Epigenetics-inspired photosensitizer modification for plasma membrane-targeted photodynamic tumor therapy

Biomaterials 224 (2019) 119497

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Epigenetics-inspired photosensitizer modification for plasma membranetargeted photodynamic tumor therapy

T

Hong Chengb,1, Gui-Ling Fana,1, Jing-Hao Fana, Ping Yuana, Fu-An Denga, Xiao-Zhong Qiub, Xi-Yong Yua, Shi-Ying Lia,∗ a

Key Laboratory of Molecular Target & Clinical Pharmacology and the State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences & the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, 511436, PR China b Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering, Biomaterials Research Center, School of Biomedical Engineering, Southern Medical University, Guangzhou, 510515, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Epigenetics Amino acid Photosensitizer Plasma membrane targeting Tumor therapy

In recent years, epigenetics has attracted great attentions in the field of biomedicine, which is used to denote the heritable changes in gene expression without any variation in DNA sequence, including DNA methylation, histone modification and so on. Inspired by it, a simple and versatile amino acids modification strategy is proposed in this paper to regulate the subcellular distribution of photosensitizer for plasma membrane targeted photodynamic therapy (PDT). Particularly, the plasma membrane anchoring ability and photo toxicity of the photosensitizer against different cell lines could be effectively manipulated at a single amino acid level. Systematic researches indicate that the number and variety of amino acids have a significant influence on the plasma membrane targeting effect of the photosensitizer. Furthermore, after self-assembling into nanoparticles, the obtained nano photosensitizers (NPs) also exhibit a good biocompatibility and plasma membrane targeting ability, which are conducive to enhancing the PDT therapeutic effect under light irradiation. Both in vitro and in vivo investigations confirm a robust tumor inhibition effect of NPs with a good biocompatibility. This epigenetics-inspired photosensitizer modification strategy would contribute to the development of structure-based drug design for tumor precision therapy.

1. Introduction The subcellular availability and accessibility of therapeutic targets to drugs determine the treatment efficiency as well as the potential sideeffects [1–4]. In the past decades, photodynamic therapy (PDT) has been recognized as a promising interventional treatment strategy by photosensitized oxidation of lipids, DNA and proteins in the presence of photosensitizers, oxygen and light [5–10]. Although PDT possesses some inimitable advantages such as noninvasiveness and on-demand light controllable capabilities, it is still seriously limited in tumor therapy due to the short lifespan, limited diffusion distance and rapid deactivation of toxic reactive oxygen species (ROS) in cytoplasm before it comes to the site of action [11–14]. Definitely, enhancing the spatiotemporal interaction between photosensitizer and its designated target will dramatically improve the therapeutic effect of PDT. Recently, targeting delivery of photosensitizer to subcellular organelles, such as mitochondrion [15–18], nucleus [19–21], endoplasmic

reticulum [22–24], was reported to significantly improve the PDT efficiency by generating ROS in situ. However, the photosensitizer-target interaction was always impeded owing to the intracellular sequestration after cellular uptake, which would further compromise the effectiveness of PDT [25–28]. As the boundary of cells, plasma membrane plays a crucial role in maintaining cellular homeostasis, preserving cell integrity and regulating nutrients transport [29–31]. The oxidation of unsaturated lipid by ROS could trigger the conformation changes of lipid membranes and cause cell death [32–34]. Apparently, plasma membrane-targeted PDT would be a robust strategy to disrupt the cell integrity by photosensitized lipid oxidation [35–37]. More importantly, plasma membrane-targeted delivery of photosensitizer could take effect without the endocytosis and bypass the various biological barriers [38–40]. However, it should be noted that this contact-dependent reaction between photosensitizer and plasma membrane needs to fully compromise membrane functions [41,42]. Directing the photosensitizer to localize

∗

Corresponding author. E-mail address: [email protected] (S.-Y. Li). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biomaterials.2019.119497 Received 9 May 2019; Received in revised form 8 September 2019; Accepted 12 September 2019 Available online 16 September 2019 0142-9612/ © 2019 Elsevier Ltd. All rights reserved.

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Scheme 1. Schematic illustration of epigenetics-inspired amino acids regulated plasma membrane targeting of photosensitizer for enhanced photodynamic therapy (PDT). A) The altered non covalent bonding behaviors between histone and DNA induced by the epigenetics changes (methylation, acetylation or phosphorylation) of the amino acids in histone. B) Chemical structures of amino acids modified photosensitizer and schematic illustration of plasma membrane targeted PDT. C) Selfassembly of the modified photosensitizer to prepare nano-photosensitizers (NPs). D) Tumor targeted delivery of NPs by enhanced penetration and retention (EPR) effect. E) Plasma membrane targeted photosensitizer delivery and PDT-induced plasma membrane rupture.

Fmoc-Lys(Dde)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Glu(otBu)-OH and 2chlorotrityl chloride resin were purchased from GL Biochem Ltd. (Shanghai, China). Analytical grade of N,N′-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH), methanol and dichloromethane (DCM) were provided by Shanghai Chemical Co. (China). Trifluoroacetic acid (TFA), trypan blue, o-benzotriazoleN,N,N′,N'-tetramethyluroniumhexaflorophosphate (HBTU), diisopropylethylamine (DIEA), triisopropylsilane (TIS), hydrazine hydrate, acetic anhydride and protoporphyrin IX (PpIX) were obtained by Aladdin Reagent Co. Ltd. (China). Annexin V-FITC, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), propidium iodide (PI), 2′,7′-dichloroflorescein diacetate (DCFH-DA), Calcein-AM, Hoechst 33342 and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were provided by R&D-SYSTEMS. Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), Roswell Park Memorial Institute 1640 (RMPI 1640), trypsin, penicillin-streptomycin, Dulbecco's phosphate buffered saline (PBS), MitoTracker Green, LysoTracker Green, and fetal bovine serum (FBS) were purchased from Invitrogen Corp. Electrospray ionization mass spectrometry (ESI-MS, ThermoFisher Scientific) was used to measure the molecular weight of the photosensitizer conjugates. The size, zeta potential and stability of the particles were investigated by Nano-ZS ZEN 3600 (Malvern) and transmission electron microscope (TEM, JEOL-1400 PLUS). The UV–vis absorbance and fluorescence was analyzed by Lambda 35 (PerkinElmer) and LS55 luminescence spectrometer (Perkin-Elmer), respectively. The cellular uptake behaviors, subcellular distribution of the photosensitizer conjugates and live/dead cell staining were observed by confocal laser scanning microscope (CLSM, LSM 880, Carl Zeiss). The cellular fluorescence analysis and Annexin V-FITC/PI staining analysis were performed by flow cytometry (Amnis, Merck millipore). Photodynamic therapy was conducted using 630 nm LED light (light power intensity: 29.8 mW/cm2) in vitro and 630 nm He-Ne laser (laser intensity: 250 mW/cm2) in vivo, respectively. MTT assay was conducted

