Reservas del Campo Santuario

normalized to the untreated control. Values represent the average ± SD (n=3). Statistical significance was compared between micellar DOX treated group and free DOX treated control: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

2.4.9 Dose-dependent apoptosis by DOX-loaded crosslinked Lys-Phe-PEAcou- PEG micelles

To determine whether the overproduction of ROS and mitochondria damage could actually lead to increased apoptosis in HCT116 cells treated with micellar DOX, we further examined the dose dependent apoptosis. Figure 2.11A shows the confocal images of the apoptotic HCT116 cells by annexin-V

staining. In the apoptotic HCT116 cells induced by DOX-loaded crosslinked Lys-Phe-PEAcou-PEG micelles, the red fluorescence from DOX still distributed in the cytoplasm, while apoptotic cells treated with free DOX exhibited DOX fluorescence only in the nuclei. Micellar DOX with higher DOX concentration (5-10 μM) also exhibited similar distribution in the cytoplasm instead of in the nuclei (data not shown). These results suggest that, instead of intercalating in nuclear DNA, DOX from crosslinked micelles stayed away from the nuclei throughout the apoptotic process, which can be a possible indication of potentially different pathway in inducing apoptosis between micellar DOX and free DOX. Similar results were also reported by Kopeck et al. [43, 44]_{, in which} DOX bounded with N-(2-hydroxypropyl) methacrylamide (HPMA) polymer showed different distribution pattern and induced apoptosis in ovarian carcinoma cells by the disruption of mitochondrial function.

The data in Figures 2.11C shows the percentage of early apoptotic cells detected with flow cytometry. For DOX-loaded crosslinked Lys-Phe-PEAcou- PEG micelles, the percentage of apoptotic cells was much lower than free DOX, when the DOX concentration is less than 2 μM. The result is consistent with the lower cytotoxicity data shown in Figure 2.6B, suggesting that at lower DOX concentration, free DOX is more lethal toward HCT116 cells than micellar DOX. However, the difference in apoptosis percentage disappeared at 2 μM DOX concentration as no statistical difference between free DOX and micellar DOX was observed. This trend was reversed when the DOX concentration was > 2 μM, as significantly increased percentage of apoptosis was detected in HCT116 cells treated with micellar DOX. Free DOX, demonstrated by Yokochi et al. [25], induces necrosis at higher dose in HCT116 cells, which explains the reduced apoptosis percentage detected in cells treated with free DOX with concentration > 2 μM. Micellar DOX, on the other hand, was not observed to induce necrotic cell death in the concentration range between 2 to 10 μM.

Cleaved caspase 3 (17-19 kDa) is an active enzyme in the execution of apoptosis. Western blot data in Figure 2.11B suggest that the cleaved caspase 3

was present in HCT116 cells treated with micellar DOX with the DOX concentrations of 2 to 8 μM, or free DOX treated cells with the DOX concentration of 0.5 to 2 μM. The caspase 3/7 activity was further quantified and the data in Figure 2.11D is consistent with the result of apoptosis percentage. For HCT116 cells treated with free DOX, increased caspase 3/7 activity was detected only when DOX concentration was between 0.5-2 μM, and was significantly lower than DOX-loaded crosslinked Lys-Phe-PEAcou- PEG micelles at the DOX concentration > 2 μM. The result of dose-dependent apoptosis in HCT116 cells shows the similar trend with the data in Figure 2.9 and 2.10, which suggests the correlation between apoptosis, MPTP, ROS and MMP, and that the concentration range to induce apoptosis in HCT116 cells is different between micellar DOX and free DOX. Yokochi et al. [25] _{reported the} conditional apoptosis by free DOX in several cancer cell lines, and related the dose-dependent behaviour to DNA methyltransferase. However, detailed molecular biological characterization are required to further elucidate the mechanism of how DOX loaded in crosslinked Lys-Phe-PEAcou-PEG micelles translocated to the mitochondria, and their specific signalling pathway in inducing apoptosis, which would be the focus of our following study.

