Design of an Amphiphilic iRGD Peptide and Self-assembling

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Functional Nanostructured Materials (including low-D carbon)

Design of an Amphiphilic iRGD Peptide and Self-assembling Nanovesicles for Improving Tumor Accumulation and Penetration and the Photodynamic Efficacy of the Photosensitizer Yue Jiang, Xin Pang, Ruiling Liu, Qicai Xiao, Pan Wang, Albert Wingnang Leung, Yuxia Luan, and Chuanshan Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11699 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 22, 2018

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ACS Applied Materials & Interfaces

Design of an Amphiphilic iRGD Peptide and Self-assembling Nanovesicles for Improving Tumor Accumulation and Penetration and the Photodynamic Efficacy of the Photosensitizer Yue Jiang,†,‡ Xin Pang,‡ Ruiling Liu,† Qicai Xiao,‡ Pan Wang,‡ Albert Wingnang Leung,ǁ Yuxia Luan,† Chuanshan Xu∗*,‡,§ §Key Laboratory of Molecular Target and Clinical Pharmacology, School of Pharmaceutical Sciences & Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 511436, P. R. China †School of Pharmaceutical Science, Shandong University, 44 West Wenhua Road, Jinan, 250012, Shandong, P. R. China ‡School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, 000000, P. R. China ǁDivision of Chinese Medicine, School of Professional and Continuing Education, The University of Hong Kong, Hong Kong, 000000, P. R. China *Email: [email protected] (C. Xu)

Keywords: iRGD, peptide amphiphiles, self-assembly, tumor targeting and penetration, fluorescence imaging, photodynamic therapy

Abstract Photodynamic therapy (PDT) is a minimally invasive treatment for many diseases, including infections and tumors. Nevertheless, clinical utilization of PDT is severely

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restricted due to the shortcomings of the photosensitizers, especially their low water solubility and poor tumor selectivity. iRGD(internalizing RGD, CRGDKGPDC), a nine-unit cyclic peptide, has been applied as an active ligand to realize tumor homing and tissue penetration. Herein, we innovatively fabricated a novel OFF-ON mode iRGD-based peptide amphiphile (PA) to self-assemble into spherical nanovesicles to enhance the tumor-targeting and tumor-penetrating efficacy of PDT. To introduce the self-assembling feature into iRGD, a hydrophilic arginine-rich sequence and hydrophobic alkyl chains were sequentially linked to the iRGD motif. A short proline sequence was selected to control the morphology of the self-assembled aggregates. Next, the photosensitizer hypocrellin B (HB) was encapsulated into PA vesicles with a high loading efficiency. The aggregation-caused quenching (ACQ) effect inactivated HB in the PA vesicles; however, the iRGD-peptide-based material was able to be selectively degraded in tumor cells. Thus, the HB fluorescence was recovered to achieve tumor-targeted imaging. This approach endows HB-loaded PA vesicles (HB-PA) with tumor-targeted activation, preferable tumor accumulation and deep tumor penetration, thus leading to an excellent fluorescence-imaging-guided photodynamic efficacy both in vitro and in vivo. These amphiphilic iRGD aggregates provide a novel strategy for improving the accumulation, penetration and imaging-guided photodynamic efficacy of photosensitizers.

1. Introduction Photodynamic therapy (PDT) is a promisingly alternative tumor treatment that can effectively eliminate tumors via reactive oxygen species (ROS) induced by


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photosensitizers (PSs) under light irradiation.1,2 However, clinical application of PDT is severely restricted due to the shortcomings of PSs, including their low water solubility and poor tumor selectivity. In fact, most PSs often accumulate in the skin and healthy tissues, resulting in non-specific phototoxic damages.3-5 In this regard, PSs that are specifically activated to produce ROS only in targeted areas are highly desirable because they offer selectivity and increase the safety of PDT.6 Therefore, various nanoscale delivery systems have been designed to address these problems based on passive and active tumor-targeting strategies.7-10 On the other hand, some efforts are ongoing to control the activity of PSs using an OFF-ON mode.11-13 For this purpose, the commonly used approach is based on the aggregation-caused quenching (ACQ) effect. PSs are pre-quenched and presented in the OFF state during circulation. When the system reaches the targeted tumor tissues or cell interior, PSs exert their effect in the monomeric state due to dissociation induced by the tumor microenvironment. Thus, the photosensitization activity of the system switches ON and provides high ROS production in specific tumor tissues.14-16 Nevertheless, most of the widely used carriers are prepared via tedious processes or exhibit poor physicochemical stability, low drug-loading efficiency and, especially, insufficient penetration into tumor tissues (only 3-5 cell diameters).17,18 Therefore, an easily fabricated delivery system with specific PS accumulation, activation and high tumor penetration is highly desirable. iRGD (internalizing RGD, CRGDKGPDC), a nine-unit cyclic tumor-homing and tissue-penetrating peptide, can bind to integrin receptors (αvβ3, αvβ5, and αvβ1), which



