Mixed alkyltrimethylammonium-alkylcarboxylate Surfactants Systems: A Potential Anti-Proliferation Agent for MDA-MB-231 Cancer Cell Line Xiou

Series of catanionic based alkyltrimethylammonium-alkylcarboxylate surfactants systems having different degree of alkyl chain length asymmetry were prepared and tested for their cytotoxicity against both fibroblast 3T3 and breast cancer MDA-MB231 cell lines. Catanionic surfactant with the highest alkyl chain length possessed significant potent cytotoxicity, with a half maximal inhibitory concentration (IC50) of 4.04 ± 0.06 μM on MDA-MB-231 cell line. Combination of anti-cancer agent (tocotrienol rich fraction,TRF; and curcumin, respectively)with the catanionic at different weight ratios was found to exert lower cytotoxicity on both cell lines as compared to the treated cells with catanionic only. Noticeably, combination of TRF withcatanionic showed higher anti-cancer activities than the incorporation of curcumin with catanionic (resulted low IC50 value). Combination index (CI) of both anti-cancer agent (TRF and curcumin) and catanionic mixtures indicated antagonistic effect (more than 1), respectively, which may explained catanionic surfactant having long alkyl chain length has the potential to act as anti-cancer agent in its pristine form and less effective with other anti-cancer agents combination.


Introduction
As it is widely known, surface active agents or surfactants having unique properties are been enormously utilized in cosmetic and personal care, chemical processes as well as health and pharmaceutical sectors 1 . Apart from the common classes of surfactants (anionic, cationic, nonionic and zwitterionic) , there is a new class of surfactant known as catanionic surfactants that are produced from mixing of two oppositely charged ionic surfactants at equimolar ratio 2 . This type of surfactant exhibited very interesting properties and unique self-aggregation behaviour into various aggregates such as vesicles, micelles, hollow icosahedra and planar bilayers [3][4][5] .
Interestingly, a recent review highlighted that the vesicles formed in catanionic surfactants have been correlated with anti-cancer activities in vitro 6 , representing a useful finding for pharmaceutical industries. Treatment with catanionic surfactant vesicles produced from sodium dodecylsulfate and hexadecyltrimethylammonium bromide (TA 16 ) induced apoptosis in murine macrophage-like cells but no apoptosis occurred in normal cells 7 . The cell proliferation of cancer cells was much more inhibited than that of normal mouse fibroblast cells when treated with catanionic surfactant vesicles, this was explained by different intrinsic cell membrane permeability of cancer cells in relative to normal cells 8 . Besides that, synthesis of chlorambucil prodrug-containing catanionic vesicles that acted as both carrier and active ingredients at the same time exhibited desirable prolonged drug release and enhanced anti-cancer activities 3 . On the other hand, catanionic solid lipid nanoparticles constructed from catanionic microemulsion efficiently transported chemotherapy drug across blood brain barrier for the treatment of malignant brain tumour in central nervous system 9,10 . Novel catanionic microemulsion containing curcumin displayed higher cytotoxicity in vitro and effectively suppressed tumour growth in vivo through intravenous or oral administrations 11 . Further studies are required to support the anti-cancer activities of catanionic surfactants.
Cancer is a disease with immense public health significance suffered by over millions of patients in the world. Breast cancer is the most frequent invasive cancer among women in both developed and developing countries 12 . Breast cancer is a highly metastatic cancer which is capable to spread from one site to another site very rapidly. Triple negative breast cancer accounts for 15% to 20% of all breast cancers 13 . The treatment for this highly invasive triple negative breast cancer is very challenging as there is still lacking of targeted treatment site for targeted therapy and available targeted therapeutic agent 14 . Furthermore, adverse effects of cancer treatment and anti-cancer drugs that damages both tumour and normal cells at the same time still remained unresolved 15 . A recent review concluded that 70% of articles reported enhanced chemotherapy efficiency with the presence of antioxidants 15 . This makes antioxidants such as tocotrienol, quercetin, curcumin, carotene, vitamin C, glutathione, coenzyme Q10 and selenium gaining popularity in anticancer researches [15][16][17][18] .
Other than that, hexadecanoic acid (C 16 ) and octadecanoic acid (C 18 ) which are found abundantly in palm oil fractions were discovered to have anti-cancer activities [19][20][21] . C 16 and C 18 were found to inhibit the colony-forming ability of certain cancer cells in vitro and inhibit tumour development in vivo [22][23][24] , but not the healthy normal fibroblasts in dose-dependent manner 22 . Studies showed the inhibition of cancer cell proliferation of C 16 and C 18 was caused by apoptosis 19,20 . However, another study demonstrated C 18 inhibited cancer cell proliferation by inhibiting cell adhesion that involved a laminin integrin receptor mechanism 25

