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Combined Photodynamic and Photothermal Therapy Using Cross-Linked Polyphosphazene Nanospheres Decorated with Gold Nanoparticles Xuan Wei, Hongzhong Chen, Huijun Phoebe Tham, Nan Zhang, Pengyao Xing, Guangcheng Zhang, and Yanli Zhao ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00776 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 19, 2018

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ACS Applied Nano Materials

Combined Photodynamic and Photothermal Therapy Using Cross-Linked Polyphosphazene Nanospheres Decorated with Gold Nanoparticles

Xuan Wei,†,‡ Hongzhong Chen,‡ Huijun Phoebe Tham,‡ Nan Zhang,†,‡ Pengyao Xing,‡ Guangcheng Zhang,†,* Yanli Zhao*,‡

†

Key Laboratory of Applied Physics and Chemistry in Space (Ministry of Education),

Department of Applied Chemistry, School of Natural and Applied Science, Northwestern Polytechnical University, Xi’an, 710129 China ‡

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical

Sciences, Nanyang Technological University, Singapore 637371

ABSTRACT. A rational combination of photodynamic therapy (PDT) and photothermal therapy (PTT) could achieve a synergistic therapeutic effect to enhance therapeutic efficacy in the cancer treatment. Herein, polyphosphazene nanospheres as a novel kind of photosensitizer carriers were synthesized by anchoring and isolating photosensitizing porphyrin monomers covalently in the cross-linked structure. Gold nanoparticles were then immobilized on the surface of the nanospheres to introduce the photothermal performance. The hybrid system was eventually conjugated by polyethylene glycol to decrease its cytotoxicity. The morphology analysis showed that the nanospheres were covered by homogeneously dispersed gold nanoparticles. In this way, the resulted system could afford efficient photodynamic and photothermal effect simultaneously, as confirmed by the cancer cell killing studies. The cell experiments demonstrated that the as-prepared polyphosphazene nanospheres with inherent fluorescence could be internalized by HeLa cells, showing high performance of combined PDT and PTT under suitable light irradiation. Thus, this integrated system presents its effectiveness to achieve combined PDT and PTT for enhanced cancer therapeutics.

KEYWORDS: combined therapy, gold nanoparticles, photodynamic therapy, photothermal therapy, polyphosphazene nanospheres 1

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1. INTRODUCTION Photodynamic therapy (PDT) is known as a palliative cancer therapeutic technique, which has minimally invasive property, reduced side effects and improved selectivity as compared with traditional chemotherapy and radiotherapy.1-4 In the treatment process, reactive oxygen species (ROS) such as singlet oxygen for inducing the cell apoptosis is generated by the photosensitizer molecules under the light illumination with appropriate wavelengths.5,6 As a type of PDT photosensitizers, porphyrin and its derivatives are organic heterocyclic macrocycles with high phototoxicity.7,8 Similar to most of the photosensitizers, however, porphyrins are lipophilic compounds and essentially insoluble, unstable, and easily aggregated in aqueous solution under physiological conditions.9,10 Furthermore, relatively low accumulation selectivity to tumors is another barrier hindering their wide clinical applications.11 To overcome these limitations, liposomes,12 silica nanoparticles,13,14 polymeric nanoparticles,15 and many other nanomaterials have been developed as carriers for the porphyrin photosensitizers. On the other hand, these methods usually require complicated materials preparation and photosensitizer-loading process, and the aggregation issue of the photosensitizers still cannot be fully solved.7,8 Cyclomatrix-type polyphosphazenes are organic-inorganic hybrid macromolecules that have attracted a lot of attention during the last decade.16,17 In a standard procedure, the cyclotriphosphazene rings are linked by nucleophilic substitution of aromatic organic monomers bearing dual/multi-nucleophilic groups through precipitation polycondensation to form cross-linked polyphosphazenes. By using this strategy, some monomers such as 4,4’-sulfonyldiphenol,18,19 4,4’-diaminodiphenyl ether,20 phloroglucinol,21 and fluorescein22 were employed to prepare cross-linked polyphosphazene materials with micro/nano-sized morphologies including nanotubes,16 nanofibers,23 and microspheres.24 Therefore, the porphyrin derivatives bearing phenolic hydroxyl groups could also serve as co-monomers to cross-link with hexachlorocyclotriphosphazene (HCCP) for being immobilized and isolated in stable cyclomatrix-type structure.25 In this way, the porphyrin moiety could still keep its monomeric state for efficient PDT. As a novel carrier of photosensitizers, such cross-linked polyphosphazenes have abundant active groups on the surface, which could be further 2