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are highly expressed in tumor endothelial cells (TECs) and tumor cells.19,20 After binding, iRGD can be cleaved by the tumor-associated protease, which exposes its C-terminal CendR motif (CRGDK/R). This motif can bind to the neuropilin-1 receptor (NRP-1), a trans-membrane receptor glycoprotein shared by TECs and tumor cells that mediates its penetration into the interior of tumors.21,22 iRGD has been applied in delivery systems to improve the tumor-targeting and tumor-penetrating ability

of

drugs

through

several

approaches,

such

as

co-administration,

post-modification, and co-precipitation of iRGD with other materials.23-27 Among these strategies, the fabrication processes are often tedious and/or they may require many other excipients to obtain the expected formulations. Self-assembly is an effective and versatile method to fabricate novel nanoformulations.28-30 However, self-assembling iRGD-based amphiphilic molecules have rarely been reported. In this sense, amphiphilic iRGD self-assembled aggregates may serve as a highly advantageous platform for the targeted delivery of PSs. Herein, an iRGD peptide amphiphile (PA) was designed to entrap HB, a photosensitizer isolated from Hypocrella bambusae, into assembled vesicles to improve its tumor accumulation, penetration and antitumor efficacy. To achieve self-assembly, the iRGD targeting motif was chemically modified with a hydrophilic arginine-rich sequence and hydrophobic alkyl chains sequentially. A short proline sequence was inserted to obtain the expected spherical assemblies (Scheme 1). We further hypothesize that ROS production via HB would be significantly inhibited due to the ACQ effect induced by the extremely high HB loading efficiency. After selective delivery to tumor tissues,


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the aggregates are dissociated by tumor proteases, such as matrix metalloproteinase and cathepsin B, and the encapsulated HB are released and its photosensitization recovered. In this manner, HB-loaded PA aggregates (HB-PA) are able to realize tumor-specific activation of PS, as well as improved tumor penetration and an improved photodynamic efficiency. In breast tumor models, as expected, the OFF state HB-PA accumulated in the targeted tumor sites and HB photosensitization was switched

ON,

which

resulted

in

a

significant

fluorescence-image-guided

photodynamic efficacy. This work provides an efficient strategy to solve the common bottleneck encountered by PDT and realizes an excellent tumor-targeted and fluorescence-image-guided photodynamic therapy.

2. Results and discussion Design strategy of the iRGD-based amphiphilic peptide To achieve the self-assembly feature, the amphiphilic iRGD peptide was designed to contain both hydrophobic and hydrophilic regions (Scheme 1). A cationic arginine-rich (R6) sequence was used as the hydrophilic head group to improve water solubility and aid intracellular lysosomal escape. The hydrophobic domain was composed of two stearic acid chains to ensure sufficient tail volume according to the packing parameter theory. Although several studies focus on cylindrical nanotubes and nanofibers with high aspect ratio, spherical particles are preferred in many biological processes when efficient cell internalization and trafficking is required.31 To date, biocompatible and biodegradable vesicular systems are the most successful carriers.32 Their enclosed bilayers enable them to simultaneously encapsulate both



hydrophilic and hydrophobic agents. In this study, we focus on a vesicular system for delivering photosensitizers. A short proline sequence (P4) is inserted between the hydrophobic and hydrophilic segments as a spacer to obtain spherical self-assembled aggregates.

Scheme 1. Schematic representation of the iRGD-based peptide amphiphile (PA) and the encapsulation of HB.

Preparation and characterization of PA The designed peptide amphiphile is obtained via an effective and easy solid phase method. The chemical structure of the synthesized self-assembling iRGD peptide


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amphiphile (PA) was characterized via MALDI-TOF-MS and 1H-NMR (Figure S1-S3, Supporting Information). Circular dichroism was used to determine the secondary structures. The peak (Figure 1a) at 200 nm revealed PA-adopted random-coil structures.33 There was no hydrogen bonding (β-sheet) among the peptide units; therefore, electrostatic repulsion and hydrophobic interactions were the major driving forces for the self-assembled structures. Critical aggregation concentration (CAC) is an important solution property of amphiphiles. Figure 1b shows the fluorescence intensity ratios of pyrene emission spectra versus logarithm of PA concentration levels. The CAC value of our PA was determined to be approximately 5×10-6 mol·L-1, which was much lower than that of conventional surfactants. When the PA concentration was above the CAC, spherical vesicles formed with a mean diameter of 212.7 nm (Figure 1c). The PA solution exhibited a high degree of positive potential (52.08 ± 2.03 mV) that was attributed to the arginine-rich sequence of the molecules, which provides the possibility for improved tumor penetration, lysosomal escape and gene delivery.



Figure 1. (a) Circular dichroism spectra of PA and HB-PA; (b) determination of the CAC of PA; (c) size distribution of PA and HB-PA.

HB encapsulation and HB-PA characterization HB was then entrapped into the assembled vesicles, and it exhibited an average diameter of 183.4 nm. The 30 nm decrease in size after loading HB was most likely due to the electrostatic attraction between anionic HB (hydroxyl-group-derived negative charge) and cationic PA in the solution, which could make the vesicle structure more intact. Recent studies show that nanoparticles with diameter larger than 100 nm have a limited penetration in hypovascular and hypopermeable tumors.34 There are several elements other than particle size and surface charge that govern penetration depth, such as particle shape, rigidity and targeting ligand. In particular,


