Self-Assembly of Monomeric Hydrophobic Photosensitizers with Short

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Biological and Medical Applications of Materials and Interfaces

Self-Assembly of Monomeric Hydrophobic Photosensitizers with Short Peptides Forming Photodynamic Nanoparticles with RealTime Tracking Property and without the Need of Release in Vivo Jieling Li, Anhe Wang, Luyang Zhao, Qianqian Dong, Meiyue Wang, Haolan Xu, Xuehai Yan, and Shuo Bai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09933 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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ACS Applied Materials & Interfaces

Self-Assembly of Monomeric Hydrophobic Photosensitizers with Short Peptides Forming Photodynamic Nanoparticles with Real-Time Tracking Property and without the Need of Release in Vivo Jieling Li1, Anhe Wang1, Luyang Zhao1, Qianqian Dong1, Meiyue Wang1, Haolan Xu2, Xuehai Yan1* and Shuo Bai1* 1

State Key Laboratory of Biochemical Engineering

Institute of Process Engineering, Chinese Academy of Sciences No.1 North 2nd Street, Zhongguancun, 100190 Beijing, China E-mail: [email protected]; [email protected] 2

Future Industries Institute

University of South Australia Mawson Lakes, South Australia 5095, Australia

KEYWORDS: short peptide, self-assembly, photosensitizer, photodynamic nanoparticles, realtime tracking

ACS Paragon Plus Environment

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ABSTRACT: Employing nano-scaled materials as photosensitizer (PS) carriers is an effective strategy to solve the problem of poor solubility and low tumor selectivity of hydrophobic PS in photodynamic therapy (PDT), which compulsorily requires the PS release in PDT implementation. However, the complicated environment in vivo makes it difficult to precisely design and control the release process and the delivery process requires real-time tracking. Developing a delivery strategy of hydrophobic PS in the monomeric form with fluorescent emission and without consideration of the PS release in the PDT process, is in urgent demand. Herein, we report a versatile and potent strategy for fabrication of photodynamic nanoparticles (nanoPSs) with featuring the monomeric PS based on aromatic peptide-modulated self-assembly of porphyrin derivatives. Aromatic peptides within nanoPSs can isolate hydrophobic porphyrins from each other, resulting in monomeric porphyrin delivery with real-time fluorescence tracking property and avoiding self-aggregation and hence porphyrin release. Moreover, partially charged porphyrins tend to expose on the surface of nanoPSs, facilitating production and diffusion of 1O2. The highest 1O2 yield can be achieved with porphyrin loading as low as 6 wt%, reducing side effects of excessive porphyrin injection. The nanoPSs show enhanced PDT efficacy in vitro and in vivo leading to complete tumor eradication. This study highlights opportunities for development of active photodynamic nanoparticles and provides an alternative strategy for delivery of hydrophobic photosensitive drugs with enhanced therapeutic effects.


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With the advantages of minimal invasiveness, little systemic toxicity as well as high therapeutic selectivity and efficacy, photodynamic therapy (PDT) has emerged as an effective treatment for tumor eradication.1-2 In PDT process, photosensitizers (PS) can be activated under light irradiation with a proper wavelength to produce reactive oxygen species (ROS), particularly singlet oxygen (1O2) to induce cell death.3 The antineoplastic effects of PDT depend greatly on light absorptivity, ROS productivity and tumor accumulation of photosensitizers.4-5 Recently, despite the great progress in developing high-efficient photosensitizers especially porphyrin derivatives for PDT, the strong hydrophobicity of porphyrin renders them high propensity to self-aggregate in physiological conditions resulting in reduced bioavailability, limited light absorptivity, inefficient 1O2 productivity and poor tumor accumulation,6 which impedes PDT in further clinical application.7-8 Thus, to achieve a satisfying therapeutic effectiveness, excessive injection of porphyrins is required causing side effects such as skin phototoxicity which in turn restricts dose escalation and reducing antineoplastic effects.9 Various attempts have been reported to face this challenge among which employing nanoscaled carriers stands out to be an effective way.9-11 The nano-carriers may solve the problem of PS solubility and facilitate tumor accumulation through the enhanced permeability and retention effect (EPR effect).8,10 Yet for traditional nanocarriers, they have been plagued by long-term biosafety due to the non-bio-derivation and non-biodegradable feature for inorganic nanocarriers; or inefficient PS loading/releasing and poor cell uptake for organic nanocarriers.6 Besides, porphyrins encapsulated in nanocarriers still stand too close to each other causing selfaggregation problem and the generated 1O2 by the deep encapsulated porphyrin may not be able to diffuse and reach the cancerous tissue owing to its poor diffusion ability.9,12 To address the above problems, it is routine to employ external forces such as pH, magnetic field, enzymes,