Figure 2.11. A) Annexin V staining of apoptotic HCT116 cells treated with DOX- loaded crosslinked Lys-Phe-PEAcou-PEG micelles or free DOX for 48 hrs with equivalent DOX concentration of 2 μM. Red fluorescence is from DOX and the green fluorescence is from annexin V-FITC. Scale bar represents 20 μm. B) Western blot to identify the presence of cleaved caspase 3 (17 – 19 kDa) in the lysate of HCT116 cells

after incubation with DOX-loaded crosslinked Lys-Phe-PEAcou-PEG micelles or free DOX. Untreated cells or cells incubated with blank crosslinked micelles were also characterized. C) Percentage of early apoptotic HCT116 cells (annexin V positive and SYTOX Red negative cells) detected by flow cytometry. HCT116 cells were treated with blank crosslinked micelles, DOX-loaded crosslinked Lys-Phe-PEAcou-PEG micelles or free DOX with various DOX concentrations for 48 hrs. D) Caspase 3/7 activity assay for HCT116 cells treated with blank crosslinked micelles, free DOX or DOX-loaded crosslinked Lys-Phe-PEAcou-PEG micelles with various concentrations of DOX for 48 hrs. Values represent the average ± SD (n=3). Statistical significance was compared between micellar DOX treated group and free DOX treated control: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

2.5 Conclusions

A lysine-phenylalanine based pseudo-protein micelles with photo- crosslinkable methylcoumarin moieties were synthesized for the intracellular delivery of DOX. The major conclusions of this study are: A) The crosslinked micelles exhibited significantly better structural stability in serum containing media and were able to counter the dilution-induced micelle disintegration; B) The in vitro release of DOX was not hindered by crosslinking, and can be accelerated by enzymatic degradation due to the biodegradability of the pseudo protein micelles, suggesting the stabilization-on-demand capability; C) DOX- loaded crosslinked Lys-Phe-PEAcou-PEG micelles co-localized to a large extent with mitochondria and endolysosomes in HCT116 cells, while free DOX localized only in the nuclei; D) DOX-loaded crosslinked Lys-Phe-PEAcou-PEG micelles induced apoptosis in HCT116 cells at higher and broader drug concentration range than free DOX, and the difference in inducing apoptosis showed a correlation with mitochondrial depolarization, MPTP opening and ROS generation. This research brings a novel perspective in the study of in vitro therapeutic effect of micellar drug delivery systems. The pseudo protein

micelles also exhibit versatile promise as a “stabilization-on-demand” delivery carrier, and can be further functionalized with targeting moieties and other bioactive agents. Furthermore, this study provides researchers with new insight into the design of micellar delivery vehicles and could have the potential in developing new nanocarriers for the mitochondria-targeting delivery of payloads.

References:

[1] K. Miyata, R. J. Christie and K. Kataoka, Reactive and Functional Polymers, 2011, 71, 227-234.

[2] A. P. R. Johnston, G. K. Such, S. L. Ng and F. Caruso, Current Opinion in

Colloid & Interface Science, 2011, 16, 171-181.

[3] Y. H. Bae and H. Yin, Journal of Controlled Release, 2008, 131, 2-4.

[4] T. Riley, T. Govender, S. Stolnik, C. D. Xiong, M. C. Garnett, L. Illum and S. S. Davis, Colloids and Surfaces B: Biointerfaces, 1999, 16, 147-159.

[5] A. Rösler, G. W. M. Vandermeulen and H.-A. Klok, Advanced Drug Delivery

Reviews, 2012, 64, 270-279.

[6] J. Zhang, H. Sun and P. X. Ma, ACS Nano, 2010, 4, 1049-1059.

[7] J. Jiang, B. Qi, M. Lepage and Y. Zhao, Macromolecules, 2007, 40, 790-792. [8] J. He and Y. Zhao, Dyes and Pigments, 2011, 89, 278-283.

[9] Q. Jin, X. Liu, G. Liu and J. Ji, Polymer, 2010, 51, 1311-1319.

[10] J. W. Chung, K. Lee, C. Neikirk, C. M. Nelson and R. D. Priestley, Small, 2012, 8, 1693-1700.

[11] J. He, X. Tong and Y. Zhao, Macromolecules, 2009, 42, 4845-4852.

[12] Y. Shao, C. Shi, G. Xu, D. Guo and J. Luo, ACS Applied Materials &

Interfaces, 2014, 6, 10381-10392.

[13] A. S. Hoffman, Advanced Drug Delivery Reviews, 2013, 65, 10-16.

[14] H. Wei, R. X. Zhuo and X. Z. Zhang, Progress in Polymer Science, 2013, 38, 503-535.

[15] X. Shuai, H. Ai, N. Nasongkla, S. Kim and J. Gao, Journal of Controlled

Release, 2004, 98, 415-426.