to the plasma membrane of cells by a simple but versatile strategy remains a huge challenge. Epigenetics, which describes the phenotype of heritable changes without the change of underlying DNA sequences, has attracted great attentions in the last decade [43–46]. Among which, the methylation, acetylation or phosphorylation of amino acids in histone proteins induced chromatin structure variation could initiate a domino effect to cause various epigenetics diseases (Scheme 1A) [47–50]. Inspired by this, we surmise that a simple modification with amino acids may be utilized to regulate the subcellular distribution of photosensitizer by enhancing the noncovalent interaction between the photosensitizer and plasma membrane. To verify this idea, we proposed a versatile strategy to realize the plasma membrane targeting of photosensitizer at a single amino acid level for enhanced PDT against tumor. Firstly, a photosensitizer protoporphyrin IX (shorted as PpIX), linked onto the ε-amine of lysine and modified with arginine (designated as Ac-K(PpIX)-Rn) or glutamic acid (designated as Ac-K(PpIX)-En), was constructed to evaluate the plasma membrane targeting ability (Scheme 1B). Compared with glutamic acid, arginine was confirmed for regulation of the plasma membrane anchoring ability of PpIX at a single arginine level. Besides, after self-assembly, the obtained nano photosensitizers (NPs) could achieve the tumor targeted delivery through enhanced penetration and retention (EPR) effect of tumor tissues (Scheme 1C-1D). Finally, the NPs with plasma membrane targeting ability also exhibited an enhanced PDT effect under light irradiation (Scheme 1E). This simple and versatile strategy would inspire the development of structure-based drug design for site-specific delivery and tumor precision therapy.

2. Materials and methods 2.1. Materials and methods N-fluorenyl-9-methoxycarbonyl (Fmoc)-protected amino acids of 2

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acids on the plasma membrane targeting ability, 20 μM of Ac-K(PpIX)R2, Ac-K(PpIX)-R4, PpIX-KR4, Ac-K(PpIX)-R6, Ac-K(PpIX)-R8 and Ac-K (PpIX)-E4 were incubated with 4T1 cells for 4 h, respectively. Then 4T1 cells were washed with PBS and stained with DiO for CLSM observations. As the control, 20 μM of free PpIX, Ac-K(PpIX)-R, Ac-K(PpIX)-E, Ac-K(PpIX)-R4, Ac-K(PpIX)-E4, Ac-K(RB)-R4, Ac-K(FAM)-R4, Ac-K (PpIX)-RERE and Ac-K(PpIX)-RFRF were incubated with 4T1 cells for 2 h and then observed by CLSM. To investigate the effect of incubation time on the plasma membrane targeting ability, 20 μM of Ac-K(PpIX)-R, Ac-K(PpIX)-R2, Ac-K (PpIX)-R4, PpIX-KR4, Ac-K(PpIX)-R6, Ac-K(PpIX)-R8, Ac-K(PpIX)-E and Ac-K(PpIX)-E4 were incubated with 4T1 cells for 1 h, 2 h and 3 h, respectively. Subsequently, the cells were washed by PBS and observed by CLSM. To study the effect of the concentration on the plasma membrane targeting ability, 4T1 cells were treated with Ac-K(PpIX)-R, Ac-K(PpIX)R2, Ac-K(PpIX)-R4, Ac-K(PpIX)-R6, Ac-K(PpIX)-E and Ac-K(PpIX)-E4 at the concentration of 5 μM, 10 μM and 40 μM for 2 h, respectively. Then the cells were washed by PBS and observed by CLSM. To evaluate the versatile plasma membrane targeting abilities of these photosensitizer conjugates to various cells types, 20 μM of Ac-K(PpIX)-R2, Ac-K(PpIX)R4, Ac-K(PpIX)-R6 and Ac-K(PpIX)-E4 were incubated with 4T1 cells, HeLa cells and A549 cells for 2 h, respectively. After that, the cells were washed by PBS and observed by CLSM. Besides, the subcellular distributions of PpIX, Ac-K(PpIX)-R4 and Ac-K(PpIX)-R8 were also studied by incubating with 4T1 cells for 30 min at the concentration of 100 μM for CLSM observations.

on microplate reader (Mithras2 LB 943, BERTHOLD). Trypan blue staining cells and hematoxylin-eosin (H&E) staining tissues were imaged by inverted microscope (Revolve FL, ECO Laboratories). The real time fluorescence images of 4T1 tumor bearing mice and ex vivo fluorescence images of the sacrificed tissues were analyzed by In-Vivo FX Pro (Bruker). 2.2. Synthesis of photosensitizer conjugates All of the photosensitizer conjugates, including Ac-K(PpIX)-R, Ac-K (PpIX)-R2, Ac-K(PpIX)-R4, PpIX-KR4, Ac-K(PpIX)-R6, Ac-K(PpIX)-R8, AcK(PpIX)-E and Ac-K(PpIX)-E4, were synthesized by standard solid phase peptide synthesis strategy. In brief, Fmoc protected amino acids were coupled onto the 2-chlorotrityl chloride resin by using HBTU and DIEA as coupling agents. Piperdine/DMF (20%, V/V) and hydrazine hydrate/ DMF (2%, V/V) were employed to remove the Fmoc and Dde protecting group respectively. The photosensitizer conjugates were cleaved from the resin by using the cocktail of TFA/TIS/H2O (95%/2.5%/2.5%, V/V/ V). The obtained products were stocked at −20 °C and their molecular weights were measured by ESI-MS. 2.3. Preparation and characterization of NPs-E4 and NPs-R4 NPs-E4 and NPs-R4 were obtained by respectively adding Ac-K (PpIX)-R4 (74 μM in 0.1% DMSO) and Ac-K(PpIX)-E4 (74 μM in 0.1% DMSO) into the distilled water under ultrasonication. After that, the particle size distribution and zeta potential were analyzed by dynamic light scattering (DLS), and the morphology was observed by TEM. 100 μM of PpIX, Ac-K(PpIX)-R4 and Ac-K(PpIX)-E4 in the distilled water containing 0.1% DMSO were prepared for tyndall phenomenon. The UV–vis absorbance of NPs-E4 (25 μM containing 0.1% DMSO), NPs-R4 (25 μM containing 0.1% DMSO) or the equivalent concentration of PpIX (25 μM containing 0.1% DMSO) was measured by UV–vis spectrophotometer.

2.7. ROS production measurement The ROS generated by PpIX, NPs-E4 or NPs-R4 was measured via CLSM using DCFH-DA as the sensor. Briefly, 4T1 cells were incubated with PpIX, NPs-E4 and NPs-R4 at the concentration of 74 μM for 4 h. After which, the cells were washed by PBS and incubated with DCFHDA (5 μM) for another 20 min. Subsequently, the cells were irradiated with LED light (power intensity: 29.8 mW/cm2) for 2 min or incubated in the shield of light. Finally, the ROS production was evaluated by CLSM.

2.4. Determination of critical micelle concentration (CMC) By using pyrene as the hydrophobic fluorescent probe, the CMC of the Ac-K(PpIX)-R4 and Ac-K(PpIX)-E4 was determined according to the fluorescence spectra. Briefly, pyrene solution in acetone (1.2 × 10−5 M, 0.05 mL) was added in tubes. After the evaporation of acetone from tubes, 1 mL of Ac-K(PpIX)-R4 and Ac-K(PpIX)-E4 at various concentrations were added and mixed. After 24 h, the solution was detected by fluorescence spectra (emission wavelength: 393 nm, excitation wavelength range: 300–360 nm). The fluorescent intensity ratio was plotted against the logarithm of the concentration of Ac-K(PpIX)-R4 or Ac-K (PpIX)-E4. The CMC value was obtained by the intersection of the tangent to the curve at the inflection with the horizontal tangent through the point at low concentration.