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redox reaction or laser irradiation to stimulate the release of PS from nanocarriers13-17 which requires complex design and multi-steps fabrication of carrier systems. However, it is rather difficult to precisely control the release of porphyrin due to the complicated environment in vivo, individual differences and other uncontrollable factors, especially when it comes to practical clinical application. Thus, to monitor a satisfying drug delivery, tumor accumulation and cell endocytosis, loading of dyes in carriers is required for real-time tracking. Therefore, there is a desperate demand to develop monomeric porphyrin delivery that can not only avoid selfaggregation with fluorescent emission property, but more importantly eliminate the premature PS release process. Owing

to

the

facile

structural

programmability,

excellent

biocompatibility

and

biodegradability, short peptides, especially aromatic short peptides that are capped with the Fluorenylmethoxycarbonyl (Fmoc) group at the N-terminus, are capable of self-assembling into various supramolecular structures18-21 for applications in drug delivery, optoelectronic devices and cell culture22-26. In consideration of the super assembly capability, we herein introduce aromatic short peptide (Fluorenylmethoxycarbonyl-Leucine-Leucine-Leucine-OMe (Fmoc-L3OMe)) to co-assembly with porphyrin derivative meso-Tetra(p-hydroxyphenyl) porphine (mTHPP) forming supramolecular nanoparticles for enhanced antitumor treatment through solventshifting method (Scheme 1 and Figure S1 A). Similar to natural porphyrin/protein composites, the strong π-π stacking and hydrophobic interactions can trigger the co-assembly of peptide and porphyrin with optimized molecular arrangement to obtain functional optimization of porphyrin. The co-assembly of aromatic peptides and porphyrins results in photodynamic nanoparticles (denoted as nanoPS) within which porphyrin molecules are kept in monomeric form by the isolation of peptides, avoiding porphyrin aggregation and eliminating the necessity of stimuli-


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release process. More interestingly, porphyrin molecules show more tendency to expose on the surface of nanoPS owing to their partial protonation during the assembly process. This exposure facilitates light absorption, 1O2 productivity and diffusion, improves porphyrin bioavailability and further weakens the requirement of porphyrin release. The nanoPSs can achieve a high 1O2 productivity at a very low PS loading amount (6 wt%), which benefits for reducing side effects from the excess uptake of PS.

Scheme 1. Illustration of fabrication of nanoPSs and their application in PDT. The nanoparticles were prepared through the solvent shifting method, which involves three components, a hydrophobic component, an amphiphilic organic solvent that is fully miscible with water and a nonsolvent (water). First, hydrophobic component is dissolved in the amphiphilic solvent. Then the solution is quickly mixed with a large amount of water. Owing to the quick interdiffusion of the amphiphilic organic solvent and water, nucleation and growth of hydrophobic component spontaneously happens during the mixing process, leading to the