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modified easily to gain better properties and more functions. In

previous

studies,

Lu

et.

al.

poly(cyclotriphosphazene-co-4,4’-sulfonyldiphenol)

found

that

microspheres

the were

cross-linked excellent

core

materials for the growth of metal nanoshells on their surface.26 As a class of versatile micro/nano-materials having excellent solvent resistance, thermal stability, biodegradability, and water dispersion, polyphosphazenes are rich in N and P atoms and phenolic hydroxyl groups capable of coordinating with gold nanoparticles.27-29 These gold nanoparticles exhibit significant surface plasmon resonance in the near infrared region. Furthermore, they can effectively induce a quick increase of local temperature under laser irradiation to kill tumor cells rapidly, indicating their potential applications in photothermal therapy (PTT).30,31 It has also been proven that the combination of different therapies such as PDT and PTT could achieve synergistic therapeutic effects to enhance therapeutic efficacy beyond separate mode of treatments.8,32-34 Theoretically, the photothermal effect could result in a local temperature increase signally, thereby speeding up blood flow to attract more oxygen for photodynamic effect.35,36 In addition, the heat generated by PTT could enhance the permeability of tissues and cell membranes to improve the delivery efficiency of nanoparticles and cellular uptake of photodynamic agents, significantly increasing subsequent PDT efficacy because of short lifetime and limited effective range of singlet oxygen.37,38 Based

on

abovementioned

considerations,

herein,

poly(cyclotriphosphazene-co-tetraphenylporphyrin-co-sulfonyldiphenol)

we

prepared nanospheres

(CP-TPP) with regular spherical appearance, smooth surface, relatively narrow size distribution, and good dispersion by the precipitation copolymerization of HCCP with a porphyrin monomer in a covalently cross-linked manner. Then, polyethylenimine (PEI) was attached onto the surface of CP-TPP nanospheres by N,N’-carbonyldiimidazole (CDI) activation. PEI could alter the surface potential of the nanospheres, making a better condition for the adsorption of HAuCl4.39 The combination of PDT and PTT was realized by the growth of Au nanoparticles on the surface of PEI-covered CP-TPP nanospheres. Since poly(ethylene glycol) (PEG) is a flexible hydrophilic polymer that could endow nanoparticles with better biocompatibility, the nanospheres bearing gold nanoparticles were further coated by PEG in the final step to give rise the hybrid CP-TPP/Au/PEG for combined PDT and PTT in the 3



cancer treatment.

2. EXPERIMENTAL METHODS Synthesis of CP-TPP nanospheres. Tetra(4-hydroxyphenyl) porphyrin (TPP, 10 mg, 14.7 mmol) and HCCP (10 mg, 28.8 mmol) were added to a flask containing acetonitrile (50 mL), followed by sonication for 15 min. After injecting triethylamine (TEA, 1 mL) into the solution, the polycondensation reaction was carried out in an ultrasonic bath (1000 W, 50 Hz) at room temperature for 15 min. Subsequently, bisphenol S (BPS, 5 mg, 20 mmol) and TEA (1 mL) were added into the system. The solution was sonicated under the same conditions for another 6 h. The precipitates were collected by centrifugation (9000 rpm), washed for three times using deionized water and ethanol (50 mL) respectively, and dried at 50 °C in vacuum overnight to afford CP-TPP as a magenta powder. Surface modification of CP-TPP nanospheres. In a typical procedure, the surface of the nanospheres was firstly activated by CDI. CP-TPP nanospheres (10 mg) were dispersed in anhydrous ethanol (5 mL) by sonication. Then, CDI (2.5 mg) was added into the solution and the mixture was stirred at room temperature for 2 h. The precipitates were centrifuged, washed twice with ethanol, and dispersed again in deionized water (5 mL). Branched PEI (2.5 mg) was added to the nanosphere suspension followed by stirring overnight. Finally, the PEI modified CP-TPP nanospheres were collected by centrifugation (9000 rpm), washed with 50 mL ethanol and deionized water, and ultrasonically suspended in water to form a suspension at the CP-TPP equivalent concentration of 0.5 mg/mL. Synthesis of CP-TPP/Au nanospheres. The aqueous dispersion of the obtained PEI modified CP-TPP nanospheres (20 mL, 0.5 mg/mL) and HAuCl4 aqueous solution (2 mL, 24.0 mM) were mixed and stirred for 30 min, which were then centrifuged and washed with water. The resulting nanospheres bearing the AuCl4- ion on the surface were redispersed in sodium citrate aqueous solution (40 mL, 1 wt.%). After stirring for 10 min, NaBH4 (2 mL, 0.1 wt.%) solution was added, and the mixture system was vigorously stirred for additional 3 min. The solution color turned from red wine to reddish brown. The obtained CP-TPP/Au seeds were collected by centrifugation, washed with ethanol and deionized water, and ultrasonically suspended in water to form a suspension with the CP-TPP equivalent concentration of 1 4


