Chapter V: Intra mitochondrial assembly of peptide amphiphile via pH induced disassembly of micelle in
5.3. Results and Discussion
Mito-FF is synthesized as described in our previous article using standard solid phase peptide synthesis procedure and purified with high pressure liquid chromatography (HPLC).7 Mito-FF is converted into Mito-SA by reacting with 100 eq of succinic anhydride, in DMSO for 12 h (Fig. 5-2a).
The conversion of Mito-FF to Mito-SA is monitored by HPLC at different time point, in which the peak of Mito-FF appeared at 31 min upon elution with ACN/Water mixture in C-18 column which showed partial conversion at 6 h with the appearance of new peak at 35 min for Mito-SA and then completely transformed to Mito-SA within 12 h indicated by the disappearance of peak at 31 min and appearance of new peak at 35 min (Fig. 5-2b). The formation of Mito-SA was confirmed with mass analysis using MALDI-TOF/TOF (Experimental; Fig. 5.6-1). The unreacted Mito-FF later removed by dialysis against water using membrane cut off 500 D whenever necessary. Using the reaction mixture itself for the further experiments also does not make a difference when the precursor materials are pure.
Mito-SA micelle cleaving only in tumor tissue Blood vessel
Normal tissue Tumor tissue
Abolishing cancer
Intra mitochondrial assembly followed by micelle disassembly
Page 94 of 106 Figure 5-2. (a) Schematic representation for the conversion of Mito-FF to Mito-SA. Mito-FF was treated with succinic anhydride in DMSO for 12 h, the formation of Mito-SA was confirmed by mass analysis. (b) The HPLC traces of Mito-FF conversion to Mito-SA at different time point.
10 15 20 25 30 35 40 Mito-FF + SA (0 h)
Time (min)
10 15 20 25 30 35 40 Mito-FF + SA (6 h)
Time (min)
10 15 20 25 30 35 40 Mito-FF + SA (12 h)
Time (min) 12 h/DMSO
a)
b)
Page 95 of 106 The morphology of Mito-SA is confirmed using electron microscopic analysis (TEM) which showed micellar like aggregates of 50 nm diameter (Fig. 5-3a). The size distribution analysis were done using dynamic light scattering (DLS) measurements which showed consistent result with a diameter of
~ 50 nm (Fig. 5-3b). The critical aggregation concentration (CAC) of micelle is analyzed using pyrene excitation method, showed a CAC of 5 μM (Fig. 5-3c). The lower CAC of Mito-SA might be because of the strong zwitter ionic interaction between the positive groups of triphenyl phosphonium (TPP) and negative charge of acid group, which allow them to aggregate at lower concentration, which is much lower than the CAC of Mito-FF (~ 60 μM) (Fig. 5-3d). To further confirm the formation of Mito-SA, surface charge distribution is analyzed in water and showed a potential of -Ve or 0 mV, indicating the formation of neutral micellar aggregates, in contrast Mito-FF under similar condition showed a charge distribution of + 40 mV.
Figure 5-3. (a) TEM image of Mito-SA. (b) DLS analysis of Mito-SA. (c) CAC determination of Mito- SA using pyrene excitation method (left). The plot of I344/I339 gives the CAC of 5 μM (right).
0 20 40 60 80 100
0 10 20 30 40
Number PSD
Diameter (nm)
1 10
I 344/ I 339
I 344/I 339
Log [C] (M) -100 -50 0 50 100
Mito-SA Mito-FF
Intensity (a.u)
Size (nm)
TEM image DLS showing size distribution
CAC determination Surface charge analysis
Page 96 of 106 The pH induced hydrolysis of Mito-SA was analyzed using the analytical column under different conditions. Mito-SA in PBS (pH 7.4) appeared around 9 min which shifted to 2 min in pH 6.5 after 24 h, similar to the peak of Mito-FF (Fig. 5-4a). However, at pH 7.4 Mio-SA showed no peak shifting after 24 h suggesting the higher stability of Mito-SA (Fig. 5-4b). The TEM images after treating with pH 6.5 for 24 h showed fibers, indicating the conversion of Mito-FF to Mito-SA (Fig. 5-4d).
Figure 5-4. (a) The analytical HPLC trace of Mito-SA (upper), Mito-SA after 24 h incubation in pH 6.5 (middle) and Mito-FF in pH 6.5 (Final). (b) Mito-SA in pH 7.4 after 24 h incubation. (c) TEM image of Mito-SA before cleavage (d) after cleavage.
