Protein Shell-Encapsulated Pt Clusters as Continuous O2-Supplied

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Biological and Medical Applications of Materials and Interfaces 2

Protein Shell Encapsulated Pt Clusters as Continuous O-Supplied Biocoats for Photodynamic Therapy in Hypoxic Cancer Cells Yahang Li, Xiaoxia Jian, Shanshan Zhou, Yongxin Lu, Chenxi Zhao, Zhida Gao, and Yan-Yan Song ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02484 • Publication Date (Web): 22 Apr 2019 Downloaded from http://pubs.acs.org on April 22, 2019

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ACS Applied Materials & Interfaces

Protein Shell Encapsulated Pt Clusters as Continuous O2-Supplied Biocoats for Photodynamic Therapy in Hypoxic Cancer Cells Yahang Li, Xiaoxia Jian, Shanshan Zhou, Yongxin Lu, Chenxi Zhao, Zhida Gao and Yan-Yan Song*

Department of Chemistry, Northeastern University, 110004 Shenyang, China.

KEYWORDS. Pt Clusters, Protein Shell, Biocoats, O2 evolving, Photodynamic Therapy, Hypoxic cells.

ABSTRACT. As a highly oxygen-dependent process, the effect of photodynamic therapy (PDT) is often obstructed by the premature leakage of photosensitizers and the lack of oxygen in hypoxic cancer cells. To overcome these limitations, this study designs serum albumin protein (BSA) encapsulated Pt nanoclusters (PtBSA) as O2-supplied biocoats, and further cooperates with mesoporous silica nanospheres (MSNS) to develop intelligent

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nanoaggregates for achieving improved therapeutic outcomes against hypoxic tumors. The large number of amino groups on BSA can provide sufficient functional groups to anchor tumor targeting agents, and thus enhance the selective cellular uptake efficiency. Owing to the outstanding biocompatibility features of BSA and the state-of-the-art catalytical activity of Pt nanocluster, the nanocomposites have lower dark cytotoxicity, and continuous O2 is evolved via the decomposing H2O2 in a tumor microenvironment. Both in vivo and in vitro experiments indicate that the resulted nanocomposites can effectively relieve hypoxic condition, specifically induce necrotic cell apoptosis, and remarkably hinder tumor growth. Our results illuminate the great potential of BSA encapsulated Pt nanoclusters as versatile biocoats in designing intelligent nanocarriers for hypoxic-resistant photodynamic therapy.

1. Introduction

Photodynamic therapy (PDT) is known as an attractive strategy for anti-tumor1-5 and antibacterial6-8 treatments. Upon irradiation, excited photosensitizers (PSs) transfer energy to the available O2 and generate cytotoxic singlet oxygen (1O2), which has been


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demonstrated to be effective in causing the apoptosis and necrosis of cancer cells via multiple pathways, e.g., stimulating the inflammatory and immune responses to cancer cells, destroying tumor microvasculature, and reacting with intracellular proteins.9-14 However, the local lack of oxygen (O2) in tumors and premature drug release are two of the major obstacles of PDT,15-20 which frequently leads to poor therapeutic outcomes.2122

Therefore, the development of safe and activatable PSs delivery systems that

overcomes these obstacles remains a critical, albeit unmet, scientific goal.

An ideal PSs delivery system for PDT needs to combine multiple functions, such as good biocompatibility, effective PSs encapsulation, carrier stability, tumor-targeting ability, and sufficient

1O 2

production.23-26 It is generally accepted that the

microenvironment of cancer cells is more acidic than that of normal cells. In addition, the glutathione (GSH) concentration in cancer cells is ~3-fold higher than that in normal cells.27,

28

To date, such pH and GSH differences in tumor microenvironments have

utilized for the design of PSs delivery systems.29-32 However, since the local production of a large amount of 1O2 appears to be important during PDT therapy, the activity of PSs


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largely relies on the O2 concentration in the intracellular environment.33 Nanomaterials (i.e. CaO2 and MnO2) that exhibit O2-generation abilities have been introduced into hypoxic tumor tissues along with PSs to increase the concentration of O2 in cancer cells.4, 34-40

As the O2-generation depends on redox reactions between H2O2 and nanomaterials,

the generation of O2 would significantly decrease in response to the consumption of these materials during redox reactions. Platinum (Pt) nanomaterials, as the state-of-the-art catalysts, can break the oxygen-oxygen bond of H2O2 and ultimately lead to the generation of O2.41, 42 However, due to the undesired aggregation and contamination in a complex biological environment, most of the metal nanoclusters or nanoparticles usually present reduced O2 production.43, 44

In this study, we designed an O2-supplied biocoats with continuous O2-evolving and pH/GSH dual-responsive properties to achieve an improved hypoxia-resistant photodynamic therapy. Compared to traditional sole-core delivery systems, these biocoats covered nanocarriers exhibit unique advantages.45 For example, the core and biocoats can exert separate functions without mutual hindrance. In addition, both parts


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may also work in a concerted fashion. As illustrated in Scheme 1, disulfide-bridged mesoporous silica nanospheres (MSNS) serve as the nanocarrier to accommodate methylene blue (MB), which is a well-studied photosensitizer with a high quantum yield ( ~0.5) in the 1O2 production. BSA-encapsulated Pt nanoparticles (PtBSA) act as biocoats. The as-prepared FA/PtBSA@MB-MSNS nanocomposites exhibit three distinct features: i) cargo release and O2 generation are only active in cancer cells; ii) in addition to providing various functional groups for the anchoring of tumor targeting agents, the BSA scaffold protects Pt clusters from aggregation and inactivation in complex intercellular microenvironments, thus retaining excellent catalytic activity characteristics; iii) due to the excellent biocompatibility of BSA, the as-designed covered nanocarrie did not exert cytotoxic effects toward normal cells.46