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mg/mL. K-gold (gold salt in potassium-basic solution) was prepared by stirring HAuCl4 (0.23 g) and K2CO3 (0.5g) in deionized water (10 mL) overnight and stored for subsequent experiments. CP-TPP/Au seed suspension (1 mL) and K-gold solution (1 mL) were added to a round-bottomed flask containing deionized water (20 mL). After stirring for 5 min, aqueous formaldehyde solution (0.2 mL, 37 wt.%) was added to perform the reduction by stirring for another 15 min. The solution color turned from reddish brown to dark brown, indicating the formation of Au nanoparticles. After the centrifugation and washing, the purified CP-TPP/Au was stored in water as a suspension at the CP-TPP equivalent concentration of 1 mg/mL. Synthesis of CP-TPP/Au/PEG nanospheres. To further immobilizing PEG on the surface of the nanospheres, mPEG-COOH (0.01 g) was dissolve in DMSO (4 mL), and then 1-ethyl-3[3-dimethylaminopropyl]

carbodiimide

hydrochloride

(6

mg)

and

N-hydroxysuccinimide (18 mg) were added. After stirring in the dark at room temperature for 3 h, CP-TPP/Au (1 mg) diluted in aqueous solution (3 mL) was added. Then, the system was treated with NaOH (0.2M) to adjust the pH to 8 and stirred for overnight. The precipitates were collected by centrifugation (9000 rpm), washed for three times using ethanol and deionized water (50 mL) respectively, and stored in water as a suspension with the CP-TPP equivalent concentration of 1 mg/mL. Singlet oxygen generation of CP-TPP and CP-TPP/Au/PEG nanospheres. The 1O2 generation efficiency of CP-TPP and CP-TPP/Au/PEG nanospheres was detected by using 1,3-diphenylisobenzofuran (DPBF) as a chemical indicator. By following a typical procedure, DPBF aqueous solutions containing CP-TPP and CP-TPP/Au/PEG were respectively irradiated with a 630 nm LED light at the power density of 50 mW/cm2 for different periods of time. The generation of 1O2 would lead to a reduction of optical absorption density at 417 nm. The DPBF solution was also measured as a control. Photothermal conversion. Aqueous suspensions containing CP-TPP or CP-TPP/Au/PEG nanospheres in different concentrations were irradiated using an 808 nm laser (1.5 W) at a distance of 5 cm for different periods of time. The temperatures of the solutions were measured. In vitro cytotoxicity study. HeLa cells and human umbilical vein endothelial cells (HUVECs) 5



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were incubated in Dulbecco’s modified eagle medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator maintained at 37 °C (95% atmosphere, 5% CO2).

The

in

vitro

cytotoxicity

was

evaluated

using

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the cells were seeded in 96-well plates (about 104 cells in 100 µL medium per well) and incubated for 24 h. Then, the medium was replaced using fresh one and the samples were added at the final CP-TPP equivalent concentrations of 10, 25, 50, or 100 µg/mL. After another 48 h incubation, fresh medium and MTT solution were added and the samples were further incubated for 4 h. Then, the medium was removed and DMSO was added. The absorbance of the samples was measured by a microplate reader at 570 nm. The PDT effect on HeLa cells was studied using MTT assay. After the incubation of the cells with different concentrations of the nanospheres in 96-well pates for 24 h, the cells were irradiated by 630 nm LED (50 mW/cm2) for 8, 15, and 20 min, respectively. Then, the cells were incubated for another 24 h and measured using the MTT method described above to obtain the cell viability values. The PTT effect on HeLa cells was studied through the same procedure, using the 808nm laser to irradiate for 15 min. As for the synergistic effect of PDT and PTT, in the irradiation step, the cells were irradiated using the 630 nm LED immediately after irradiated by 808 nm laser. All the cytotoxicity experiments were carried out at least in quadruplicate. Values are mean ± standard deviation (SD). Cellular uptake study. The uptake of the nanospheres by HeLa cells was investigated using a confocal laser scanning microscope (CLSM) method. HeLa cells were seeded in a 6-well plate (about 2×105 cells in 2 mL medium per well) and incubated for 24 h. Then, the medium was replaced using fresh one and the samples were added at the final CP-TPP equivalent concentration of 50 µg/mL. After the incubation for additional 4 and 24 h respectively, the medium was removed, and the cells were washed using phosphate-buffered saline for three times. Subsequently, the cells were fixed, stained with 4',6-diamidino-2-phenylindole (DAPI), and then imaged by CLSM. Intracellular ROS generation study. The procedure for the incubation of HeLa cells with the samples was similar to the cellular uptake study. After incubation with the nanospheres for 24 h, the cells were irradiated by 630 nm LED (50 mW/cm2) for 15 min. Then, the cells were 6


