Synthesis of the amphiphilic conjugate
Rh123 was linked with the PEG chain of amphiphilic polymer PEG-PE via amide bond formation (). The polymer was characterized by 1H-NMR in d-chloroform using a Varian 500 MHz spectrometer.
Synthesis of Rh123-PEG3400-DOPE.
1H-NMR: δ 0.86–0.92 (t, 6H, [[CH2]n–CH3]2 of lipophilic chain in phospholipid), 1.28–1.31 (m, [[C H2]n–CH3]2 of DOPE), 2.01–2.02 (m, 8H, [C H2–CH=CH–CH2]2 of DOPE), 2.29–2.32 (m, from DOPE), 2.38–3.85 (m, PEG, DOPE), 4.06–4.48 (2 bs, 1 m, DOPE), 5.25 (bs, 1H,-O–CH2–CH (CH2–)–O– of DOPE), 5.35–5.37 (m, 4H, [CH2–CH=CH–CH2]2, 6.91–7.07 (m, 4H, Ar-H of Rh123), 7.41–7.43 (m, 2H, Ar-H of Rh123), 7.66–7.80 (m, 1H, Ar-H of Rh123), 8.07–8.08 (d, J = 8.5 Hz, Ar-H of Rh123), 8.30–8.32 (d, 2H, J= 9.0 Hz, Ar-H of Rh123). The presence of new peaks in the aromatic region from the aromatic protons of Rh123 indicated successful conjugation.
Characterization of liposomes
The synthesized amphiphilic copolymer Rh123-PEG-PE was incorporated into the liposomal lipid bilayer. For cell-based experiments, the surface modification of liposomes with 1 mol% of Rh123 polymer was enough to impart a targeting effect. Size distribution, mean diameter, and zeta potential values for targeted and nontargeted liposomes as well as for PCL-loaded targeted and nontargeted liposomes were all rather similar ( and ).
Size distribution of PL, Rh123-L, PCL-PL, and PCL-Rh123-L. No significant change in size distribution was observed after PCL incorporation.
FACS analysis of cellular uptake of liposomes
Uptake of liposomes by HeLa and B16F10 cells was performed under the same conditions. In all fluorescence experiments, PL were modified with the green fluorescence marker NBD-PE, and the fluorescence of PL was normalized prior to cell treatment by adjustment of the amount of NBD-PE in the liposomal formulation to produce an equal intensity of fluorescence with the Rh123-L. The fluorescence intensity of PL modified with 0.78 mol% of NBD-PE corresponded to the fluorescence intensity of Rh123-L modified with 1 mol% of Rh123-PEG-PE.
Flow cytometry data revealed a significantly higher uptake of Rh123-L as little as 15 min of treatment compared with PL (). The targeted liposomes were internalized in significantly higher amounts at all time points compared with PL (P < 0.001). A time-dependent increase in cell-associated fluorescence of Rh123-L occurred in both cell lines as seen from the comparison of the geometric mean of fluorescence at the three time points of 15, 30 min, and 1 h (). However, no time-dependent increase occurred in the uptake of PL after 1 h.
Figure 3 Cell uptake of Rh123-L and PL at different time points using two different cell lines (HeLa and B16F10) by flow cytometry. Cells were incubated with the Rh123-L or PL at lipid concentrations of 0.2 mg/mL for 15 min, 30 min, and 1 h. After incubation, (more ...)
Figure 4 Cellular internalization of Rh123-L and PL in HeLa and B16F10 cells. Geometric mean fluorescence obtained from flow cytometry data of (A) HeLa cells and (B) B16F10 cells. Cellular internalization estimated from the fluorescence after incubation for 1, (more ...)
Estimation by cell lysis
Cellular internalization of formulations was also studied with a cell lysis assay after cell treatment with liposome preparations for 1, 2, and 4 h, and subsequent analysis of samples by fluorescence spectroscopy of HeLa and B16F10 cell lines (). The data are presented as fluorescence intensity/microgram of protein versus the incubation time. The uptake pattern was similar to the result obtained using FACS analysis. Rh123-L were taken up by cells in a significantly higher amount than by PL at all time points. In fact, the uptake of PL did not change significantly over 4 h time period in both the cell lines. However, it was not certain whether the formulation was associated with the cell membrane or internalized and directed into the subcellular compartments.