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formation of nanoparticles. Specifically, in this work, Fmoc-L3-OMe and m-THPP (hydrophobic oil) were first dissolved in DMSO (amphiphilic organic solvent) with different molar ratios. Then 50 µL of the above mixture was quickly added to 1000 µL water (nonsolvent) resulting in a homogeneous dispersion of peptide/porphyrin co-assembled nanoparticles with an obvious Tyndall Effect (Figure S1 B i). However, Fmoc-L3-OMe or m-THPP alone could not form stable nanoparticles as Figure S1 B ii and iii showed, which suggested that the interaction between peptide and porphyrin assists the formation and stabilization of the nanoparticles. The morphology of nanoPSs (sample 4) was characterized with scanning electron microscopy (SEM) (Figure 1A), transmission electron microscopy (TEM) (Figure 1B) and laser scanning confocal microscopy (CLSM) (Figure 1C), showing that the nanoPSs has an average diameter of about 160 nm which is in good consistence with the Dynamic Light Scattering (DLS) result (inserted in Figure 1C). The nano-scaled size was beneficial for nanoPSs to permeate and accumulate in tumor tissue, and the fluorescence signal (Figure 1C) endowed intracellular tracking and tumor diagnostic function of nanoPSs.


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Figure 1. Morphology characterization of nanoPSs (sample 4): A) SEM image, B) TEM image and C) CLSM image; the insert image is a DLS result; D) Zeta potential of samples with different molar ratio, E) Molar ratio of Fmoc-L3-OMe and m-THPP in different nanoPSs. The co-assembly of Fmoc-L3-OMe and m-THPP resulted in monodispersed nanoPSs due to the similar hydrophobicity. When a less hydrophobic molecule such as Fmoc-L-COOH (Figure S1 A) was used instead of Fmoc-L3-OMe, the fabrication of stable nanoparticles was frustrated, resulting in only fibrillar aggregation (Figure S1 B iv). Also, it has been reported that when hydrolyzing Fmoc-L3-OMe into Fmoc-L3-COOH, nanofibers instead of nanoparticles were obtained.27 The comparison suggested the importance of hydrophobic interaction between peptide and porphyrin in the formation and stabilization of nanoPSs.


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The zeta potential was used to investigate the stability of nanoPSs. By increasing the molar ratio between m-THPP and Fmoc-L3-OMe, as table in Figure 1E showed, the nanoPSs became positively charged with an increasing tendency along with the increase of m-THPP ratio (Figure 1D). For sample 1-3 (table in Figure 1E), owing to insufficient surface charge, the samples tended to aggregate in solution (Figure S2). However, with the increase of the m-THPP component, the nanoPSs exhibited great stability in water which reveals that the nanoPSs were stabilized by electrostatic repulsion. X-ray photoelectron spectroscopy (XPS) explained electropositivity of nanoPSs. The molecular structure (Figure S1 A) showed that there are equally two kinds of chemical environments for the N element in m-THPP. Thus, theoretically the XPS peak areas of N with the two chemical environments should be equal. The larger area at 399.53 eV (N-H in m-THPP) compared with that at 397.39 eV (N-C in m-THPP) in Figure S3 revealed that m-THPP in nanoPSs was partially protonated, causing the excess positive charge of nanoPSs. The precipitation of nanoPSs in PBS or NaCl solution (Figure S4 A) owing to the charge shielding effect further proved the importance of electrostatic interactions in nanoPSs stability. However, nanoPSs were stable in cell culture medium. This was probably caused by the adsorption of proteins onto the surface of nanoPSs, which led to charge reversal from positive to negative and size increase of nanoPSs (Figure S4 B). To investigate the influence by Fmoc-L3-OMe on the dispersion of m-THPP within nanoPSs, fluorescence spectra of nanoPSs were recorded with pure m-THPP dissolved in water as comparison. As Figure S5 A showed, the strong hydrophobicity of pure m-THPP resulted in severe self-aggregation without fluorescence emission (curve b in Figure S5 A), while a strong fluorescence can be detected for nanoPSs (curve a in Figure S5 A). The strong emission could be attributed to the fact that hydrophobic aromatic moieties of Fmoc-L3-OMe were able to isolate