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ligand-installation plays a critical role in the tumor distribution of nanoparticles. The presence of iRGD provides a driving force that promotes inward penetration in proportion to superficial iRGD density and the surface area of the nanoparticles. Given that HB-PA bears efficient iRGD molecules, it is expected to exhibit promotive intratumoral infiltration regardless of its slightly large size. The HB loading content was optimized to (43.44 ± 1.01)% with a much lower positive charge (30.35 ± 4.06 mV) than that of blank PA vesicles (Table S1, Supporting Information). HB and HB-PA exhibited negligible hemolysis toxicity (less than 5%) in the concentration range studied, which indicated good biocompatibility and biosafety for HB-PA (Figure S4, Supporting Information). The slight increase in hemolytic toxicity of high-concentration HB and HB-PA was mainly caused by the increase in UV signals at 540 nm with increase in concentration. The low hemolytic toxicity of HB-PA could be explained by the high drug loading capacity of HB, which significantly decreased the concentration of cationic PA used in the final formulation. Transmission electron microscopy (TEM) clearly showed spherical vesicles with a diameter of approximately 200 nm (Figure 2a). Differential scanning calorimetry (DSC) was performed to test the effect of incorporation of HB in the thermotropic behavior of vesicles. In Figure 2b, the melting point of crystalline HB appeared at 249.8 °C. PA gave rise to endothermic peaks, a small peak at approximately 156.7 °C and a broad endothermic peak at 252.0 °C. The thermogram of the physical mixture was similar to that of individual HB and PA, which indicated that there may be a lack of interaction between HB and PA in the physical mixture. When HB molecules were



embedded in PA, the sharp endothermic peak at approximately 250 °C that corresponded to the melting of HB almost disappeared, which demonstrated that HB was converted from the crystalline to the amorphous form and was successfully entrapped into the nanovesicles. The UV-vis curve of HB-PA was identical to the shape of the curve for HB-DMSO solution, which indicated that PA could successfully entrap HB and significantly increase its solubility in aqueous solution (Figure 2c). A slight red shift was possibly due to the change in the molecular environment surrounding HB. In Figure 2d, fluorescent signals of HB-PA switched OFF in solution, which was caused by the large amount of HB molecules entrapped in PA vesicles. ROS generation was analyzed via 9, 10-anthracenediyl-bis (methylene) dimalonic acid (ABDA) quenching at 380 nm with light-irradiation time. The results (Figure 2e) indicated that the HB-PA produced considerably less ROS under light irradiation compared with those produced in the HB-Tween-80 solution, but produced a slightly higher amount of ROS than those produced by HB in the DMSO/H2O mix solvent. This result could be explained by the incomplete quenching of HB-PA, which was likely attributed to the HB molecules that absorbed on the outer surface of the PA vesicles.


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Figure 2 (a) TEM images of HB-PA, left: low-power microscopic appearance, right: higher power microscopic appearance; (b) DSC curves of PA, HB, HB and PA mixture (HB+PA) and HB-PA; (c) UV-Vis absorption and fluorescence spectra (d) of HB and HB-PA; (e) ROS levels of each group monitored by the decay of ABDA.



Dark toxicity and intracellular fate of HB and HB-PA Dark toxicity of PA, HB and HB-PA to MDA-MB-231 tumor cells and NIH-3T3 normal cells was analyzed before the PDT analysis. Figure 3a shows that toxicity of PA to the two cell lines was not obvious in the dark. The viability of PA-treated NIH-3T3 cells was higher than that of MDA-MB-231 cells, which could be explained by the increased intracellular PA accumulation in tumor cells due to the iRGD motif. Although high-positive-charge carriers often cause high toxicity, the dose of PA that we used in the system was very limited because the drug loading was extremely high. Thus, the toxicity of PA in normal cells could be minimized even if the drug concentration was 64 µM. The viability of cells treated with HB and HB-PA was dose-dependent. Free HB exhibited toxicity to both tumor and normal cells due to its poor selectivity. As expected, HB-PA was more toxic to tumor cells than NIH-3T3 cells. For the following photodynamic study, we set the concentration as 2 µΜ because HB and HB-PA proved safe for both tumor and normal cells at 2 µΜ. Confocal laser scanning microscopy (CLSM) was used to investigate the intracellular accumulation of HB-PA and free HB in 2D cells. Results (Figure 3b) showed that the fluorescence intensity of HB-PA-treated MDA-MB-231 cells was much higher compared with that in HB-treated cells. The bright fluorescent signal of HB-PA-treated cells demonstrated that the fluorescence of PS turned ON after HB-PA was transported into the cells. Additionally, pre-incubation with the iRGD peptide inhibited the uptake of HB-PA, which indicated that HB-PA was transported into tumor cells via receptor-mediated endocytosis. Compared with tumor cells, NIH-3T3


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cells showed a weaker uptake of HB and HB-PA. Flow cytometry provided similar results (Figure S5, Supporting Information). We also quantitatively studied the intracellular HB release of HB-PA at different time points by detecting the intrinsic fluorescence signals of HB in cells. The results (Figure S6, supporting information) indicated that the PA vesicles were gradually degraded in tumor cells and that they released HB into the interior of the cells, which exhibited a signal that was two times higher than that of normal cells. This result indicated that PA could be specifically degraded in tumor cells, which selectively switched the fluorescent signals ON. Mitochondria are recognized as the preferential subcellular targets for phototherapy, while lysosomes are also attractive antitumor targets that are involved in cancer progression and drug resistance.35,36 Therefore, light-induced damage to lysosomes and mitochondria is a promising strategy to eradicate cancer cells. Co-localization experiments (Figure 3c) showed that both HB and HB-PA localized within lysosomes and mitochondria. HB distributed mainly in mitochondria, which was consistent with the results from previous studies.37 HB-PA exhibited stronger lysosomal signals with a yellow or orange fluorescence in the merged images, which indicated that HB-PA was transported into the cells via receptor-mediated endocytosis. In lysosomal degradative environments, the HB-PA were turned ON to exhibit its imaging signals, further allowing the subsequent distribution of HB-PA in the mitochondria. Therefore, the accumulation of HB-PA in both lysosomes and mitochondria was considerably more than that of HB.



Figure 3. (a) Dark toxicity (%) of PA, HB, and HB-PA in tumor and normal cells under different concentrations. (b) Intracellular uptake of HB and HB-PA in two cell lines. Cell nuclei were counter-stained with Hoechst 33342. (c) Intracellular localization of HB and HB-PA in tumor cells after a 4 h incubation.