Fiber at 6.5 Fiber at 7.4
Page 97 of 106 This was very surprising observation that the succinyl amides of amine undergoes hydrolysis under acidic condition, since it could happen barely according to the previous reports. Here, we postulated that the presence of TPP facilitates the cleavage of succinyl moiety under acidic condition (Fig. 5-5). It is well known that the oxygen-phosphorous dative bonding is quite feasible under any condition when they are in close proximity, in which the lone pair of oxygen donates to the antibonding orbital of phosphorous as per the molecular orbital theory. The succinyl amide bond (-NC=O) of Mito- SA is stable under normal conditions. However, once the intermediate is established as shown in the schematic diagram (step 2), the tertiary amine group thus formed is prone to protonation under acidic condition which leads to the conversion of compound back to Mito-FF in acidic condition (step 3).
Therefore, we concluded that the presence of TPP facilitate the acid hydrolysis of succinyl amide of Mito-SA. The amidization of Mito-FF with maleic anhydride is found to be unstable (data not shown).
Figure 5-5. The proposed mechanism for the cleavage of Mito-SA under acidic condition. The cleavage of succinyl amide group of Mito-SA is facilitated by the presence of TPP moiety as a consequence of oxygen to phosphorous dative bond.
Acidic pH
Page 98 of 106 The internalization of Mito-SA is confirmed in HeLa cells, after incubating 10 μM for a time period of 12 h in pH 6.5 medium (Fig. 5-6a). The blue fluorescence was appeared from the HeLa cell mitochondria suggesting that Mito-SA undergo cleavage and internalize inside the cell to target mitochondria as shown in schematic representation (Fig. 5-6b). However in the normal cell line, NIH 3T3, there appeared no fluorescence from the cells after treating with Mito-SA for 12 h in pH 7.4 or normal cell culturing media, suggesting that no cellular entry of Mito-SA happened (Fig. 5-6c). We assume that, Mito-SA could not transform to Mito-FF under normal condition, so that cellular entry is prohibited suggesting highly cancer specific internalization of Mito-SA (Fig. 5-6d).
Figure 5-6. (a) The cellular internalization of Mito-SA in HeLa cells. The medium of the pH is changed to pH 6.5. (b) The schematic representation of Mito-SA cellular entry. It is expected that the micellar like aggregates dis-assemble near the tumor and internalize as Mito-FF inside the cell. (c) The normal cells do not showed any blue fluorescence after treating with Mito-SA, indicating that, the Mito-SA, as aggregates does not internalize inside the normal cells. (d) The schematic diagram showing no cellular entry of Mito-SA inside the normal cells.
Mito-SA – 12 h HeLa
NIH3T3
Schematic illustration
a) b)
c) d)
Page 99 of 106 The mitochondria dysfunction analysis induced by Mito-SA is analyzed in both cancer and normal cells. To analyses the mitochondrial fragmentation, cells were labelled with Mito Tracker Red FM, and analyzed using the confocal microscope after 12 h of treatment period. The microscopic analysis showed that the mitochondria were severely fragmented with a leakage of Mito Tracker from the mitochondria to the cytoplasm for the cell treated with Mito-SA in the HeLa cells (Fig. 5-6a).
Figure 5-7. The mitochondria dysfunctional study in HeLa cells (a) Mitochondrial fragmentation monitored with Mito-Tracker Red FM showing drastic fragmentation after treating with Mito-SA. (b) The ROS generation monitoring with DHE. (c) Mitochondrial ROS generation monitored with Mito- SOX Red. (d) Membrane depolarization checked with TMRM. The FACS data is provided on the right for each. The same experiment repeated on normal cell, NIH 3T3 with (e) Mito Tracker Red FM. (f) DHE. (g) Mito-SOX. (h) TMRM.
Control Mito-SA –12 h Mito Tracker
Control Mito-SA –12 h Mito Tracker
DHE DHE
Mito-SOX Mito-SOX
TMRM
HeLa NIH 3T3
TMRM a)
b)
c)
d)
e)
f)
g)
h)
Page 100 of 106 However, the normal cells under similar condition does not showed any mitochondrial fragmentation and leakage (Fig. 5-6e). The cells were labelled with Mito SOX Red dye which shows red fluorescence from the mitochondria upon the production of mitochondrial ROS. HeLa cells showed appearance of red fluorescence form the mitochondria after treating with Mito-SOX, suggesting the production of mitochondrial ROS prior to the cell death (Fig. 5-6b). In contrast, the NIH 3T3 cells does not showed any red fluorescence from the cells, suggesting no damage to the cells (Fig. 5-6f). We have also analyzed the production ROS from the cell upon treatment with dihydroethidium (DHE), an ROS sensor which shows red fluorescence in presence of ROS upon intercalation with DNA, in turn indicates the cellular damage. HeLa cells showed red fluorescence form the cell nucleus suggesting that the DHE could permeable inside the nucleus, to intercalate with nuclear DNA to show red fluorescence (Fig. 5-6c). The NIH 3T3 does not showed any red emission from the nucleus suggesting lack of cellular damage (Fig.
