Photosensitizer Complex

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Biological and Medical Applications of Materials and Interfaces

Lipid-wrapped Upconversion Nanoconstruct /Photosensitizer Complex for Near-Infrared Light-mediated Photodynamic Therapy Pounraj Thanasekaran, Chih-Hang Chu, Sheng-Bo Wang, Kuan-Yu Chen, Hua-De Gao, Mandy Manchi Lee, Shih-Sheng Sun, Jui-Ping Li, Jiun-Yu Chen, Jen-Kun Chen, Yu-Hsu Chang, and Hsien-Ming Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07760 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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ACS Applied Materials & Interfaces

Lipid-wrapped Upconversion Nanoconstruct/Photosensitizer Complex for Near-Infrared Light-mediated Photodynamic Therapy Pounraj Thanasekaran,† Chih-Hang Chu,† Sheng-Bo Wang,§ Kuan-Yu Chen,§ Hua-De Gao,†,‡ Mandy Manchi Lee,† Shih-Sheng Sun,† Jui-Ping Li,# Jiun-Yu Chen,# Jen-Kun Chen#,*, Yu-Hsu Chang§,*, Hsien-Ming Lee†,* †

Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan Department of Materials and Mineral Resources Engineering, Institute of Mineral Resources Engineering, National Taipei University of Technology, Taipei 10608, Taiwan ‡ Department of Chemistry, National Taiwan University, Taipei 106, Taiwan # Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli 350, Taiwan §

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ABSTRACT: Photodynamic therapy (PDT) is a non-invasive medical technology that has been applied in cancer treatment where it is accessible by direct or endoscopeassisted light irradiation. To lower phototoxicity and increase tissue penetration depth of light, great effort has been focused on developing new sensitizers that can utilize red or near-infrared (NIR) light for the past decades. Lanthanide-doped upconversion nanoparticles (UCNPs) have a unique property to transduce NIR excitation light to UVVis emission efficiently. This property allows some low-cost, low-toxicity, visible light commercially available sensitizers, which originally is not suitable for deep tissue PDT, to be activated by NIR light, and has been reported extensively in the past few year. However, some issues still remain in UCNP-assisted PDT platform such as colloidal stability, photosensitizer loading efficiency, and accessibility for targeting ligand installation, despite some advances in this direction. In this study, we designed a facile phospholipids-coated UCNP method to generate a high-colloidally stable nanoplatform that can effectively load a series of visible light sensitizers in the lipid layers. The loading stability and singlet oxygen generation efficiency of these sensitizers loaded lipid-coated UCNP platform were investigated. We also have demonstrated the enhanced cellular uptake efficiency and tumor cell selectivity of this lipid-coated UCNP platform by changing the lipid dopant. On the basis of the evidence of our results, the lipid-complexed UCNP nanoparticles could serve as an effective photosensitizers carrier for NIR light mediated PDT.

KEYWORDS: bioimaging, photodynamic therapy, photosensitizers, phospholipids, upconversion

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INTRODUCTION Photodynamic therapy (PDT) is a minimally invasive chemotherapy that employs photosensitizer (PS), appropriate excitation light, and cellular oxygen to generate reactive oxygen species (ROS) for cellular toxicity.1

ROS can cause oxidative

damages to protein, DNA, lipid, and consequently induce cell apoptosis or necrosis.2,3 PDT provides excellent spatial and temporal precision to treat the irradiated tissue, and hence, it is useful in skin or esophagus cancer where excitation light can be easily applied (with endoscope if needed). Although PDT has been widely used in cancer treatments for the past decades, the inaccessibility to the deep-seated tumors using visible light limits the application.4-6 On the other hand, near-infrared (NIR), though can penetrate deeper in tissues, does not have enough quantum energy to turn on many clinically safe and commercially available visible light sensitizers.7,8 Sensitizer excitation has the need to be red-shifted to NIR region. Although the energy required to sensitize oxygen from triplet to singlet can be provided with up to 1100 nm light in theory, practically, we need a light shorter than 800 nm to have enough energy to overcome rotation and vibration energy loss to excite PSs and sensitize molecular oxygen.9-12 Some new PSs that can be excited by light over 650 nm have been currently under development or on trial.13,14 In the meantime, nano-sized polymer and micelle formulated PS for PDT has also been studied extensively to overcome the hydrophobic property of PS, and to improve the targeting delivery purpose because the nanomedicine formulation

provides

better

pharmacokinetics

and

pharmacodynamics.15-18

Lanthanide-doped upconversion nanoparticle (UCNP) can serve as a nanocarrier for many other light-responsive bioeffectors,19-22 and can be an internal transducer to generate significant anti-Stokes emission upon NIR irradiation to excite the current 3



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photosensitizers, which were low-cost but only visible light-responsive.23-26 This dual advantages make UCNP a suitable carrier for PS. The upconversion phenomenon of lanthanide-doped UCNP is a nonlinear process, in which several low quantum energy photons absorbed by the particle are converted to higher energy (but fewer) photons through multiple sequential photon absorption.27-29 Many research groups have developed a variety of methodologies to produce waterdispersible UCNPs that can load photosensitizers by (i) PS-particle surface direct conjugation, and (ii) PS partition into surface coating methods. The PS to particle surface conjugation method does not have PS leaching problem, but it is very laborious in fabrication. Examples using this method include UCNP@SiO2-PS conjugates,35-37 UCNP@AEP-PS conjugates,38 and UCNP@lipid-PS conjugates.39 On the other hand, PS non-covalently loaded to particle coating using a physical adsorption method is a more facile approach. These works include PS adsorbed in porous silica,40-42 PS adsorbed in amphiphilic polymers,43-47 PS adsorbed in polymer-crosslinked lipid,48-52 and PS adsorbed in PEG53 or PEGylated lipid.54-58 No matter which approach we are using, the following three important factors need to be taken into consideration for the best performance of UCNP-assisted PDT; (i) surface coating of UCNP needs to be biologically compatible and immunologically inert for future clinical use. Polyethylene glycol (PEG) derivative coating is widely used in the interface of nanomedicine. However, increasing number of literatures has reported that healthy individuals (who never exposed to PEGylated therapeutics) produced antibodies against PEG, which can lead to the “accelerated blood clearance” of PEGylated nanomedicine.59-61 These findings could be caused by heavy use of PEG additives in daily cosmetic / body cleaning products, and may have burdened the application of PEGylated therapeutics 4