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m-THPP molecules to prevent them from aggregation. With the increase of m-THPP molar ratio, the fluorescence intensities of nanoPSs firstly increased and then decreased (Figure 2A), and the highest fluorescence intensity could be obtained when the molar ratio of m-THPP and Fmoc-L3OMe was at 1:18, with m-THPP loading amount of 6 wt% (sample 4). We speculated the great influence of Fmco-L3-OMe on the mono-dispersion of m-THPP that at low molar ratio, m-THPP could be completely isolated from each other by the far excess amount of Fmoc-L3-OMe without aggregation induced fluorescent quenching. Thus, fluorescence intensity kept increasing with the increase of the m-THPP component. However, as the molar ratio of m-THPP/Fmoc-L3-OMe further increased exceeding 1:18, Fmoc-L3-OMe were not sufficient enough to keep all m-THPP in their monomeric state that the hydrophobic m-THPP started to aggregate resulting in fluorescence quenching. It is predictable that with m-THPP/Fmoc-L3-OMe molar ratio at 1:18 (sample 4), the co-assembly of m-THPP and Fmoc-L3-OMe achieves the optimized molecular organization to keep m-THPP monomeric for optimal function in PDT. Hence, nanoPSs with this special molar ratio will be used for further investigation.


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Figure 2 A) Fluorescence spectra of nanoPSs samples with different molar ratio and the inserted image is normalized FL; B) Molecular dynamic simulation result of the peptide-porphyrin coassembly. Red, grey and blue molecules are m-THPP, Fmoc-L3-OMe and water, respectively. The inside box typically represents hydrogen bonds between the hydroxyl group of m-THPP and water with the corresponding distances (Å); C) Absorption spectra of ABDA in the presence of nanoPSs under irradiation as a function of time; D) Normalized absorbance changes of ABDA caused by 1O2 oxidation, plotted against irradiation time, at 378 nm in the presence of different samples and the percentage at the end point (irradiation at 635 nm). It’s reported that the aggregation of porphyrins may result in a broadened Soret band.28 Thus the UV-Vis spectra were used to further investigate the aggregation of m-THPP. The broadened Soret band of pure m-THPP (curve c in Figure S5 B) indicated the severe aggregation of pure mTHPP in water. By contrast, the absorption of nanoPSs (sample 4) showed a relatively sharp and strong Soret band (curve b in Figure S5 B) compared with monomeric m-THPP (curve a in


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Figure S5 B) with no obvious bathochromic shift, suggesting little aggregation of m-THPP in nanoPSs. Interestingly, the nanoPSs showed an enhanced Q-band absorption (insert in Figure S5 B). As the peak wavelength of the Q-band fell into the phototherapeutic window (600-850 nm), the enhanced intensity of Q-band absorption indicated an improved light absorption efficiency for PDT. The increase of the positive charge of nanoPSs with the increase of m-THPP ratio implied that m-THPP molecules were more likely to expose on the surfaces of nanoPSs. For further confirmation of this experimental result as well as the monomeric assembly of m-THPP within nanoPSs demonstrated by the fluorescence spectra and UV-Vis spectra, molecular dynamic (MD) simulation was employed to investigate the co-assembly behavior of m-THPP and Fmoc-L3OMe molecules with molar ratio at 1:18 (sample 4). As a qualitative model, 5 porphyrins and 90 peptide molecules were contained in a water box sized 15.9 × 15.9 × 11.2 nm3 in which the molar ratio between porphyrin and peptide corresponded to the experiment. The simulation results after running 150 ns (Figure 2B) confirmed that the Fmoc-L3-OMe and m-THPP molecules could co-assemble into nanoparticles along with m-THPP preferring a surficial position. Each m-THPP molecule formed approximately 6 pairs of hydrogen bonds with water indicating the major reason for m-THPP surficial distribution and stability of nanoPSs. More importantly, the simulation results verified that m-THPPs were rather dispersed as monomers in nanoPSs than self-aggregated to oligomers. It could be explained that m-THPP and Fmoc-L3OMe exhibited similar hydrophobicity and tended to co-assemble between each other for optimized molecular organization. The m-THPP molar ratio was much lower than that of FmocL3-OMe, rendering m-THPP well-distributed in balance within nanoPSs. The π-stacking and hydrophobic intramolecular interactions between m-THPP and Fmoc-L3-OMe played a dominant