[16] K. Kataoka, T. Matsumoto, M. Yokoyama, T. Okano, Y. Sakurai, S. Fukushima, K. Okamoto and G. S. Kwon, Journal of Controlled Release, 2000, 64, 143-153. [17] J. S. Chawla and M. M. Amiji, International Journal of Pharmaceutics, 2002, 249, 127-138.

[18] C. C. Chu, in Biodegradable Polymers: New Biomaterial Advancement and

Challenges, ed. C. C. Chu, Nova Science Publisher, 2015, vol. 2.

[19] C. C. Chu, Journal of Fiber Bioengineering and Informatics, 2012, 5, 1-31. [20] C. C. Chu, in Biomaterials – Principles and Practices, eds. J. Y. Wong, J. D. Bronzino and D. R. Peterson, CRC Press, 2012.

[21] M. Deng, J. Wu, C. A. Reinhart-King and C. C. Chu, Biomacromolecules, 2009, 10, 3037-3047.

[22] H. Song and C. C. Chu, Journal of Applied Polymer Science, 2012, 124, 3840- 3853.

[23] J. Wu, D. Yamanouchi, B. Liu and C. C. Chu, Journal of Materials Chemistry, 2012, 22, 18983.

[24] D. Q. Wu, J. Wu, X. H. Qin and C. C. Chu, J. Mater. Chem. B, 2015, 3, 2286- 2294.

[25] T. Yokochi, Molecular Pharmacology, 2004, 66, 1415-1420.

[26] R. Lüpertz, W. Wätjen, R. Kahl and Y. Chovolou, Toxicology, 2010, 271, 115- 121.

[27] P. J. G. Coutinho, E. M. S. Castanheira, M. Céu Rei and M. E. C. D. Real Oliveira, The Journal of Physical Chemistry B, 2002, 106, 12841-12846.

[28] J. Wu and C. C. Chu, Acta Biomaterialia, 2012, 8, 4314-4323.

[29] H. Silva, F. Frézard, E. J. Peterson, P. Kabolizadeh, J. J. Ryan and N. P. Farrell,

Molecular Pharmaceutics, 2012, 9, 1795-1802.

[30] D. Colin, E. Limagne, S. Jeanningros, A. Jacquel, G. Lizard, A. Athias, P. Gambert, A. Hichami, N. Latruffe, E. Solary and D. Delmas, Cancer Prevention

[31] P. H. Hoet, B. Nemery and D. Napierska, Nano Today, 2013, 8, 223-227. [32] A. Wojtala, M. Bonora, D. Malinska, P. Pinton, J. Duszynski and M. R. Wieckowski, in Methods in Enzymology, Elsevier, 2014, 542, 243-262.

[33] E. Sakurai, H. Ozeki, N. Kunou and Y. Ogura, Ophthalmic Research, 2001, 33, 31-36.

[34] P. Opanasopit, M. Yokoyama, M. Watanabe, K. Kawano, Y. Maitani and T. Okano, Journal of Controlled Release, 2005, 104, 313-321.

[35] R. Savić, T. Azzam, A. Eisenberg and D. Maysinger, Langmuir, 2006, 22, 3570-3578.

[36] B. A. Aguilar-Castillo, J. L. Santos, H. Luo, Y. E. Aguirre-Chagala, T. Palacios-Hernández and M. Herrera-Alonso, Soft Matter, 2015, 11, 7296-7307.

[37] J. Lu, S. C. Owen and M. S. Shoichet, Macromolecules, 2011, 44, 6002-6008. [38] G. Speelmans, R. W. H. M. Staffhorst, B. de Kruijff and F. A. de Wolf,

Biochemistry, 1994, 33, 13761-13768.

[39] M. Han, M. R. Vakili, H. Soleymani Abyaneh, O. Molavi, R. Lai and A. Lavasanifar, Molecular Pharmaceutics, 2014, 11, 2640-2649.

[40] S. R. Jean, D. V. Tulumello, C. Riganti, S. U. Liyanage, A. D. Schimmer and S. O. Kelley, ACS Chemical Biology, 2015, 10, 2007-2015.

[41] J. D. Ly, D. R. Grubb and A. Lawen, Apoptosis, 2003, 8, 115-128.

[42] H. U. Simon, A. Haj-Yehia and F. Levi-Schaffer, Apoptosis, 2000, 5, 415-418. [43] A. Malugin, P. Kopečková and J. Kopeček, Molecular Pharmaceutics, 2006, 3, 351-361.

[44] T. Minko, P. Kopečková and J. Kopeček, Journal of Controlled Release, 2001, 71, 227-237.