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and nanomedicine, (ii) PS needs to be spatially and stably confined sufficiently in the biocompatible surface-coating of UCNP without significant leaching for effective excitation to generate enough singlet oxygen. Hydrophobic coating enables stable PS partitioning but induces solubility problem. Switching to highly charged or highly hydrophilic coating material for high colloidal stability will inevitably hurdle the partitioning of hydrophobic aromatic sensitizer. This dilemma is a serious issue and may need amphiphilic coating to overcome, (iii) coating materials for UCNP also need to make particle colloidally stable.27,30-34 Many coating materials such as silica-,62-65 and PLA66-based nanoparticles display good water solubility but show poor colloidal stability in physiological solution. However, their bioapplications have been published despite of this important issue. Chitosan is more colloidally stable in physiological solution when only high molecular weight chitosan is used to form particle.67 Zwitterionic micelles, although they are colloidally stable, normally have higher critical micelle concentration (CMC) and make coating stability a concern.51 Hence, extra chemical transformation is needed to stabilize it. Based on the above mentioned limitations, we expect that using naturally occurring polar lipids to produce high PS loading and colloidally stable PS-UCNP should be a more practical approach. L-α-phosphatidylcholine lipid coating offers the following advantages: (i) phosphatidylcholine is the most abundant polar lipid head group in the vertebrate animal cell membrane system, so it is more biocompatible and immunologically inert compare with PEGylated derivatives and other type of coating materials, (ii) phosphatidylcholine contains a zwitterionic lipid head group, and it is colloidally stable even with very low zeta-potential68 while other type of coating materials normally require much larger magnitude zeta potential (> +30 mV or < -30 5



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mV) to keep particle well-suspended;69,70 the low zeta potential but colloidally stable phosphatidylcholine partitioning,

and

lipid (iii)

coating natural

facilitates

lipid

extract

hydrophobicity-driven such

as

Egg-yolk

sensitizer extracted

phosphatidylcholine has a balanced distribution of fatty chains in terms of chain length and saturation / unsaturation level for an optimized membrane lipid packing, bilayer elasticity, stability and fluidity.71,72 (iv) self-assembled coating such as lipid layer easily allows surface functionalization change simply by adding different lipid dopants (different charges, different targeting motifs) in a modulated fashion, while non-selfassembled type of coating needs to redo synthesis to obtain new surface functionality. Herein, we report a facile method to one-pot formulate zwitterionic lipid-coated, colloidally stable, and monodisperse UCNPs and encapsulate PS to assemble upconversion-assisted NIR PDT construct (Scheme 1). A mixture of Egg PC, cholesterol, and oleic acid-capped UCNPs is dissolved in THF, injected into water at 50 °C, and consequently, is self-assembled on to UCNP surface after nano-emulsion evaporation process. The natural zwitterionic EggPC affords biocompatibility by mimicking the composition and functionality of the cellular membrane, and therefore, it has been widely used for encapsulating and delivering drugs in vivo such as liposomes and micelles.73,74 Phospholipids also have been successively used to provide biofunctionality to various inorganic nanoparticles, such as quantum dots and iron nanoparticles for various applications.74 This method enables not only robust coating of UCNPs, but also provides surface property modularization simply by changing lipid composition, such as charged lipids, or targeting ligands and antigen labelled lipids. The zwitterionic lipid-coated particles exhibit exceptionally high dispersibility in distilled deionized water, phosphate-buffered saline (PBS), and cell culture medium. In 6


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this paper, we present: i) an approach to generate UCNPs@EggPC for stable physiological aqueous dispersion; ii) comprehensive studies of sensitizers loading and partition stability in UCNPs@EggPC system using several important sensitizers; iii) singlet oxygen generation efficiency of the sensitizers-loaded UCNPs@EggPC systems; iv) demonstration of the general and cell-selective uptake of UCNPs@EggPC and the NIR-induced cellular phototoxicity using different lipid formulation; and v) describing the PDT effect by UCNPs@EggPC intratumor injection and NIR irradiation.

Scheme 1. A schematic illustration of PS loading of UCNP@EggPC, and the upconversion light assisted oxygen sensitization.

EXPERIMENTAL SECTION Materials and Chemicals. Rare earth acetates including Yttrium(III) acetate hydrate [Y(CH3CO2)3·xH2O], ytterbium(III) acetate [Yb(CH3COO2)3·4H2O], Erbium acetate (III) [Er(CH3COO)3· xH2O] were of 99% purity, Oleic acid (technical grade, 90%), 1-Octadecene (technical grade, 90%), cholesterol, p-nitroso-dimethylaniline (RNO), imidazole, folic acid (FA), dicyclohexyl-carbodiimide (DCC), Perinaphthenone (PN), Methylene blue (MB), Rose bengal (RB), Zinc phthalocyanine (ZnPC), Aluminum phthalocyanine chloride (AlPC), Propidium Iodide (PI) and mesoTetraphenylporphine

(TPP)

were

purchased

from

Sigma-Aldrich.

L-α7



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phosphatidylcholine (Egg-PC) (95%), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and DSPE-PEG2000-Folate (DSPE-PEG2000 = 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000]) were purchased from Avanti. Dulbecco's Modified Eagle's medium (DMEM), L-15 medium and 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from GIBCO and Merck, respectively. [Re(bpy)(CO)3Br] (bpy = 2,2'-bipyridine) was synthesized based on the reported literature.75 All solvents and reagents used for synthesis, chromatography, UV–vis spectroscopy, and upconversion emission and photolysis studies were purchased from Sigma-Aldrich and used as received, unless otherwise noted.

Characterization. The morphology and size of the prepared UCNPs@EggPC were characterized by JEOL JEM-1400 high-resolution transmission electron microscope (HR-TEM). The hydrodynamic diameter of NPs was measured by a Malvern Zetasizer Nano-ZS. X-ray Diffraction (XRD) was measured utilizing a Rigaku XRD DMAX-2200VK diffractometer. Thermogravimetric analysis (TGA) was obtained by Perkin Elmer TGA 7. UV-Vis absorption spectra were measured by Cary 50 Scan UV-Vis spectrophotometer (Varian). The photoluminescence (PL) spectra were recorded by Eclipse Cary Fluorescence (Varian) with modified 980 nm diode laser attachment (8 W, Lasermate group, Inc.). Upconversion fluorescence images were acquired by optical fluorescence microscope (Olympus IX71) with modified 980 nm diode laser attachment (8 W, Lasermate group, Inc.).76 Cellular Z-axis fluorescence images were acquired by Olympus FV1000 confocal microscope equipped with a 60X, NA = 1.2 water objective and a SIM scanner.

