High Singlet Oxygen Yield Photosensitizer Based Polypeptide

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High Singlet Oxygen Yield Photosensitizer based Polypeptide Nanoparticles for Ultralow-Power Near Infrared Light Imaging–guided Photodynamic Therapy Zheng Ruan, Wei Maio, Pan Yuan, Le Liu, Lijuan Jiao, Erhong Hao, and Lifeng Yan Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00576 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 6, 2018

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Bioconjugate Chemistry

High Singlet Oxygen Yield Photosensitizer based Polypeptide Nanoparticles for Low-Power Near Infrared Light Imaging–guided Photodynamic Therapy

Zheng Ruan a, Wei Miao b, Pan Yuan a, Le Liu a, Lijuan Jiao b, Erhong Hao b,*, and Lifeng Yan a,* a

CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for

Physical Sciences at the Microscale, and Department of Chemical Physics, iCHEM, University of Science and Technology of China, Jinzai road 96, Hefei,230026, Anhui, China. E-mail: [email protected]. b

Laboratory of Functional Molecular Solids, Ministry of Education; Anhui

Laboratory of Molecule Based Materials (State Key Laboratory Cultivation Base) and School of Chemistry and Materials Science, Anhui Normal University, No.1 East Beijing Road, Wuhu, 241000, Anhui, China. E-mail: [email protected] Ruan and Miao contributed equally.

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Abstract NIR photosensitizer is attractive for photodynamic therapy (PDT). Low-power light irradiation and imaging-guided PDT makes it possible to increase tissue penetration depth. Pyrrole-substituted iodinated BODIPY (BDPI) molecule was designed and synthesized, and it possesses an intense NIR absorption and emission band, and exhibits a high singlet oxygen quantum yield (Φ∆ = 0.80) which leads remarkable cytotoxicity upon low power illumination (IC50 = 0.60 µg/mL, 6.1 mW/cm2). After being encapsulated with biocompatibility polypeptide PEG-PLys, polymeric micelles nanoparticles (PBDPI NPs) was obtained that are water-dispersed and passively tumor-targetable. Such enhanced accumulation in tumor area makes it easily traced in vivo due to its NIR fluorescence. In addition, such nanoparticles offer an unprecedented photodynamic therapeutic effect by using a low-power irradiation light, which makes it possible to kill cancer cells in deep tissue efficiently.

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INTRODUCTION

Photodynamic therapy (PDT), as a promising minimally invasive alternative to conventional approaches, has been widely exploited over the past years.1 PDT requires three elements simultaneously: a photosensitizer (PS), molecular oxygen (3O2) and light source.2 When the PS was activated by the irradiation of light with specific wavelengths, it will transfer its excited triplet state energy to the neighboring molecular oxygen or other substrates, thereby producing cytotoxic reactive oxygen species (ROS) like singlet oxygen (1O2) for tissue damage. Much effort has been developed to improve the therapeutic efficacy of PDT, especially highly quantum yield of singlet oxygen under low power density of NIR light to penetrate deep-tissue and reduce the side-effect of light.3-4 The direct strategy is to generate sufficient ROS in tumor area by increasing the ROS yield (in NIR region) and tumor accumulation of PSs in vivo.5-8 Among the reported PDT photosensitizers, boron dipyrromethene (BODIPY) derivatives, which have many ideal photosensitizer characteristics including high molar extinction coefficients, lower photobleach, and less dark toxicity, are considered as potential candidates for synergistic-use as imaging-guided PDT therapeutic agents.9-14 However, BODIPYs usually exhibit low singlet oxygen (1O2) quantum yield (QYs, Φ∆), which greatly restricts its further application for cancer diagnosis and PDT therapeutic.15-16 Heavy atoms are acknowledged to enhance the spin−orbital coupling (SOC) and thereby promote the singlet-to-triplet intersystem crossing (ISC) rate to improve the 1O2 QYs of PSs, which is named as “heavy atom 3