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role for co-assembly process and nanoPS formation. All the above results and simulation verified the monomeric assembly of m-THPP on the surficial position of nanoPSs. The superficial assembly of monomeric m-THPP within nanoPSs could be expected to exhibit enhanced PDT antitumor efficiency. We firstly investigated the 1O2 production capacity of nanoPSs to prove the efficiency of PDT.29-30 9,10-anthracenediylbis (methylene) dimalonic acid (ABDA) was employed as 1O2 probe to investigate 1O2 productivity of nanoPSs by using pure mTHPP in water as comparison (Figure S6 B). It’s known that the absorbance intensity of ABDA at 378 nm decreases with increase of 1O2 concentration in the solution.5 As Figure 2C shows, under laser irritation at 635 nm, the relative absorbance of ABDA at 378 nm for nanoPS (sample 4) decreased rapidly to 35.7% within 30 min, which was much faster than that of pure m-THPP in water (about 82.8%) (Figure 2D and Figure S6 B) indicating the high 1O2 production efficiency of nanoPSs. The severe m-THPP aggregation should be responsible for the low 1O2 productivity of pure m-THPP in water, which is inimical for PDT application. While for mTHPP within nanoPSs, the isolation by Fmoc-L3-OMe assisted m-THPP to exist as monomers to maintain the photoactivity and promote 1O2 production. The 1O2 productivity of sample 6 nanoPSs with higher porphyrin/peptide ratio was also measured for comparison. As Figure 2C, D and Figure S6 A shown, although the amount of m-THPP in sample 4 was smaller than that in sample 6, the 1O2 yield of sample 4 was dramatically enhanced. The low 1O2 yield of sample 6 further proved the aggregation induced inactivation of photosensitizers and the necessity of monomeric photosensitizer delivery for effective PDT. It is not necessary to use the highest amount of PS but the proper amount for an effective PDT cancer treatment. Our result suggested that we could achieve a highly effective PDT with PS loading amount as low as 6 wt%, reducing side effects of excessive PS injection.


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The strong fluorescence emission (Figure 1C) endowed the nanoPSs with intracellular tracking as well as tumor diagnosis properties. We next utilized the fluorescence signals to exploit endocytosis behavior of nanoPSs. After co-culturing with nanoPSs, the cytoplasm of the MCF-7 cells showed strong red fluorescence emission (Figure 3A, left) indicating that a large amount of nanoPSs could be captured by cancer cells and nanoPSs were most likely localized in the cytoplasm. The observation of red emission in both XZ and YZ cross section in the 3D reconstruction image (Figure 3A, right) further confirmed the successful uptake of nanoPSs into cancer cells. We further exploited the 1O2 production ability of nanoPSs in cells with 2,7Dichlorodihydrofluorescein diacetate (DCFH-DA), a redox sensitive fluorescence probe, which could be degraded in cells to produce DCFH (2,7-Dichlorodihydrofluorescein). The DCFH could further be oxidized by 1O2 into DCF (2,7-Dichlorofluorescein), exhibiting a strong green fluorescent signal.31 As shown in Figure S7 A, without laser illumination the DCFH-DA stained cells exhibited no fluorescence signal. After irradiation at 635 nm for 20 min, a strong green fluorescence could be observed (Figure S7 B). The well-defined boundary between the green (with light) and dark (without light) areas (Figure S7 C) suggest efficient 1O2 production ability of nanoPSs under light illumination and little dark toxicity of nanoPSs.