2. Experimental Section

Preparation of MSNS and MB-MSNS: The MSNS NPs were prepared from an emulsion system using TEOS as silica source. Typically, 0.2 M TEOS was dissolvedin 15 mL npentanol and 15 mL cyclohexane. Then, 15 mL aqueous urea (0.30 g) solution containing


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0.90 g hexadecyl trimethyl ammonium bromide (CTAB) were dropply added to above solution under vigorous stirring. The resulted mixture was then transferred to a Teflonlined autoclave and heated at 130 °C for 2 h. After quickly cooling to room temperature, the product was washed with ethanol and water three times and dried in a vacuum freeze dryer. Finally, MSNS was obtained after annealing in air at 600 °C for 4 h. The obtained MSNS was then thiolated by MPTMS. Briefly, 500 mg MSNS was dissolved in 1.325 g mL-1 (3-mercaptopropyl)-trimethoxysilane (MPTMS) ethanol solution and refluxed at 80 °C for 8 h. The resulted product was washed with ethanol three times and the obtained MSNS-SH material was stirred with synthetic py-ss-COOH overnight to yield MSNS-ssCOOH.

MB was loaded onto MSNS by electrostatic adsorption between MB and carboxyl on the surface of MSNS. Briefly, 1 mL of 0.1 mg mL-1 MB was mixed with 9 mL of an aqueous solution containing 50 mg MSNS. After stirred for 2 h at 20 oC, the excess MB was removed by centrifugation and then tested by UV-vis spectrum to evaluate the loading mass of MB in MSNS.


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Preparation of PtBSA and FA/PtBSA: PtBSA was synthesized using an established strategy.41 Briefly, 5 mL of BSA (50 mg mL-1) and 0.5 mL NaOH (1.5 M) was added to 5 mL H2PtCl6 (16 mM).41 The mixture was undergone constant stirring for 2 h at 80 °C. The resulting PtBSA was washed by ultrafiltration (Millipore, 30 kDa) to a neutral pH value, and then dried in a vacuum freeze drier. The as-formed PtBSA was linked with folic acid (FA) via amide bond. Typically, 1 mL of FA (6.8 mM) aqueous solution was mixed with 1 mL of 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimidehydrochloride (EDC, 0.04 M) aqueous solution at room temperature under constant stirring for 15 min. After that, 5 mL PtBSA (4.76 mg mL-1) was added to the solution under stirring for 2 h to anchor FA. FA functionalized PtBSA (FA/PtBSA) was then purified by ultrafiltration and dried in a vacuum freeze drier.

Preparation of FA/PtBSA@MB-MSNS: FA/PtBSA was successively loaded onto MSNS via amide bond. Typically, 10 mL aqueous solution containing MB-MSNS (5.0 mg mL-1) and EDC (0.004 M) was stirred for 15 min; after that, the as-prepared FA/PtBSA (2.5 mL,


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5.0 mg mL-1) was added to the above solution under constant strirring for another 2 h. Excess FA/PtBSA was then removed by centrifugation.

MB Release from FA/PtBSA@MB-MSNS: FA/PtBSA@MB-MSNS was used to deliver MB to cancer cells. The loading capacity was calculated using equation (1) as follows:

Loading capacity 

m MB  m unabsorbed MB m MSNS

The mMB, munasorbed

MB

(1)

and mMSNS represent the mass of MB, unabsorbed MB and

MSNS. The masses of MB and unabsorbed MB were calculated based on a standard curve by monitoring the absorbance of MB at 665 nm. The MB loading capacity of MSNS was 1.47 mg g-1. The release kinetics of MB from FA/PtBSA@MB-MSNS was determined by centrifugation of the FA/PtBSA@MB-MSNS suspensions in PBS. Briefly, the NPs (25 mg) and GSH (1 mM or 10 mM) were mixed in 10 mL PBS (either pH 5.5 or 7.4) and stirred at 37 °C. The samples NPs were tested by UV-vis spectroscopy at different intervals after high-speed centrifugation (1000 rpm for 2 min).


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Extracellular O2 Production stimulated by H2O2: The extracellular production of O2 from H2O2 was evaluated by the quenching of fluorescence signal from [Ru(dpp)3]Cl2 (RDPP) at 615 nm (ex = 455 nm), which is one of the well-known O2 probe. Typically, 100 µL RDPP ethanol solution (, 1 mM) was added to 2 mL PBS buffer (10 mM, pH 5.5) containing MB-MSNS or FA/PtBSA@MB-MSNS (20 µg mL-1). After the addition of 100 μM H2O2, the fluorescence intensity of RDPP was monitored. Groups of MB-MSNS without H2O2, FA/PtBSA@MB-MSNS without H2O2, and 100 M H2O2 without NPs were employed as references to evaluate the O2 generation.

Extracellular H2O2-Enhanced 1O2 Production: 1,3-diphenylisobenzofuran (DPBF) was employed as the probe to detect extracellular 1O2 production via monitoring the absorption intensity at 411 nm. Typically, 2 mL DPBF (55 μM) acetonitrile solution of was mixed with 0.5 mL suspensions of MB@MSNS or FA/PtBSA@MB-MSNS (125 g mL-1) in PBS (10 mM, pH 5.5). After the addition of 100 M H2O2, the suspensions were irradiated by a 635 nm laser (6 mW cm-2). The absorption intensity at 411 nm was monitored by UV-vis spectroscopy. The groups of nanoparticles without H2O2 and H2O2 without nanoparticles


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were tested as controls. To investigate the influence of the biological tissue on the PDT conversion effect of FA/PtBSA@MB-MSNS, fresh skin from as-killed chickens was employed as both a biological tissue model and as barrier layers. A 10 mm quartz cuvette containing FA/PtBSA@MB-MSNS was coated with chicken skin on the 635 nm laser pathway. Then, a similar procedure was used to measure the 1O2 production by DPBF assay.