Photodynamic efficacy of HB-PA under light irradiation


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Photodynamic activity of HB and HB-PA in 2D cancer cells was found to be dependent on the light dose (Figure 4a). HB or HB-PA pre-incubated cancer cells began to die obviously when they were irradiated for more than 4 s. The photodynamic activity of HB-PA was considerably higher than that of HB. After pre-incubation with iRGD for an hour, the photocytotoxicity of HB-PA was distinctly inhibited. These results demonstrated that the improved photodynamic activity of HB-PA was derived from the enhanced intracellular accumulation caused by the tumor-targeting ability of iRGD. ROS production was evaluated, and the results are illustrated in Figure 4b. The groups of untreated cells, PS-treated cells and light-treated cells produced negligible ROS. A marked right shift of HB and HB-PA with light irradiation could be observed. The HB-PA/PDT treatment group showed substantially higher fluorescence intensity. The results also indicated that HB-PA could turn ON the fluorescent and photodynamic activity and generate more ROS after transportation into the cells.

Figure 4. (a) Photocytotoxicity of HB and HB-PA in MDA-MB-231 cells, *P < 0.05. (b) Intracellular ROS generation measured via flow cytometry.



Mitochondria are organelles that produce ROS, and they play a key role in PDT-induced apoptosis. Therefore, the mitochondria membrane potential (MMP) after PDT was evaluated via flow cytometry using a cationic dye, DiOC6(3). Figure 5a presents a remarkable attenuation of MMP in cells treated with PS (HB or HB-PA) and light irradiation compared with that in other groups, which indicates that mitochondrial function was impaired more severely by photo-activated HB-PA in MDA-MA-231 cells. PDT-induced apoptosis was detected using Hoechst 33342 nuclear staining (Figure S7, Supporting Information) and Annexin V-FITC/PI kit. In Figure S7, typical features of apoptosis, such as cell shrinkage and nuclear condensation, presented 16 h after PDT treatment. Flow cytometric analysis (Figure 5b) revealed that early- and late-phase apoptosis significantly increased, and the normal cells remarkably reduced after PDT treatment. HB-PA-mediated PDT markedly increased late apoptosis in cells compared to that in the HB-mediated group, which indicated that HB-PA-mediated PDT exhibited a better therapeutic efficacy than that of free HB.


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Figure 5. (a) Mitochondrial dysfunction and (b) apoptosis in tumor cells after PDT treatment.

HB-PA penetration in 3D tumor spheroids The restricted interstitial penetration of therapeutic agents is a major factor for the failure of tumor treatment.38 Multicellular tumor spheroids (MCTSs), which are a 3D tumor model that mimic the actual pathophysiological aspect of solid tumor tissues, have been proven to provide accurate predictions in evaluating drug penetration into tumor tissues.39,40 Herein, we constructed 3D breast cancer spheroids to investigate the penetration of HB and HB-PA into spheroids using CLSM. Figure 6a shows that HB fluorescence signal declined dramatically at a z-axis distance of 40 µm from the periphery of the spheroids. In contrast, the HB-PA group demonstrated a penetration



depth that was approximately 3 times that observed for HB (at an approximate depth of 120 µm), which implied that HB-PA exhibited a better tumor penetration ability than that of free HB. Notably, HB distributed on the peripheral area of the MCTSs at a z-axis of 80 µm with very weak signals. In contrast, HB-PA distributed throughout the MCTSs at the same z-axis distance (Figure 6b, Figure S8, Supporting Information). The distribution of HB and HB-PA in the MCTSs was further evaluated by detecting the fluorescence intensity of the central area, as indicated in Figure 6c. HB-PA accumulation at 70 µm was slightly higher than the maximum HB accumulation in the central area at 20 µm z-distance, which suggested that HB-PA significantly improved the tumor penetration and accumulation. To confirm the role of iRGD, iRGD pre-incubation was performed, which blocked the penetration of HB-PA into spheroids (30-40 µm, Figure S9, Supporting Information); this revealed that the iRGD targeting moiety of PA could significantly facilitate HB-PA penetration into the 3D tumor spheroids. In this work, the addition of iRGD peptide blocked the penetrating ability of HB-PA, which differed from co-administration of iRGD with other non-iRGD-modified carriers. Because the PA is based on iRGD, the addition of a high concentration of iRGD could saturate the integrin receptors on the cell surfaces, thus, blocking the penetrating ability of the iRGD motif in HB-PA. K. Wang et al also reported similar results; intracellular uptake of their iRGD-PPCD (conjugations) was significantly inhibited by free iRGD.41 To investigate the growth of spheroids after PDT treatments, the optical images of spheroids were monitored for 7 days. The results indicated (Figure 6d) that tumor


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spheroids grew fast and became more compact in the absence of any treatment. In contrast, HB-mediated PDT-treated spheroids became distorted, with some cells dissociating from the main region, which indicated that the outer layer cells were destroyed due to the cytotoxic effect of HB-PDT. It was observed that the HB-PA-mediated PDT treatment exhibited a more significant inhibition on spheroid growth. The spheroids disintegrated and almost lost their 3D structure. The results suggested that PDT utilizing the tumor-penetrating HB-PA displayed a considerably stronger growth-inhibitory effect on 3D tumor spheroids compared with that of PDT utilizing free HB.