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Synthesis of NaYF4:20%Yb3+/2%Er3+ NPs.

NaYF4:Yb3+/Er3+ particle was

synthesized based on literature reported protocol.77 Briefly, three-necked flask containing 2 millimoles of lanthanide (Y3+, Yb3+, Er3+) acetate salts along with 12 mL of oleic acid and 30 mL of 1-octadecene was heated to 120 °C under vacuum for 1.5 h. After cooling to 50 °C, the reaction flask was placed under nitrogen gas flow. Methanol (20 mL) containing ammonium fluoride (0.2964 g, 8.0 millimoles) and sodium hydroxide (0.2 g, 5.0 millimoles) were slowly added and stirred for 1 h. The reaction was then heated to 80 °C to evaporate methanol from the reaction mixture with nitrogen gas flow. Subsequently, the reaction temperature was increased to 310 °C quickly and maintained at this temperature for 1 h under the nitrogen gas flow. After heating, the reaction mixture was cooled down to room temperature. The mixture was precipitated by the addition of ethanol and collected by centrifugation at 8000 g. The resulting pellet was then dispersed by hexane (10 mL) and the trace amount of oversized particle was removed by centrifugation at 500 g. The suspension was dried by rotary evaporation and stored. Preparation of Phospholipids Coated UCNPs. For the preparation of UCNPs@EggPC by the solvent evaporation method,78 UCNPs (1 mg), lipid (2.68 mg, EggPC, 50% mole ratio) and cholesterol (1.32 mg, 50% mole ratio) were first added into a solution of tetrahydrofuran (THF, 100 μL). The solution was then rapidly added into H2O (2 mL) at 50 °C with vortex for 30 s. The THF solvent was removed from the colloidal suspension by rotary evaporation, and then the residual suspension was dispersed by a probe-type sonicator at 35 W output for 60 s. Lipid-coated particle was harvested by centrifugation at 8000 g for 20 min and washed with water two times. For using DOTAP (1% mol) or DSPE-PEG2000-Folate (1% mol) as a lipid dopant, they were dissolved in THF with EggPC and followed the same procedure above to generate 9



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UCNPs@EggPC-DOTAP or UCNPs@EggPC-FA, respectively. The UCNPs@EggPC centrifuge force was increased to 12000 g to overcome charge repulsion or PEGgrafting caused density decrease. Preparation of Photosensitizers (PSs) Loaded UCNP and Encapsulation Efficiency Studies. PSs loading to lipid layer of UCNPs@EggPC (EggPC only and with DOTAP as a dopant) were carried out by mixing each PSs [PN, MB, RB, TPP, ZnPC, AlPC, and Re(CO)3(bpy)Br] at specified concentrations with UCNPs@EggPC (0.5 mg/mL) in 1 mL of water. The PSs / UCNP@EggPC mixture was stirred at 500 rpm at room temperature for 16 h. PSs-loaded UCNP@EggPC were pelleted by centrifugation at 8000 g for 20 min and PSs-loaded particles were washed with water twice. To estimate the loading efficiency, 0.5 mg PSs-loaded UCNPs@EggPC were lyophilized and re-dissolved in THF, and the concentration of the extracted PSs was quantified by their signature absorption wavelengths and extinction coefficients (PN = 350 nm, 16240 M-1cm-1; MB = 660 nm, 101640 M-1cm-1; RB = 560 nm, 140200 M1

cm-1; TPP = 420 nm, 371200 M-1cm-1; AlPC and ZnPC = 670 nm, 194000 and 223240

M-1cm-1, respectively; and Re(CO)3(bpy)Br = 390 nm, 3238 M-1cm-1). To load the photosensitizer TPP into the UCNPs@EggPC-FA, TPP (the final loading concentration = 6.25 μM) was mixed with UCNPs@EggPC-FA (0.5 mg/mL) in water (1 mL) and the mixture was stirred at 500 rpm for 16 h at room temperature. The unpartitioned TPPs were removed by centrifugation at 12000 g for 20 min and the resulting UCNPs@EggPC-FA-TPP pellet was washed with water twice. Encapsulation efficiency (ee) was calculated by the following equation:

ee% = [amount of PS extracted from extraction step UCNPs@ EggPC / total amount of PS used in partition step] × 100. 10


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Partition Stability Studies of UCNPs@EggPC-PSs. The UCNPs@EggPC-PSs (3 mg) in Tris-HCl (3 mL, 20 mM, pH 7.4) solution was loaded into a 12000-14000 MWCO cellulose ester membrane dialysis bag (Membrane Filtration Products, Inc., USA). The dialysis bag was gently stirred in Tris-HCl buffer (3 L) at 37 °C. A 250 μL UCNPs@EggPC-PSs inside dialysis bag was collected at a standing time of 2, 4, 6, 8, 24, 48, 72, 144, 168 h. The collected samples were then centrifuged at 8000 g for 20 min, and the pellets were freeze-dried. The lyophilized pellets were redissolved in THF, and the PS remained in the particle lipid layer was measured by absorbance, as described above. In vitro Determination of Singlet Oxygen. Singlet oxygen generated by UCNPs@EggPC-PSs was determined by RNO bleaching method.41 Briefly, UCNPs@EggPC-PSs (105 μL, 0.5 mg/mL) was mixed with RNO (1 mM, 3 μL) and imidazole (80 mM, 105 μL) in Tris-HCl buffer (20 mM, pH 7.4) up to 1 mL, and irradiated by a 980 nm laser (5 W/cm2) for different periods of time. The generated singlet oxygen resulted in the bleaching of RNO followed by decreasing the absorbance at 440 nm. Cellular Experiments Cell Culture. HeLa (human cervical cancer), KB (subline of the ubiquitous KERATIN-forming tumor cell line HeLa) and REF52 (rat embryonic fibroblast) cells were cultured in normal Dulbecco’s modified eagle media (DMEM) with a mixture of 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin unless otherwise specified, and maintained at 37 °C in a humidified atmosphere containing 5% CO2.