2.8. Subcellular distributions of NPs-E4 and NPs-R4 Subcellular distributions of NPs-E4 and NPs-R4 were investigated by CLSM. Briefly, 4T1 cells were incubated with 74 μM of NPs-E4 or NPs-R4 for 4 h. And then, the cells were washed thrice by PBS and stained by DiO, MitoTracker Green, LysoTracker Green or Hoechst 33342. Finally, the cells were washed by PBS and the intracellular distribution was measured and analyzed by CLSM. 2.9. Plasma membrane targeted PDT in vitro The dark toxicities of the photosensitizer conjugates were evaluated against 4T1 cells by MTT assay. Briefly, 4T1 cells were seeded in 96well plates. Subsequently, gradient concentrations of Ac-K(PpIX)-R, AcK(PpIX)-R2, Ac-K(PpIX)-R4, PpIX-KR4, Ac-K(PpIX)-R6, Ac-K(PpIX)-R8, Ac-K(PpIX)-E or Ac-K(PpIX)-E4 were added into each well and co-cultured for 24 h. After which, MTT (20 μL, 5 mg/mL in PBS) was added into each well. After treatment for another 4 h, the supernatant was replaced by DMSO (150 μL) and the optical density (at 570 nm) was measured by a microplate reader. The cell viability (%) calculated as the survival cells after various treatments per nontreated cells. As the control, the photo toxicity and dark toxicity of free PpIX, Ac-K(PpIX)RFRF and Ac-K(PpIX)-RERE against 4T1 cells were carried out by MTT assay using the similar methods. Also, the photo toxicity and dark toxicity of Ac-K(PpIX)-R4 and Ac-K(PpIX)-E4 against 3T3 cells were performed by MTT assay and live/dead cell staining. The photo toxicities of Ac-K(PpIX)-E and Ac-K(PpIX)-R were evaluated by MTT assay. Briefly, gradient concentrations of Ac-K(PpIX)-E or

2.5. Cell culture Murine mammary carcinoma (4T1) cells and colorectal cancer (CT26) cells were cultured in RPMI-1640 medium in an atmosphere of 5% CO2 at 37 °C. Human cervical carcinoma (HeLa) cells, human lung adenocarcinoma (A549) cells and mouse embryo fibroblasts (3T3) cells were cultured in DMEM medium in an atmosphere of 5% CO2 at 37 °C. The medium contained 10% FBS and 1% antibiotics. 2.6. Plasma membrane targeting ability of photosensitizer conjugates Plasma membrane targeting abilities of photosensitizer conjugates were evaluated by CLSM. Firstly, 20 μM of Ac-K(PpIX)-R and Ac-K (PpIX)-E were incubated with 4T1 cells, HeLa cells and A549 cells for 4 h, respectively. Then the cells were washed with PBS and stained with DiO for CLSM observations. To further investigate the effect of amino 3

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was collected via centrifugation for 5 min at 3000 r min−1. Finally, the amount of PpIX was measured by using fluorescence spectrum (Excitation wavelength: 405 nm). To calculate the tumor accumulation amounts of photosensitizer, the tumor tissues were sacrificed after intravenous injection of NPs-R4 or NPs-E4 for 6 h. And then, the tumor tissues were homogenized. Subsequently, 200 μL of PBS was added and the supernatant was collected via centrifugation for 5 min at 3000 r min−1. Finally, the amount of PpIX was measured by using fluorescence spectrum.

Ac-K(PpIX)-R were co-cultured with 4T1 cells, HeLa cells and A549 cells in 96-well plates. After treatment for 4 h, the cells were irradiated with LED light for 1 min. And then, the cells were further incubated in the shield of light for 20 h. Finally, the cell viability was measured by using MTT and microplate reader. The photo toxicities of NPs-E4 and NPs-R4 were also investigated against 4T1 cells, HeLa cells, A549 cells and CT26 cells by MTT assay using the similar method. Besides, The PDT therapeutic effects of Ac-K(PpIX)-E, Ac-K(PpIX)-R, NPs-E4 and NPs-R4 were also investigated against 4T1 cells by trypan blue staining, flow cytometry and live/dead cell staining. For trypan blue staining assay, Ac-K(PpIX)-E (60 μM), Ac-K(PpIX)-R (60 μM), NPsE4 (74 μM) or NPs-R4 (74 μM) was co-cultured with 4T1 cells for 4 h. Then the cells were irradiated with LED light for 2 min or incubated in the shield of light. After that, 4T1 cells were treated with trypan blue (4 mg/mL) for 5 min and then imaged by inverted microscope. 4T1 cells incubated with trypan blue in the presence or absence of light were used as the blank control. For flow cytometry assay, on the one hand, 4 μM of Ac-K(PpIX)-E or Ac-K(PpIX)-R was co-cultured with 4T1 cells for 4 h in 6-well plates. Subsequently, the cells were irradiated with LED light for 3.5 min or incubated in the shield of light. On the other hand, 20 μM of NPs-E4 or NPs-R4 was co-cultured with 4T1 cells for 4 h in 6well plates. Subsequently, the cells were irradiated with LED light for 2 min or incubated in the shield of light. Then the cells were washed with PBS and stained with Annexin V-FITC and PI for flow cytometry analysis. For live/dead cell staining assay, on the one hand, 4 μM of AcK(PpIX)-E or Ac-K(PpIX)-R was co-cultured with 4T1 cells for 4 h. Then the cells were irradiated with LED light for 3 min or incubated in the shield of light. On the other hand, 74 μM of NPs-E4 or NPs-R4 was cocultured with 4T1 cells or 3T3 cells for 4 h. Then the cells were irradiated with LED light for 1.5 min or incubated in the shield of light. After that, the cells were treated with Calcein-AM (2 mM) and PI (1.5 mM) for 25 min and observed by CLSM. 4T1 cells and 3T3 cells incubated with Calcein-AM and PI in the presence or absence of light were used as the blank control.

2.12. Anti-tumor studies of NPs-E4 and NPs-R4 in vivo 4T1 tumor bearing mice were randomly divided into five groups (5 mice in each group), including PBS group, NPs-E4 group, NPs-R4 group, NPs-E4 with light group and NPs-R4 with light group. Subsequently, the mice were intravenously injected with 200 μL of PBS or NPs-E4 and NPs-R4 at an equivalent PpIX concentration of 6 mg/kg per mouse, respectively. After 12 h, the mice in light groups were exposed to 630 nm He-Ne laser for 5 min. During the treatments, the tumor volume (V) and body weight (M) of mice were monitored every other day. The tumor volume was calculated as follows: V= (tumor width)2 × (tumor length)/2. The relative tumor volume and relative body weight were respectively defined as V/V0 and M/M0, in which V0 and M0 represented the tumor volume and body weight of the mice at the first day when without any treatments. After 15 days, 4T1 tumor bearing mice were sacrificed. The blood of the mice in various groups was collected for blood routine analysis and blood biochemistry test. The tumors were harvested for weighing and imaging. The heart, liver, spleen, lung, kidney and tumors were obtained for Hematoxylin/eosin (H&E) staining analysis. 2.13. Statistical analysis Statistical analysis was conducted using a Student's t-test. The differences were considered to be statistically significant for #P < 0.05, *P < 0.005, &P < 0.001 and §P < 0.0005.

