Nanoscale OrganicâInorganic Hybrid Photosensitizers for Highly

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Nanoscale Organic-Inorganic Hybrid Photosensitizer for Highly Effective Photodynamic Cancer Therapy Jia Chen, Yu Xu, Yu Gao, Dongliang Yang, Fei Wang, Lei Zhang, Biqing Bao, and Lianhui Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15581 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 15, 2017

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ACS Applied Materials & Interfaces

Nanoscale Organic-Inorganic Hybrid Photosensitizer for Highly Effective Photodynamic Cancer Therapy Jia Chen, Yu Xu, Yu Gao, Dongliang Yang, Fei Wang, Lei Zhang, Biqing Bao*, and Lianhui Wang* Key Laboratory for Organic Electronics and Information Displays (KLOEID) & Jiangsu Key laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergistic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts and Telecommunications (NUPT), Nanjing 210023, China

ABSTRACT: Recently, photodynamic therapy (PDT) has attracted significant attention as a minimally invasive approach for cancer treatment. Clinical applications of current photosensitizers are often limited by their poor water solubility, low singlet oxygen (1O2) quantum yields, long-term toxicity, instability and complex nanostructures. Here we report a rational design of polyhedral oligomeric silsesquioxanes (POSS)-based porphyrin (PPP5000) used as intrinsically nanoscale photosensitizer. In this strategy, inorganic 3D rigid block POSS not only act as anti-aggregate units but also provide conjugating reactive sites for further chemical modification. Without additional carrier and formulation process, PPP5000 intrinsically shows high water solubility (~40 mg/mL), good PDT efficiency and more excellent anticancer performance compared to THPP (the parent compound of m-THPC, Foscan®). Considering the organic nature of porphyrin and the biodegradable property of inorganic POSS scaffolds at physiological conditions, the present work may lead to a new generation of biodegradable and intrinsically PDT agents with overall performance superior to conventional agents in terms of 1O2 production efficiency, water solubility, structurally stability, photostability, and biocompatibility.

Key Words: polyhedral oligomeric silsesquioxanes (POSS), porphyrin, anti-aggregate, enhanced 1O2 efficiency, photodynamic therapy have poor water solubility, which leads to their

Introduction Recently, photodynamic therapy (PDT) is emerging as one of the significant therapies for cancer treatment due to its minimally invasive nature, higher therapeutic efficacy and reduced systemic toxicity.1-2 In PDT, photosensitizers are activated by a specific wavelength of light, and consequently sensitizing the triplet oxygen to singlet oxygen, resulting in the death of tumor cell via apoptosis or necrosis.3-5 Thus the PDT efficiency depends to a large extent on the property and dose of photosensitizer used. However, traditional photosensitizer especially porphyrin and its derivatives are hydrophobic and

aggregation

in

solutions.6

aqueous

The

aggregation of porphyrin photosensitizer will induce significant fluorescence quench as well as reduced

the

generation

efficiency

by

oxygen

of

singlet

sensitization,

oxygen which

compromise their ability of imaging and PDT efficiency.

A

variety

of

photosensitizer

nanoparticles and polymeric photosensitizers, such

as

liposomes,7

phthalocyanines-layered

polymer-Ce6,8

double

hydroxide

composites,9 and porphyrin-based metal-organic framework,10 have recently been proposed to improve the photochemical activity of porphyrin 1

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photosensitizer. Unfortunately, most of these

rendering

POSS

derivatives

nanoscale photosensitizers show relatively large

scaffolds with potentially reduced risk of renal

size (∼100 nm) and are widely fabricated through

accumulation.20 Owing to the ease in its

encapsulation of porphyrin into various inorganic

modification on the periphery of POSS, it is very

framework.11-12 They are non-biodegradable and

promising as a versatile nano-building block that

thus their long-term in vivo toxicity is a major

act as a rigid structural scaffold to carry diverse

concern. Other strategies such as porphysome

functionalities.21-23

also give rise to concerns about unstable

hybrids are found to have fast uptake kinetics by

nanostructures and in vivo dissociation, causing

cells and can efficiently penetrate into cells due

the inadequate PDT efficiency, poor bio-

to their small and compact size, making these

distribution of nanoparticles and also escalating

organic-inorganic hybrids promising delivery

the pharmacokinetic complexity.13-14 Hence, it is

platform for nano-therapeutics.24

In

biodegradable

addition,

POSS-based

of great significance to fabricate intrinsically

In this perspective, a new organic-inorganic

theranostic nano-photosensitizer exhibiting high

hybrid photosensitizer was synthesized, in which

water

5,10,15,20-tetra(m-hydroxyphenyl)

solubility,

small

size,

excellent

porphyrin

biocompatibility and precise surface functionality

(m-THPP) and biodegradable POSS units were

to optimize their efficacy and safety in cancer

connected via “click” chemistry. An important

treatment.

