Table 1.
List of ELP-lipid conjugates.
Table 2.
List of all simulations.
Figure 1.
Schematic diagram of e-TSL and the chemical structure of ELP-lipid conjugates.
Figure 2.
Transmittance profile of ELP-lipid conjugates of (A) 1 mM, (B) 0.5 mM, (C) 0.2 mM, and (D) 0.1 mM in PBS at pH 7.4., and transition temperature as function of the concentration (mM) of various ELP-lipid conjugates (E).
The turbidity of ELP-lipid conjugates were characterized by monitoring transmittance at 280 nm as a function of temperature. Solution of ELP-lipids conjugates were heated at a constant rate of 1°C/min. The transition temperature (Tt) was defined as the temperature at which the solution of ELP-lipid conjugate reached 50% of transmittance. Data is shown as mean ± S.D. (n = 3).
Figure 3.
Calcein release profile of the liposome with the formulations of varied phospholipid compositions.
Phospholipid (DPPC/DSPC = 100-X/X):DSPE-PEG:cholesterol:SA-V6 = 55∶2∶10∶0.55 (X = 0, 25, 50, 75, and 100). Samples were measured after 5 min incubation at desired temperatures from 25 to 55°C by fluorometry at Ex. 493 nm/Em. 513 nm. Data is shown as mean ± S.D. (n = 3).
Figure 4.
DOX release profile of the liposome with the formulations.
(A) DPPC:DSPE-PEG:cholesterol:SA-V3 = 55∶2:X:55 and (B) DPPC/DSPC (75/25):DSPE-PEG:cholesterol:SA-V3 = 55∶2:X:55 (X = 10, 15, or 20). The amounts of DOX release were measured after 5 min incubation at desired temperature from 25 to 55°C by fluorometry at Ex. 490 nm/Em. 615 nm. Data is shown as mean ± S.D. (n = 3).
Figure 5.
DOX release profile of the liposome with the formulations.
(A) DPPC:DSPE-PEG:cholesterol:SA-V3 = 55∶2∶15:Y1 (Y1 = 0.28, 0.41, or 0.55) and (B) DPPC/DSPC (75/25):DSPE-PEG:cholesterol:SA-V3 = 55∶2∶10:Y2 (Y2 = 0.28, 0.55, or 1.1). The amounts of DOX release were measured after 5 min incubation at desired temperature from 25 to 55°C by fluorometry at Ex. 490 nm/Em. 615 nm. Data is shown as mean ± S.D. (n = 3).
Figure 6.
DOX release profile of the liposome with the formulations.
DPPC:DSPE-PEG:cholesterol = 55∶2∶15 in the presence/absence of SA-V3 (molar ratio∶0.41). The amounts of DOX release were measured after 5 min incubation at desired temperature from 25 to 55°C by fluorometry at Ex. 490 nm/Em. 615 nm. Data is shown as mean ± S.D. (n = 3).
Figure 7.
Cryo-TEM images with varying amounts of DOX in the liposome.
DPPC:DSPE-PEG:cholesterol:SA-V3 = 55∶2∶15∶0.41, with (A) liposome only, (B) 300 µg DOX/mL and (C) 500 µg DOX/mL.
Figure 8.
Cryo-TEM images of the liposome at (A) 25°C, (B) 37°C, and (C) 42°C.
Liposomes were composed of the formulation DPPC:DSPE-PEG:cholesterol:SA-V3 = 55∶2∶15∶0.41 and heat-treated for 5 min at indicated temperature in advance and followed by general cryo-TEM measurement.
Figure 9.
Snapshots at the (A) beginning (0 ns) and (B) end (2 µs) of the simulation of the bilayer system.
The molar ratio of liposome formulation is DPPC:DSPE-PEG:cholesterol:SA-Vn 55∶2∶15∶0.41. Gray, red, dark-, and light-blue colors respectively represent the lipid-head phosphate, PEG, ELP head (peptide), and tail (carbon chain) groups. For clarity, lipid tail, water, and ion beads are omitted. The images were created with Visual Molecular Dynamics [29].
Figure 10.
Average lateral diffusion coefficients (D) of DPPC lipids.
D was calculated from the slopes of the mean-square displacements in the xy-plane (the direction perpendicular to the bilayer normal), for the simulation systems with different ELP lengths (SA-V1, -V2, and V3) as functions of the molar ratio of (A) ELP and (B) cholesterol.
Figure 11.
The protocol of the drug accumulation study with e-TSL-DOX and free DOX, administered under mild hyperthermia.
Figure 12.
DOX accumulation in the tumor at 1, 6, and 12 hr after i.v. injection combined with preheating.
*, p<0.005, #, p<0.001, significant difference compared to e-TSL without preheating and free DOX with preheating.
Figure 13.
Intratumoral distribution of DOX. Red, DOX; green, CD31 (endothelial marker); blue, Hoechst (nuclear marker). Scale bar, 100 µm.
Figure 14.
Antitumor efficacy of e-TSL and free DOX in the presence/absence of mild hyperthermia.
e-TSL and free DOX were administered (5 mg DOX/kg) into tumor bearing BALB/c mice 5 min after preheating (30 min of water bath) and followed by mild hyperthermia (42°C, water bath) after 6 hrs. *, p<0.05, significant difference compared to free DOX and PBS (control).