2.10. Penetration in 4T1 tumor spheroids

3. Results and discussion

Avascular 4T1 tumor spheroids were obtained according to previous reports. Above all, the 96-well plate was coated with 1% (w/v) agarose gel to prevent cell adhesion. Subsequently, 4T1 cells were seeded into each well at a density of 1000 cells per well. After gently mixed for 5 min, 4T1 cells were incubated at 37 °C for 7 days. The uniform and compact multicellular spheroids were obtained for penetration tests. 60 μM of PpIX, Ac-K(PpIX)-E4 or Ac-K(PpIX)-R4 were added and incubated for 10 h for CLSM observations.

3.1. Single amino acid-regulated plasma membrane-targeted PDT of PpIX Inspired by epigenetics, the plasma membrane targeting ability of photosensitizer PpIX was expected to be regulated at a single amino acid level. Above all, the PpIX conjugates of Ac-K(PpIX)-R and Ac-K (PpIX)-E were synthesized by standard solid phase peptide synthesis. Their molecular weights were characterized by electrospray ionizationmass spectrometry (ESI-MS) (Fig. S1). Their plasma membrane targeting ability were evaluated against different cell types by confocal laser scanning microscope (CLSM). As shown in Fig. 1A–C, after treatment with Ac-K(PpIX)-R, an intense red fluorescence of PpIX was found to overlap well with the green fluorescence of DiO on the plasma membrane of murine mammary carcinoma (4T1) cells, human cervical carcinoma (HeLa) cells and human lung adenocarcinoma (A549) cells, indicating a good membrane targeting ability of Ac-K(PpIX)-R. Conversely, very weak red fluorescence of Ac-K(PpIX)-E was observed on the plasma membrane of different cell types, which suggested a disappointing membrane location capability. These significant difference could be also demonstrated by the fluorescence profile analysis and Zstack observations (Figs. S2–S3), further verifying the effective regulation to the plasma membrane targeting ability of PpIX at a single amino acid level. The main reason might be the altered noncovalent interaction between the photosensitizer conjugates and plasma membrane. The disruption of plasma membrane would cause a serious cell damage [51,52]. The success of plasma membrane targeting regulation to PpIX encouraged us to further explore its PDT effect at a cellular

2.11. Biodistributions and pharmacokinetic study of NPs-E4 and NPs-R4 in vivo Biodistributions of NPs-E4 and NPs-R4 were studied by establishing 4T1 tumor model on female BALB/c mice. All of the in vivo experiments were carried out according to the guidelines of the Institutional Animal Care and Use Committee of the Animal Experiment Center of Guangzhou Medical University (Guangzhou, China) as well as the Regulations for the Administration of Affairs Concerning Experimental Animals. Briefly, 4T1 tumor bearing mice were intravenously injected with 200 μL of NPs-E4 or NPs-R4 at an equivalent PpIX concentration of 6 mg/kg per mouse. At the predetermined time, the mice were observed and imaged by the small animal imaging system. After 24 h post-administration, the mice were sacrificed. The heart, liver, spleen, lung, kidney and tumors were obtained and imaged for fluorescence analysis. For pharmacokinetic study, the mice were injected with NPs-R4 or NPs-E4 via tail vein and the blood samples (20 μL) were taken at the preset times. After which, the blood samples were diluted with PBS (180 μL) and then repeatedly freeze-thawed. Furthermore, the blood samples were suffered from ultrasound for 5 min and the supernatant 4

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Fig. 1. Plasma membrane targeted PDT of PpIX regulated at a single amino acid level. Plasma membrane targeting abilities and photo toxicities of Ac-K(PpIX)-R (20 μM) and Ac-K(PpIX)-E (20 μM) against A) 4T1 cells B) HeLa cells and C) A549 cells by CLSM and MTT assays. Scale bar: 10 μm. D) Inverted microscope images, E) flow cytometry assay and F) live/dead cell staining analysis of 4T1 cells after treatment with Ac-K(PpIX)-R (4 μM or 60 μM) or Ac-K(PpIX)-E (4 μM or 60 μM) in the absence or presence of light irradiation. Scale bar: 100 μm. G) Schematic illustration of amino acid-regulated plasma membrane-targeted PpIX delivery for enhanced 5

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plasma membrane targeting ability was also evaluated on the normal 3T3 cells (Fig. S14). As expected, Ac-K(PpIX)-R4 and Ac-K(PpIX)-R exhibited the better plasma membrane anchoring effect rather than Ac-K (PpIX)-E4 and Ac-K(PpIX)-E, which further verified the universality and non-selectivity of this membrane anchoring strategy. To further investigate the plasma membrane anchoring mechanism, Ac-K(RB)-R4, Ac-K(FAM)-R4, Ac-K(PpIX)-RERE and Ac-K(PpIX)-RFRF were prepared and incubated with 4T1 cells for CLSM observations. As illustrated in Fig. S15, compared with Ac-K(PpIX)-R4, Ac-K(FAM)-R4 and Ac-K(RB)R4 exhibited the disappointing plasma membrane anchoring ability, indicating that the hydrophilicity of the fluorophores had a negative effect on the plasma membrane anchoring ability. Moreover, Ac-K (PpIX)-RERE and Ac-K(FAM)-RFRF also exhibited no obvious plasma membrane anchoring effect, indicating that the negative charged and hydrophobic amino acids sequence would also reduce the plasma membrane targeting ability. These results indicated that the plasma membrane anchoring mechanism of photosensitizer conjugates was ascribed to the synergistic effect of hydrophobic insertion of PpIX and electrostatic interaction of the positively charged amino acids with plasma membrane [53]. In order to systematically study the cellular uptake behaviors of these plasma membrane targeted PpIX conjugates, 4T1 cells were incubated with them for various time at various concentrations for CLSM observations. As suggested in Fig. 2B, as the incubation time went on, an increasing red fluorescence was found in 4T1 cells, which illustrated an incubation time-dependent cellular uptake of these PpIX conjugates. However, due to the lack of plasma membrane targeting, there were a small amount of Ac-K(PpIX)-E and Ac-K(PpIX)-E4 uptake by cells. Especially, the latter possessed much worse uptake ability than the former, implying that the modification of PpIX with glutamic acid might inhibit the interaction between the photosensitizer and cells. Furthermore, with the increase of PpIX conjugates concentration, a visible enhancement of red fluorescence was observed in 4T1 cells, verifying a concentration-dependent cellular uptake by cells (Fig. 2C). A simple but versatile plasma membrane anchoring strategy was necessary for an ideal membrane-targeted PDT. In this work, the universality of PpIX conjugates for plasma membrane targeting was investigated by CLSM. As demonstrated in Fig. 2D, after modified with different numbers of arginine, the obtained Ac-K(PpIX)-R2, Ac-K(PpIX)R4 and Ac-K(PpIX)-R6 also exhibited similar plasma membrane anchoring abilities on different cell types such as 4T1 cells, HeLa cells and A549 cells, which suggested the great advantage of this versatile amino acid modifying strategy for regulating the plasma membrane targeting of photosensitizer. As the control, Ac-K(PpIX)-E4 was found to have a poor plasma membrane targeting and cellular uptake ability against various cell types. Moreover, after modified with arginine, the solubility as well as the plasma membrane targeting ability of PpIX were also found to be dramatically enhanced (Fig. S13). These significant differences further highlighted the potential applications of this photosensitizer modifications by specific amino acids. Besides, just like arginine, it could be speculated that other positively charged amino acids such as histidine, lysine, glutamine and asparagine with similar structure might be also used for photosensitizer modifications to regulate its subcellular distributions. We believe that this simple but versatile plasma membrane anchoring strategy would open a window in the development of structure-based drug design for site-specific delivery.