additional design feature is surface modification

To overcome the non-biodegradability of

of POSS by hydrophilic polyethylene glycol

inorganic nanoparticles retain in body organs,

(PEG5000)

silica nanoparticles have been widely developed

biocompatibility and extend the blood circulation

biomedical applications.15 Polyhedral oligomeric

periods.25-28 The hybrid photosensitizer is highly

silsesquioxanes

fascinating

water-dispersible and small with an average

nanostructured materials that consist of a

hydrodynamic diameter of ∼28 nm. As shown in

nanometer-sized siloxane cube.16-17 Organic-

Scheme 1, the dispersion of porphyrin in the

inorganic hybrid materials based on POSS have

POSS framework inhibits its aggregation, and the

attracted much attention due to their well-defined

PEG branches facilitate water solubility and

3D structure, oriented functional groups, facile

further suppress its aggregation, accounting for

chemical

good

its excellent 1O2 generation capability. In vitro

biocompatibility.18-19 The inorganic POSS core

and in vivo studies reveal a satisfactory PDT

has low toxicity and can decompose into primary

effectiveness and anticancer performance of the

silsesquioxanes

organic-inorganic

(POSS)

modification

at

are

and

physiological

conditions,

to

provide

aqueous

hybrid

solubility,

photosensitizer 2


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(PPP5000). More importantly, PPP5000 also show

Results and Discussion

the advantages of non-toxicity and the ability of hydrolytic

degradation

at

physiological

conditions to give main small molecule products.

Scheme 1. Schematic illustration of the fabrication process and photodynamic therapy of PPP5000 and THPP.

Design and Synthesis of PPP5000

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Reagents and conditions: (i) a, 1eq TfOH, CH2Cl2, 3 h; b, wet acetone, CH2Cl2, 3 h; (ii) 4-pentynoic acid, DMAP, DIPC, CH2Cl2, 24 h; (iii) 1,2-dibromoethane, DMF, 70℃, 4h; (iv) sodium azide, DMSO, 75℃, 4 h; (v) a, BF3·OEt2, Argon, CH2Cl2, 2 h; b, DDQ, 1 h; (vi) CuBr, PMDETA, Argon, THF, 24 h; (vii) SH-PEG5000, DMPA, UV 365 nm, chloroform, Argon, 20 min. Scheme 2. The synthetic route of PPP5000.

The synthetic route of Porphyrin-POSS-

“click” chemistry from (Vinyl)7-POSS-alkyne

PEG5000 (PPP5000) is shown in Scheme 2. The

and Porphyrin-N3 in 71% yield. As depicted in

synthesized

the 1H,

PPP5000

is

composed

of

a

13

C NMR and MALDI-TOF spectra of

tetra(hydroxyphenyl)porphyrin (THPP) core and

Porphyrin-POSS, four POSS units were bound to

four POSS as scaffolds with hydrophilic PEG

one porphyrin core (Figure S7-S8). Porphyrin-

groups on its periphery. Briefly, monohydroxyl

POSS-PEG5000 (PPP5000) was synthesized by

heptavinyl substituted POSS ((Vinyl)7-POSS-

convenient thiol-ene “click” reaction between

OH) was obtained from the octavinyl-POSS.

Porphyrin-POSS and SH-PEG5000. The IR and 1H

Then it was esterified with 4-pentynoic acid

NMR spectra of PPP5000 are shown in Figure S9

leading to (Vinyl)7-POSS-alkyne in 85% yield

and S10 respectively. The proton signals at d =

according to a reported procedure.29 Porphyrin-

5.92-6.05 ppm and 3.35-3.64 ppm are ascribed to

POSS was synthesized by copper-catalyzed

vinyl of Porphyrin-POSS and the methoxyl 4


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groups of PEG, respectively. The IR and 1H