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labeled by 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) for the detection of the ROS generation. The cells were finally fixed, stained with DAPI, and imaged by CLSM. The HeLa cells without any treatment and the cells with different kinds of samples but without the light irradiation were conducted as control groups. HCCP

TPP

BPS

CP-TPP

Scheme 1. Synthetic route and proposed chemical structure of cross-linked CP-TPP nanospheres.

3. RESULTS AND DISCUSSION The synthetic procedure for the preparation of CP-TPP nanospheres is presented in Scheme 1. TEA as an acid acceptor was added excessively to a flask containing HCCP and TPP with a molar ratio of 2:1 in acetonitrile. Under a high-intensity ultrasonic bath, the hydroxyl groups on TPP were activated to interact with the chlorine groups on HCCP. The polycondensation led to the generation of oligomers and HCl byproduct that can be adsorbed by TEA to accelerate the polymerization. Because of the steric hindrance effect, only one chlorine atom on each phosphorus atom would be substituted by TPP. As a result, BPS could react with remaining chlorine atoms in the system. Although chlorine atoms on the cyclotriphosphazene ring are not replaced completely, this situation does not affect the formation of the highly cross-linked structures with spherical morphology, and the obtained nanospheres would not be broken by aqueous media. The formation of CP-TPP nanospheres follows an oligomeric species adsorbing mechanism in a standard procedure. The polycondensation between HCCP and TPP forms oligomers at initial stage of the precipitation reaction. These oligomers aggregate to form unstable nucleus particles and then converge to 7



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generate stable cross-linked particles. These particles have high surface energy, so that they would grow in size by seizing more oligomers having -OH groups. Finally, the nanospheres with cross-linked structures are formed.

CP-TPP

CP-TPP/Au

CP-TPP/PEI

CP-TPP/Au/PEG

Scheme 2. Synthetic procedure for preparing CP-TPP/Au/PEG nanospheres.

The synthesis of CP-TPP/Au/PEG nanospheres is illustrated in Scheme 2. The surface of CP-TPP nanospheres is rich in heteroatoms (such as N, P, and S) and phenolic hydroxyl groups, and these atoms and groups have strong coordination ability with gold, providing the possibility for the immobilization of Au nanoparticles. Theoretically, gold nanoparticles could also be encapsulated into the CP-TPP nanospheres. However, the encapsulation may reduce the photothermal efficiency of the resulted materials.40-42 When gold nanoparticles were attached onto the CP-TPP nanospheres by in situ reduction of HAuCl4 in aqueous suspension of CP-TPP, aggregated Au nanoparticles on the nanosphere surface were obtained. The reason is that the surface of CP-TPP is negatively charged due to the presence of unreacted phenolic hydroxyl groups (Figure S1), showing the zeta potential of -22. In order to solve this problem, the PEI functionalization on the CP-TPP nanospheres was performed through the CDI surface activation method. The zeta potential of CP-TPP/PEI turned out to be +25, confirming the successful modification of PEI on CP-TPP. HAuCl4 was then located on the surface of the 8


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CP-TPP/PEI nanospheres by the electrostatic interaction between PEI and AuCl4-, which was reduced into Au nanoparticles in situ. The generated Au nanoparticles were homogeneously distributed on the surface of CP-TPP/PEI nanospheres. To further reduce the cytotoxicity, the obtained CP-TPP/Au nanospheres were finally covered with PEG. The nanospheres are quite stable in aqueous solution without obvious aggregation.