Cell Culture and Confocal Fluorescence Imaging (CLSM): The in vitro studies were determined by HeLa and A549 cells. The cells were incubated in a traditional complete medium including 90% Dulbecco's Modified Eagle's medium, 10% fetal bovine serum and 1% antibiotics (penicillin/streptomycin). The cell culture condition is control at 37 °C under under 5% CO2.. After washed with PBS buffer (10 mM, pH 7.4) for three times, the CLSM images of cells were conducted with a confocal microscope (FV1200, Olympus, Japan).

In Vitro Photodynamic Therapy: For the photodynamic therapy, the toxicity and phototoxicity properties of nanoparticles were evaluate by 3-(4,5-dimethylthiazol-2-yl)-


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2,5-diphenyl-tetrazolium bromide (MTT) assay. Typically, HeLa cells were first seeded into two 96-well plates in 100 μL complete medium with the account of 1 × 104 cells per well; cells were then incubated for 24 h (37 oC, 5% CO2). After washing with PBS, 100 μL of fresh incubation media containing FA/PtBSA@MB-MSNS were added into the wells, and then HeLa cells were incubated for 6 h. The media was then removed by washing with PBS for three times, and subsequently the resulted cells were irradiated using a 635 nm laser for a certain period. One plate remained in the dark and was used as reference sample. Then, 10 μL of 5 mg mL-1 MTT solution in PBS (pH 7.4) was added to each well, and then incubated for 4h. After that, the incubation medium was removed carefully and the generated formazan crystals were dissolved by 100 μL dimethyl sulfoxide. The absorbance of MTT at 490 nm was read by a plate reader and the cell viability was compared to the control cells (untreated) and determined by using equation (2) as follows:

Cell viability (%) =

A t reatment group - A Blank 1 A control - A Blank 2

× 100

(2)


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The Ātreatment group represents mean absorbance of MTT incubated with nanocomposites and cells, Ācontrol group represents mean absorbance of MTT incubated only with cells, ĀBlank

1

represents the absorbance of MTT incubated with nanoparticals without cells and ĀBlank

2

represents MTT incubated with PBS without cells.

The cell apoptosis assay was tested by Annexin V-FITC/PI staining. First, HeLa cells were seeded into the culture disk and maintained for 24 h. Then, after removing the media and washing with PBS, the cells were incubated with 2 mL MB-MSNS or FA/PtBSA@MBMSNS (125.0 mg mL-1) for another 6 h. After washing carefully with PBS buffer, cells were irradiated by a 635 nm laser (45 mW cm-2) for 30 min. Subsequently, 25 μL of Annexin VFITC staining solution was added to the CLSM-specific culture dish and stained for 5 min. PBS was used to remove any redundant dye. Then, 10 L of PI was added and stained for 10 min. Redundant dye was removed via PBS washing for three times. The cells were observed by CLSM.

In Vitro Phototoxicity Assay: HeLa cellswere first seeded into two 96-well plates (cell density: 1 × 104 cells per well) in 100 μL complete medium, followed with a 24 h


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incubation (37 °C, 5% CO2). After adherence, the incubation medium was removed and HeLa cells were then washed with PBS for three times and then incubated with 10 group samples (PtBSA, FA/PtBSA, PtBSA@MSNS, FA/PtBSA@MSNS, MB, MSNS, MBMSNS, PtBSA@MB-MSNS, FA/PtBSA@MB-MSNS, and control) at the identical dose of 125.0 g mL-1 FA/PtBSA@MB-MSNS or equal to the loading mass (25.0 g mL-1 PtBSA, 25.0

g

mL-1

FA/PtBSA,

125.0

g

mL-1

PtBSA@MSNS,

125.0

g

mL-1

FA/PtBSA@MSNS, 0.18 g mL-1 MB, 100.0 g mL-1 MSNS, 100.0 g mL-1 MB-MSNS, and 125.0 g mL-1 PtBSA@MB-MSNS) for 6 h. The cells were washed by PBS and then irradiated by 635 nm laser (45 mW cm-2) for 30 min. A further plate remained in the dark and MTT assays were conducted as described above to quantitively evaluate the cytotoxicity.

In Vitro Cytotoxicity Assay: A longer incubation period was also tested to further study the toxicity of FA/PtBSA@MB-MSNS. HeLa cells were incubated with PtBSA, FA/PtBSA, PtBSA@MSNS, FA/PtBSA@MSNS, MB, MSNS, MB-MSNS, PtBSA@MB-MSNS,


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FA/PtBSA@MB-MSNS, or complete medium (control) for 48 h in the absence of irradiation and MTT assays were conducted as described above.

Selectivity of FA/PtBSA@MB-MSNS: HeLa and A549 cells were used to certify the selectivity of FA/PtBSA@MB-MSNS. HeLa cells and A549 cells were first seeded into 96well plates (1 × 104 cells per well) in 100 μL complete medium and then incubated for 24 h (37 oC, 5% CO2). After washing with PBS buffer for three times, HeLa and A549 cells were incubated with 100 μL incubation media containing serial concentrations of FA/PtBSA@MB-MSNS for 6 h. After removing the media and washing with PBS, the cells were irradiated by 635 nm laser (45 mW cm-2) for 30 min and then MTT assays were conducted as described above. HeLa cells and A549 cells were also seeded into a CLSMspecific culture disk and maintained for 24 h. Then, HeLa cells were incubated with FITC modified FA/PtBSA@MB-MSNS both with and without FA for 6 h. A549 cells were incubated with FITC modified FA/PtBSA@MB-MSNS with FA for 6 h. All the cells were observed by CLSM.