stronger than that of Porphyrin-N3. The observed

NMR results clearly confirmed the successful

fluorescence

incorporation of PEG5000 and the degree of

ascribed to the weakened interaction between the

PEGylation is greater than 70%. Moreover,

porphyrin

PPP5000 exhibits excellent water solubility of ~40

suppressed self-quenching of the excited state

mg/mL. The aqueous solution of PPP5000 is

and recovery of the fluorescence of porphyrins.31-

transparent and no obvious precipitation can be

32

observed after 6 months. We consider that

(EPR) spectroscopy was also employed to

porphyrin core maybe molecularly dissolved in

monitor the 1O2 generation ability of Porphyrin-

water and no or less aggregation occurs even in

POSS and Porphyrin-N3. As shown in Figure

high concentration due to the steric hindrance

S12, the of EPR signal of Porphyrin-N3 and

effect of POSS scaffolds and PEG branches

Porphyrin-POSS both increase gradually upon

(Figure 1a).

white-light irradiation, indicating 1O2 molecules

Photophysical

and

Photochemical

enhancement

molecules

and

should

leading

also

to

be

the

Furthermore, electron paramagnetic resonance

were generated. Besides, the 1O2 generation rate for Porphyrin-POSS is obviously faster than

Properties

Porphyrin-N3, which reveals that Porphyrin-

The UV-vis absorption spectra of PorphyrinPOSS and Porphyrin-N3 in acetone are compared in Figure S11a. Both porphyrin-POSS and porphyrin-N3 show a Soret band at 424 nm and four Q-bands spread from 525 nm to 660 nm. Importantly, Porphyrin-POSS exhibits two-fold increase in the extinction coefficient of the lowest-energy Q band relative to Porphyrin-N3, which is beneficial for PDT efficiency and may be due to the increased distance of porphyrin.30 Furthermore, fluorescence spectra of PorphyrinPOSS and Porphyrin-N3 was also investigated to support this assumption. As shown in Figure S11b, Porphyrin-N3 exhibits a fluorescence peak at ∼ 665 nm in acetone solution, and the fluorescence of Porphyrin-POSS is ∼ 6-fold

POSS has a stronger 1O2 generation ability than Porphyrin-N3. In order to achieve a high water solubility, we introduced high density of hydrophilic PEG groups to the eight arms of POSS to fabricate the organic-inorganic hybrid photosensitizer PPP5000. THPP, the parent compound of m-THPC (Foscan®) was introduced for contrast. THPC is the second-generation photosensitizer, which has already been shown to be more potent than photofrin.33-34 As shown in Figure 1b and 1c, the molar extinction coefficient of PPP5000 shows significant

increase

relative

to

THPP.

Furthermore, THPP shows very weak emission in DMSO solution, while PPP5000 shows intensified fluorescence than that of THPP in aqueous 5



Page 6 of 19

solution. These results indicate that the aggregate

PPP5000 with average diameters as small as ~ 20

of PPP5000 NPs is insignificant, which is

nm can be observed, which is smaller than that of

consistent with our speculation that POSS

previously reported nanoscale porphyrin-based

scaffolds and PEG branches could efficiently

photosensitizer.37

suppress the π-π interaction and hydrophobicity

nanoparticles with small diameters are highly

of porphyrin. We also compared the singlet

desirable

oxygen generation efficiencies of PPP5000 NPs

nanoparticles with relative large size may cause

with THPP using SOSG as 1O2 indicator. As

problems such as poor tissue penetration and

illustrated in Figure 1d and Figure S13, a widely

nonspecific clearance by the reticuloendothelial

used quantitative 1O2 indicator, Singlet oxygen

system (RES).38-39 Moreover, the particle size

sensor green (SOSG) was used to detect and

obtained from DLS indicated that the majority of

measure the singlet oxygen.35-36 The oxidation of

PPP5000

SOSG at the presence of PPP5000 NPs results in

diameters of ∼ 28 nm, which is consistent with

increased fluorescence intensity which indicates

the results of TEM.40

for

It

is

worth

noting

nano-therapeutics

possessed

average

that

because

hydrodynamic

the generation of 1O2. The 1O2 production of

The photostability of PPP5000 was then

PPP5000 is ~3 times as efficient as THPP, which is

compared with that of its analogue THPP and

ascribed to the increased extinction coefficient of PPP5000 and the suppressed self-quenching of the excited states of porphyrins. To

investigate

absorption spectra of PPP5000, THPP or RB upon repetitive UV irradiation. PPP5000 exhibits no

and

obvious changes in the absorption spectra under

morphologies, the as-prepared PPP5000 was

UV irradiation, while UV illumination could

determined

electron

cause 18% and 29% reduction in the absorption

microscope (TEM) and dynamic light scattering

intensity of THPP and RB respectively. These

(DLS). Figure 1e shows the representative TEM

results indicate that PPP5000 NPs were much more

data of PPP5000 in aqueous solution. Remarkably,

resistant to photo-bleaching than THPP and RB.