Figure 1. FTIR spectra of HCCP, TPP, BPS monomer, and CP-TPP nanospheres.

The chemical structure of CP-TPP nanospheres was characterized by Fourier-transform infrared spectroscopy (FTIR, Figure 1). The two strong absorption peaks at around 3400 cm-1 in the spectra of TPP and BPS are attributed to the Ph-OH groups. On the contrary, a single broad band at about 3437 cm-1 in the spectrum of CP-TPP corresponds to the Ph-O-P unit. In addition, the absorption at 1260 cm-1 assigned to the Ph-OH group disappeared in the CP-TPP spectrum, and a new peak at 960 cm-1 attributed to the Ph-O-P group appeared. Other characteristic peaks could also be observed in the spectrum of CP-TPP. The peak at 1207 cm-1 is assigned to the P=N stretching of HCCP, and the absorption peaks at 1591 cm-1 and 1489 cm-1 correspond to the aromatic groups. Therefore, it could be concluded that the polycondensation of TPP, BPS and HCCP was successfully achieved. The presence of gold nanoparticles on the CP-TPP/Au/PEG nanospheres was confirmed by powder X-ray 9



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diffraction (XRD) measurements (Figure S2). The CP-TPP shows one broad diffraction peak corresponding to the amorphous nature of the nanospheres. On the other hand, all the peaks in the powder XRD pattern of CP-TPP/Au/PEG nanospheres could be assigned to literature values for Au (JCPDS Card No. 04-0784), suggesting the existence of face-centered cubic gold nanoparticles in the CP-TPP/Au/PEG nanospheres.

a

b

CP-TPP/Au Seeds

CP-TPP

d

c

CP-TPP/Au

CP-TPP/Au/PEG

e

Figure 2. (a-d) TEM images and (e) size distribution of CP-TPP, CP-TPP/Au seeds, CP-TPP/Au, and CP-TPP/Au/PEG nanospheres based on DLS.

10


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The morphology and size of the as-prepared nanospheres were investigated by transmission electron microscopy (TEM) and dynamic light scattering (DLS) as displayed in Figure 2. The CP-TPP nanospheres show smooth surface, good dispersion, and relatively narrow size distribution (Figure 2a), with an average diameter of 196 ± 20 nm (Figure 2e). After the in situ reduction of HAuCl4, it is obvious that the nanospheres with uniformly distributed gold seeds on the surface were obtained (Figure 2b). Then, the seed growth on the CP-TPP/PEI spheres was accomplished by reducing the K-gold solution to form gold nanoparticles. Figure 2c clearly reveals that gold nanoparticles with a diameter of 3.2 nm were distributed on the nanosphere surface. Consistently, the size of CP-TPP/Au/PEG nanospheres shows a right shift as compared with CP-TPP nanospheres determined by DLS. The experimental results are consistent with the inference that the amount of gold nanoparticles on the nanosphere surface is positively correlated with the concentration of the K-gold added. When an excessive amount of K-gold was added, CP-TPP nanospheres were fully covered by gold nanoparticles (Figure S3). Since the full coverage of gold nanoparticles could reduce the photodynamic effect of the CP-TPP core, partially covered nanospheres were employed to present sufficient photothermal effect. The compositional features of CP-TPP, CP-TPP/Au seeds, and CP-TPP/Au nanospheres were determined by energy-dispersive X-ray spectroscopy (EDS, Figure S4). Signals for Si, C, O, P, and Cl were observed in the EDS spectrum of CP-TPP nanospheres. The Si signal is from the silicon wafer used for dropping the sample, while the P signal in the spectrum should come from the cyclotriphosphazene skeleton. The presence of the Cl element signal proves that the cross-linking reaction was incomplete, mainly due to the steric hindrance of TPP molecule and only some of the Cl atoms on HCCP were replaced. Au signals were observed in the EDS spectra of CP-TPP/Au seeds and CP-TPP/Au nanospheres. The atomic percentages of Au atoms are 0.4 and 0.7 respectively, indicating that the gold seeds were successfully attached onto the surface of the CP-TPP nanospheres and further grown into gold nanoparticles. The emission spectra of TPP, CP-TPP, and CP-TPP/Au/PEG nanospheres were then measured to compare their fluorescence properties (Figure 3a). As for the small molecule TPP, its emission peak at 668 nm was obtained when excited at 580 nm. The CP-TPP and 11



CP-TPP/Au/PEG nanospheres show weaker emission peaks at 723nm. The obvious red shift of the fluorescence emission is mainly because of the chemical environment change of the TPP after being fixed in the cross-linked structure, leading to the electronic transition change of TPP. The strong fluorescence emission of CP-TPP nanospheres indicates good isolation of TPP unit in the structure to avoid the fluorescence quenching.

a

Wavelength (nm)

b

Wavelength (nm)

Figure 3. (a) Fluorescence spectra and (b) UV-vis spectra of TPP solution (0.05 mg/mL), CP-TPP (0.1 mg/mL), and CP-TPP/Au/PEG (CCP-TPP = 0.1 mg/mL) suspension in deionized water.