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DAPI and actin-tracker green were loaded onto MSNS together with MB; then, PtBSA with and without FA were coated on them, called PtBSA@Nucleusdye-MSNS, FA/PtBSA@Nucleusdye-MSNS, PtBSA@Actin-MSNS, and FA/PtBSA@Actin-MSNS. HeLa cells were first seeded into the CLSM-specific culture disk and maintained for 24 h. Subsequently, HeLa cells were incubated in the presence of PtBSA@Nucleusdye-MSNS, FA/PtBSA@Nucleusdye-MSNS, PtBSA@Actin-MSNS, or FA/PtBSA@Actin-MSNS for another 6 h, and then washed by PBS buffer for three times. All cells were observed by CLSM.

In Vivo Photodynamic Therapy: All animal experiments strictly followed the guidelines of the “National animal management regulations of China” and were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of Simcere. For in

vivo test, six-week old female Bab/c nude mice (weighing 12-18 g) were chosen to establish the xenograft cancer cell (human cervical cancer cell lines-HeLa cells) tumor models. Hela cells (~106) in 100 μL saline were injected into the hind limb of mice, and the tumors were allowed to grow to a similar size (~100 mm3) before use. Then, the mice


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were divided into four groups (each group consists six mice). For each group, 500 L of (i) saline (control), (ii) 0.735 g mL-1 MB, (iii) 0.5 mg mL-1 MB-MSNS, and (iv) 0.5 mg mL-1 FA/PtBSA@MB-MSNS were injected into the mice via the tail vein every two days. After the injection treatment for 24 h, a 635 nm laser (100 mW cm-2) was employed to irradiate the tumors in mice for 15 min. Following the PDT treatment, the body weights were monitored and the tumor size was recorded with digital calipers in two dimensions. Tumor volumes were determined according to equation (3):

Volume =

Tumor length × Tumor width 2 2

(3)

To evaluate the therapeutic efficacy of the different groups, all animals were sacrificed on the 15th day and the tumor tissues were harvested.

H&E and HIF-1α Staining: The tumor tissues harvested from the sacrificed mice were first fixed in formalin (4%) and then treated in paraffin. Then, the tissues for the four groups were cut to slices of 4 μm thicknesses for H&E and HIF-1α staining. All tissue slices were observed via a digital microscope.


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3. Results and Discussion

3.1. Synthesis and Characterization of FA/PtBSA@MB-MSNS

The transmission electron microscopic (TEM) image of silica nanoparticles in Figure 1A shows an average particle diameter of 40-50 nm (Figure S1A), large surface area (219.0 m2 g-1, Figure S2A), and mesoporous characteristic (pore diameter ~2.54 nm, Figure S2B). The resulted MSNS was firstly thiolated by (3-mercaptopropyl)-trimethoxysilane (MPTMS), and subsequently combined with 2-carboxyethyl 2-pyridyl disulfide (py-ssCOOH) to yield MSNS-ss-COOH, which results in a negatively charged surface (zeta potential was -18.5 mV) at pH 7.4. MB is linked to disulfide-bridged MSNS via electrostatic interaction (details are described in experimental section).47-50 The characteristic absorbance peaks of MB in the supernatant solution clearly decreased (Figure S3), which was used to calculate the 1.8 mg MB loading in 1.0 g MSNS. The successful loading of MB onto MSNS could also be demonstrated by the positive shift of the zeta potential from -18.5 mV to +39.0 mV and the color changes from white (MSNS) to blue (MB-MSNS) in inset of Figure S3.


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The FA/PtBSA nanoparticles were applied as corresponding biocoats. Folate acid (FA), a widely used recognizer of human female cervical cancer cell lines (HeLa cells),51 was linked on the PtBSA biocoats via amide bond. FA decorated PtBSA nanoparticles (FA/BSA) exhibit the typical absorption peak of FA in UV-vis spectra (~280 nm, Figure 2A). Figure 1B is the high resolution TEM (HR-TEM) images of FA/PtBSA, which shows the lattice planes of Pt (111) and PtO (111). Subsequently, FA/PtBSA integrated with the MB-MSNS core via the formation of an amide bond,52 which is stable in acidic tumor microenvironments. The TEM (Figure 1C) and HR-TEM (Figure 1D) images show that the mesoporous structure of MSNS became obscure after PSs loading, and the lattice planes attributed to PtO (111) could be distinguished on the surface of MSNS, which matched with the HR-TEM of FA/PtBSA (Figure 1B). EDX elemental mapping images (Figures 1E-J) exhibit the presence of Pt, Si, S, O, C, and N elements in the FA/PtBSA@MB-MSNS. X-ray photoelectron spectroscopy (XPS, Figure 2B) analysis confirms the existence of both Pt and PtO in the product. The splitting of the 4f doublet of Pt was 3.35 eV, indicating that metallic Pt was mainly present.53,

54

Furthermore, the

successful formation of FA/PtBSA@MB-MSNS was also demonstrated by Fourier


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transform infrared (FT-IR) spectroscopy (Figure S4). The bands at 1652 cm-1 and 1560 cm-1 are ascribed to the amide I and amide II of BSA, respectively. The Pt loading amount in FA/PtBSA@MB-MSNS is determined as 2.52 mg g-1 by using atomic absorption spectroscopy.