level. As illustrated in Fig. 1A–C, both Ac-K(PpIX)-E and Ac-K(PpIX)-R exhibited a concentration-dependent photo toxicity against 4T1 cells, HeLa cells and A549 cells. However, the PDT effect of Ac-K(PpIX)-R was always better than that of Ac-K(PpIX)-E, which might be ascribed to its more accurate regulation of membrane localization of PpIX. To confirm this speculation, the plasma membrane integrity of 4T1 cells was then investigated after treated with Ac-K(PpIX)-E or Ac-K(PpIX)-R under light irradiation. As suggested in Fig. 1D, when without light irradiation, Ac-K(PpIX)-E and Ac-K(PpIX)-R had a negligible dark toxicity, implying a good biocompatibility of them in vitro. While in the presence of light, an obvious plasma membrane destruction was found, especially 4T1 cells treated with Ac-K(PpIX)-R. And the cells treated with light only were uninjured, demonstrating the harmlessness of light irradiation. As the control, the cytotoxicity of free PpIX was also detected by MTT assay. As shown in Fig. S4, although PpIX had a robust PDT effect, it also exhibited a relatively high dark toxicity, which implied a poor biocompatibility. Together with the previous investigations (Fig. 1A–C), it could be concluded that this membrane localization regulation strategy could achieve a good PDT effect by efficiently destroying the plasma membrane structure with low side effects. Subsequently, the cytotoxicity was also evaluated by flow cytomety and live/ dead cell staining assay. As shown in Figs. 1E and 4T1 cells treated with Ac-K(PpIX)-E or Ac-K(PpIX)-R had a very high survival rate in the absence of light irradiation. With the addition of light, 4T1 cells viability decreased, which was even less than 20%. Similarly, by live/dead cell staining assay, almost no red fluorescence was found in 4T1 cells in the absence of photosensitizer or light, which suggested the necessity of them for PDT (Fig. 1F). However, there was still very weak red fluorescence in 4T1 cells treated with Ac-K(PpIX)-E and light, indicating a disappointing therapeutic effect. Notably, under light irradiation, a significantly enhanced red fluorescence was found in 4T1 cells treated with Ac-K(PpIX)-R, suggesting that the robust PDT of Ac-K(PpIX)-R caused lots of cells death. Based on the above, one could draw a conclusion that the subcellular distribution of photosensitizer could be regulated at a single amino acid level for plasma membrane targeted PDT (Fig. 1G). This simple but effective modification strategy would contribute to the development of structure-based drug design for tumor precision therapy. 3.2. Amino acids-regulated plasma membrane targeting evaluation To further examine the influence of amino acids on the membrane anchoring ability of photosensitizer, various PpIX conjugates including Ac-K(PpIX)-R2, Ac-K(PpIX)-R4, PpIX-KR4, Ac-K(PpIX)-R6, Ac-K(PpIX)R8 and Ac-K(PpIX)-E4 were designed and synthesized by standard solid phase peptide synthesis. The chemical structures and the molecular weights of these PpIX conjugates were also characterized by ESI-MS (Figs. S5–S7). And their plasma membrane targeting ability were firstly investigated against 4T1 cells by CLSM. As reflected in Fig. 2A and Figs. S8–S11, similar to 4T1 cells treated with Ac-K(PpIX)-R, an obvious plasma membrane anchoring effects were found on the cells after treatment with Ac-K(PpIX)-R2, Ac-K(PpIX)-R4, Ac-K(PpIX)-R6 or Ac-K (PpIX)-R8. As the control, 4T1 cells treated with free PpIX were also observed by CLSM. As shown in Fig. S12, the red fluorescence was found to mainly distribute in cytoplasm rather than on plasma membrane, which might be ascribed to its poor membrane location effect. Moreover, with the number of arginine mounting up, the red fluorescence in 4T1 cells seemed to gradually increase, resulting from that the enhanced noncovalent interaction between the peptide sequence and plasma membrane improved the cellular uptake of PpIX. Besides, PpIXKR4 also preferred to accumulate on the plasma membrane (Fig. 2A and Fig. S13), confirming that the conjugation site of photosensitizer did not affect the plasma membrane anchoring ability. By contrast, Ac-K (PpIX)-E4 was found to have a poor plasma membrane targeting ability, demonstrating that the species of amino acids had a significant influence on the subcellular localization of photosensitizer. Besides, the

3.3. Preparation and characterization of nano photosensitizers (NPs) Low dark toxicity is very important for PDT therapeutic agents in clinical practice. Prior to further exploring the biomedical applications of these PpIX conjugates, their potential dark toxicities were examined above all by MTT assay. As shown in Fig. S16, Ac-K(PpIX)-R, Ac-K (PpIX)-R2, Ac-K(PpIX)-R4, Ac-K(PpIX)-E and Ac-K(PpIX)-E4 were found to have much lower dark toxicities than Ac-K(PpIX)-R6, Ac-K(PpIX)-R8 and PpIX-KR4. In view of the plasma membrane targeting property, Ac6

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Fig. 2. Amino acids regulated plasma membrane targeting of PpIX. A) CLSM images and fluorescence profile analysis of 4T1 cells after treatment with 20 μM of Ac-K (PpIX)-R, Ac-K(PpIX)-R2, Ac-K(PpIX)-R4, PpIX-KR4, Ac-K(PpIX)-R6, Ac-K(PpIX)-R8, Ac-K(PpIX)-E or Ac-K(PpIX)-E4 for 4 h and stained by DiO. B) CLSM images of 4T1 cells after treatment with 20 μM of Ac-K(PpIX)-R, Ac-K(PpIX)-R2, Ac-K(PpIX)-R4, PpIX-KR4, Ac-K(PpIX)-R6, Ac-K(PpIX)-R8, Ac-K(PpIX)-E or Ac-K(PpIX)-E4 for 1 h, 2 h and 3 h respectively. C) CLSM images of 4T1 cells after treatment with Ac-K(PpIX)-R, Ac-K(PpIX)-R2, Ac-K(PpIX)-R4, Ac-K(PpIX)-R6, Ac-K(PpIX)-E or Ac-K(PpIX)-E4 for 2 h at the concentration of 5 μM, 10 μM and 40 μM respectively. D) CLSM images of 4T1 cells, HeLa cells and A549 cells after treatment with 20 μM of Ac-K(PpIX)R2, Ac-K(PpIX)-R4, Ac-K(PpIX)-R6, Ac-K(PpIX)-E4 for 2 h. Scale bar: 20 μm.