The UV-vis absorption of TPP and CP-TPP was also employed to study the arrangement state of the TPP unit in the nanospheres (Figure 3b). The TPP solution exhibits the characteristic absorption maximum at 416 nm, while the CP-TPP solution shows a similar absorption peak, suggesting that most of the TPP unit in the cross-linked structure is isolated. The UV-vis absorption of CP-TPP/Au/PEG nanospheres also shows a similar absorption peak 12


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at 416 nm. On the other hand, a slight enhancement in broad NIR region was observed due to the surface plasmon resonance of Au nanoparticles, which is consistent well with the color change from magenta to dark brown after the formation of gold nanoparticles.

a y = -0.003x + 0.240 R2 = 0.972

every 2 min

b

y = -0.096x + 0.757 R2 = 0.997

every 1 min

c

y = -0.091x + 0.731 R2 = 0.989

every 1 min

Figure 4. Absorbance of DPBF after irradiated by 630 nm LED (50 mW/cm2) over different periods of time for (a) control, (b) in the presence of CP-TPP, and (c) in the presence of CP-TPP/Au/PEG nanosphere suspension in deionized water (CCP-TPP = 0.1 mg/mL).

13



The singlet oxygen generation efficiency of the CP-TPP and CP-TPP/Au/PEG nanospheres under light irradiation was determined by using the DPBF indicator. The time-dependent absorbance of DPBF at 417 nm indicates the generation of 1O2. As a control, the absorption intensity of the DPBF solution without the addition of any photosensitizer was measured ever 2 min under the irradiation of 630 nm LED (50 mW/cm2, Figure 4a). Negligible changes of the absorption were observed within 6 min, indicating the stability of DPBF under experimental conditions. Figure 4b,c shows the absorption spectra of DPBF in the presence of CP-TPP and CP-TPP/Au/PEG nanospheres, respectively. In both cases, the absorption was measured ever 1 min after the light irradiation. It was observed that the DPBF absorption peak was significantly decreased over time because of the 1O2 generation by the nanospheres. Since Au nanoparticles could absorb near-infrared light, the heat is generated through plasma resonance or energy transition, causing an increase in surrounding temperature. A near-infrared laser with a power of 1.5 W and a wavelength of 808 nm was used as a light source to irradiate CP-TPP, CP-TPP/Au and CP-TPP/Au/PEG aqueous suspensions in order to investigate their photothermal conversion properties. As shown in Figure 5a, the coating of PEG has almost no effect on the photothermal properties of the nanospheres. CP-TPP/Au/PEG nanospheres show a significant rise in temperature especially within the first 6 min under the irradiation by 808 nm laser. The temperature of CP-TPP/Au/PEG nanosphere suspensions increased rapidly to over 70 °C and 55 °C within 5 min at relatively high concentration of 1 and 0.5 mg/mL, respectively. Even at a low concentration of 0.1 mg/mL, the temperature also rose to 47 °C in 5 min. On the other hand, the CP-TPP nanospheres, even at a high concentration, show weak photothermal effects provided by the porphyrin unit after 808 nm NIR laser irradiation and 630 nm LED light irradiation. After 5 cycles of irradiation repeatedly, no significant loss of photothermal heating capability was observed (Figure 5b), indicating that the CP-TPP/Au/PEG nanospheres have a good photothermal stability. In addition, CP-TPP/Au/PEG nanospheres exhibit a similar temperature increase under the irradiation by 808 nm laser before and after four weeks of storage (Figure S5). Therefore, the introduction of Au nanoparticles on the surface of the nanospheres makes the system a suitable photothermal agent. 14


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a

b

Figure 5. (a) Photothermal properties of CP-TPP/Au/PEG and CP-TPP nanospheres under 808 nm laser (1.5W) irradiation. (b) Photothermal performance of CP-TPP/Au/PEG nanosphere suspension (0.5 mg/mL) after 5 cycles of irradiation (1.5W, 5 min each).