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Upconversion Imaging for UCNP@EggPCs Cellular Uptake. Since upconversion emission, in general, is dimmer, it is necessary to incubate the cells for a longer period of time to visualize the particle uptakes clearly. For general and folate receptor mediated uptake studies, UCNPs@EggPC, UCNPs@EggPC-DOTAP and UCNP@EggPC-FA were incubated with folate receptor positive HeLa cells. Initially, HeLa cells were cultured on a 35 mm glass dish at a density of 2 × 105 cells and preincubated for 24 h in DMEM medium. Then the DMEM medium was replaced by 1 mL of fresh DMEM medium containing UCNPs@EggPC, UCNPs@EggPC-DOTAP and UCNP@EggPC-FA for 24 h incubation to uptake particle. After that, the cells were washed, and the medium was replaced by L-15 medium for microscopy observation under ambient condition. For the cell-selective uptake studies with particle containing TPP, more folate receptor expressed KB cells were used. UCNPs@EggPC-FA-TPP was incubated with KB, folate receptor blocked KB, and REF52 cells. The KB and REF52 cells were cultured on a 35 mm glass dish at a density of 2 × 105 cells and pre-incubated for 24 h in DMEM medium. The medium was then replaced by folate and FBS deficient DMEM for additional 12 h. For folate receptor blocked experiment, KB cells were incubated with folate-rich (1 mM) medium for additional 1 h. The DMEM medium was replaced by 1 mL of fresh DMEM medium containing 100 μg/mL of UCNPs@EggPC-FA-TPP for incubation on a shaker at 100 rpm for 2 h at 37 °C, and then, the cells were washed three times with the PBS. The medium was replaced by L-15 medium for microscopy observation under ambient condition. The cells were imaged in bright field and upconversion luminescence mode with 980 nm NIR excitation.

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Cytotoxicity Assay. Cytotoxicity of lipids-coated UCNPs was evaluated on HeLa cells by standard MTT assay. In brief, HeLa cells were seeded in a 12 well cell culture dishes at a density of 5 × 104 cells/dish for 24 h incubation. The HeLa cells were then incubated with various concentrations of UCNPs@EggPC, UCNP@EggPC-DOTAP, UCNPs@EggPC-TPP, and UCNsP@EggPC-FA-TPP (0, 10, 50, 100, 200, 1000 μg/mL) for 2 h at 37 °C. The cells were washed with phosphate-buffered saline (PBS) three times to remove the non-internalized NPs and were then incubated in DMEM at 37 °C for additional 16 h for toxicity screening. A 1 mL of MTT reagent (0.5 mg/mL) containing medium was added into each well and incubated for another 4 h to generate purple formazan crystals. After removing the medium, the wells were washed by PBS, and then the intracellular formazan crystals were extracted with 500 μL of dimethyl sulfoxide (DMSO). The absorbance of cell lysate was measured at 540 nm by UV-Vis spectrophotometer. Propidium iodide (PI) staining was used to visualize the upconversion-assisted PDT induced cell death.79 To evaluate the NIR induced PDT effect, HeLa cells were seeded and incubated for 24 h. The medium was then replaced with 1 mL of DMEM medium containing UCNPs@EggPC-DOTAP-RB and UCNP@EggPC-DOTAP-TPP (100 μg/mL) without 10% FBS. After 2 h incubation for particle uptake, the cells were washed three times, incubated in DMEM medium, and were exposed under 980 nm laser irradiation (power density = 5 W/cm2) for 60 min total NIR irradiation dose (six 10 min irradiation with 5 min intervals). The cells were further incubated for 1 h and 16 h before PI staining. For the folate receptor targeting NIR assisted PDT experiment, KB (folate receptor positive) and REF52 (folate receptor negative) cells (2 × 105 cells/dish) were cultured 13



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in DMEM for 24 h. The medium was then replaced by DMEM without folate and FBS for additional 12 h. The UCNPs@EggPC-FA-TPP (100 μg/mL) were gradually added in folate-free DMEM and incubated on a shaker at 100 rpm for 2 h particle uptake at 37 °C, and then, the cells were washed three times with PBS followed by DMEM medium incubation. The cells were exposed under 980 nm laser irradiation (power density = 5 W/cm2) for 60 min total NIR irradiation dose (six 10 min irradiation with 5 min intervals). The cells were further incubated for 16 h before PI staining. In vivo Safety Evaluation. For the preliminary evaluation of in vivo safety of nanoconstructs employed in this work, mice (n = 5) were subcutaneously injected with 50 μL of UCNPs@EggPC-TPP suspension (25 μg/mL). Plasma was separated from blood collected from facial vein at one day before and 4 days after administration of nanoconstructs. Plasma samples were spotted onto slides of Dri-Chem (Fujifilm, Kanagawa, Japan), including glutamate oxaloacetate transaminase (GOT, for liver function), glutamate pyruvate transaminase (GPT, for liver function), lactate dehydrogenase (LDH, for cytotoxicity) and creatine phosphokinase (CPK, for kidney function) assays, then were quantified by a biochemical analyzer (Fujifilm Dri-Chem 4000i; Fujifilm, Kanagawa, Japan) according to the manufacturer’s instructions. Inflammation markers, monocyte chemotactic protein-1 (MCP-1) and chemokine ligand-1 (CXCL-1) in plasma, were determined by commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. In vivo PDT Experiment. Female Bala/c Nude mice were purchased from BioLASCO (Taipei, Taiwan) and acclimated for 2 weeks in the animal facilities at National Health Research Institutes (NHRI), Taiwan. All animal treatments and experimental protocols for this study were reviewed and approved by Institutional 14


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Animal Care and Use Committee at NHRI. CT-26wt tumor cells were inoculated by subcutaneous injection of 5 × 106 cells in 100 μL of serum-free medium onto the back of each nude mouse. The mice were treated when the tumor volumes approached 50 mm3. For the PDT treatment, the CT-26wt tumors-bearing mice were divided into 5 groups (n = 5 per group) and were intratumorally injected with 50 μL saline, UCNPs@EggPC-DOTAP,

UCNPs@EggPC-TPP

and

UCNPs@EggPC-FA-TPP

suspension where particle concentration was 25 µg/mL. A 980 nm laser was used to irradiate tumors at a power density of 1 W/cm2 for 30 min (1 min interval after each minute of irradiation). The tumor sizes were measured by a caliper every the other day and calculated as the volume ＝ (tumor length) × (tumor width)2/2. Relative tumor volumes were calculated as V/V0 where V0 was the tumor volume when the treatment was initiated.