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Figure 3 A) CLSM images of MCF-7 cells stained with nanoPS suspensions after 4 h of coculturing (left); 3D reconstruction image of the cells with nanoPSs (the orthogonal section images at the bottom and on the right were recorded along the yellow lines) (right); B) Viability of MCF-7 cells with (red) and without (black) 635 nm wavelength light irradiation; C) The CLSM images of MCF-7 cells stained with propidium iodide and treated with (i) laser irradiation and nanoPSs; (ii) laser irradiation only (left of i and ii: images collected at 575-630 nm; right i and ii: transmission light images; area in the red frame was treated with 635 nm light) (NanoPSs of sample 4 were used in the above experiments). The antitumor efficiency of nanoPSs was next proved in vitro with MCF-7 cancer cells by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide cell survival assay (MTT). As Figure 3B showed, after laser illumination for 10 min, the photo-cytotoxicity of nanoPSs enhanced with the increase of nanoPSs concentration suggesting a high anti-tumor efficacy of nanoPSs. The high cell viability in dark situation indicated low cytotoxicity and great biocompatibility of nanoPSs. The anti-tumor efficacy of nanoPSs in vitro was further evaluated by fluorescence imaging under laser excitation at 635 nm. For easy observation of the cell apoptosis, propidium iodide (PI) which can specifically stain dead cells was used. After exposure to 635 nm laser light for 20 min, the irradiated area of cells changed a lot in morphology and gave a strong red fluorescence emission (Figure 3Ci, area within the red frame), indicating that the irradiation of laser induces the death of cancer cells. Cells with nanoPSs without laser treatment showed no significant morphology change and fluorescence emission indicating low dark toxicity of nanoPSs (Figure 3Ci, area out of the red frame). As comparison, cells with no nanoPSs uptake showed no fluorescence signal after laser exposure (Figure 3C ii) proving that laser alone cannot cause cell death. Further, the anti-tumor efficacy of nanoPSs was evaluated in vivo by using the MCF-7 cancer bearing mice model. It’s prerequisite that PS could selectively accumulate at tumor sites. Guiding from the result of real-time imaging in vivo, we found that nanoPSs were able to selectively accumulate at the tumor site in 8 h after injection (Figure 4A) and the fluorescence


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signal at the tumor sites was sustained for at least 24 h (Figure 4A) indicating the accumulation and retention of nanoPSs in tumor tissues. After irradiation with 635 nm light for 10 min, the tumors of nanoPS-treated mice were suppressed and completely eradicated within two weeks (Figure 4B and D). No significant body weight change (Figure 4C) and organ damage (Figure S8) were observed after PDT treatment, confirming the good biocompatibility and biosafety of the nanoPSs. Thus, both the in vitro and in vivo anti-tumor experiments confirmed that without complicated design for PS release, we achieved a high PDT anti-tumor efficiency simply through the monomeric delivery of porphyrin with the assistance of peptide self-assembly.

Figure 4 A) Body fluorescence image of MCF-7 cancer bearing mice at different times; B) The curve of tumor growth inhibition; C) The curve of body weight changes; D) Representative photos of mice after various treatments at different time points (NanoPSs of sample 4 were used in the above experiments).


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In conclusion, the aromatic peptide Fmoc-L3-OMe and porphyrin derivative m-THPP are coassembled to fabricate photodynamic reactive nanoparticles, denoted as nanoPSs. By regulating the co-assembly molar ratio, we could obtain an optimal molecular organization between mTHPP and peptide, where m-THPP molecules could maintain as monomers within nanoPSs by the hydrophobic peptide isolation to achieve monomeric porphyrin delivery. Owing to the partial protonation during the assembly process, m-THPP is more likely to expose on the surface of nanoPSs which is confirmed by both experiments and molecular simulation. As there is no selfaggregation of m-THPP within nanoPSs, the requirement of porphyrin release could be eliminated and the fluorescence emission of monomeric PSs endowed real-time tracking and tumor diagnostic function of nanoPSs. The exposure of m-THPP on the nanoparticle surface is in favorable for light absorption, 1O2 production and diffusion, improving bioavailability of mTHPP. The highest 1O2 productivity can be achieved with a rather low PS loading (6 wt%), reducing side effects from the excess uptake of PS. Both in vitro and vivo experiments demonstrate the excellent biocompatibility, good tumor selectivity and high anti-tumor efficiency. There are opportunities to design peptides to fully biocompatible molecules by replacing the aromatic components or using unmodified self-assembling peptides. This could extend the possibilities to facilitate encapsulation and compartmentalization of hydrophobic analogues such as hydrophobic drugs with applications in drug delivery and cancer therapy in potential clinical use. ASSOCIATED CONTENT Supporting Information. The following files are available free of charge.