effect”.3,

8, 17-19

The most frequently encountered is halogenation (Br, I), which

converts the low triplet transition molecule to photosensitizer with competent intersystem crossing capacity.12, 20 In addition, iodine atom shows higher efficiency than bromine atom which is found in most researches.10 Several reports of iodine BODIPYs produced singlet oxygen, but these dyes had low ultraviolet absorption that restricted the potential application of them.8, 21-23 Besides the high 1O2 QYs, the tumor accumulation should be also taken into consideration. Most BODIPYs are hydrophobic and the free PS molecules are easily eliminated during the blood circulation.12, 19, 24 Owing to the enhanced permeability and retention (EPR) effect, nanoparticles (NPs) with proper size rather than free PS molecules prefer to accumulate at tumor site.25-27 After the NPs’ enrichment in tumor area, the irradiation permeation into tissues is another obstacle for efficient damage. Notably, near infrared (NIR) light in the region of around 650–1000 nm, recognized as “the NIR-I window” or “biological transparency window”, exhibits deeper issue penetration than UV or Vis light.28 The clinical use of PDT agents for deep-tissue tumor curing remains challenging; e.g., the FDA-approved PDT agent PpIX whose absorption is quite weak in the NIR region,29-30 and most BODIPYs used for PDT are tend to be activated by short wavelength light which hardly travel far into biological tissues.10 Considering that, more effort has lately emphasized on to shift the excitation wavelength to the NIR region for cancer treatment at the deep-tissue level.31-35 To date, although several azo-BODIPY molecules as PDT photosensitizer and their carrier were declared to be activated to NIR light,36-39 the PDT efficiency reported in vivo 4


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still requires irradiation with relatively high power density (>100 mW/cm2), and such utilization was only limited to frequent drug feeding during the whole treatment period. The main obstruction is still the low singlet oxygen yield of PS, especially under weak NIR light irradiation (most light will be adsorbed by the tissue). Here, an iodinated BODIPY (BDPI) has been designed and synthesized with high singlet oxygen quantum yield (Φ∆=0.80). Its absorption and emission are both in near infrared (NIR) region, which could show minimum photodamage to cells and excellent tissue penetration. Di-iodinated BDPI presents the highest 1O2 QY of 0.80 with the lowest half maximal inhibitory concentration (IC50) of only 0.60 µg/mL (0.93 µM) on HepG2 cells with a low power density (6.1 mW/cm2). Moreover, PEG initiated poly (ε-carbobenzoxy-L-lysine) as biodegradable nanocarrier were prepared by ring opening polymerization for BDPI delivery. And the BDPI loaded in amphiphilic polypeptide to could be self-assembled into micelles with suitable size (about 110 nm) as PEG-PLys@BDPI nanoparticles (PBDPI NPs) for prolonged blood circulation and tumor accumulation (Scheme 1). Moreover, in vivo PDT result suggests that PBDPI NPs we prepared have negligible dark toxicity, high phototoxicity, as well as good biocompatibility under a low-power near infrared lamp light.

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Scheme 1. Schematic illustration of high ROS generation micelles PEG-PLys@BDPI nanoparticles for PDT in vivo.

RESULTS AND DISCUSSION

Pyrrole

HIO3,I 2 N

N

B F F

I

EtOH

N I

1a

B F F

PEG-PLys I

85 ℃

O

O

NH2

I

N

NH

HN BDPI

BDPI

O O

NH

N H

112

DMF 72 h mPEG-NH 2

N

B F F

I

2a

O O

I

N

O

O

N H O 112

H N O

N H H 19

PEG-PLys

Figure 1. Synthesis route of the iodinated BODIPY loaded polymeric micelles PEG-PLys@BDPI.

As shown in Figure 1, according to the literature we synthesized Bodipy 1a,40 Then 1a and excess iodine and iodic acid were refluxed in ethanol to get 2a. The 1H 6


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NMR spectra of 2a was shown in Figure 2a. BODIPY 2a and pyrrole reacted at 85°C to get BDPI and the 1H NMR spectra of BDPI was shown in Figure 2b. As shown in the 1H NMR spectra of BDPI, there are two hydrogen at 10.18 ppm, indicating two pyrrole NH exist. Also, the

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C NMR spectra of 2a and BDPI, the

HRMS for 2a and BDPI was show in Fig. S2-S5. As shown in Figure 2c, d and Table 1, the UV absorption of BDPI is from 670 to 692 nm in various solvents, which were red-shifted 22 nm from acetonitrile to toluene. Similar results were obtained for fluorescence, which were all in the near infrared region for potential deep penetration imaging-guided PDT. Besides, we have also checked the photo stability of photosensitizers in CH3CN and water with laser irradiation (Figure S5). The absorption data of BDPI showed no obvious change after irradiation, which confirmed its stability in different solvents.

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Figure 2. 1H NMR spectra of 2a (a), 1H NMR spectra of BDPI (b), Absorption (c) and emission (d) spectra of BDPI recorded in different solvents. Excited at 620 nm.

Table 1. Photophysical properties of BDPI in different solvents at room temperature.