RESULTS AND DISCUSSION Synthesis and Characterization of UCNPs and UCNPs@EggPC: A highly and luminescently efficient, oleic acid-capped UCNP (NaYF4:20%Yb3+/2%Er3+) was synthesized via the thermal decomposition process as described.77 Powder X-ray diffraction pattern confirmed that the UCNPs were well-defined and agreed with the standard pattern of hexagonal NaYF4 phase (JCPDS No. 16-0334) (Figure S1). After the above oleic acid-capped UCNPs were coated with zwitterionic phospholipids (1:1 molar ratio of Egg PC and cholesterol), excellent aqueous-dispersible UCNPs@EggPC was generated. The UCNPs@EggPC remained colloidally stable and well-dispersed, without size change during the observation period of 7 days (Figure S2). Fourier 15



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transform infrared (FTIR) spectra (Figure S3) showed new peaks at 1250, 1100 and 900 cm-1 for UCNPs-EggPC compared with UCNP-oleic acid, indicating the successful coating of EggPC onto the surface of UCNPs.80 TEM image of conventional UCNP (Figure 1a) and UCNP@ EggPC (Figure 1b) showed the dispersed and uniform size UCNPs with an average diameter of 27 ± 3 and 40 ± 2 nm, respectively. Magnified TEM image (Figure 1b, insert) confirmed that the lipid shell of UCNPs@EggPC was in uniform size and approximately 5 nm in thickness. Dynamic light scattering (DLS) measurement indicated that UCNPs@EggPC in PBS had a mean hydrodynamic diameter of approximately 195 nm (Table S1). In comparison with the size of UCNPs@EggPC under TEM which is around 40 nm (Figure 1b), the size discrepancy was probably due to the slight, but limit, inter-particle aggregation, which can also be observed in Figure 1b. Thermogravimetric analysis (TGA) curves showed a significant weight loss (～40 %) of the UCNPs@EggPC compared with the original oleic acidcapped UCNPs (～9 %) at a temperature ranging from 200 to 500 °C (Figure S4). The weight loss difference between UCNP and UCNPs@EggPC was attributed to the successful lipid coating, suggested that the phospholipid (EggPC) and cholesterol have self-assembled on to the particle surface.

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Figure 1. TEM images of UCNPs (a) NaYF4:Yb3+/ Er3+ with oleic acid coating (suspended in THF), and (b) NaYF4:Yb3+/ Er3+@EggPC using cholesterol (suspended in water). There are some particle aggregation, which might cause the larger diameter estimation in DLS.

Sensitizer Encapsulation Efficiency and Releasing Rate of PSs in UCNPs@EggPC: The zwitterionic lipid head group drastically improve the colloidal stability of particle in physiological solution, and the hydrocarbon fatty region of lipid is also a good carrier for photosensitizers. The loading of PS is mainly driven by the hydrophobic interaction between photosensitizer and the lipid hydrocarbon layer. To screen the photosensitizers that can be efficiently partitioned into phospholipid layer in UCNPs@ EggPC, several representative photosensitizers including rose bengal (RB), tetraphenylporphyrin (TPP), zinc phthalocyanine (ZnPC), aluminium phthalocyanine chloride (AlPC), fac-(2,2’-bipyridine)tricarbonylbromorhenium(I) (Re(bpy)(CO)3Br), perinaphthenone (PN) and methylene blue (MB) were examined to quantify their loading efficiency. Figure S5 shows the different colors and transparent solution of PSs loaded with UCNPs@EggPC in 20 mM Tris-HCl buffer (pH 7.4) under the ambient light and 980 nm laser excitation. Based on the logP value, we divided the 17



Page 18 of 42

photosensitizers into “hydrophobic” and “less hydrophobic” categories. As shown in Figure 2a, the loaded amount of PSs in lipid coating increased with increasing the concentration of PSs in loading solution, and attained saturation when the concentration of hydrophobic PSs such as TPP/ZnPc/AlPC/RB in loading solution reached 50 µM. The loading plateau denotes the loaded amount of PSs was close to saturation, and excess PSs might be difficult to be entrapped further into the UCNPs@EggPC. For the less hydrophobic PSs such as Re(bpy)(CO)3Br/PN/MB, loading saturation in lipid coating cannot be reached even the PSs concentration of loading solution was up to 300 µM (Figure 2b). To the best of our knowledge, no systematic studies have been performed till date that explores suitable the class of sensitizers for this kind of loading approach. Here, we systematically studied UCNP lipid coating partition capability against an array of photosensitizer to obtain a partition equilibrium of PS in solution phase and in lipid coating as shown in Figure 2a and 2b. The partition screening of the seven representative sensitizers (with the given calculated hydrophobicity) has concluded that the loading capacity of hydrophobic porphyrin-based PSs is higher compared to less hydrophobic PSs in lipid coating of UCNP. TEM pictures of PS loaded UCNP@EggPC showing in Figure S6 indicated no significant morphological change compared with UCNP@EggPC. To make sure that the sensitizers can be confined in the lipid layer to be excited by the upconverted light, the release (departition) kinetics of the loaded sensitizers were determined. Not surprisingly, when UCNP@EggPC-PS suspended in dialysis environment, all the PSs diffusion obeys the Fick’s law and has two stage patterns showing an early burst-release stage and a later slow sustain-release stage. Hydrophobic sensitizers exhibited a slower releasing kinetics while less hydrophobic sensitizers had 18


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a fast early burst-release rate in the first 20 h (Figure S7). In particular, the release of photosensitizers (TPP, ZnPC, AlPC) was minimized via hydrophobic interaction between PS and lipid layer, and more than 50% PSs can be retained in the lipid layer over 72 hours (RB, although is a hydrophobic sensitizer, has an unexpected fast release rate). Compared to other PSs, the partitioned hydrophobic non-metallated porphyrin derivative TPP can be retained in the lipid layer for a long time to effectively produce singlet oxygen, thereby it was selected for the following cell experiments. Other PSs rapidly leached from the surface of lipid coating, generating insufficient singlet oxygen and were excluded for the cellular studies (Figure S9 and S10).