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Materials and supplementary methods, supplementary data and figures(PDF)

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]; [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Jieling Li and Shuo bai conceived and designed the experiments, and Jieling Li performed the fabrication and characterization of the nanoparticles. Luyang Zhao contributed the molecular dynamics simulation. Jieling Li, Qianqian Dong, and Meiyue Wang conducted the antitumor experiments. Anhewang, HaolanXu, Shuo Bai and Xuehai Yan were responsible for the statistical analysis. Jieling Li, Shuo Bai and Xuehai Yan cowrote the manuscript. Notes The authors declare no competingfinancial interest. ACKNOWLEDGMENT We greatly thank Dr. Dongdong Qi at University of Science and Technology Beijing for a grant use of computations. The authors acknowledge ﬁnancial support from the National Natural Science Foundation of China (Project No. 21522307, 21774132, 21703253, 21644007 and 21473208) and the Talent Fund of the Recruitment Program of Global Youth Experts.


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ABBREVIATIONS Fmoc-L3-OMe, Fluorenylmethoxycarbonyl-Leucine-Leucine-Leucine-OMe; m-THPP, mesoTetra(p-hydroxyphenyl) porphine; ABDA, 9,10-anthracenediylbis (methylene) dimalonic acid; DCFH-DA, 2,7-Dichlorodihydrofluorescein diacetate. REFERENCES [1] Brown, S. B.; Brown, E. A.; Walker, I. The Present and Future Role of Photodynamic Therapy in Cancer Treatment. Lancet Oncol. 2004, 5, 497-508. [2] Dolmans, D.; Fukumura, D.; Jain, R. K. Photodynamic Therapy for Cancer. Nat. Rev. Cancer. 2003, 3, 380-387. [3] Pass, H. I. Photodynamic Therapy in Oncology-Mechanisms and Clinical Use. J. Natl. Cancer. 1993, 85, 443-456. [4] Zou, Z.; Chang, H.; Li, H.; Wang, S. Induction of Reactive Oxygen Species: An Emerging Approach for Cancer Therapy. Apoptosis. 2017, 22, 1321-1335. [5] Zhou, Z.; Song, J.; Nie, L.; Chen, X. Reactive Oxygen Species Generating Systems Meeting Challenges of Photodynamic Cancer Therapy. Chem. Soc. Rev. 2016, 45, 6597-6626. [6] Rajora, M. A.; Lou, J. W. H.; Zheng, G. Advancing Porphyrin's Biomedical Utility Via Supramolecular Chemistry. Chem. Soc. Rev. 2017, 46, 6433-6469. [7] Chatterjee, D. K.; Fong, L. S.; Zhang, Y. Nanoparticles in Photodynamic Therapy: An Emerging Paradigm. Adv. Drug. Deliver. Rev. 2008, 60, 1627-1637. [8] Bechet, D.; Couleaud, P.; Frochot, C.; Viriot, M. L.; Guillemin, F.; Barberi-Heyob, M. Nanoparticles as Vehicles for Delivery of Photodynamic Therapy Agents. Trends in Biotechnol. 2008, 26, 612-621.


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Self-Assembly of Monomeric Hydrophobic Photosensitizers with Short

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