fluorescent probe (Fig. S17). Then, their morphologies were characterized by transmission electron microscope (TEM) (Fig. 3B and C). Dynamic light scattering (DLS) suggested that the hydrodynamic sizes of NPs-R4 and NPs-E4 were about 159.5 nm and 312.6 nm and both of them possessed favorable dispersibility (Fig. 3D and E). Moreover, the hydrodynamic size and polydispersity index (PDI) of NPs-R4 were found to have no obvious changes in seven days, implying a good stability of nanoparticles in aqueous solution (Fig. S18). Furthermore, the zeta potential of NPs-R4 and NPs-E4 was also detected. As shown in Fig. S19, the zeta potential of NPs-R4 and NPs-E4 was 16.6 mV and −25.8 mV,

K(PpIX)-R, Ac-K(PpIX)-R2 and Ac-K(PpIX)-R4 might be the ideal therapeutic agents for effective PDT. In this paper, low toxic Ac-K(PpIX)-R4 were chosen for following studies because it could self-assemble into spherical nanoparticles (designated as NPs-R4) in aqueous solution (Fig. 3A), facilitating its preferential tumor accumulation after intravenous injection through the enhanced penetration and retention (EPR) effect. Ac-K(PpIX)-E4 with low dark toxicity, which could also form into nanoparticles (designated as NPs-E4), was used as the control. To validate it, critical micelle concentration (CMC) of Ac-K(PpIX)-R4 and Ac-K(PpIX)-E4 was calculated by using pyrene as the hydrophobic 7

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Fig. 3. The preparation and characterization of NPs. A) Chemical structures of amino acids-modified PpIX and schematic illustration of the preparation of NPs by selfassembly. TEM images of B) NPs-R4 and C) NPs-E4. Scale bar: 200 nm. The particle size distributions of D) NPs-R4 and E) NPs-E4. F) UV–vis absorbance of PpIX, NPsR4 and NPs-E4. G) CLSM images of 4T1 cells after treatment with 74 μM of NPs-E4 and NPs-R4 in the presence or absence of light. 4T1 cells without any treatments were employed as the blank control. Scale bar: 10 μm.

might be ascribed to its strong hydrophobicity and self-quenching effect. Notably, upon light irradiation, NPs-E4 also reflected a disappointing ability of generating ROS while NPs-R4 exhibited an excellent productive competence, which could be attributed to the significantly improved water solubility and cellular uptake of PpIX with the modification of tetra-arginine rather than tetra-glutamic acid. This exciting result suggested a great potential of NPs-R4 for robust PDT against tumor cells.

respectively. Besides, the UV–vis spectra of NPs-R4 and NPs-E4 were also detected while PpIX was used as the control. As shown in Fig. 3F, compared with PpIX, both of NPs-R4 and NPs-E4 exhibited a sharp Soret band around 400 nm, which suggested the negligible existence of π-π stacking-induced aggregation. To verify it, the aqueous solutions of PpIX, Ac-K(PpIX)-R4 and Ac-K(PpIX)-E4 were observed for tyndall effect. As shown in Fig. S20, compared with Ac-K(PpIX)-R4 and Ac-K (PpIX)-E4, a serious aggregation behavior was observed in PpIX solution, demonstrating that amino acids modification could really improve the solubility and stability of PpIX. To investigate the effect of the improved physicochemical property on the capability of PpIX to generate ROS, NPs-R4 and NPs-E4 were incubated with 4T1 cells in the presence or absence light irradiation for CLSM observations. PpIX with light irradiation was used as the control and DCFH-DA was used as the sensor. As demonstrated in Fig. 3G, when without light irradiation, NPs-R4 and NPs-E4 hardly seemed to have generated ROS in cells, which confirmed the necessity of light in PDT. However, PpIX with light irradiation still failed to produce ROS, which

3.4. Subcellular distributions and penetration in multicellular tumor spheroids To study the subcellular distribution of NPs-R4 and NPs-E4 after cellular uptake, 4T1 treated with NPs-R4 or NPs-E4 were further stained with MitoTracker Green, LysoTracker Green or Hoechst 33342 for CLSM observations. As displayed in Fig. S21, the red fluorescence of NPs-R4 was not matched very well with MitoTracker Green, demonstrating the off-target property of NPs-R4 to mitochondria. However, 8

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Fig. 4. PDT effect of NPs in vitro. A) Schematic illustration of NPs-R4 and NPs-E4 with or without plasma membrane targeting ability for PDT in vitro. B) Inverted microscope images of 4T1 cells after treatment with NPs-E4 (74 μM) or NPs-R4 (74 μM) in the absence or presence of light irradiation. Photo toxicities of NPs-R4 and NPs-E4 against 4T1 cells, HeLa cells, A549 cells and CT26 cells respectively by MTT assay. D) Flow cytometry assay and E) live/dead cell staining analysis of 4T1 cells after treatment with NPs-E4 (20 μM or 74 μM) or NPs-R4 (20 μM or 74 μM) in the absence or presence of light irradiation. Scale bar: 100 μm.

3.5. Anti-tumor studies of NPs-R4 and NPs-E4 in vitro

NPs-R4 was found to have a considerable overlap with LysoTracker Green, implying the unavoidable endocytosis of plasma membrane targeted NPs-R4 by 4T1 cells. Besides, there was little overlap between the red NPs-R4 and blue Hoechst 33342, demonstrating that NPs-R4 was primarily anchored on the plasma membrane or internalized in the cytoplasm rather than in cell nucleus. For NPs-E4, due to the limited cellular uptake, few of it was found to match with MitoTracker Green or Hoechst 33342. Notably, NPs-E4 without plasma membrane targeting ability exhibited a good overlap with LysoTracker Green, verifying that it entered into 4T1 cells by the endocytosis pathway. The similar results could be observed by the fluorescence profile analysis. Despite of the inevitable endocytosis of NPs-R4 by cells, an obvious plasma membrane targeting ability of NPs-R4 could be still observed, which was of great significance to the subsequent in situ PDT on plasma membrane of tumor cells. The permeability of NP-R4 was evaluated by establishing multicellular tumor spheroids model. NP-E4 and free PpIX were used as the controls. As illustrated in Fig. S22, positive NPs-R4 with membrane targeting ability exhibited the best permeation effect compared with NPs-E4 and free PpIX, which was advantageous for its tumor therapy in vivo. However, negative NPs-E4 had the worst permeation effect. It could be concluded that the amino acids-regulated plasma membrane targeting had a significant effect on the permeability of the photosensitizer for enhanced PDT effect.