BDPI

solvent

λmax(nm)

λem(nm)

logεmaxa

φb

Ssc

dichloromethane hexane toluene acetonitrile

679 687 692 671

712 707 715 707

4.81 5 4.84 4.68

0.18 0.22 0.17 0.13

683 412 465 759

a

Molar absorption coefficient are in the maximum of the highest peak. bFluorescence quantum yields of BDPI were calculated using 1,7-diphenyl-3,5-di(p-methoxyphenyl)-azadipyrromethene (φ = 0.36 in chloroform) as the reference. cStokes shift.

Measurement of singlet oxygen quantum yield (Φ∆). The generation ability of singlet oxygen was further studied with the singlet-oxygen trap molecule 1, 3-diphenylisobenzofuran (DPBF). Under irradiation at 0.5 mW/cm2, iodinated BODIPY BDPI sensitizes molecular oxygen to generate singlet oxygen. The DPBF could react with the singlet oxygen, leading a decrease in the absorbance of DPBF at 414nm. Meanwhile, the methylene blue (MB) was used as reference compound whose singlet oxygen quantum yield is known as 0.52 (in acetonitrile) or 0.57 (in DCM). The absorption spectra changes of MB and BDPI was shown in Figure 3. Sharp decrease of peaks at 414nm with BDPI was found during the whole irradiation process. The Φ∆ of BDPI in acetonitrile and DCM were calculated as 0.80 and 0.76, respectively, which could serve as a potential photosensitizer with high efficiency for PDT.

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Figure 3. Absorption spectra changes of DPBF upon irradiation in the presence of reference compound methylene blue (MB) or BDPI and the decrease rate plots (recorded at 10 s interval) in CH3CN (a, MB, b, BDPI, c, plots) and DCM (d, MB, e, BDPI, f, plots). Irradiation: 635 nm laser with 0.5 mW/cm2.

Preparation and characterization of PEG-PLys@BDPI (PBDPI). Owing to the hydrophobicity of free BDPI molecules, we have designed some kind of soluble drug delivery platform to realize more accumulation in tumor area for further application. Here we chose the polypeptide as the photosensitizer carrier. The amphiphilic shell PEG-PLys was synthesized via ring opening polymerization initiated by mPEG-NH2. The 1H NMR spectra were shown in Figure 4a & b and the appearance of phenyl peaks around 7.5 ppm indicates the formation of the amphiphilic block. The degree of polymerization (DP) of PLys segment was determined to be 19 according to the 1H NMR analysis. The macro-initiator and diblock polymer were also measured by DMF GPC and the obtained amphiphilic polypeptide showed a narrow molecular weight 9



distribution with Mw/Mn of 1.16 (Figure 4c). The polymeric micelles PBDPI was self-assembled by dialysis. TEM image exhibited a spherical and compact morphology with a slightly smaller average diameter (about 100 nm, seen in Figure 4d). Meanwhile, the average size of the obtained PBDPI NPs were calculated by DLS measurement as 110 nm in PBS (Figure 4e) compared with TEM results, suggesting the existence of hydrophilic PEG corona in aqueous solution. In Figure 4f, the micelles was also proved to be stable in DMEM with 10% FBS which was used as the cell culture medium (average diameter is about 116 nm). Moreover, the probable size of PEG shell prohibited aggregation of these photosensitizer nanoparticles, which potentially prolonged the blood circulation and enhanced the retention of nanoparticles following systemic in vivo administration.

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Figure 4. 1H NMR spectra of the initiator mPEG-NH2 (a) and the diblock polymer PEG-PLys (b). GPC curves of the polymers (c). TEM image of PBDPI NPs, the scale bar is 100 nm (d). Size distribution by DLS measurement in PBS (e) and DMEM with 10% FBS (f).

Anticancer activity with BDPI in low irradiation power. To evaluate the PDT efficiency of BDPI with high singlet oxygen quantum yield, we first check the phototoxicity of free BDPI molecules against HepG2 cancer cells with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. MTT assay 11