Based on their inherent fluorescent properties, CP-TPP and CP-TPP/Au/PEG nanospheres could be used directly as suitable fluorescent agents for cell imaging. To confirm whether the two kinds of nanospheres were internalized by cancer cells, the confocal microscope was employed to explore the cellular fluorescence (Figure 6). The HeLa cells were incubated with CP-TPP and CP-TPP/Au/PEG nanospheres in a concentration of 50 µg/mL for 4 and 24 h respectively, and then fixed for the characterization. The nuclei of the cells emit blue fluorescence under the excitation of the 405 nm laser, while the CP-TPP and CP-TPP/Au/PEG nanospheres exhibit red fluorescence when excited by a 561 nm laser. As shown in Figure 6, the cell uptake performance of the two nanospheres was similar. The intensity of intracellular red fluorescence increased from 4 h to 24 h for both CP-TPP and CP-TPP/Au/PEG incubated cells. These observations indicate that the synthesized nanospheres could enter the cells upon time. The fluorescence distribution localized inside HeLa cells is probably owing to the endocytosis of CP-TPP and CP-TPP/Au/PEG nanospheres by cells.

15



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a

b

c

d

e

f

g

h

i

j

k

l

Figure 6. Confocal microscopy images of HeLa cells incubated with (a-f) CP-TPP and (g-l) CP-TPP/Au/PEG nanospheres at an equivalent CP-TPP concentration of 50 µg/mL for (a-c, g-i) 4 h and (d-f, j-l) 24 h. Images from left to right present cell nuclei stained by DAPI (blue), incubated by microspheres (red), and merged images. Scale bar = 50 µm.

16


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a

b

c

d

e

f

g

h

i

Figure 7. Confocal microscopy images of carboxy-H2DCFDA stained HeLa cells (a-c) without and (d-i) with CP-TPP/Au/PEG nanospheres at equivalent CP-TPP concentration of 50 µg/mL (a-c, g-i) under 630 nm LED irradiation (50 mW/cm2) for 15min or (d-f) incubated in the dark. Images from left to right present cell nuclei stained by DAPI (blue), cells treated by carboxy-H2DCFDA as a fluorogenic marker for ROS generation (green), and merged images. Scale bar = 50 µm.

The intracellular singlet oxygen generation of the CP-TPP and CP-TPP/Au/PEG nanospheres was measured using 5-(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) staining. As depicted in the cell images taken by confocal microscope (Figures 7 and S6), HeLa cells incubated without any nanospheres and then irradiated by 17



630nm LED did not exhibit evident green fluorescence. The cells incubated with the nanospheres but without the light irradiation also showed no green fluorescence. On the contrary, HeLa cells incubated with CP-TPP or CP-TPP/Au/PEG nanospheres and then treated with 630 nm LED for 15 min emitted green signals, indicating that the singlet oxygen was generated inside the cells. The MTT cytotoxicity assay was conducted to quantitatively assess the cytotoxicity of the CP-TPP, CP-TPP/Au, and CP-TPP/Au/PEG nanospheres (Figure 8). Firstly, the CP-TPP nanospheres without any modification on the surface in a wide concentration range (10-100 µg/mL) were incubated with HeLa cells for 48 h. It turned out that such cross-linked polyphosphazene nanospheres did not show obvious cytotoxicity. The survival ratio of the cells was higher than 80% for all cases. Hence, the CP-TPP itself could be used safely as a new type of photosensitizer for PDT of cancer. In another case, HeLa cells were incubated with CP-TPP/Au nanospheres and undergone the MTT assay. When the concentration of the CP-TPP/Au nanospheres was less than 50 µg/mL, the cell viability was higher than 80%. When 100 µg/mL CP-TPP/Au was added, the cell viability dropped to 74%, indicating that the CP-TPP/Au nanospheres has relatively weak cytotoxicity probably due to the presence of the hyperbranched PEI. To reduce the cytotoxicity, mPEG-COOH was used to modify the nanosphere surface. The modification was meaningful. The cell viability was higher than 80% when HeLa cells were incubated with CP-TPP/Au/PEG nanospheres in the concentration range from 10 to 100 µg/mL. The MTT assay was also conducted to quantitatively assess the cytotoxicity of the nanospheres in HUVECs (Figure S7). The test results were similar to those in HeLa cells. The cell viability was higher than 80% when HUVECs were incubated with CP-TPP and CP-TPP/Au/PEG nanospheres in the concentration range from 10 to 100 µg/mL. Therefore, the CP-TPP/Au/PEG nanospheres were employed in subsequent investigations of combined PDT and PTT.

