270.86 / 4.960.82 / 4.990.77

(b)

n / Avg. / Max. / Min. / Std. Dev. / Valid E range
EED / 2.19 / 2.86 / 1.78 / 0.32 / E > Et,EED
EP / 6.21 / 9.33 / 4.47 / 1.67 / E > Ec,EP

(c)

Phase Diagrams of EED and EP

After quantitative analysis on more than 2,000 single-cell data points, the Et,EED and Ec,EP, with different pulse duration (tD) and total electric treatment time (tT), were determined, respectively. We constructed a series of Ea–tDEa–tT phase diagrams to distinguish EED from EP, as shown in Fig. S2. The solid blue line is the Et,EED, which delineates the boundary of areas for weak EED (dIavg,FM / dEa≤0.120.05 cmkV-1) and enhanced EED (dIavg,FM / dEa ≥ 0.570.08 cmkV-1). The black dashed EP critical curve delineates the Ec,EP. The window area outlined by EED and EP curves can be regarded as the optimized electric fields for the uptake of molecules through enhanced EED (dIavg,FM / dEa ≥ 0.570.08 cm/kV, dIavg,PI / dEa ≤ 0.050.02 cm/kV). When using shorter tDor tT, higher Eawas need to efficiently enhance EED efficiency to a significant value; on the contrary, lower Ea is sufficient when adding the pulse train with longer tD or extending tT. Similarly, longer tDor tT are necessary for charging a plasma membrane to the threshold electroporation potential when Ea is comparatively weak; but larger Ea may effectively curtail applied tDand tT for EP. In addition, from the comparison between Fig. S2 (d)-(f) and (a)-(c), we discovered that the effects of tT on Et,EED and Ec,EP curvesare more important than that of tD.

Scaling Analysis

Scaling analysis was utilized to quantify how significantly the average electric field strength (Ea) influences average fluorescent intensity (Iavg) both in the EED and EP processes. At the range when EaEt,EED (or Ec,EP), assuming, we determined each n value for both EED and EP, and calculated the average, the range, and standard deviation of n from a set of single-cell experimental data with various pulse duration (tD) and total electric treatment time (tT), as listed Table S1.

The increase of EED efficiency, labeled from the average fluorescent intensity of FM4-64, shows dependence on electric field strength (Ea)with the exponent n = 2.190.32 (n2), while the exponent for EP is 6.211.67 (n6). This indicates that the EED and EP have distinct electric field dependence. This scaling analysis clearly shows that EED and EP have different mechanisms.

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