was conducted to measure the cells viability after treatment with the BDPI under NIR irradiation. After incubation for 24 h without extra treatment, negligible cytotoxicity was exhibited indicating good biocompatibility of BDPI. For the irradiation power selection, according to our previous work, we have chosen 20 mW/cm2 for 10 min with a 660 nm spectral output lamp at first. After that living cells were rarely found (Figure S6) owing to the superior ability of singlet oxygen generation. Then we tried to reduce the irradiation power and duration time (to 5 min). Upon exposure to the NIR laser for 5 min, HepG2 cells incubated with 2 µM showed about 90% growth inhibition ratio even with 6.1 mW/cm2 (Figure 5). The results were compared with BODIPY in our previous work (singlet oxygen quantum yield, Φ∆=0.36). There are over 70% cell survival rate with 2 µM BODIPY incubation, which was predictable due to the low efficiency of single oxygen generation (Figure S7). The IC50s of BDPI under different irradiation power were roughly calculated as 3.70 µM (2.40 µg/mL, 0.5 mW/cm2), 3.14 µM (2.04 µg/mL, 1.0 mW/cm2), 2.30 µM (1.50 µg/mL, 2.0 mW/cm2), 1.86 µM (1.21 µg/mL, 3.0 mW/cm2), 0.80 µM (0.60 µg/mL, 6.1 mW/cm2) and 0.71 µM (0.46 µg/mL, 10.2 mW/cm2). The data above was fitted as curve in Figure 5b (each fitting line seen in Figure S8). We can infer that photosensitizer dosage and irradiation power are both important factors that should be put more emphasis on.

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Figure 5. MTT results of BDPI under different low power density (a) and its IC50 fitting curve toward different irradiation intensity (b).

PDT induced cytotoxicity of PBDPI nanoparticles. Inspired by the effective growth inhibition behavior of BDPI, the phototoxicity of polymeric micelles PBDPI were evaluated with two different cell lines which are HepG2 (hepatocellular carcinoma cells from human) and EMT6 cells (mammary carcinoma cells from mouse). In Figure 6a, these cells showed similar growth inhibition ratio against PBDPI not only at low concentration of photosensitizer but also with low power of light. Less than 20% cells still alive at 2 µM with 6.1 mW/cm2 NIR light. Similar results could be found towards the EMT6 cells (Figure 6b). The dosage and light

power we used here were much weaker than previous PDT reports as far as we know.

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Figure 6. Cell viability evaluation of gradient concentration of PBDPI NPs towards HepG2 (a) and EMT6 (b) cells upon various power density illumination.

A fluorescein diacetate (FDA) and propidium Iodide (PI) staining was conducted to confirm the MTT result. The red signal (PI) stands for dead cells and the green (FDA) for alive ones. From the fluorescence images, we could directly confirm the good biocompatibility of PBDPI without NIR light (Figure S9). With 3.1 mW/cm2, a large area of dead cells were noticed in both HepG2 and EMT6 cells. Most of cells were killed with 6.1 mW/cm2, suggesting the forceful damage to different cell lines even with such weak NIR light (Figure 7a & b).

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Figure 7. Dead/live staining of HepG2 (a) and EMT6 (b) cells with PBDPI (at a concentration of 2 µM BDPI) upon different NIR laser power density (3.1 and 6.1 mW/cm2). Scale bar = 200 µm.

Cellular ROS detection and nanoparticles uptake. To elucidate the mechanisms of high cytotoxicity of PBDPI-micelle under NIR light, 2, 7-dichlorofluorescin diacetate (DCFH-DA) as the 1O2 sensor, which could be rapidly oxidized to emit green fluorescence. DCFH-DA, as ROS probe, was utilized to detect the intracellular ROS concentration. Owing to the short diffusion distances and half-life of ROS, 15



nanoparticles uptake into tumor cells is vital for the PDT agent to cause cell killing via oxidative stress. Upon NIR light after incubation for 24 h, BDPBr (used in our previous work20) with EMT6 cells exhibited slightly stronger fluorescence intensity than PBS control (Figure S10), which is presumably attributed to the limited ROS generation ability under irradiation. Compared with BDPBr, markedly higher ROS level with strong green fluorescence signal was observed inside EMT6 cells incubated with PBDPI NPs upon exposure to NIR illumination (Figure 8a). Fluorescence microscope was further used to evaluate the cellular uptake of PBDPI NPs directly against HepG2 and EMT6 cell lines. 4’-6-diamidino-2-phenylindole (DAPI) staining nucleus exhibited blue fluorescence while the red signal represented the emission of BDPI with NIR activation. Distinct fluorescence of photosensitizer BDPI was observed well-dispersing throughout the cytoplasm. Similar intracellular fluorescence signals of BDPI were observed in the EMT6 cells (Figure 8b). Time-depend fluorescence images of BDPI in HepG2 cells was taken to check the gradual uptake of the nanoparticles (Figure S11). These results clearly demonstrated that incubation with PBDPI had comparable intracellular uptake and bioimaging ability in different cell lines.