by

their

Rose Bengal (RB). Figure 1f shows the changes of

both

particle

size

Transmission


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Figure 1. (a) Photographs of Porphyrin-POSS, PPP5000 at solid state and dispersed in PBS buffer. UV-vis absorbance spectra (b) and fluorescence spectra (c) of PPP5000 NPs and THPP, the corresponding inset images are photographs and fluorescence pictures under a hand-held UV set, respectively. (d) The change of PL intensity of the SOSG peak with irradiation time. (e) DLS results and representative TEM images of PPP5000 (inset Figure). (f) Photostability comparison among PPP5000, THPP and RB in aqueous solution.

NPs and THPP in the concentration from 1.25 to

In Vitro PDT Performance The highly water-soluble and photostable

20 µM and subsequently treated with white light

PPP5000 can also be used as fluorescence indicator

irradiation (50 mW/cm2) for 7 min. As

for live cell imaging and image-guided PDT

illustrated in Figure 2, PPP5000 have little effect

therapy. As illustrated in Figure S14, after

on the cell viability in the dark even at a

incubation with HeLa cells at 37 °C, PPP5000 NPs

concentration of 20 µM. While PPP5000 can kill

were efficiently taken up by cells and primarily

about 48% and 82% of HeLa cells at a

localized in cytoplasm.

concentration of 2.5 µM and 10 µM under white light irradiation. In contrast, a smaller cell

Considering that negligible dark cytotoxicity and

significant

irradiation

is

cytotoxicity necessary

for

under

light

an

ideal

photosensitizer, MTT assays were performed to quantitatively evaluate the PDT efficiency and cytotoxicity of the PPP5000 compared with THPP. HeLa cells were incubated with PPP5000

viability of 70% was obtained for 5 µM THPP even in the dark condition. And only 10% and 53% of the cancer cells were killed for 2.5 µM and 10 µM THPP under the same irradiation conditions. The cellular endocytosis of PPP5000 and THPP has also been investigated. As shown in Figure S14 and S15, PPP5000 could be 7



Page 8 of 19

effectively taken up by HeLa cells while most of

compared to THPP, which may be attributed to

THPP form aggregates extracellularly. These

anti-aggregation and biocompatibility of POSS

results indicate that PPP5000 have superior

scaffolds and PEG branches.

photodynamic efficiency and lower cytotoxicity

Figure 2. Cell viability of HeLa cells treated with PPP5000 and THPP (a) without irradiation and (b) with irradiation.

Furthermore, the cell death induced by PPP5000

the apoptotic and necrotic cells were observed for

NPs mediated PDT treatment was determined by

cells treated with PPP5000 NPs than cells treated

flow

with THPP upon light irradiation. These results

cytometry

apoptosis kit.

41

using

Annexin

V-FITC/PI

The majority (97.4%) of cells

also

indicate

that

PPP5000

NPs

show

were viable and no obvious apoptosis was

extraordinarily higher anticancer behavior in

observed for cells treated with PPP5000 NPs. On

PDT as well as lower cytotoxicity compared to

the other hand, significantly higher proportion of

commercial photosensitizer THPP.


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Figure 3. Flow cytometry analysis of HeLa cells induced by PPP5000 and THPP with (+) or without (-) light irradiation for 7 min at 50 mW/cm2, all cells were stained by Annexin V-FITC/PI kit.

The Annexin V-FITC/PI apoptosis detection of

PPP5000 NPs mediated PDT could effectively

PPP5000 NPs induced cell necrosis upon light

cause cell apoptosis and necrosis. However, in

irradiation was subsequently investigated by

control experiment, almost no fluorescence had

confocal laser scanning microscopy (CLSM).

been detected, which reveals that PPP5000 NPs are

After cells were stained with Annexin V–

highly biocompatible. Meanwhile, when PPP5000

FITC/PI, a long-term observation mode via

NPS were coincubated with reductive L-Ascorbic

CLSM was adopted to monitor the apoptosis

Acid (Vitamin C), the irradiation-induced cell

process.

green

apoptosis or death could be effectively prevented.

fluorescence intensity of Annexin V-FITC and

These results indicated that the generated reactive

red fluorescence signal of PI was observed after

oxygen species (ROS) in PPP5000 mediated PDT

irradiation of white light at the rate of 50

was responsible for the cell death.