Figure 6. (a) CLSM examination of the HB and HB-PA distributions in MCTSs. The z-stack images were scanned from the top to the middle of the spheroids per 10 µm. (b) 2.5D model of the HB and HB-PA distributions at 80 µm of MCTSs. (c)


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Fluorescent intensity of MCTS’s central region versus the z-axis distance. (d) Growth inhibition of the tumor spheroids with HB- and HB-PA-mediated PDT.

In vivo imaging and biodistribution of HB-PA To further assess the tumor accumulation of HB-PA, in vivo fluorescence imaging of HB and HB-PA was monitored at different time points after intravenous injection. As shown in Figure 7a, HB distributed quickly throughout the whole body within 1 h and was also cleared rapidly. The fluorescent signals for HB-PA mainly accumulated at the tumor site and the liver, and the signal intensity was much stronger than that of free HB. HB-PA gradually localized in tumor tissues from 1 h and tended to reach a maximum at 4 h. This HB-PA accumulation could be attributed to the iRGD-enhanced tumor targeting and penetration. Moreover, the HB-PA exhibited lasting fluorescence at the tumor sites with only a minor decrease in intensity after 12 h of injection, which indicated the long circulation and retention effect of HB-PA at the tumor sites. The β-phase half-lives (t1/2β) were (1.26 ± 0.07) and (7.08 ± 1.49) h for free HB and HB-PA, respectively, according to the pharmacokinetic profile (Figure S10). The pharmacokinetic data supported that the long circulation of HB-PA and sustained HB release might be one reason for the slight background of the in vivo imaging, however, which also contributed to the increased tumor accumulation and long-time tumor retention of HB. Ex vivo fluorescence images of major organs at 24 h post-injection are also presented in Figure 7b. Although some liver accumulation was observed, the major fluorescence was still observed in the tumor tissue, and the fluorescent intensity



of HB-PA was much stronger than that of HB, which was in accordance with the results from in vivo imaging. All the above results indicated that HB-PA with a suitable size and iRGD targeting motif could selectively accumulate at tumor sites through the EPR and active-targeting effects. Based on the results presented in Figure 7, the optimum photodynamic effects of HB-PA and HB could most likely be achieved through light irradiation at 4 h and 1 h post-injection, respectively.

Figure 7. (a) In vivo image of HB and HB-PA at various time points. Tumor sites are marked with a black circle. (b) Ex vivo images of the major organs and tumors at 24 h after HB-PA and HB injection. Number 1-6 represents the heart, liver, spleen, lung, kidney and tumor, respectively.

In vivo antitumor efficacy of HB-PA


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The in vivo PDT efficiency was investigated using a breast tumor xenograft model in balb/c mice. As shown in Figure 8a, after three treatments, light or photosensitizer alone showed negligible tumor inhibition, while HB-PA together with light irradiation exhibited the highest tumor suppression compared with those of all of the other groups. To confirm the role of iRGD, the iRGD-blocked group was set, and the result proved that the improved antitumor effect of HB-PA-mediate PDT was largely attributed to iRGD (Figure S11). During the treatment course, each group exhibited no significant body weight loss, which implied a good tolerance to the PDT treatments in mice (Figure 8b). At the end of the treatment, the tumors of each group were weighed, and the tumor inhibition rates were calculated (Figure 8c, d). HB-mediated PDT inhibited only approximately 60% of the tumor growth, most likely due to poor tumor accumulation of HB. HB-PA with light irradiation displayed the highest antitumor efficiency (~92%) owing to efficient tumor accumulation and penetration. Hematoxylin-eosin (H&E) staining revealed obvious tumor cell necrosis and membrane lysis with HB-PA PDT treatment (Figure 8e). Negligible histological damage of the major organs (i.e., heart, liver, spleen, lung, and kidney) confirmed good biosafety of each group (Figure S12). Although HB-PA exhibited some untargeted distribution (e.g., liver and kidney), the HB-PA-induced PDT could be limited to the tumor sites by controlling the irradiation light via fluorescent imaging. This HB-PA-mediated PDT could maximize the benefits of the therapy and minimize the side effects.



Figure 8. In vivo antitumor studies. (a) Changes in the tumor volumes following treatment with a) saline, b) light alone, c) HB alone, d) HB-PA alone, e) HB + light and f) HB-PA + light within 21 days. (b) Body-weight changes for each group during the treatments. (c) Tumor weight and (d) tumor-inhibition rate of different treatments. (e) H&E features of the tumors in different groups.

3. Conclusion In summary, a novel iRGD-based nanoplatform was designed and synthesized to self-assemble into vesicles for enhancing tumor-targeted accumulation, activation,


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and penetration, and as a result, the nanoplatform improved the image-guided photodynamic anticancer efficacy of the photosensitizer. This peptide-based system exhibits considerably better biocompatibility and biodegradability compared with those of the most popular inorganic systems, such as the metal-organic framework (MOF), graphene oxide (GO), mesoporous silica nanoparticles (MSN), and upconversion nanoparticles (UCNP).10,42-44 Notably, unlike the widely used liposomal and micellar carriers, which have tedious preparation processes and a low drug-loading efficiency,11,45 the synthesized PA was able to self-assemble into vesicular structures with an extremely high drug-loading content (43.44 ± 1.01) %. More importantly, HB-PA showed a three-fold higher penetration depth and more widespread signals in tumor tissues than those of the control group, which proved the improved tumor-penetration ability conferred by PA. The 2D and 3D experiments showed that the as-prepared HB-PA nanovesicles achieved high tumor accumulation and significant PDT activity. The in vivo results proved that the assembled HB-PA nanovesicles exhibited an excellent photodynamic anticancer efficacy with negligible side toxic effect. In the present study, we highlighted a novel nanoplatform with enhanced tumor penetration and improved anticancer activity, which had been successfully fabricated through self-assembling of our designed iRGD-based amphiphilic peptides. However, the as-prepared iRGD-based nanovesicles had a positively charged surface, which might affect in vivo biodistribution of the nanovesicles. Therefore, continued research of rationally changing the ratio of the hydrophilic and hydrophobic segments, using the negatively charged hydrophilic



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chains or introducing tumor microenvironment responsive materials to neutralize the positive charges, will be conducted to further improve the in vivo biodistribution of the nanovesicles in our future investigation. Thus, given the potential for versatile selection of hydrophobic and hydrophilic components, the iRGD-peptide-based nanoplatform reported in this study provides a novel strategy for effective and precise photodynamic therapy in breast cancer.