Preparation of Catanionic Surfactants
Neat catanionic systems were prepared by using a modified version of a previously published procedure 27  water bath with magnetic stirrer at temperature below 55°C to ensure neat catanionic surfactant was fully dissolved. The surfactants were sterilized by filtration using 0.2 µM pore size cellulose acetate syringe filters prior to use.

Cell Lines and Cell Culture
The mouse embryo fibroblast cell line 3T3 was obtained from cell bank of  The stock solutions were further diluted with fresh media to achieve final concentrations of 0 µM to 100 µM. After subjected to different treatments as described above, the treated cells were incubated for 24 hours post-treatment. Each assay and concentration were tested in triplicate (n = 3). Control cells were exposed to culture medium only. The cell density after 24 hours post-treatment was observed using Zeiss Primovert inverted light microscope (Jena, Germany).

MTT Cytotoxicity Assay
The cytotoxicity of individual parent surfactants, catanionic surfactants, TRF, curcumin, different weight ratios of C 18

Combination Index Analysis
Combination Index of TRF:C 18 TA 18 and curcumin:C 18 TA 18 was calculated using the method described in previous studies 30, 31 . A combination index of more than, equal or less than 1 were defined as antagonistic, additive and synergistic effect, respectively 30,32 . The combination index was calculated according to the following formula: Equation (2) Where, C A,50 and C B,50 are the concentration required for C 18  for single compound to achieve the same effect.

Statistical Analysis
All experiments were performed in triplicates and the results were expressed as means ± standard deviation. The one-way Analysis of Variance was used to determine the statistical significance difference. Tukey's and Bonferroni's Multiple Comparison Test was conducted subsequently for significant main effects. The GraphPad Prism Version 7.04 software was used for statistical analyses. All results were considered to be statistically significant at p < 0.05.

Data Availability
All data generated and analysed during this study are included in this article. Tables 1 and 2

Combination Analysis of C 18 TA 18 With Other Anti-Cancer Agents
Cytotoxicity of TRF and curcumin as anti-cancer agents are investigated and shown in Table 5. Similar as previous studies, TRF and curcumin effectively inhibited cell proliferation of cancerous cell compared with healthy normal cell [16][17][18] .
From the results, TRF displayed significantly higher cytotoxicity than curcumin on both cell lines. IC 50 of TRF on 3T3 cell line was 28.39 µg mL -1 ± 0.89 µg mL -1 , slightly higher than that of MDA-MB-231 cell line with IC 50 of 22.77 µg mL -1 ± 0.34 µg mL -1 . Treatment with TRF on MDA-MB-231 cell line gave IC 50 that was almost twice lower than that in curcumin. Results indicated that TRF was more potent in the inhibition of cancer cell proliferation than curcumin.
In the previous section, C 18  that, incorporation of TRF into C 18 TA 18 resulted lower cell density than that of curcumin at the same treatment ratio. Therefore, TRF was more suitable to be incorporated with C 18 TA 18 in order to kill the cancer cells at greatest magnitude but with minimal damage on healthy cells.

Conclusion
The present study suggests catanionic surfactant is able to inhibit the proliferation of        The data were expressed as means ± standard deviation (n = 3). Data with different small letters within the same group were significantly different (p < 0.05) by comparing with C 18 TA 18 . Data with different capital letters within the same group were significantly different (p < 0.05) between the same anti-cancer agent treatment.