As

shown

in

Figure

4,

mW/cm2 for 10 min ， which indicated that



Page 10 of 19

Figure 4. Confocal fluorescence images of HeLa cells stained with Annexin V–FITC/PI after cells were incubated with PPP5000 NPs and then irradiated under white light. Ex = 488 nm. Scale bars: 40 µm.

mediated PDT, 2,7-dichlorifluoresceindiacetate

Figure 5. Confocal fluorescence images of HeLa cells treated with 5 µM PPP5000 and stained by DCFH-DA. Ex = 488 nm. scale bars: 40 µm.

(DCFH-DA) was employed to monitor the 1O2

In vivo PDT performance

To clarify the role of ROS in PPP5000 NPs

generation of PPP5000 NPs in living cells. DCFH-

Encouraged by the above investigations, in

DA can be converted to DCFH when enter into a

vivo PDT therapy was performed using HeLa

cell, followed by being oxidized to strong

tumor-bearing mice. All animal operations were

fluorescent 2,7-dichlorofluorescein (DCF) at the

approved by the China Committee for Research

42

presence of singlet oxygen. As shown in Figure

and Animal Ethics in compliance with the law.

5, strong green fluorescence can be observed

The mice were random subjected to 3 groups and

when the cells were treated with PPP5000 NPs

given Saline, THPP (0.4 mg/kg) and PPP5000 (0.4

followed by white light irradiation at the rate of

mg

20 mW/cm2 for 2 min, indicating that PPP5000

respectively. After 1-hour drug-light interval,

NPs mediated PDT is in fact responsible for the

PDT was performed by irradiating the tumor

generation of singlet oxygen in cells.

region with white light irradiation at the power of

THPP/kg)

by

intratumor

injection,

80 mW/cm2 for 15 min (72 J/cm2).

The

photodynamic therapeutic effects of PPP5000 NPs and THPP in vivo were assessed by monitoring respective relative tumor volumes. As show in Figure 6a and 6b, the tumors from PPP5000 treated group began to regress after 2 days and shrink into small nodules gradually in 15 days, while 10 ACS Paragon Plus Environment

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less efficient therapeutic effect was observed for

indicate that PPP5000 has a better PDT efficiency

THPP treated group. In a sharp contrast, the

than THPP does, which is in perfect accordance

tumors from the saline treated group increased

with the in vivo observation from the tumor

significantly. The life span of mice in the saline

growth curves in Figure 6a.

treated control group was no more than 24 days

In vivo safety evaluation of PPP5000 NPs were

with the tumor volumes reaching up to 1000

also conducted. No obvious weight loss and other

mm3, while the mice in the PPP5000 and THPP

abnormal behavior were observed after the mice

treated groups all survived over 30 days (Figure

treated with PPP5000 NPs upon irradiation, which

S16). The treatment efficacy in terms of tumor

means no obvious side effects and negligible

cell death was characterized by hematoxylin-

systemic toxicity of PPP5000 NPs to mice (Figure

eosin (H&E) and TdT-mediated dUTP-biotin

S17). Furthermore, at the day 30 after treatment,

43-44

nick end labeling (TUNEL) staining.

As

the major organs such as heart, liver, spleen, lung,

shown in Figure 6c, PPP5000 treated mice show

and kidney were collected and then sliced for the

more obvious necrosis as compared to those of

histological examination. The results showed no

THPP-injected mice, and no obvious apoptosis or

obvious tissue damage and inflammatory lesion

necrosis is observed for PBS treated group at day

in PPP5000 NPs treated mice compared to the

3 after treatment. These histological results

control groups (Figure S18).



Page 12 of 19

Figure 6. (a) Changes of relative tumor volume (V/V0) after mice were treated with saline, THPP and PPP5000. ** P < 0.01 compared to other groups using Student’s t-test. (b) Representative photos of mice after treatment. (c) TUNEL and H&E staining of tumor slices at day 3 after the PDT treatment with saline, THPP and PPP5000 NPs. Scale bars: 100 µm.