(A)

30

Apparent conc. of hydrophobic PSs loaded on lipid layers, µM

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UCNPs@EggPC-TPP UCNPs@EggPC-ZnPC UCNPs@EggPC-AlPC UCNPs@EggPC-RB

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Apparent conc. of less hydrophobic PSs loaded on lipid layers, µM


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UCNPs@EggPC-PN UCNPs@EggPC-MB UCNPs@EggPC-Re(CO)3(bpy)Br

30

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Figure 2. Plot of the apparent concentration of (A) hydrophobic PSs and (B) less hydrophobic PSs loaded on lipid layers in solution vs. different loading concentration of PSs in aqueous phase. Experiments were triplicated. For TPP-loaded UCNP@EggPC, the PS release profile indicated that the leaching was only 10% over 7 days, as shown in Figure S7. This result implied that TPP can be stably confined in the EggPC-coated UCNP in physiological solution. Hydrophilic polymer coating such as chitosan had less stable confinement. For example, in chitosan coating, TPP-like sensitizer (Ce6) leached over 65% within 6 h, and had to be conjugated to chitosan layer to slow down the PS leaching rate (less than 10% over 24 h).81 For Lyso lipid coating, due to its high critical micellar concentration (CMC), the coating itself needs to be crosslinked to maintain the coating stability. It also cannot stably confine TPP-like PS (ZnPc) and leached over 50% within 6 h.51 Mesoporous silica coating is somehow quite interesting, it can stably encapsulate both hydrophilic methylene blue40 and hydrophobic ZnPc41 without noticeable leaching.

20


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Singlet Oxygen Production of PS-loaded UCNPs@EggPC:

Generation of

cytotoxic singlet oxygen (1O2) is the core of PDT because singlet oxygen can cause DNA, lysosomes, mitochondria or membrane damaged, resulting in cell death.82 In this UCNPs@EggPC-PSs complex system, oxygen sensitization efficiency is determined not only by sensitizer concentration and particle-sensitizer proximity on the UCNP membrane, but also the spectral overlap and quantum efficiency of PS. As evidenced in Figure S8, the absorption spectra of the chosen PSs overlapped very well with the upconversion luminescence bands of UCNPs@EggPC, which ensured that PSs could be effectively excited by the emitted upconverted light from UCNPs@ EggPC, and produce singlet oxygen upon NIR irradiation. Hence, we quantified the singlet oxygen production of each UCNPs@EggPC-PSs complex after 7 days dialysis to remove the unbound and loosely associated sensitizers on the lipid layer. The yield of 1O2 at dialysis-equilibrated UCNPs@EggPC-PSs under the 980 nm laser irradiation is shown in Figure 3. The production of 1O2 from UCNPs@EggPC-TPP, UCNPs@EggPC-ZnPC and UCNPs@EggPC-RB was more effective than others within the same irradiation time. A probable explanation for the high production of 1O2 is that these PSs might obtain more energy from the UCNP to produce more singlet oxygen because of not only the larger spectral overlap between these PSs and the UCNP but also the higher PSs loading amounts. It is worth mentioning that although AlPC had a higher loading and less release than RB in buffered solution, the overall apparent singlet oxygen generation efficiency of UCNPs@EggPC-AlPC was actually lower than that of UCNPs@EggPC-RB even though both PSs had a similar spectral overlap with UCNP. This could be due to RB has better quantum yield in generating singlet oxygen.83 Singlet oxygen generation of various loading concentration of PSs in UCNPs@EggPC under the 980 nm laser irradiation for 20 min is shown in Figure S9 and S10. No singlet 21



oxygen was generated in negative control experiments where UCNPs@EggPC-PSs was not irradiated by 980 nm light and UCNPs@EggPC without loading PSs or PS without UCNP. It indicates the importance of cooperation between UCNPs and PSs to produce singlet oxygen under NIR light. Among these seven UCNPs@EggPC-PSs, we chose UCNPs@EggPC-TPP and UCNPs@EggPC-RB for further cell experiments because of their high 1O2 generation and distinctive yet representative structural difference.

0.20

Absorbance difference in RNO (∆A440 nm)

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UCNP@EggPC UCNP@EggPC-PN UCNP@EggPC-MB UCNP@EggPC-RB UCNP@EggPC-Re(CO)3(bpy)Br UCNP@EggPC-TPP UCNP@EggPC-ZnPC UCNP@EggPC-AlPC

0.16 0.12 0.08 0.04 0.00

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Figure 3. Production of singlet oxygen from UCNPs@EggPC-PSs after dialysis for seven days. The particle suspensions were irradiated by a 980 nm laser (5 W/cm2) for different periods of time. Experiments were reproduced 3 times.

Cellular Uptake Studies: Singlet oxygen has to be generated intracellularly for cytotoxicity, hence the first investigation is to understand the relationship between cellular uptake and lipid composition of the PSs delivering vesicle. To study the singlet oxygen production in cells, the zwitterionic EggPC coated particle (UCNPs@EggPC), and cationic lipid DOTAP doped UCNPs@EggPC-DOTAP were incubated with HeLa 22


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cells to monitor the uptake. To enhance the PDT tumor cells selectivity and to reduce the damage to normal cells, a targeting lipid, DSPE-PEG2000-folate was also used to formulate UCNPs (UCNPs@EggPC-FA) and tested its selective uptake by HeLa cells. The particle internalization was observed directly using upconversion compatible fluorescence microscopy. After incubation of UCNPs@EggPC and UCNPs@EggPCDOTAP nanocomposites with HeLa cells for 24 h, the cells were excited by 980 nm light and observed the upconverted 460 nm emission from the internalized particles. To understand the effect of positively charged lipid formulation, bright-field (Figure 4a,c) and upconversion luminescence images (Figure 4b,d) of HeLa cells treated with lipidcoated UCNPs were acquired. Figure 4b, d clearly shows that the uptake of UCNPs@EggPC-DOTAP was more efficient than that of UCNPs@EggPC, indicating that the electrostatic interaction between positively charged DOTAP and negatively charged polysaccharide (such as sialic acid) coated cell membrane enhanced the nanocarrier uptake by the cells. The HeLa cells were also incubated with UCNPs@EggPC-FA to evaluate the uptake. The UCNPs@EggPC-FA were able to enter into folate receptor-positive HeLa cells possibly through endocytosis, and a strong upconversion emission was observed as shown in Figure 4e,f. The cytotoxicity of UCNPs@EggPC,

UCNPs@EggPC-DOTAP,

UCNPs@EggPC-TPP

and

UCNsP@EggPC-FA-TPP was also examined as shown in Figure S11, and all showed very high cellular viability even up to 1000 μg/mL in particle concentration. To ensure that the UCNPs@EggPC-FA is internalized instead of just binding on the cell surface receptor, UCNPs@EggPC-FA-TPP were prepared, incubated with HeLa cells, and examined the location of UCNPs@EggPC-FA-TPP in HeLa cells along the z-axis by confocal microscopy. A strong TPP fluorescence of UCNPs@EggPC-FA23



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TPP from the focal plane of cell bottom can be seen in upward z-direction, indicating that UCNPs were uptake and located in cytoplasm (Figure S12).