To assess more closely the intracellular trafficking of the liposomal formulations, confocal microscopy was performed with stained mitochondria and co-localization of the labeled formulations was followed with mitotracker. Overlaid multichannel confocal fluorescence micrograph showed significantly higher co-localization of Rh123-L in the mitochondria compared with PL as indicated by yellow color in the merged picture of deep red-stained mitochondria and green fluorescence-labeled formulations in the same field (). Analysis of co-localization (ImageJ software) showed significant accumulation of targeted liposomes in the mitochondria (Pearson’s coefficient 0.55, Mander’s coefficient 0.75 for Rh123-L compared with Pearson’s coefficient −0.083, Mander’s coefficient 0.007 for PL). Thus, the confocal microscopy co-localization data stronglysupportsourhypothesisthattheRh123-conjugated pharmaceutical nanocarrier targets mitochondria.
Figure 5 Intracellular co-localization by fluorescence confocal microscopy. (A) HeLa cells treated with PL and (B) cells treated with Rh123-L. Left panel: Cell treatment with PL or Rh123-L in the green channel (ex. 505 nm, em. 530 nm); Middle panel: Cell staining (more ...)
Mitochondria were isolated from the cultured HeLa cells after Rh123-L and PL treatment for 4 h, and mitochondrial-associated fluorescence was measured (). Significantly higher fluorescence occurred in the mitochondrial fraction isolated after Rh123-L treatment compared with control mitochondria and PL-treated mitochondria, which again clearly demonstrated the mitochondrial-targeting property of the Rh123-L formulation. The fluorescence of the cytosol fraction was also significantly higher in Rh123-L-treated cells compared with PL-treated ones.
Figure 6 Fluorescence associated with isolated mitochondrial fractions of HeLa cells treated with Rh123-L or PL for 4 h. One-way ANOVA analysis was performed followed by Bonferroni’s multiple comparison test that indicated that differences of control versus (more ...)
To demonstrate that specific delivery of the drug to the desired subcellular compartment can significantly enhance drug action, the mitotic inhibitor PCL was used since much evidence of PCL acting on and altering the function of mitochondria to produce apoptotic action have been observed (André et al., 2002
; Kidd et al., 2002
; Ferlini et al., 2009
). PCL-Rh123-L treatment at PCL concentration 1.5 μg/mL produced significantly higher cytotoxicity than free PCL and PCL-PL (). The 35–40% reduction of cell survival was observed with PCL-Rh123-L compared with nontargeted PCL formulations.
Figure 7 Cytotoxicity of PCL in targeted and nontargeted formulations on HeLa cells by trypan blue assay. Cells were treated with free PCL, PCL-PL, or PCL-Rh123-L for 4 h followed by 24 h incubation before assessment of cell viability. Two-way ANOVA analysis was (more ...)
PCL is also well-known as microtubule-destabilizing agent (Schiff et al., 1979
). However, apart from its interaction with beta tubulin, PCL also activates the intrinsic mitochondrial apoptotic pathway leading to opening of the permeability transition pore channel (PTPC) and subsequent release of proapoptotic factors, which ultimately leads to apoptosis (Bhalla, 2003
; Ferlini et al., 2009
). PCL directly inhibits the anti-apoptotic protein BCl-2 in the loop domain that participates in the regulation of PTPC (Shimizu et al., 1998
) and thereby facilitates the initiation of apoptosis. Therefore, much evidence indicates a possible apoptotic action of PCL mediated by alteration of mitochondrial function. This study demonstrated the efficient delivery of the therapeutic cargo PCL into the intracellular site of action as evidenced by remarkable increase in PCL-associated cytotoxicity with mitochondrial-targeted PCL-Rh123-L.