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Figure 8. (a) Intracellular ROS generation of EMT6 cell under NIR illumination. After incubation for 24 h, the residual PBDPI NPs were removed by adding fresh medium, and then the cells were further incubated with DCFH-DA (10 µM) for 25 min, exposed to a 660 nm laser for 5 min (6.1 mW/cm2). The scale bars are 50 µm. (b) Fluorescence microscope images of HepG2 and EMT6 cells after incubation with PBDPI NPs for 24 h. Blue: DAPI, red: BDPI. And the scale bars are 20 µm.

Enhanced permeability and retention (EPR) effect and biodistribution. The results above strongly proved the NIR imaging ability in vitro. According to former 17



researches, nanoparticles with 50-500nm were confirmed to have enhanced permeability and retention (EPR) effect to accumulate at tumor site because of the loose blood vessels.25 Owing to the complex situation of blood circulation and diffusion, the tumor accumulation and biodistribution of PBDPI were also taken into consideration comparing with the hydrophobic BDPI molecules. For in vivo bioimaging, PBS, free BDPI molecules (in 1% DMSO) and PBDPI NPs (at a concentration of 2.5 mg/kg BDPI) were injected intravenously into EMT6 tumor-bearing mice. The fluorescence signal was detected at predominant time points (2 h, 6 h, 12 h, 24 h and 48 h) by a Xenogen IVIS® Lumina system. As shown in Figure 9a, after treatment with PBDPI NPs, the fluorescence intensities of BDPI gradually enhanced in the tumor site at the first 12 h which were stronger than in other organs. Subsequently, the BDPI fluorescence signals were gradually reduced till the 48 h. In contrast, the highest fluorescence intensities at tumor site of mice treated with free BDPI were emerged at 6 h post-injection, then it rapidly decreased (Figure 9b). For 48 h post-injection, the mice were sacrificed for ex vivo imaging. Main organs and tumors were collected. In Figure 8c it was noticed that the stronger BDPI fluorescence signal treated with PBDPI NPs than the free BDPI groups in the tumor. The much better accumulation at tumor site of PBDPI NPs was attributed to the EPR effect of nanoparticles with proper size. The free BDPI molecules could be wiped out quickly during blood circulation. In conclusion, the PBDPI NPs we prepared showed the capacity for prolonged blood circulation and significant tumor accumulation.

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Figure 9. Time-dependent in vivo NIR fluorescence images of EMT6 tumor-bearing mice after intravenously injection (con. 2.5 mg/kg BDPI), free BDPI as negative group while PBDPI NPs as positive group. The tumor sites are denoted by white circles (n=2). (b) Total radiant efficiency values of the tumors in free BDPI and PBDPI NPs treated mice at 2 h, 6 h, 12 h, 24 h and 48 h. (c) Ex vivo images of main organs and tumors excised after 48 h injection. (d) Quantification of BDPI fluorescence in the main organs and tumors by average counts.

PDT performance in vivo. Inspired by the excellent accumulation at tumor site, the final antitumor efficacy were systemically evaluated by treating mice with NIR irradiation. In brief, when the tumors grew to about 100 mm3 (Figure 10a), we 19



injected free BDPI and PBDPI NPs (with a same BDPI concentration of 2.5 mg/kg) to mice bearing EMT6 tumors on both sides. After 12 h accumulation, only the right tumors were given NIR light from a 660nm laser (25 mW/cm2 for 10 min, total 15 J/cm2) while the tumors on the left as control. Due to the high singlet oxygen quantum yield of BDPI (0.80), the light power we used here was considerable low among reported PDT researches (Table S1). The tumor sizes were measured every other day during the therapeutic process (Figure 10b) to record the tumor growth. And the injection with following illumination was just given three times at the beginning (Figure 10c). As shown in Figure 10 b, the tumors in PBS group with or without irradiation showed rapid expedition. For the group of BDPI or PBDPI without laser treatment (the left side), tumors grew quite fast and reached similar level compared with the PBS group. Tumors treated with free BDPI and laser showed moderate inhibition. The limited PDT efficiency was attributed to the restricted accumulation of the hydrophobic BDPI molecules. Significantly, the group of PBDPI NPs with laser exposure ablated tumors most effectively (volume reduced about 80 %) and some of tumors were cured to disappear even without wound (PBDPI group in Figure 10a). After 21 days post-injection, mice were sacrificed and the tumors were collected. The digital photographs (Figure 10d) and wet weight analysis (Figure 10e) of each tumor also confirmed the high PDT efficiency of PBDPI NPs. The difference of tumors in each group was accord with the tumor growth during the whole treatment. The potential toxic side effects of PBDPI on EMT6 tumor-bearing mice were investigated. Body weight reduction was recognized as an indicator for 20


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treatment-induced toxicity. No significant weight loss was observed, indicating that the anticancer effect was not caused by the toxicity (Figure 10f).