4. Experimental section Materials:

2-Chlorotrityl

N-Fluorenyl-9-methoxycarbonyl (Fmoc-Asp(Otbu)-OH, Fmoc-Arg(Pbf)-OH,

chloride (FMOC)

Fmoc-Pro-OH, Fmoc-Cys(trt)-OH,

resin

(H-Cys(Trt)-2Cl),

protected

Fmoc-Gly-OH,

amino

acids

Fmoc-Lys(Boc)-OH

Fmoc-Lys(Fmoc)-OH),

stearic

acids,

O-(Benzotriazol-1-yl)-N, N,N’,N’- tetramethyluronium hexafluorophosphate (HBTU) and piperdine were purchased from GL Biochem Ltd. (China). Triisopropylsilane (TIS) and pyrene were provided by Sigma-Aldrich and were used directly. Subcellular trackers, such as ER-Green and Mito-Green, were purchased from Life Technologies (USA). All other chemical reagents and solvents were directly used as commercially received. Synthesis of the peptide amphiphiles (PA): The PA was synthesized following a standard Fmoc solid-phase peptide synthesis method. H-Cys(Trt)-2Cl resin (0.4 mmol/g substitution) resin was used as a solid support. The resin was immersed in


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dichloromethane (DCM) for 15 min before use. After draining off DCM, a mixture of Fmoc-Asp(Otbu)-OH (2 equiv), HBTU (2 equiv) and DIEA (2 equiv) solution was added to the resin, which was followed by nitrogen agitation. One hour later, the solvent was removed, and the resin was washed with DMF for three times. Then, Fmoc group was deprotected using 20% piperidine and shaken for 20 min, followed by draining-washing nine times. Each acylation and deprotection circle was monitored by performing the Kaiser test. After the repetition of deprotection and acylation reaction, the PA was cleaved from the resin. Cleavage was performed by treating the peptide with trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/water at a ratio of 95:2.5:2.5 for 2-2.5 h. Later, the product was obtained by washing with cold diethyl ether several times and was subsequently dried under vacuum. The precipitate was dissolved in water, and trimethylamine was used to neutralize the residual TFA. Finally, the product was concentrated via rotary evaporation and freeze-dried. The purity of PA was determined using the Agilent 100 series HPLC system (USA). Characterization of PA: The chemical structure of PA was confirmed by Mass spectral analysis (AXIMA Assurance MALDI-TOF, Shimadzu, Japan) and 1H-NMR (Bruker Avance 600 NMR instrument, Bruker Corp, USA). Circular dichroism spectrum was recorded using JASCO J-810 CD to provide secondary information regarding the obtained PA. Wavelength scans were performed at 0.1 nm intervals between 260 and 200 nm. The CAC value was determined using a pyrene probe. The working solution was excited at 335 nm, and the emission spectra were scanned from



350 to 450 nm. The CAC value was determined from the fluorescence intensity ratio at 373 and 384 nm (I1/I3) versus logarithm of concentration curve. Preparation of HB-loaded peptide-amphiphile-based nanovesicles (HB-PA): Stock solutions of PA and HB in DMSO were mixed together in a glass vessel and sonicated for 15 min in a water bath. After the sample was returned to room temperature, the solution was dialyzed (1000 kD cut-off) with 2000 mL distilled water, which was refreshed 6 times every 4 h. Next, the HB-PA obtained was passed through a 0.22 µm filter to remove insoluble HB and was stored at 4 °C in the dark. Characterization of HB-PA: The drug-loading (DL) and entrapment efficiency (EE) of HB-PA were optimized using a UV-1800PC spectrophotometer (Shanghai Mapada Instruments Co., Ltd, China). The morphology was observed via TEM at 30 kV (Hitachi, Japan). The size distribution and zeta potential of PA and HB-PA were characterized using the DelsaTM Nano C Particle Analyzer (Beckman Coulter, Inc.). DSC measurements were performed using a PerkinElmer DSC 6000 (PerkinElmer, Hong Kong). Samples (2.0 mg) were placed in aluminum pans and were scanned from 30 °C to 350 °C at a heating rate of 10 °C⋅min-1. Detection of ROS: 9,10-Anthracenediyl-bis (methylene) dimalonic acid (ABDA) was used to evaluate ROS generation. In a typical process, ABDA solution was prepared by dissolving 20 µM ABDA in PBS. The HB stock solution and HB-PA were diluted using the ABDA solution. Next, a 630 nm LED was selected to irradiate the obtained working solutions at an energy density of 106 mW⋅cm-2, and the UV-absorbance at 380 nm was recorded at 0, 8, 15, 30, 60, 120, 180, 240, 300 s, respectively, using a