5-6g). Finally, the mitochondrial membrane depolarization ability of Mito-SA is evaluated using the membrane depolarization dye, Tetramethyl rhodamine, Methyl ester (TMRM) which fluoresce under normal condition and vanishes the red emission from the membrane upon membrane damage. As expected, the HeLa cells after treating with Mito-SA showed the dis appearance of red fluorescence from the mitochondrial membrane (Fig. 5-6d), but the normal cells remained fluoresce after treatment with TMRM suggesting that Mito-SA is inactive on the normal cells, NIH 3T3 normal cells (Fig. 5-6h).
Figure 5-8. (a) Cell viability of Mito-SA in HeLa cells after 24 h. (b) The cell viability in NIH 3T3 cells. (c) Cellular apoptosis monitored with FI-TC Annexin V and PI staining in HeLa cells. (d) In NIH 3T3 cells.
0 5 10 15 20
20 40 60 80 100 120
Mito-SA; 7.4 Mito-SA; 6.5 Mito-FF
Cell Viability (%)
Concentration () NIH 3T3; 24 h
0 5 10 15 20
20 40 60 80 100 120 140
Mito-SA; 7.4 Mito-SA; 6.5 Mito-FF
Cell Viability (%)
Concentration (M) HeLa; 24 h
PI Annexin V
PI Annexin V
B.F Merge
B.F Merge
HeLaNIH 3T3
a) b)
c)
d)
Page 101 of 106 The cellular cytotoxicity were evaluated in the HeLa cells after treating with Mito-SA, which showed good cell killing ability with an IC-50 of 17 μM, which is similar to Mito-FF. However, in the NIH 3T3 cells, the toxicity were not shown suggesting higher tumor specificity of Mito-SA. The cellular apoptosis study were done in HeLa and NIH 3T3 cells with staining with apoptotic detector dye FITC-Annexin V and PI. The membrane impermeable PI discriminates live or early apoptotic cells from late apoptotic or necrotic cells that lose membrane integrity. Annexin V stains both apoptotic cells, which expose phosphatidylserine extracellularly, and necrotic cells, which lose membrane integrity. The confocal imaging showed the appearance of green fluorescence from the HeLa cells, but not red fluorescence of PI. The normal cells, NIH 3T3 does not showed both color emission, suggesting lack of cellular apoptosis.
Figure 5-9. (a) The images showing in vivo tumor reduction induced by Mito-SA in HT-29 tumor bearing mouse. (b) Systematic analysis of in vivo tumor reduction induced by Mito-SA, showing better tumor reduction ability of Mito-SA than Mito-FF.(c) The body weight lose measurement after treating with Mito-SA, Mito-FF and control showing no fetal toxicity induced by mitochondrial assembling compounds.
The in vivo tumor reduction ability of Mito-SA was measured by monitoring the degree of tumor suppression upon repeated administration of Mito-SA at 5 mg/kg of the mouse dosage. Mito-FF at same concentration was used as a control. The peptides were peritumorally injected around HT-29 tumor xenografts (volume; 70-100 mm3) every other day, 7 times in total, and the degree of tumor suppression was monitored for 2 weeks. The peptide-treated groups displayed apparent signs of tumor apoptosis (i.e. redness, swelling, and scab formation at tumor tissues) and suppression of tumor growth compared to the control group (PBS-treated mice), evidencing potential therapeutic efficacies of the peptides. During the treatment period, all experimental groups displayed no noticeable weight loss compared to the control group, revealing the minimal in vivo toxicity of the peptides. The result showed higher tumor reduction property for Mito-SA compared with Mito-FF which might be indicating the higher tumor uptake of Mito-SA than Mito-FF, since Mito-SA could be cleaved and internalized only in the tumor.
Dose: 5 mg/kg/2d, n=3.
Yellow circle: location of tumor HT-29, BALB/c nude mice male.
Before 4 day 12 day
Mito-FFMito-SA
0 2 4 6 8 10 12
0 100 200 300 400 500 600
Control Mito-FF Mito-SA
Tumor Volume (mm3)
Time (day)
0 2 4 6 8 10 12
0 5 10 15 20 25
Control Mito-SA Mito-FF
Body Weight (g)
Time (day)
a. b. c.
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