Figure 10. (a) Images of EMT6 tumor-bearing mice at beginning and different groups after 21 days treatment. (b) The EMT6 tumor growth curves of different groups of mice after the corresponding treatments indicated. (c) Drug injection and illumination time points during the treatment. Digital images (d) and wet weight (e) of mice tumors after treated with PBS (L- & L+), free BDPI (L- & L+) and PBDPI NPs (L- & L+). The white dash circle means no tumor left. (f) Body weights changing of mice in each group during treatment. 21



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CONCLUSIONS In this study, a high singlet oxygen quantum yield (0.80) iodinated pyrrole-BODIPY (BDPI) has been synthesized and used as near infrared photosensitizer encapsulated by polypeptide for photodynamic therapy. The obtained polymeric micelles PEG-PLys@BDPI nanoparticles has a diameter of about 110nm which significant prolonged the blood circulation and increased the tumor accumulation of BDPI. The micelles showed excellent intracellular ROS generation and cellular uptake. Comparing with most PDT researches, the PBDPI NPs could effectively kill cancer cells and inhibit tumor growth upon extremely low power and dosage in the NIR light region. The reduction of light power or dosage means more biocompatible during tumor growth inhibition, exhibiting great potential of such powerful photosensitizer carrier for improved PDT therapeutic efficacy.

EXPERIMENTAL

Materials and characterization. All reagents and solvents mentioned were obtained from J&K (China), Aladdin Corporation (China) or and Sinoreagent Corporation.

Tetrahydrofuran

(THF),

dichloromethane

(DCM)

and

N,

N-dimethylformamide (DMF) were dehydrated with CaH2. Dialysis bags (cut off Mw = 3000/7000/10000) were bought from Bomei Biotechnology Corporation. Ultrapure water was obtained from a Milli-Q Synthesis System (Millipore, Bedford, MA, USA). HepG2 and EMT6 cancer cells from ATCC (American Type Culture Collection) were 22


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cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Hyclone, USA) with 10% fetal bovine serum (FBS, ExCell Bio, Shanghai, China) at 37 °C with 5% CO2. 1

H NMR and

13

C NMR spectra were scanned on Bruker AC 300/400/500

spectrometer with dimethyl sulfoxide-d6 (DMSO-d6) or chloroform-d (CDCl3) as solvent. The molecular weight distribution (Mw/Mn) was measured by gel permeation chromatography (GPC) with a refractive index detector (RID-10A, Shimadzu). The eluent was N, N-dimethylformamide (DMF). The particle sizes and its distributions were detected by a zeta-potential analyzer with dynamic laser light scattering (DLS) by a Malvern Zetasizer Nano ZS90 machine containing a He–Ne laser. UV-vis absorption spectra and fluorescence spectra were recorded by an UV1700pc (Shanghai AuCy Scientific Instrument Co., Ltd) ultraviolet spectrophotometer and an F97pro fluorescence spectrophotometer (Shanghai Lengguang Industrial Co. Ltd), respectively.

Synthesis of 2a. Iodic acid (918mg, 5.2 mmol) dissolved in a minimum amount of water was added dropwise to a solution of 1a (350mg, 1.3 mmol) and iodine (3.3g, 13 mmol) in EtOH 40 mL. This mixture was refluxed for 5 hours. The cooled crude mixture was poured into water (100 mL), extracted with CH2Cl2 (3×75 mL). Organic layer was collected, dried over anhydrous Na2SO4, and evaporated under vacuum. The residue was purified through column chromatography on silica, from which the desired product 2a was obtained in 72% yield (726mg).

1

H NMR (500 MHz, CDCl3)

δ 7.62 (t, J = 7.5 Hz, 1H), 7.54 (t, J = 8.0 Hz, 2H), 7.47 (m, 2H), 6.95 (s, 2H). 23


13

C


NMR (125 MHz, CDCl3) δ 140.0, 139.3, 137.6, 131.9, 131.3, 130.3, 128.8, 116.5, 91.1. HRMS (ESI) m/z calcd for C15H7BFI4N2+ (M)+ 752.68596, found 752.68610.