According to the previous study, Ac-K(PpIX)-R4 possessed a good plasma membrane targeting ability and a low dark toxicity. After assembling into nanoparticles, the obtained NPs-R4 was expected to have the similar property for enhanced PDT of tumor (Fig. 4A). The PDT effect of NPs-R4 was evaluated in vitro using NPs-E4 as the control. As shown in Fig. 4B, the light and NPs-E4 had an ignorable influence on the integrity of plasma membrane. Additionally, a few of 4T1 cells were destructed after treatment with NPs-E4 upon light irradiation, which could be ascribed to the limited cellular uptake in the absence of plasma membrane targeting. By contrast, NPs-R4 with light irradiation could wreak havoc on the membrane integrity, suggesting the great advantage of plasma membrane targeted PDT. Despite of the enhanced cellular uptake by the plasma membrane anchoring, NPs-R4 had no obvious impact on the membrane integrity, implying a low dark toxicity as well as a good biocompatibility in vitro. Moreover, the photo toxicities of NPs-R4 and NPs-E4 were further investigated against various cell lines by MTT assay. As reflected in Fig. 4C, NPs-E4 had a relatively low photo toxicity to all of the cell lines including 4T1 cells, HeLa cells, A549 cells and colorectal cancer (CT26) cells. However, NPs-R4 with light irradiation exhibited a strong cytotoxic effect in a concentrationdependent manner, demonstrating a potential advantage of this amino acids modification method as a versatile plasma membrane targeting strategy. Besides, the cytotoxicity of NPs-R4 and NPs-E4 were also tested 9

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Fig. 5. Biodistribution of NPs in 4T1 tumor-bearing mice. A) Real-time fluorescence images of 4T1 tumor-bearing mice after intravenous injection with NPs-R4 or NPs-E4 for 1 h, 3 h, 6 h, 9 h, 12 h and 24 h. The circle of dots represented the tumor tissue. B) Ex vivo tissue imaging and C) the mean fluorescence intensity analysis of sacrificed tumor tissues and main organs at the 24th h after postinjection. He, Li, Sp, Lu, Ki, and Tu represent heart, liver, spleen, lung, kidney and tumor, respectively. #P < 0.05 were tested via a Student's t-test.

treated with NPs-R4 or NPs-E4 was monitored at the predetermined time. After postinjection for 1 h, the fluorescence of PpIX was found to aggregation due to the EPR effect of nanoparticles. Over time, the fluorescence accumulations in tumor tissues were strengthened and then weakened, which could be ascribed to the ceaseless blood circulation and metabolism. Even so, the preferential fluorescence accumulations could be still observed after 24 h, suggesting an extended halflife and enhanced tumor retention capability. Notably, NPs-E4 seemed to show a better tumor accumulation than NPs-R4, which might be attributed to the alleviative immune clearance in vivo endowed by negative nanoparticles of NPs-E4. To verify it, the blood retention kinetic study of NPs-R4 was performed, which exhibited a short blood circulation time compared with NPs-E4 (Fig. S26). After postinjection for 24 h, the mice were sacrificed, and the tumors and main organs (heart, liver, spleen, lung and kidney) were harvested for optical imaging (Fig. 5B). Although there were massive fluorescence aggregations in the primary metabolic organs of liver and kidney, the considerable accumulations of NPs-R4 and NPs-E4 were still observed in tumor tissues. Similar results could be also found in the mean fluorescence intensity analysis and quantitative PpIX fluorescence analysis (Fig. 5C and Fig. S27). Since reliable investigations had confirmed the good biocompatibilities and low dark toxicities of NPs-R4 and NPs-E4, the undesirable aggregations of them were supposed to cause an insignificant system toxicities on mice.

against the normal 3T3 cells. As shown in Fig. S23, NPs-R4 exhibited the higher photo toxicity than NPs-E4, suggesting its membrane targeting ability enhanced PDT effect on 3T3 cells. Also, this result implied that the membrane targeting ability of NP-R4 was non-selective to tumor cells. Even so, both NPs-R4 and NPs-E4 had a low dark toxicity, suggesting a good biocompatibility. Based on this, it could be concluded that this plasma membrane targeted PDT would improve the effect of tumor inhibition while not increase the side effects. As the control, the cytotoxicity of Ac-K(PpIX)-RERE and Ac-K(FAM)-RFRF against 4T1 cells was also measured by MTT assay. As illustrated in Fig. S24, due to the absence of plasma membrane targeting ability, they exhibited a worse PDT effect compared with Ac-K(PpIX)-R4. Besides, the PDT effect was also assessed by flow cytomety (Fig. 4D) and live/dead cell staining analysis (Fig. 4E and Fig. S25). As expected, only NPs-R4 with light irradiation caused a large number of cells death, further verifying the overwhelming superiority of this plasma membrane targeting strategy for enhanced photodynamic tumor therapy. 3.6. Biodistributions of NPs-R4 and NPs-E4 in vivo Prior to evaluating the PDT effect of NPs-R4 in vivo, the biodistribution was investigated by optical imaging of 4T1-tumor bearing mice after tail vein injection. Intravenous injection with NPs-E4 was used as the control. As shown in Fig. 5A, 4T1 tumor bearing mouse 10

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Fig. 6. In vivo anti-tumor study of NPs against 4T1 tumor-bearing mice via intravenous injection. A) Relative tumor volume changes in 15 days after treated with NPsR4 or NPs-E4 in the presence or absence of light irradiation. Mice treated with PBS were used as the negative control. B) Representative tumor images and C) average tumor weight of the mice on the 15th day. D) Relative body weight changes during the treatments. E) H&E staining of the sacrificed tumor tissues after treatment for 15 days. F) Alanine aminotransferase (ALT), G) aspartate transaminase (AST), H) blood urea nitrogen (BUN) and I) uric acid (UA) analysis in the serum of the mice on the 15th day. J) Hematological parameters of the mice on the 15th day. #P < 0.05, *P < 0.005, &P < 0.001 and §P < 0.0005 were tested via a Student's t-test.

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anchoring behavior could be applied to different cell lines with the incubation time- and concentration-dependent manners. Furthermore, the self-assembled nano photosensitizers (NPs) possessed a good stability and biocompatibility as well as the improved capacity in generating intracellular reactive oxygen species (ROS). Benefiting from its efficient plasma membrane anchoring ability, the NPs could destroy the membrane integrity and achieve an enhanced PDT effect under light irradiation by the in situ generation of ROS on plasma membrane. Besides, sufficient in vivo investigations verified that the NPs could realize a significant tumor accumulation and effective tumor inhibition with a minimal systemic toxicity. We believe that this epigenetics-inspired amino acids-regulated plasma membrane targeting strategy opens a window in the development of structure-based drug design for site-specific delivery and tumor precision therapy.