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a

b

c

Figure 8. (a) Viability of HeLa cells incubated with CP-TPP, CP-TPP/Au, and CP-TPP/Au/PEG nanospheres at different concentrations for 48 h. (b) Viability of HeLa cells incubated with CP-TPP nanospheres at different concentrations under the irradiation of 630 nm LED. (c) Viability of HeLa cells incubated with CP-TPP/Au/PEG nanospheres at different concentrations under the irradiation of 808 nm laser and/or 630 nm LED.

19



The viability of HeLa cells showed a negligible change when irradiated by the light sources (Figure 8b,c). After incubated with different concentrations of CP-TPP nanospheres for 24 h and then irradiated under 630 nm LED for 8, 15, and 20 min respectively, the cell viability was significantly decreased. Especially when the concentration of the nanospheres was 100 µg/mL and the irradiation time was over 15 min, the cell viability decreased to about 35%, meaning that CP-TPP nanospheres are a type of an effective photosensitizer for PDT. The CP-TPP/Au/PEG nanospheres exhibited similar but slightly weakened PDT effect as compared with that of CP-TPP nanospheres (Figure 8c). After irradiated for 15 min under 630 nm LED, the viability of HeLa cells incubated with 100 µg/mL CP-TPP/Au/PEG was 40%. As for the PTT effect, the cell viability of the HeLa cells incubated with different concentrations of the CP-TPP/Au/PEG nanospheres and then treated with 808nm laser for 15 min was investigated. Obviously, the resulted hyperthermia caused a certain percentage of cell apoptosis. The cell viability decreased to 45% when the HeLa cells were treated with 100 µg/mL CP-TPP/Au/PEG nanospheres under the laser irradiation. To investigate the synergistic effect of combined PDT and PTT, the cytotoxicity of the CP-TPP/Au/PEG nanospheres was analyzed with the MTT assay in HeLa cells under sequential irradiation of 630 nm LED and 808nm laser, each for 15min. The cell viability was lower than individual PDT or PTT under the concentrations of CP-TPP/Au/PEG nanospheres from 10 to 100 µg/mL. The cell viability decreased to about 10% when the nanospheres used were at the highest concentration, indicating that combined PDT and PTT could achieve a synergistic effect to enhance the therapeutic ability on cancer. Thus, the CP-TPP/Au/PEG nanospheres are an effective single system to realize these two types of phototherapies synergistically.

4. CONCLUSIONS In summary, we have successfully synthesized polyphosphazene nanospheres CP-TPP with porphyrin units covalently immobilized in the cross-linked network. As a novel photodynamic carrier, CP-TPP has shown efficient singlet oxygen generation capability and photodynamic cancer cell killing efficacy. Moreover, gold nanoparticles were anchored on the surface of the nanospheres by the in situ reduction to achieve a synergistic PDT/PTT effect. The nanospheres were partially covered by Au nanoparticles for sufficient photothermal effect, 20


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while the photodynamic effect would not be significantly affected. The final CP-TPP/Au/PEG nanospheres have exhibited low cytotoxicity when incubated with HeLa cells in the dark, while showing significant cytotoxicity when treated with 808nm laser for PTT and 630nm LED for PDT, respectively. Importantly, combined PDT and PTT by CP-TPP/Au/PEG have presented a synergistic effect to enhance the therapeutic ability on cancer. In addition, these nanospheres possess the cell imaging capability based on their inherent fluorescent properties. Therefore, CP-TPP/Au/PEG nanospheres offer a new opportunity for bioimaging guided combinational cancer treatment.

ASSOCIATED CONTENT Supporting Information Zeta potential, SEM images, EDS and elemental mapping of CP-TPP, CP-TPP/Au seeds, CP-TPP/Au, and CP-TPP/Au/PEG nanospheres, TEM image of CP-TPP nanospheres fully covered by gold nanoparticles, confocal microscopy images of CP-TPP nanospheres for singlet oxygen generation, and cell viability of HUVECs.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] Notes The authors declare no competing financial interest. Acknowledgements This research is supported by the Singapore Academic Research Fund (No. RG121/16, RG11/17, and RG114/17) and the Singapore National Research Foundation Investigatorship (No. NRF-NRFI2018-03).

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