Synthesis of BDPI. The 2a (77mg, 0.10 mmol) and Na2CO3 (21mg, 0.20 mmol) was added to a 50 mL straight Schlenk reactor and dissolved with 2 mL pyrrole. The reaction was heated to 85°C for 20 hours under the protection of argon. The cooled crude mixture was poured into water (100 mL), extracted with CH2Cl2 (3×75 mL). Organic layer was collected, dried over anhydrous Na2SO4, and evaporated under vacuum. The residue was purified through column chromatography on silica, from which the desired product BDPI was obtained in 26% yield (17 mg). 1H NMR (300 MHz, CDCl3) δ 10.18 (s, 2H), 7.50-7.56 (m, 7H), 7.13 (s, 2H), 7.10 (s, 2H), 6.43-6.44 (m, 2H).

13

C NMR (125 MHz, CDCl3) δ 147.3, 138.7, 136.6, 136.2, 133.7, 130.4,

130.2, 128.6, 123.7, 122.4, 118.0, 111.5, 110.5. HRMS (APCI) calcd. for C23H15I2BF2N4[M+H]+ 650.9521, found 650.96209.

Singlet Oxygen Trap Experiments. In singlet oxygen measurements, 1, 3-diphenylisobenzofuran (DPBF) was used as a singlet oxygen trap molecule. Methylene blue (MB) was used as the reference compound with singlet oxygen quantum yield as 0.52 (in acetonitrile) and 0.57 (in DCM). In brief, photosensitizer (O.D ~ 0.5) and a trap molecule (O.D ~ 1.5) were mixed acetonitrile or DCM, followed by irradiation (635 nm laser with 0.5 mW/cm2) at a 10 s interval. Absorbance decrease of DPBF at 414 nm was monitored and recorded indicating 24


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singlet oxygen generation and consumption in the presence of light and BDPI. The singlet oxygen quantum yield ( ) of BDPI was calculated by the formula below.

k 1 − 10 = 1 − 10 * is the singlet oxygen quantum yield of the MB in acetonitrile (0.52) or DCM (0.57), and are slopes of plots, which represent the absorbance at 414 nm of DPBF in the BDPI and MB, respectively. and is absorbance at the irradiation wavelength (635nm) of the MB and BDPI at the beginning.

Synthesis of mPEG5000-NH2. The mPEG5000-NH2 (PEG) was obtained by modifying the terminal group of mPEG-OH according to the literatures.7, 41

Synthesis of ε-carbobenzoxy-L-lysine N-carboxy anhydride (Lys-NCA). In argon, ε-carbobenzoxy-L-lysine (2.00g, 7.1 mmol) and triphosgene (2.31g, 1.1 eq.) was mixed in 30 mL dry THF for 2 h at 45 °C. After that, the solution was precipitated in anhydrous hexane to obtain white crystals in ice water bath. The obtained mixture was recrystallized for another two times. Then the white crystals were dried in vacuum as Lys-NCA (1.8 g, 82% yield) for immediate use.

Synthesis of mPEG5000-b-poly (ε-carbobenzoxy-L-lysine) (PEG-PLys). In a flame dried and argon purged Schlenk tube, mPEG-NH2 (1.55g, 0.31 mmol) was pre-cooled and dissolved in dry DMF. 1.8 g Lys-NCA (20 eq.) in 10 mL DMF was 25



added in argon atmosphere. The mixture was sealed for 3 days stirring at 0 °C then 12 h at room temperature. Further purification was performed by extensive dialysis against deionized water. Removing water by freeze-drying gives 2.77 g product as a white solid (PEG-PLys, 90% yield).

Preparation and characterization of polymeric micelles PEG-PLys@BDPI (PBDPI). 300mg PEG-PLys and 10mg BDPI was dissolved in 3 mL DMF for high speed stirring. 12 mL deionized water was poured into the solution for self-assembling. After 30 min continuous stirring, the mixture was dialyzed as PBDPI NPs and freeze dried as green solid (PBDPI, 270mg, 87% yield). The photosensitizer (BDPI) load content (PLC) and efficiency (PLE) calculated by the equation below were 1.07% and 28.9%, respectively.  weight of loaded photosensi tizer   × 100 % PLC (wt% ) =  weight of nanopaticl es    weight of loaded photosensi tizer   × 100 % PLE (wt% ) =   weight of photosensi tizer fed 

The size distribution and micelles stability test of PBDPI NPs was performed in PBS and DMEM with 10% FBS.