Conclusions To summarize, we developed a new-generation

and the hydrophilic PEG branches make THPP

organic-inorganic hybrid photosensitizer based

molecules well-isolated in the framework to

on biodegradable POSS scaffolds and THPP core.

avoid aggregation and self-quenching of the

The

porphyrin-derived

excited states. The organic-inorganic hybrid

photosensitizer into the robust POSS scaffolds

PPP5000 NPs thus possesses uniform particle size

incorporation

of


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(hydrodynamic diameter ∼28 nm), high water

PPP5000 NPs also exhibit advantages such as their

solubility,

good

well-defined molecular structures, good synthetic

oxygen

reproducibility and the potentially improved

production efficiency. In vivo studies indicate

biodegradability due to the intrinsic theranostic

that the photosensitizer PPP5000 show excellent

property of PPP5000 and biodegradable property

PDT performance with a low dose (0.4 mg/kg)

of POSS scaffolds.

excellent

biocompatibility,

and

stability, high

singlet

and light density (72 J/cm2). More importantly, performed with a Bruker autoflex III system

Materials and Methods

MALDI-TOF

mass

spectrometer.

UV-Vis

Reagents and Chemicals. Octvinyl-POSS

absorption spectra were obtained on a UV-3600

was purchased from Hybrid Plastics (Hattiesburg,

Shimadzu

MS). SH-PEG5000 was obtained from ToYong

Photoluminescence spectra were measured using

Bio. Co. Ltd. (Shanghai, China). Dulbecco’s

an

modified Eagle’s medium (DMEM), 3-(4,5-

permeation chromatography (GPC) analysis was

dimethylthiazol-2-yl)-2,5-diphenyl

conducted on a Shim-pack GPC-80X column

tetrazolium

UV-Vis

RF-5301PC

Spectrophotometer.

spectrophotometer.

Gel

bromide (MTT), trimethylamine, penicillin–

using

streptomycin solution, fetal bovine serum (FBS)

tetrahydrofuran

and trypsin–EDTA solution were purchased from

Transmission electron microscopy (TEM) was

KeyGEN Biotech. Co. Ltd. (Nanjing, China). 2’,

performed on a Hitachi HT7700 operating at 100

7’-dichlorfluorescein-diacetate

(DCFH-DA),

kV accelerating voltage. Dynamic light scattering

Annexin V-FITC/propidium iodide (PI) cell

(DLS) spectra were carried out on a Brookhaven

apoptosis kit and Hoechst 33342 (H 33342) was

Zeta PALS with a He-Ne laser (633 nm) and 90

purchased

Biotechnology

° collecting optics. Laser confocal scanning

(Shanghai, China). All other chemicals were

microscope (CLSM) images of Porphyrin-POSS-

bought from Sigma-Aldrich (St. Louis, USA).

PEG5000 were taken on Olympus Fluoview 1000

from

Beyotime

Instrumentation and Methods. 1H and 13C NMR spectra were recorded on a Bruker Ultra

polystyrene

(Olympus,

as

(THF)

Japan).

a as

Electron

standard the

and eluent.

paramagnetic

resonance (EPR) measurements were performed by using the Bruker EMX-10/12 apparatus.

1

Shield Plus 400 MHz NMR instrument ( H: 400 MHz,

13

C: 100 MHz). Mass spectra were ACS Paragon Plus Environment

13


Electron

Paramagnetic

Resonance

(EPR) Spectra. EPR spectroscopy has been employed for detection of singlet oxygen generated by oxygen sensitization of PorphyrinN3

and

Porphyrin-POSS.

The

diamagnetic

2,2,6,6-tetramethylpiperidine (TEMP) was used as

1

O2 trapper by yielding a paramagnetic

Page 14 of 19

prepared and set at the concentration of 5 µM (for PPP5000, the concentration was calculated as porphyrin unit), then the SOSG stock solution was added (final concentration = 2 µM) before the fluorescence measurement. Fluorescence intensity

were

recorded

on

a

fluorospectrophotometer with excitation at 504 nm every 30 seconds.

nitroxide radical TEMPO. The EPR spectral pattern of three lines of equal intensity, which is