3.2. Generation of O2 and 1O2 in the presence of FA/PtBSA@MB-MSNS

Sufficient O2 supply is a prerequisite for the reduction of hypoxia and the resistance to PDT. It is widely accepted that both Pt and PtO nanoparticles are excellent catalysts for the production of O2 via catalytic decomposition of H2O2.55, 56 To evaluate the catalytic effect of PtBSA, the time-dependent O2 evolution after the addition of PtBSA was monitored by RDPP. The fluorescence intensity of RDPP was demonstrated to be quenched by O2.4 As seen in Figure 3A, FA/PtBSA can produce O2 via catalytic decomposition of H2O2. Noticeably, the amount of O2 generated by FA/PtBSA@MBMSNS exceeded that of FA/PtBSA at the same H2O2 concentrations. This phenomenon could be originated from the aggregation of FA/PtBSA nanoparticles in PBS (Figure S1C and S1D), thus reducing the H2O2 catalytic activity of FA/PtBSA. In addition, the RDPP


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declines in the most at the dose of 20 μg mL-1 (Figure S5A). When the dose of FA/PtBSA@MB- MSNS is further increased (i.e. 50 μg mL-1), the RDPP decline rate slows down, which would come from the faster O2 evolving at a larger Pt dose. In this case, some of the generated O2 cannot react with RDPP before it escape out the solution. Since 1O 2

generation is the key characteristic that assesses PDT efficiency, a sufficient O2

supply will benefit a more effective hypoxia modulation. Therefore, the 1O2 concentration was further evaluated while using FA/PtBSA@MB-MSNS via an established method based on DPBF.57 As a typical 1O2 probe, DPBF can be irreversibly oxidized by 1O2, which results in the decreased absorbance intensity at 411 nm. As illustrated in Figure 3B, the absorbance intensity of DPBF rapidly decreased to 5.50% within 2 min for FA/PtBSA@MB-MSNS group in the presence of and H2O2 under irradiation. This is much faster than the time required by the MB-MSNS group (the absorbance of DPBF decreased to 24.20%). To further confirm the generation of 1O2 by FA/PtBSA@MB-MSNS under irradiation, tryptophan, used as the physical quencher of 1O2, was added to the system (Figure S5).57-60 The characteristic absorbance of DPBF at 411 nm followed a lower decreasing tendency with increasing tryptophan concentration, suggesting that part of the


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1O 2

produced by FA/PtBSA@MB-MSNS was quenched by tryptophan instead of reacting

with DPBF. These results confirmed the mass generation of 1O2 originated from PtBSA triggered O2 production.

To evaluate the PDT efficiency under visible-light irradiation and after nanodrug delivery at biological tissue sites, chicken skin with a thickness of ~1 mm was used to imitate a biological tissue barrier for 1O2 generation (Figure 3C). DPBF in aqueous dispersion of FA/PtBSA@MB-MSNS (125.0 g mL-1) decreased to 72.15% after 120 s (Figure 3D) under irradiation with a 635 nm laser (6 mW cm-2). This result confirmed that FA/PtBSA@MB-MSNS could produce 1O2 under the skin, highlighting its application potential for the killing of cancer cells in tumor treatments. It should also be mentioned in this context that DPBF consumption was found to decrease from 94.50% in the absence of skin (Figure 3B) to 27.85% in the presence of skin (Figure 3D). Most likely, this reduction is originated from the partly absorbed and scattered irradiation light by chicken skin.

3.3. Tumor Microenvironment Stimulated MB Release


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The controllable delivery of PSs represents one of the most valuable characteristics to achieve a satisfying efficiency in the killing of PDT induced cancer cells with low side effects. The intercellular high concentration of GSH of cancer cells can effectively break the disulfide bonds to achieve the MB release.29 To quantify the MB release under overexpressed GSH in tumor microenvironments, the dependence of the MB release kinetics on GSH concentration was studied in both netural (pH 7.4) and acidic (pH 5.5) solutions. For this purpose, the absorbance intensity of MB at 665 nm in PBS solution was measured. As shown in Figure 4A, 80.40% MB was found to be released within 5 min at pH 5.5 and in the presence of 10 mM GSH. The latter conditions are typically used to mimic the microenvironment of tumor tissues.61,

62

However, only 39.50% MB was

determined to be released at physiological pH and in the presence of 10 mM GSH. Moreover, the obtained MB release percentages were 45.10% and 15.60% at pH 5.5 and pH 7.4 and in the presence of a low GSH concentration (1 mM), respectively. More importantly, only 5.28% PtBSA of the total PtBSA loading amount (measured by atomic absorption spectrometry) was found to have detached from the MSNS core during this process. These results demonstrate that the loaded PSs could be selectively delivered at


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the target location while the as-designed biocoats covered nanocarrier remained stable in the tumor microenvironment. In addition, for physical absorbed MB, only 24.23% MB at pH 5.5 and 16.12% MB at pH 7.4 were found to have released, even in the presence of 10 mM GSH after 6 h (Figure 4B).