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UV-Vis spectrophotometer. Additional solutions containing HB and HB-PA but no ABDA served as the reference samples for each group. Decrease in the absorbance of ABDA, which was caused by photo bleaching, was measured as control. Cell culture: Breast tumor MDA-MB-231 cells and normal fibroblast NIH-3T3 cells were cultured at 37 °C in a humidified incubator with 5% CO2. The medium used was DMEM

that

contained

10%

fetal

bovine

serum

and

1%

Penicillin-Streptomycin-Neomycin (PSN) antibody. Dark toxicity: Toxicity of free HB and HB-PA (0.5, 1, 2, 4, 8, 16, 32 and 64 µM) for MDA-MB-231 and NIH-3T3 cells was evaluated under dark conditions. In these experiments, the cells were incubated with free HB and HB-PA in the dark for 24 h. Next, the MTT assay was conducted using a Bio-Tek µQUENT MQX200 spectrophotometric microplate reader (Bio-Tek Instruments, USA) at 570 nm. Uptake of HB-PA by monolayer cells: MDA-MB-231 or NIH3T3 cells (2×104 cells/chamber) were incubated with HB-PA or free HB at 2 µM for 4 h in a chamber slide (SPL life science, Korea). To better understand the cellular uptake of HB-PA, free iRGD (500 µM) was used for pre-incubation with the two cell lines for 2 h before addition of HB or HB-PA. After incubation, the medium was discarded, and the cells were washed with PBS three times. Next, the cell nuclei were counter-stained with Hoechst 33342 (10 µg⋅mL-1) for 15 min. After washing, the stained cells were imaged using a laser confocal microscope (Nikon D-Eclipse C1, Japan) using 405 nm and 488 nm wavelength lasers for excitation of the Hoechst 33342 and HB channels, respectively.



Intracellular localization: Tumor cells (1×104 cells/chamber) were incubated with HB or HB-PA (2 µM) for 4 h without light exposure. The thoroughly washed cells were stained using 0.5 mL of MitoTracker Green FM (10 nM) or LysoTracker Green DND-26 (50 nM) for 30 min. The washed cells were observed under CLSM at the excitation wavelength of 488 nm. Photocytotoxicity assay: Before light treatment, tumor cells were incubated with HB and HB-PA (2 µM) for 4 h. The cells were washed with PBS and were maintained in a fresh medium. Next, light at 630 nm wavelength and 106 mW⋅cm-2 was used to irradiate the cells for 4 s, 8 s, 16 s, 32 s and 64 s, respectively. After irradiation, the cells were incubated for 18 h for further MTT assay. Intracellular ROS assessment: Intracellular ROS production was measured via 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) using flow cytometry. When taken up by cells, DCFH-DA will be deacetylated by the intracellular esterases to form dichlorodihydrofluorescein (DCFH). DCFH can transfer to fluorescent dichlorofluorescein (DCF) in the presence of ROS. MDA-MB-231 cells (4×105 cells/dish) were first incubated with HB or HB-PA for 2 h in the dark. PBS-washed cells were treated with 10 µM DCFH-DA for 15 min. After thorough washing, the cells were exposed to light irradiation (106 mW⋅cm-2, 16 s) and were analyzed using a flow cytometer (BD FACSCalibur, USA). In each analysis, 10,000 cells were assayed. Estimation of the mitochondrial function: Dysfunction of mitochondria after PDT was evaluated using the cationic dye DiOC6 (3,3'-Dihexyloxacarbocyanine iodide).


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3×105 cells were seeded in 35×10 mm dishes and incubated overnight. HB and HB-PA solutions were incubated with the cells for another four hours in darkness. The thoroughly washed cells were exposed to 630 nm light for 16 s. Sixteen hours after the treatments, the cells were harvested and suspended in 10 nM DiOC6(3). Thirty minutes later, the cell suspensions were centrifuged and gently resuspended in pre-warmed PBS. Following the wash steps, the cell suspensions were evaluated using flow cytometry at excitation and emission wavelengths of 488 nm and 525 nm. In each analysis, 10,000 cells were assayed. Cell apoptosis: Tumor cells were treated with HB and HB-PA for 4 h in darkness. Unbound drug was rinsed away, and the cells (except for control groups) were exposed to 630 nm LED light (106 mw⋅cm-2) for 16 s. After 16 h incubation, the cells were stained using the Annexin V-FITC/PI apoptosis detection kit (Life Technologies Inc., USA) and analyzed via flow cytometry. Signals were acquired in the FL1 (530 nm) and FL2 (585 nm) channels. Nuclear staining was also performed to study the tumor cell apoptosis after PDT. MDA-MB-231 cells (2×105 cells/well) were further incubated for 16 h after HB- or HB-PA-mediated PDT treatment. The cells were stained with Hoechst 33342 (1 mg⋅mL-1) for 15 min, were gently washed with PBS and were visualized immediately using a fluorescence microscope. Emission signals were collected at 460 ± 20 nm upon excitation at 405 nm. 3D multicellular spheroid culture: MDA-MB-231 spheroids were constructed via the liquid overlay method. Briefly, monolayer cells were digested and diluted to 2.5×104 cells/mL. Then, the reconstituted basement membrane (rBM; BD Biosciences,