3.7. Anti-tumor studies of NPs-R4 and NPs-E4 in vivo After verifying the feasible tumor accumulations of NPs-R4 or NPsE4 after tail vein injection, the PDT induced by them were further investigated in vivo. Above all, 4T1 tumor bearing mice model was established as our previous report.6,33 Subsequently, these 4T1 tumor bearing mice were randomly divided into five groups, including PBS, NPs-E4, NPs-R4, NPs-E4 with light irradiation and NPs-R4 with light irradiation. After intravenous injection with PBS, NPs-E4 or NPs-R4, the tumor volume of the mouse was monitored every other day. As shown in Fig. 6A, compared with PBS, NPs-E4 and NPs-R4 without light irradiation simply could not inhibit the tumor growth, suggesting the inappreciable dark toxicities of them. However, once exposed to light, both NPs-E4 and NPs-R4 exhibited a good inhibitory effect on tumor growth. Especially, although NPs-E4 had a better tumor accumulation effect than NPs-R4 (Fig. 5A), the best therapeutic effect was found in NPs-R4 group rather than NPs-E4 group. The possible reason should be the poor plasma membrane targeting ability and the limited cellular uptake of NPs-E4 (Fig. 2). After treatment for 15 days, the tumor tissues were harvested for imaging and weighing (Fig. 6B and C), which agreed well with the above results. And the PDT effect of NPs-R4 exhibited a significant difference in comparison to other groups, which further highlighted the enormous advantage of plasma membrane targeted NPs-R4 for enhanced PDT regardless of the weaker tumor accumulation ability. The body weight monitored during the treatments had no obvious changes, implying a good biocompatibility and low side effects of NPs-R4 and NPs-E4 in vivo (Fig. 6D). After various treatments, the obtained tumor tissues were also stained with hematoxylin and eosin (H& E) for histological examinations. As demonstrated in Fig. 6E, compared with those groups without light, many damaged tumor cells without nuclei were found in the groups with light due to the participation of PDT. Among which, NPs-R4 induced PDT effect was most powerful, further confirming the enhanced anti-tumor efficacy by regulating the subcellular localization of photosensitizer using amino acids modification strategy. In view of the obvious accumulation of NPs-R4 and NPs-E4 in normal tissues after tail vein injection (Fig. 5B and C), H&E staining analysis of the harvested organs including heart, liver, spleen, lung and kidney was also performed at the 15th day. As suggested in Fig. S28, no apparent pathological changes were found in these organs and the physiological morphologies were roughly normal, implying that the undesired accumulations of NPs-R4 and NPs-E4 had no obvious side effects on these normal tissues owing to the absence of light irradiation. Moreover, to further assess the biocompatibility of NPs-R4 and NPs-E4 in vivo, biochemical indexes and blood routine examination of the mice with various treatments were also detected. As reflected in Fig. 6F-I, the concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and uric acid (UA) between different groups had no significant differences. Additionally, both of NPs-R4 and NPs-E4 with light irradiation exhibited no obvious anomalies in blood routine test, illustrating a good biocompatibility and a low system toxicity of them (Fig. 6J). In a word, these above investigations demonstrated that this amino acids-regulated plasma membrane targeting of photosensitizer had a robust PDT efficiency under light irradiation with an excellent biosecurity.

Acknowledgements We are grateful for the financial support of National Natural Science Foundation of China (81330007, U1601227, 51802049, 51803086), the Natural Science Foundation of Guangdong Province (2018030310283), the Science and Technology Programs of Guangdong Province (2015B020225006), the Science and Technology Programs of Guangzhou (201904010324), the Young Elite Scientist Sponsorship Program by CAST (2018QNRC001) and the Open Research Fund of the Key Laboratory of Molecular Target & Clinical Pharmacology (Guangzhou Medical University). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biomaterials.2019.119497. References [1] L. Rajendran, H.J. Knölker, K. Simons, Subcellular targeting strategies for drug design and delivery, Nat. Rev. Drug Discov. 9 (2010) 29–42. [2] M. Murakami, H. Cabral, Y. Matsumoto, S.R. Wu, M.R. Kano, T. Yamori, N. Nishiyama, K. Kataoka, Improving drug potency and efficacy by nanocarriermediated subcellular targeting, Sci. Transl. Med. 3 (2011) 64ra2. [3] R.I. Benhamou, M. Bibi, J. Berman, M. Fridman, Localizing antifungal drugs to the correct organelle can markedly enhance their efficacy, Angew. Chem. Int. Ed. 130 (2018) 6338–6343. [4] W.H. Chen, G.F. Luo, X.Z. Zhang, Recent advances in subcellular targeted cancer therapy based on functional materials, Adv. Mater. 31 (2019) 1802725. [5] X.S. Li, S. Lee, J. Yoon, Supramolecular photosensitizers rejuvenate photodynamic therapy, Chem. Soc. Rev. 47 (2018) 1174–1188. [6] S.Y. Li, H. Cheng, B.R. Xie, W.X. Qiu, J.Y. Zeng, C.X. Li, S.S. Wan, L. Zhang, W.L. Liu, X.Z. Zhang, Cancer cell membrane camouflaged cascade bioreactor for cancer targeted starvation and photodynamic therapy, ACS Nano 11 (2017) 7006–7018. [7] M.L. Li, J. Xia, R.S. Tian, J.Y. Wang, J.L. Fan, J.J. Du, S. Long, X.Z. Song, J.W. Foley, X.J. Peng, Near-infrared light-initiated molecular superoxide radical generator: rejuvenating photodynamic therapy against hypoxic tumors, J. Am. Chem. Soc. 140 (2018) 14851–14859. [8] D.E. Dolmans, D. Fukumura, R.K. Jain, Photodynamic therapy for cancer, Nat. Rev. Cancer 3 (2003) 380–387. [9] S.K. Li, Q.L. Zou, Y.X. Li, C.Q. Yuan, R.R. Xing, X.H. Yan, Smart peptide-based supramolecular photodynamic metallo-nanodrugs designed by multicomponent coordination self-assembly, J. Am. Chem. Soc. 140 (2018) 10794–10802. [10] K. Liu, R.R. Xing, Q.L. Zou, G.H. Ma, H. Mohwald, X.H. Yan, Simple peptide-tuned self-assembly of photosensitizers towards anticancer photodynamic therapy, Angew. Chem. Int. Ed. 55 (2016) 3036–3039. [11] Z.X. Zhou, J.P. Liu, T.W. Rees, H. Wang, X.P. Li, H. Chao, P.J. Stang, Heterometallic Ru-Pt metallacycle for two-photon photodynamic therapy, Proc. Natl. Acad. Sci. U.S.A. 115 (2018) 5664–5669. [12] Z.J. Zhou, J.B. Song, L.M. Nie, X.Y. Chen, Reactive oxygen species generating systems meeting challenges of photodynamic cancer therapy, Chem. Soc. Rev. 45 (2016) 6597–6626. [13] J.Y. Zeng, M.Z. Zou, M.K. Zhang, X.S. Wang, X. Zeng, H.J. Cong, X.Z. Zhang, πextended benzoporphyrin-based metal-organic framework for inhibition of tumor metastasis, ACS Nano 12 (2018) 4630–4640. [14] P. Agostinis, K. Berg, K.A. Cengel, T.H. Foster, A.W. Girotti, S.O. Gollnick, S.M. Hahn, M.R. Hamblin, A. Juzeniene, D. Kessel, M. Korbelik, J. Moan, P. Mroz, D. Nowis, J. Piette, B.C. Wilson, J. Golab, Photodynamic therapy of cancer: an update, CA-Cancer, J. Clin. 61 (2011) 250–281.

4. Conclusions Summarily, inspired by epigenetics, we proposed a versatile strategy to realize the plasma membrane targeting of photosensitizer for enhanced tumor photodynamic therapy (PDT). For the first time, the plasma membrane anchoring and PDT efficiency of photosensitizer was effectively manipulated at a single amino acid level. Moreover, it was found that the number and variety of amino acids for modifications had an impact on the plasma membrane targeting effect of the photosensitizer. Importantly, this amino acids-regulated plasma membrane 12

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