3.4. Cancer-Cell Killing Performance of FA/PtBSA@MB-MSNS

To evaluate the PDT effect of the induced cancer cell killing ability, HeLa cells were used as model cell line. Fluorescein isothiocyanate (FITC) marked FA/PtBSA@MB-MSNS were employed to investigate the cellular uptake efficiency. Confocal fluorescence images showed clear green fluorescence in the cytoplasm of HeLa cells after 6 h of incubation (Table S1), indicating the successful cellular nanoparticle uptake. The methyl thiazolyl tetrazolium (MTT) assay showed particle dose and power density dependent cytotoxicity toward HeLa cells in response to 635 nm laser irradiation (Figure 5A-B). The optimal PDT operation conditions to study the cancer-cell killing ability were as follows: 125.0 g mL-1 nanoparticles, 635 nm laser irradiation (45 mW cm-2) for 30 min. As seen in Figure 5C, the cell viabilities in the presence of FA/PtBSA@MB-MSNS, PtBSA,


23


FA/PtBSA,

PtBSA@MSNS,

FA/PtBSA@MSNS,

MB,

Page 24 of 60

MSNS,

MB-MSNS,

and

PtBSA@MB-MSNS were studied by MTT assays (the corresponding MTT assays in the absence of cells were plotted in Figure S6). Without light irradiation, almost none of the investigated nanodrugs were toxic to tumor cells. Upon 635 nm laser irradiation, the high cell viabilities observed for PtBSA (87.22%), FA/PtBSA (78.72%), PtBSA@MSNS (85.57%), and FA/PtBSA@MSNS (74.40%) further confirm the low cytotoxicity of these samples. These results clearly indicate that light irradiation is necessary for the activation of PSs to generate cytotoxic ROS.4 This latter finding also suggests that the low cytotoxicity characteristics toward cells could be attributed to the excellent biocompatibility of the BSA scaffold. Remarkably, the cell viability of FA/PtBSA@MBMSNS was found to decrease to 2.10% in the presence of irradiation. The apoptosis of HeLa cells may be attributed to the PDT effects induced by PSs under irradiation instead of the targeted 635 nm laser energy only and/or the cytotoxicity of the FA/PtBSA@MBMSNS structures. However, even under irradiation, the cell viabilities for MB and MBMSNS only reached 50.64% and 40.69%, respectively. These cell viabilities are consistent with the O2 evolution and the 1O2 generation tendency as shown in Figures 3A


24

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and 3B. The apparent difference of PDT effect is caused by the O2 generation effect of PtBSA. After being uptaked by HeLa cells, the MB released from the cores was activated via O2, and 1O2 was subsequently produced in the acidic H2O2 microenvironment under 635 nm laser irradiation. These results indicate that PtBSA biocoats have great potential for the reduction of tumor hypoxia, thus achieving an improved cancer-cell killing effects in the PDT process. In addition, a long-term cytotoxicity test was further conducted to study nanodrug induced toxicity in cells. Figure 5D shows HeLa cells incubated with different nanomaterials or PSs for 6 h and 48 h in the dark. Without light irradiation, MTT assays showed a poor cytotoxic effect even after a longer incubation time (i.e. 48 h). Notably, the cell viabilities are higher for the PSs loaded MSNS in the presence of PtBSA (i.e. FA/PtBSA@MB-MSNS and PtBSA@MB-MSNS) than the viability for PSs loaded MSNS in the absence of PtBSA (i.e. MB-MSNS) after incubation in the absence of light irradiation for 48 h. These results suggest that PtBSA biocats can reduce the cytotoxicity of PSs when they remain in the dark.


25


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Since most of the biomedical applications require anticancer nanodrugs to be delivered via intravenous injection, the hemolysis assay with human red blood cells (RBCs) is typically employed to evaluate the cytotoxic effect of nanodrugs to healthy cells.63,

64

Figure 5E shows the hemolysis percentage for various concentrations of FA/PtBSA@MBMSNS with both positive and negative controls (experimental details presented in Supporting Information). Corresponding sample photos are shown in the inset of Figure 5E. The hemolysis percentage of RBCs was low with only 0.49%, even at high FA/PtBSA@MB-MSNS dose (1000.0 g mL-1); moreover, negligible hemolysis activity was found at other FA/PtBSA@MB-MSNS doses. This latter finding corroborates the safety of the administered as-prepared nanodrugs.

3.5. Selective Recognition of HeLa Cells

Selectivity is a further important criterion to evaluate both PDT effect and safety. To obtain information on the guiding role of FA in the selective recognition of high folate receptors expressed in tumor cells, FITC labeled FA/PtBSA@MB-MSNS were incubated with A549 cells (which is a human lung cancer cell line that expresses low levels of folate


26

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receptors).65 As shown in Figure 5F, the cytotoxicity of FA/PtBSA@MB-MSNS to A549 cells was far lower than that to HeLa cells at identical nanodrug doses under 635 nm laser irradiation. For example, the viabilities of A549 cells and HeLa cells were 69.38% and 3.36% after irradiation for 30 min, respectively. Furthermore, nanocomposites without FA grafting (PtBSA@MB-MSNS) showed a cell viability of 60.29%. As illustrated in Figure S7A-C, weak fluorescence was found in A549 cells after incubation with FA/PtBSA@MBMSNS as well as in HeLa cells treated with PtBSA@MB-MSNS, confirming the guide role of folate in the targeting specificity of FA/PtBSA@MB-MSNS toward high folate receptors expressed in tumor cells. These results suggest specific recognition as an important feature, leading to a high uptake efficiency, and thus achieving satisfying PDT outcomes.

To further evaluate the guiding role of FA in the selective recognition of high folate receptors expressed in tumor cells, the two common dyes 4',6-diamidino-2-phenylindole (DAPI) and actin-tracker green, were used as the nucleus and actin trackers, were separately loaded into MSNS instead of MB. As shown in Table S2, the FA grafted samples (FA/PtBSA@Nucleusdye-MSNS and FA/PtBSA@antintracker-MSNS) were


27


Page 28 of 60

effectively uptaken by HeLa cells and released sufficient dyes at nucleus and actins, respectively.