USA) was thawed in 4 °C refrigerator and added to the cell suspension with 2.5% concentration. Next, 200 µL of cell suspension was transferred into 2% agarose pre-coated 96-well plates. The culture medium was partially (100 µL) refreshed using fresh medium every other day. The cells were cultured for approximately 7 days for further use. Penetration of HB-PA into spheroids: When the spheroids were 7 days old, 100 µL of medium was replaced with 4 µM HB or HB-PA, yielding a final concentration of 2 µM. After a 8 h incubation, the spheroids were collected using a pipette and gently washed with PBS. The HB fluorescence in spheroids was observed via CLSM using z-stack with 10 µm intervals. The images were acquired from the periphery (z = 0 µm) toward the center of the spheroids. Next, the penetration depth and the average HB fluorescence intensity of each layer were analyzed using the ImageJ software. Growth inhibition of tumor spheroids: The tumor spheroids were prepared as described above. The spheroids of 300-350 µM in diameter were incubated with HB and HB-PA. After 32 s light irradiation, the cell morphology was observed every day using an inverted microscope with 4 × objectives. In Vivo imaging and biodistribution of HB-PA: Male balb/c mice (6-8 weeks, 18-22 g) were obtained from the animal center of Shandong University, and all the animal experiments strictly followed the ethical principles of Shandong University. The mice breast cancer cells (4T1 cells) were inoculated into the right forelimb of each mouse. Once the tumor grew to 100-200 mm3, the mice were randomly divided into two groups with five in each group. HB (2.0 mg·kg-1) and HB-PA (2.0 mg·kg-1 equivalent


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to HB) solutions were injected intravenously into the tumor-bearing mice. In vivo fluorescence imaging measurement was performed on an IVIS Kinetics system (Perkin Elmer, USA) using a 585 nm laser. The whole-body fluorescence images were collected at different time points. Finally, the tumor-bearing mice were sacrificed, and the major organs were stripped and tested for ex vivo imaging. In Vivo antitumor efficacy evaluation of HB-PA: All tumor-bearing mice were divided into six groups with five mice in each group as the tumor volume reached approximately 100 mm3. They were treated with a) saline, b) 630 nm light, c) HB, d) HB-PA, e) HB + Light, and f) HB-PA + light, respectively. According to the in vivo imaging results, free HB and HB-PA accumulated to the maximum extent in tumor sites at 1 h and 4 h after the intravenous injection, respectively. Therefore, the tumor tissues were irradiated with a 630 nm laser (100 W·cm-2) for 3 min at 1 h after HB injection and at 4 h after HB-PA injection. Each group was treated with different treatments every three days for a total of three times. The tumor volume and body weight were measured every three days. Tumor volume was calculated using the following equation: V = (a × b × b) / 2, where “a” represents the longest diameter, and “b” represents the shortest diameter of the tumor. Histological Analysis: Mice were anesthetized and sacrificed at the end of the in vivo antitumor experiment. The main organs and tumor tissues of each group were removed, washed with saline, and fixed with 10% formaldehyde solution. The tissue sections were prepared from the paraffin-embedded tissues and were stained with hematoxylin-eosin (H&E). The obtained samples were observed under a microscope



to evaluate the organ damage and tumor cell apoptosis in each group. Statistical analysis: Data are expressed as the mean ± standard deviation and were analyzed via one-way or two-way ANOVA and Tukey’s multiple comparison tests using the GraphPad Prism 5.0 software (California, USA). The level of significance was set at P < 0.05.

Supporting Information The purity of PA determined via HPLC; MALDI-TOF MS profile and 1HNMR spectrum of the synthesized PA; EE and DL of HB-PA; hemolysis ratio of PA, HB, and HB-PA at different concentrations; cell uptake of HB and intracellular HB release in two cell lines; nuclear features of tumor cells after PDT treatments; fluorescence intensity profile of HB and HB-PA at 80 µm z-distance within MDA-MB-231 MCTSs; CLSM examination of HB-PA distribution after iRGD pre-incubation in MCTSs of MDA-MB-231 cells; the histological features of the major organs after different treatments.

Acknowledgements This work was supported by a general grant fund from the Hong Kong Research Grant Committee (476912) and the Health and Medical Research Fund (13120442).

Conflict of Interest The authors declare no conflict of interest.


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Scheme 1. Schematic representation of the iRGD based peptide amphiphile (PA) and the encapsulation of HB. 67x57mm (600 x 600 DPI)


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Figure 1. (a) Circular dichroism spectra of PA and HB-PA; (b) determination of the CAC of PA; (c) size distribution of PA and HB-PA. 188x142mm (300 x 300 DPI)



Figure 3. (a) Dark toxicity (%) of PA, HB, and HB-PA to tumor and normal cells under different concentrations. (b) Intracellular uptake of HB and HB-PA in two cellines. Cell nuclei were counter-stained with Hoechst 33342. (c) Intracellular localization of HB and HB-PA in tumor cells after 4 h incubation. 295x352mm (150 x 150 DPI)


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Figure 4. (a) Photocytotoxicity of HB and HB-PA to MDA-MB-231 cells, *P < 0.05. (b) Intracellular ROS generation measured by flow cytometry. 388x133mm (144 x 144 DPI)



Figure 7. (a) The in vivo imaging of HB and HB-PA at various time points. Tumor sites are marked with a black circle. (b) The ex vivo images of the major organs and tumors at 24 h after HB-PA and HB injection. Number 1-6 represents the heart, liver, spleen, lung, kidney and tumor respectively. 137x89mm (300 x 300 DPI)


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Figure 8. The in vivo antitumor studies. (a) The change of tumor volumes treated with a) saline, b) light alone, c) HB alone, d) HB-PA alone, e) HB + light and f) HB-PA + light within 21 days. (b) Body weight changes of each group during the treatments. (c) Tumor weight and (d) tumor inhibition rate of different treatments. (e) The H&E features of the tumors in different groups. 226x218mm (300 x 300 DPI)



368x201mm (150 x 150 DPI)


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Design of an Amphiphilic iRGD Peptide and Self-assembling

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