Figure 4. Bright field and upconverted emission images of Hela cells, showing the uptake of UCNPs@EggPC (a, b), UCNPs@EggPC-DOTAP (c, d) and UCNPs@EggPC-FA (e, f). The red line rectangle shows the NIR irradiation area by a 980 nm laser. The concentration of UCNPs@EggPC, UCNPs@EggPC-DOTAP and UCNPs@EggPC-FA was 100 μg/mL. Scale bars: 50 μm. PDT of UCNP@EggPC-PSs in Cells: We chose UCNPs@EggPC-DOTAP as a sensitizer carrier for its good cellular uptake without cell selectivity. NIR induced photocytotoxicity

of

RB,

TPP

partitioned

UCNPs@EggPC-DOTAP

and

UCNP@EggPC-DOTAP without PSs were examined. For this study, HeLa cells were first incubated with 100 μg/mL of above UCNPs separately at 37 °C for 2 h in the dark for particle uptake, and then exposed to NIR irradiation for 60 min (power density = 5 W/cm2, six 10 min irradiation with 5 min intervals), followed by propidium iodide (PI) staining to quantify the cell death. Figure 5 represents the bright field and PI staining images of HeLa cells treated UCNPs@EggPC-DOTAP, UCNPs@EggPC-DOTAP-RB and UCNPs@EggPC-DOTAP-TPP before and after 980 nm laser excitation. Before NIR irradiation, no significant cell death of all HeLa cells was observed after incubated 24


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with the above UCNPs. For NIR irradiated cells containing UCNPs@EggPC-DOTAPRB, after 1 h and 16 h incubation, a 19.2 and 33.1% of cell death was observed. For cells uptaking UCNPs@EggPC-DOTAP-TPP, after 1 h and 16 h incubation, the percentage of cell death was nearly 78.5 and 100%, respectively, as shown by PIassociated red fluorescence. The reason why UCNPs@EggPC-DOTAP-RB treated cells showed a less effective upconversion assisted PDT is probably due to the fact that partitioned RB escaped fast into the medium and became insufficient to cause the rapid cell death under NIR irradiation. In combined with the control experiment, after NIR irradiation of the UCNPs@EggPC-DOTAP without the PS for 1 h and even after 16 h, cells did not show any noticeable death, indicating that the upconverted emission and particle itself has no phototoxicity, and the origin of phototoxicity comes from the upconversion assisted oxygen sensitization. (A)

25



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Cell viability (%)

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Figure 5. (A) PDT effect of the UCNPs@EggPC-DOTAP, UCNPs@EggPC-DOTAPRB and UCNPs@EggPC-DOTAP-TPP. Damaged/dead cells after upconversionassisted PDT are stained by PI, showing the red fluorescence. Scale bars: 50 μm, and (B) Bar diagram showing the cell viability of HeLa cells after treated with UCNPs@EggPC-DOTAP, UCNPs@EggPC-DOTAP-RB and UCNPs@EggPCDOTAP-TPP nanocomposites after 980 nm NIR irradiation followed by incubation for 1 h and 16 h. For UCNPs@EggPC-DOTAP, UCNPs@EggPC-DOTAP-RB and UCNPs@EggPC-DOTAP-TPP, the stained cells / total cells counting was 2/180, 36/187, 178/227 and 4/180, 62/187, 227/227 for 1 and 16 h, respectively.

Cell–selective PDT using UCNPs@EggPC-FA-TPP: Based on the previous experiment, TPP is a more effective photosensitizer in UCNP@EggPC-DOTAP system. In this study, we synthesized UCNPs@EggPC-FA-TPP to examine the cell-selective uptake and PDT efficiency. The loading efficiency of TPP in UCNPs@EggPC, UCNP@EggPC-DOTAP and UCNPs@EggPC-FA is measured, and the results are shown in Figure S13. The facile addition of lipid dopants (DOTAP and FA-DSPE), which contained only 1% in the lipid composition caused no significant difference in TPP loading, but having significant impact on cellular biodistribution. To assess the targeting ability, UCNPs@EggPC-FA-TPP were separately incubated with KB cells (FR-positive) and REF52 cells (FR-negative) for the subsequent upconversion emission imaging. Upconverted emission images showing significant UCNPs@EggPC-FA-TPP uptake by folate-receptor assisted endocytosis84 after 2 h incubation was observed (Figure 6a,b).

27



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Figure 6. Bright field and upconverted emission images of the UCNPs@EggPC-FATPP (6.25 μM, 100 μg/mL) uptake by two cells (a, b) KB cells, (c, d) FR receptor blocked KB cells and (e, f) REF52 cells. The red line rectangle shows the NIR irradiation area by a 980 nm laser. Scale bars: 50 μm. However, the intracellular uptake of UCNPs@EggPC-FA-TPP by FR-negative REF52 cells was inefficient (Figure 6 b, f). Another negative control experiment was also carried out with KB cells in which excessive folic acid (100 times of FA, 1mM) was added in the medium to block the FR receptor of KB cells before incubating with UCNPs@EggPC-FA-TPP (Figure 6c,d). Notably, few UCNPs were uptake in receptor blocked KB cells and FR-negative REF52 cells negative control experiments, which might be due to the non-receptor-mediated cellular uptake under high concentration of nanoparticles84 compared with FR-positive unblocked KB cells. Next, we examined the cell-selective PDT treatment. KB and REF52 cells were separately treated with 100 µg/mL of UCNPs@EggPC-FA-TPP and washed three times before imaging and NIR irradiation. Cells were exposed to 980 nm laser at 5 W/cm2 for 1 h (six 10 min irradiation with 5 min intervals) followed by 1 h incubation, the cell viabilities were determined from PI staining, and the result is shown in Figure 7. After the exposure to the NIR irradiation, UCNPs@EggPC-FA-TPP treated KB cells were 28


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effectively damaged (Figure 7 a,b). However, there was no significant PI red fluorescence observed in the negative control (REF52 cells) under the same irradiation condition, which indicates ineffective PDT due to its inefficiency of uptaking the UCNPs@EggPC-FA-TPP (Figure 7c,d). This result is consistent with cell-selective particle uptaking results that the UCNPs@EggPC-FA-TPP nanocomposites were hardly able to enter into REF52 cells (Figure 6e,f). It is also confirmed that the modulate installation of targeting lipid on the surface greatly enhanced the cell-selectivity of PDT.