For

comparison,

less

PtBSA@Nucleusdye-MSNS

and

PtBSA@antintracker-MSNS nanoparticles are uptaken by HeLa cells. These results further confirm the key role of FA grafting to achieve a high uptake efficiency in HeLa cells. High-light and low-dark cytotoxicities are essential requirements for the application of a photosensitizer in PDT. The cytotoxicity of FA/PtBSA@MB-MSNS towards HeLa cells was investigated in both presence and absence of 635 nm laser irradiation (45 mW cm-2) over a period of 30 min (Figure 6). After light irradiation, most HeLa cells can be stained by Annexin V-FITC/PI, a fluorescence dye that is widely used to distinguish viable cells from dead cells. The results suggest obvious apoptotic characteristics of PtBSA@MB-MSNS. Without light irradiation, the fluorescence of Annexin V-FITC/PI was very weak, which indicates that FA/PtBSA@MB-MSNS nanoparticles were basically noncytotoxic. For comparison, the cytotoxicity of MB-MSNS is also shown in Figure 6. Only part of the HeLa cells can be stained by Annexin V-FITC/PI under irradiation, indicating the great potential of PtBSA biocoats for improving the cancer-cell killing effect in the PDT process. Stained cells were not observed in the absence of FA/PtBSA@MB-MSNS and


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MB-MSNS in the laser treatment group. Taken together, these results demonstrate the excellent cytotoxicity characteristics of FA/PtBSA@MB-MSNS under visible light irradiation.

3.6. In Vivo PDT on Subcutaneous Tumor-Bearing Mice.

Encouraged by the performance of FA/PtBSA@MB-MSNS in vitro, the anticancer potential pf PDT in vivo was investigated by xenograft cancer cell (HeLa) tumor models. The nude mice were divided into four groups and injected with 500 L of: (i) saline (control), (ii) free MB, (iii) MB-MSNS, or (iv) FA/PtBSA@MB-MSNS. The tumor sizes were measured every day and the differences were used to estimate the PDT effect. Figure 7A-C shows the PDT results of the four groups. It can be seen that the growth of tumors is slightly hindered by both MB and MB-MSNS treatments. The poor therapeutic results from MB and MB-MSNS can be attributed to the reduced PDT efficiency in a hypoxia enviroment.

However,

the

tumor

growth

was

satisfactorily

inhibited

in

the

FA/PtBSA@MB-MSNS group, suggesting the high efficiency of PDT treatment. In addition, the in vivo toxicity of FA/PtBSA@MB-MSNS was evaluated by monitoring the


29


Page 30 of 60

weight change of mice during the PDT treatment. As shown in Figure 7D, almost no obvious side effects can be found. In order to further estimated the PDT therapeutic effects of each group at the cellular level, hematoxylin and eosin (H&E) staining was employed to investigate both the morphology and apoptosis of the cells of tumor tissue of different groups (collected 24 h post-treatments). As shown in Figure 7E, the cells of the saline group keep their normal morphology, and little cell necrosis is found in MB and MB-MSNS groups. On the contrary, a great number of dead cells with apparent shrinkage of nuclei and membrane destruction can be observed in the FA/PtBSA@MB-MSNS group. The significantly enhanced PDT effect of tumors that were injected with FA/PtBSA@MB-MSNS was caused by the decoration of PtBSA on FA/PtBSA@MBMSNS, which exerted catalase-like activity and supplied O2 via the decomposition of intracellular H2O2. To investigate the hypoxic conditions in the tumor tissues, HIF-1α staining assay was further carried out after 15 days PDT treatment (Figure 7E). As presented in Figure 7E, the tumor tissues from the groups of saline, MB, and MB-MSNS are stained dark-brown. This can be attributed to the accumulation of HIF-1α under the hypoxic conditions, which can lead to the expression of hypoxia-inducible factor HIF-1α.


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In contrast, due to the generation of O2 in tumor tissues by Pt-catalysis oxidation of intracellular H2O2, the group injected with FA/PtBSA@MB-MSNS shows clearly blue staining of tumor tissues, which demonstrates the reduced tumor hypoxia, which is further verified by the blue stained tumor tissue. Such an enhanced oxygen generation in tumors verifies that the PtBSA “biocoats” can decrease the hypoxia-related PDT resistance in

vivo.

4. Conclusion

In summary, a cancer cell-activatable and O2-evolving nanostructure has been developed in an effort to improve PDT efficiency. The outstanding catalytic activity of PtBSA triggered the production of O2 in tumor hypoxia microenvironments by decomposing intracellular H2O2. According to in vitro and in vivo experiments, we demonstrate that the PtBSA shell coated PDT agent has enhanced tumor targeting ability, minimal systematic toxicity, and PDT effect. We anticipate that the FA/PtBSA@MB-MSNS nanoaggregates provide a reliable strategy for designing intelligent nanodrugs to overcome hypoxiainduced PDT resistance.


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ASSOCIATED CONTENT

Supporting Information. The characteristics of FA/PtBSA, MSNS and FA/PtBSA@MBMSNS. The cell in vitro incubation analysis is also disclosed.

Figure S1. TEM of (A) MSNS, (B) FA/PtBSA@MB-MSNS, (C) FA/PtBSA in PBS, and (D) HR-TEM of FA/PtBSA in PBS.

Figure S2. (A) Nitrogen adsorption-desorption isotherms of MSNS. (B) Pore size distribution of MSNS.

Figure S3. UV-vis spectra of 0.01 mg mL-1 MB before and after incubation with 5.0 mg mL-1 MSNS for 2 h. Inset: photos of MSNS before and after MB loading.

Figure S4. FT-IR spectra of FA/PtBSA, MSNS, MB@MSNS, and FA/PtBSA@MB-MSNS.