Cytotoxicity. The cell dark toxicity and

characteristic for the TEMPO nitroxide radical,

phototoxcity of PPP5000 and THPP were assessed

was observed when of Porphyrin-POSS and

by MTT assay. HeLa cells were first seeded to

Porphyrin-N3 were irradiated in the presence of

two 96-well plates (Costar, IL, USA) in 150 µL

TEMP at room temperature. The microwave

complete medium at an intensity of 2*104

frequency of Scan condition is 9.774 GHz, center

cells/mL. After 48h incubation, the medium was

field setting at 3483 G, corresponding to the

replaced by PPP5000 solution at the concentration

reported literature. With the time white-light

of 1.25, 2.5, 5, 10, 20 µM. One plate was kept in

irradiated the air saturated photosensitizers

the dark for studying dark toxicity, another plate

acetone solution, the intensity of the EPR signal

was irradiated using a white light irradiation at a

O2

power of 50 mW/cm2 for 7 min, and all the cells

molecules are generated. The intensities of the

were incubated for another 24 h. Afterward, the

EPR peak are plotted against the irradiation time,

medium were removed and washed with pH=7.4

the slope of the plotted line can reflect the

PBS carefully. MTT (20 µL, 5 mg/mL) solution

amount of 1O2 generated in this process.

in PBS buffer was added into each well for 4-

increases gradually, indicating that the

1

hour incubation, the unreacted MTT were

Singlet Oxygen Generation. A widely used

removed and 150 µL DMSO were added into

1

quantitative O2 indicator, singlet oxygen sensor green (SOSG, Life Technologies) was used to detect the singlet oxygen. The SOSG was used according

to

the

instruction

from

the

every well and gently shaken for 10 minutes at room temperature to dissolve the produced formazan. The absorbance of MTT at 570 nm was monitored by the microplate reader (Bio-Tek

manufacturer. Briefly, PPP5000 and THPP were ACS Paragon Plus Environment

14

Page 15 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Synergy 2). The cell viability was expressed by

0.01 was considered to be significant statistically.

the ratio of the absorbance of the cells incubated

The apoptosis of tumor cancer cell was checked

with PPP5000 to that untreated cells. The

by both TdT-mediated dUTP nick end labeling

cytotoxicity of THPP were conducted as the

(TUNEL) Staining and hematoxylin & eosin

procedure of MTT assay of PPP5000.

(H&E) Staining at Day 3 post treatment. The damage to major organs was analyzed by slices

In Vitro Imaging and PDT. In cell imaging experiments, HeLa cells was incubated with 5 µM PPP5000 NPs medium at 37℃ in incubator. A commercial nuclear dye H 33342 was employed to mark the nuclear. Normally H 33342 can mark the

cell

nuclear

area

by

emitting

blue

H&E Staining. The rest mice from the different treated groups were monitored by measuring the tumor sizes for 30 d after the PDT treatment. The relative tumor volumes were calculated for every mouse as V/V0, in which V0 was the tumor volume when the treatment was initiated.

fluorescence (420-500 nm) when excited at 405 nm. 1 µL (2 mg/mL) of H 33342 stock solution was added to the

medium at the final

ASSOCIATED CONTENT Supporting Information

concentration of 2 µg/mL for staining the cell nuclear. The distribution of PPP5000 in cell was

Reagents

and

chemicals;

visualized by collecting wavelength (630-700

instruments

nm) at the excitation 559 nm by Olympus

characterization

Fluoview 1000.

property of Porphyrin-N3 and Porphyin-POSS;

and

methods; of

PPP5000;

supplementary synthesis

and

photophysical

singlet oxygen measurement; cell culture and

In Vivo Anticancer Efficacy. The tumor volumes were measured using a Vernier caliper,

cytotoxicity; cellular uptake and in vitro and in vivo PDT studies.

then calculated using the function: L×D×D/2, in which L and D refer to the longitudinal diameter and transverse diameter of the tumor in

AUTHOR INFORMATION Corresponding Author

millimeters, respectively. Specific data collected *[email protected], from the vivo experiment were analyzed by *[email protected] calculating the P-value using the Student’s t-test (two-tailed distribution and two-sample unequal

Notes

variance) to compare the treatment effects. P < ACS Paragon Plus Environment

15


The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Technology of China (2017YFA0205302),

the

Science

of

Foundation

National China

Natural

(21574069,

21475064), the Natural Science Foundation of Jiangsu Province (BK20151503), the Sci-tech Support Plan of Jiangsu Province (BE2014719), the Jiangsu National Synergistic Innovation Center for Advanced Materials (SICAM), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, YX03001).

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