Cytotoxicity of PBDPI in vitro. 3000 HepG2 or EMT6 cancer cells were seeded in a 96-well plate per well in DMEM with 10% FBS. After 24 h, medium with gradient concentration of BDPI or PBDPI took the replacement of the original for cell incubation. The laser irradiation with different power output was performed after another 24 h. Then each well was added with 0.02 mL methyl thiazolyl tetrazolium 26


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(MTT, 5 mg/mL in each well) 24 h later, followed by removal after 4 h incubation and 0.15 mL DMSO addition. The cell viability was calculated according to the absorption of each well using a Bio-rad iMark microplate reader. To observe the cytotoxicity directly, dead/live cell staining assays were conducted and performed with an Olympus U-HGLGPS fluorescence microscope. 105 HepG2 or EMT6 cells were incubated for 24 h in a 6-well plate. PBDPI in medium at a concentration of 2 µM BDPI was added into the predestined well and followed by irradiation 24 h later. Then mixture of propidium iodide (PI, 0.100 mg/mL) and fluorescein diacetate (FDA, 0.010 mg/mL) in DMEM was added to stain dead/live cells for about 20 min. After that, each well was observed by the fluorescence microscope.

Intracellular ROS Generation and cellular uptake. 105 EMT6 cells were incubated with BDP or PBDPI NPs (at a concentration of 2.0 µM photosensitizer) in 6-well plate for 24 h, and then fresh medium containing 2’ ,7’-dichlorofluorescein diacetate (DCFH-DA, 10 µM) was added, followed by irradiation with the 660 nm laser (6.1 mW/cm2 for 5 min) to detect ROS generation inside cells. The uptake of PBDPI NPs was also performed to check the near infrared imaging ability with the fluorescence microscope. The same with above, about 105 cancer cells were seeded in a 6-well plate per well for 24 h incubation. After the addition of PBDPI NPs medium, the bioimaging ability was check after 3 h, 9 h and 24 h. then 4% formaldehyde was added for cell-fixing, followed by staining with DAPI. After 27



replaced with PBS, each well was observed by the fluorescence microscope.

Tumor model. Female BALB/c mice (5 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were cared under protocols approved by the Animal Care and Use Committee in University of Science and Technology of China. To establish the tumor model, EMT6 cells (5×105 suspended in 100 µL PBS) were subcutaneously injected into the right side for imaging assay, or both sides on the back for in vivo PDT performance. The mice were further treated when the volume of tumor reach to about 100 mm3.

Biodistribution and tumor accumulation of PBDPI NPs and free BDPI molecules. BALB/c mice with tumor on the right side were randomly divided into three groups (n=2). There are groups treated with PBS, free BDPI small molecules and PBDPI NPs injection, respectively. BDPI molecule or PBDPI NPs (both with a concentration of 2.5 mg/kg BDPI) was intravenously injected to check the biodistribution and accumulation of BDPI only or loaded in micelles at different predetermined time points (2 h, 6 h, 12 h, 24 h and 48 h). After 48 h, mice were sacrificed then tumors and main organs were removed for fluorescent visualization by a Xenogen IVISs Lumina system (Caliper Life Sciences, USA).

PDT in vivo with near infrared laser irradiation. Mice bearing tumors on both sides are randomly divided into three groups (n=4) like the above. With irradiation 28


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just on right side, we could observe the different efficiency of PDT with or without laser directly. The mice in free BDPI molecule group or PBDPI NPs group were given same dosage in BDPI concentration (2.5 mg/kg). 12 h later, the right tumor site was treated with a laser (660 nm, 25 mW/cm2) for 10 min while no further irradiation to the tumors on the left side. The injection and irradiation were only conducted three times at the start of every other day. Tumor volume and body weight were recorded every other day. The tumor volume was calculated with the formula: V = (a×b2)/2, where a and b represent the maximum length and width of the tumors. 21 days later, mice were sacrificed. All tumors were collected for photograph and wet weight record.

Statistical analysis. To measure diversity in different groups, statistical analyses were performed using the Student’s t-test.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI:

1

H-NMR and MS of intermediate of photosensitizer, cell toxicity of BDPI and

BDP with/without illumination, fluorescence imaging of the PDT in vitro studies, and comparison of irradiation of output power of light in this study to others. 29



AUTHOR INFORMATION

Corresponding Authors

*

E-mail: [email protected].

*

E-mail: [email protected].

Author Contributions

Ruan (polymer synthesis, in vitro and in vivo experiments, and writing of original draft), Miao (PS synthesis, supporting), Yuan (in vitro studies, supporting), Liu (data analysis, supporting), Jiao and Hao (PS design, equal), Yan (funding acquisition, formal analysis, investigation, project administration, writing of draft, lead)

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (No. 51373162 and 51873201).

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High Singlet Oxygen Yield Photosensitizer Based Polypeptide

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