Figure 7. Cell selectivity of UCNP@EggPC-FA-TPP. (A) Merged image of bright field and PI stain of KB cells (a) before and (b) after NIR PDT treatment. The over-expressed folate receptor on KB cells facilitates the particle uptake, thereby resulting in higher cell death rate. On the other hand, REF52 cells have very minimal folate-receptor expressed, and therefore, no significant cell death rate in (c) before and (d) after NIR PDT treatment. The cell death rate (stained cells / total cells count) for KB and REF52 cells is 414/446 and 0/81, respectively. Scale bars: 50 μm.

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In vivo PDT Studies: The UCNP complex was delivered through intratumoral injection instead of intravenous injection to bypass the pharmacokinetic and biodistribution issues. Particle safety experiment in cellular level (Figure S11) and in vivo level (Figure S14) showed no significant toxicity. To further study the potential of UCNPs@EggPC-TPP and UCNPs@EggPC-FA-TPP for in vivo photodynamic therapy, mice bearing CT-26wt tumors were intratumorally (i.t.) injected with NPs (50 μL, 25µg/mL) and were exposed to NIR light (980 nm, 1 W/cm2) for 30 min (1 min interval after each minute of irradiation). The laser power density for effective penetration depth was tuned based on the reported literature.85 The tumor size of the treated mice was measured up to 10 days. As shown in Figure 8, the volume of UCNPs@EggPC-TPP and UCNPs@EggPC-FA-TPP injected tumor grew much more slowly than those of the mice group treated with saline, UCNPs@EggPC-TPP without NIR irradiation, and UCNPs@EggPC-DOTAP (without sensitizer) with NIR light irradiation. It was observed that the difference in tumor accumulation of non-folated UCNPs@EggPCTPP and folated UCNPs@EggPC-FA-TPP in FR overexpressed tumor was negligible, because those particles were given intratumorly. These data suggested that the efficiency of singlet oxygen production by NIR-induced PDT using UCNPs@EggPCTPP and UCNPs@EggPC-FA-TPP nanocomposites offered improved tissue penetration depth, highlighting the potential of these nanocomposites in PDT application.

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25

Normalized tumor volume

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20

Saline UCNPs@EggPC-TPP UCNPs@EggPC-DOTAP+980 nm laser UCNPs@EggPC-TPP+980 nm laser UCNPs@EggPC-FA-TPP+980 nm laser

15

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10

5

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____ * _ __

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___

___

0 Day 0

Day 2

Day 4

Day 6

Day 8

Day 10

Figure 8. Photodynamic therapeutic efficacy of UCNP@EggPC-TPP and UCNP@ EggPC-FA-TPP in CT-26wt tumor-bearing mice. Histograms showing the growth of CT-26wt tumors on different groups of mice measured after various treatments. The relative tumor volumes were normalized to their initial sizes (N = 5). *p < 0.05. Error bars represent standard deviation.

CONCLUSIONS In summary, self-assembling natural phospholipid to prepare PS-containing UCNP for PDT application using upconverted light has been demonstrated. The usage of natural phospholipids for UCNP coating has several significant advantages, including nonimmunogenicity, excellent colloidally stability without using high zeta potential materials, and easy surface functionalization without developing new synthetic routes. The partition study of photosensitizers-to-lipid coating generated a partition equilibrium chart between PS-in-solution phase and PS-in-lipid layer, which is an important reference information for researchers who are interested in PS structure and partition efficiency relationship. Positively charged lipid (DOTAP) doped coating 31



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showed dramatically enhancement in general cell uptake while folated lipid-doped coating was significantly enhanced the cell-specific uptake via the folate receptormediated endocytosis. When partitioned in UCNP@EggPC, non-metallated porphyrin derivative TPP was identified to be the most effective PS by singlet oxygen generation assay under 980 nm laser irradiation and was chosen for the cellular and in vivo experiments. In vitro studies indicated that UCNPs@EggPC-TPP can effectively kill cancer cells while UCNPs@EggPC-FA-TPP had potent cell-selective PDT effect by NIR irradiation. In vivo experiment also has shown that the NIR can effectively drive PDT in deep tissue. The present study promises phospholipid coating a good approach for further exploration in multifunctional imaging and photodynamic cancer therapy applications. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxxxx Figures, and table as discussed in text (PDF). AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; Fax: +886-37-586447. *E-mail: [email protected]; Fax: +886-2-27317185 *E-mail: [email protected]; Fax: +886-2-27831237

ORCID Pounraj Thanasekaran: 0000-0001-6437-7366 Kuan-Yu Chen: 0000-0001-9841-9317 32


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Hua-De Gao:

0000-0002-2617-168X

Jen-Kun Chen: 0000-0002-3433-5971 Yu-Hsu Chang: 0000-0003-2813-6383 Hsien-Ming Lee: 0000-0003-2250-8385 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We would like to thank the Biophysics Core Facility, Institute of Biochemistry, Academia Sinica and the Bioimaging Core Facility, Agricultural Biotechnology Research Center, Academia Sinica for instrumental support. We would also like to thank the Nano Science and Technology Program of Academia Sinica (2393-106-0100), and the Ministry of Science and Technology of Taiwan for the funding supports (1012113-M-001-001-MY2; 103-2113-M-001-028-MY2).

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Table of content

Lipid-wrapped Upconversion Nanoconstruct/Photosensitizer Complex for Near-Infrared Light-mediated Photodynamic Therapy Pounraj Thanasekaran, Chih-Hang Chu, Sheng-Bo Wang, Kuan-Yu Chen, Hua-De Gao, Mandy Manchi Lee, Shih-Sheng Sun, Jui-Ping Li, Jiun-Yu Chen, Jen-Kun Chen*, YuHsu Chang*, Hsien-Ming Lee*

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Photosensitizer Complex

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