Figure S5. Consumption of DPBF triggered by FA/PtBSA@MB-MSNS in the presence of 100 µM H2O2 and tryptophan with different concentration under visible-light irradiation.

Figure S6. Absorbance of NPs incubated with MTT for 4 h in dark and under irradiation.


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Figure S7. Confocal fluorescence images of HeLa cells after incubated with FITC marked FA/PtBSA@MB-MSNS (A) with and (B) without FA modification. (C) Confocal fluorescence images of A549 cells after incubation with FITC marked FA/PtBSA@MBMSNS with FA modification. Scale bars: 50 µm.

Table S1 Real-time confocal fluorescence images displaying the uptake of FA/PtBSA@MB-MSNS by HeLa cells.

Table S2. Confocal fluorescence images of HeLa cells incubated with FA/PtBSA@ Nucleusdye-MSNS, FA/PtBSA@Actintracker-MSNS, PtBSA@Nucleusdye-MSNS and PtBSA@Actintracker-MSNS. Scale bar: 50 m.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]

Funding Sources


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This work was supported by the National Natural Science Foundation of China (No. 21775016, 21874013), the Fundamental Research Funds for the Central Universities (N160502001, N170502003, N170908001). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21775016, 21874013), the Fundamental Research Funds for the Central Universities (N160502001, N170502003, N170908001). REFERENCES (1) Lucky, S. S.; Soo, K. C.; Zhang, Y. Nanoparticles in Photodynamic Therapy. Chem.

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For Table of Contents Only


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Scheme

1.

Schematic

illustration

detailing

(A)

the

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synthesis

of

activatable

FA/PtBSA@MB-MSNS nanoparticles and (B) the O2 evolving PDT process.


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Figure 1. (A) TEM of MSNS, (B) HR-TEM of FA/PtBSA, (C) TEM and (D) HR-TEM of FA/PtBSA@MB-MSNS, (E-J) elemental (Pt, Si, S, O, C and N) mappings of FA/PtBSA@MB-MSNS.


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Figure 2. (A) UV-vis spectra of FA, PtBSA and FA labelled PtBSA. (B) XPS spectrum of Pt 4f and PtO of FA/PtBSA@MB-MSNS.


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Figure 3. (A) Consumption of RDPP triggered by FA/PtBSA, MB-MSNS, and FA/PtBSA@MB-MSNS in both presence and absence of 100 µM H2O2. (B) Consumption of DPBF triggered by MB-MSNS and FA/PtBSA@MB-MSNS in both presence and absence of 100 µM H2O2 under visible-light irradiation. (C) Photographs of the 1O2 capture experiment in the presence of chicken skin and (D) the corresponding DPBF consumption curve. A 635 nm laser with a power density of 6 mW cm-2 was used as visible-light source for the DPBF experiments.


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Figure 4. (A) MB release profiles of FA/PtBSA@MB-MSNS in PBS at pH 5.5 or pH 7.4 with GSH (1 mM or 10 mM). (B) MB release profiles of FA/PtBSA@MB-MSNS with different MB binding manners in PBS (pH 5.5 or 7.4) and 10 mM GSH.


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Figure 5. Influence of (A) laser power and (B) irradiation time on the viability of HeLa cells in the presence of FA/PtBSA@MB-MSNS at different doses. (C) Viability of HeLa cells incubated with different groups at the identical dose of 125.0 g mL-1 FA/PtBSA@MSNS or equal to the loading mass (25.0 g mL-1 PtBSA, 25.0 g mL-1 FA/PtBSA, 125.0 g mL-1 PtBSA@MSNS, 125.0 g mL-1 FA/PtBSA@MSNS, 0.18 g mL-1 MB, 100.0 g mL-1 MSNS, 100.0 g mL-1 MB-MSNS, and 125.0 g mL-1 MSNS) for 6 h in the presence and absence of irradiation. (D) Viability of HeLa cells incubated with different groups at the identical dose of 125.0 g mL-1 FA/PtBSA@MB-MSNS or equal to the loading mass for 6 h or 48 h in the absence of irradiation. (E) Percentage of hemolysis of RBCs incubated with FA/PtBSA@MB-MSNS at different concentrations in the absence of light irradiation


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(inset: photographs of RBC hemolysis). (F) Viability of HeLa cells incubated with FA/PtBSA@MB-MSNS and PtBSA@MB-MSNS at a dose of 125.0 g mL-1, viability of A549 cells incubated with FA/PtBSA@MB-MSNS at a dose of 125.0 g mL-1 in both presence and absence of light irradiation. Light source: 635 nm laser at a power density of 45 mW cm-2.


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Figure 6. Confocal fluorescence images of Annexin V-FITC/PI stained HeLa cells incubated with FA/PtBSA@MB-MSNS and MB-MSNS at a concentration of 125.0 g mL-1 for 6 h either under irradiation or in the dark. The control experiment was performed in the absence of PtBSA@MB-MSNS under 635 nm laser irradiation. Scale bars: 20 µm. A 635 nm laser with a power density of 45 mW cm-2 was used as light source.


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Figure 7. (A) Representative photographs of HeLa tumor-bearing nude mice treated with saline (control), free MB, MB-MSNS, or FA/PtBSA@MB-MSNS on the 15th day, (B) Photographs of excised tumors on the 15th day, (C) Relative HeLa tumor volumes curves, and (D) Mice body weight evolution curve. (E) H&E stained and HIF-1 stained tumor


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slices from HeLa tumor-bearing mice treated with saline (control), free MB, MB-MSNS, or FA/PtBSA@MB-MSNS on the 15th day. Scale bar: 100 m.


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Protein Shell-Encapsulated Pt Clusters as Continuous O2-Supplied

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