Colloids and Surfaces B: Biointerfaces 116 (2014) 284–294

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Intracellular delivery and activation of the genetically encoded photosensitizer Killer Red by quantum dots encapsulated in polymeric micelles Muthunarayanan Muthiah a , Seung-Hwan Park b , Md Nurunnabi c , Jooyoung Lee c , Yong-kyu Lee c , Hansoo Park d , Byeong-Il Lee e , Jung-Joon Min b , In-Kyu Park a,∗ a

Department of Biomedical Science and BK21 PLUS Center for Creative Biomedical Scientists, Chonnam National University Medical School, Gwangju 501-746, South Korea b Department of Nuclear medicine, Chonnam National University Medical School, Gwangju, Republic of Korea c Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungbuk 380-702, South Korea d School of Integrative Engineering, Chung-Ang University, Dongjak-gu, Seoul 156-756, South Korea e Medical Photonics Research Center, Korea Photonics Research Center, Buk-go, Gwangju 507-779, South Korea

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Article history: Received 25 August 2013 Received in revised form 26 December 2013 Accepted 1 January 2014 Available online 17 January 2014 Keywords: Quantum dot Polymeric micelles Killer Red Breast cancer Intracellular activation

a b s t r a c t We have prepared polymeric micelle-encapsulating quantum dots (QDots) for delivering the optically activatable protein Killer Red (KR) as a plasmid to cancer cells. QDots absorb light at a lower wavelength and emit light at a higher wavelength in the cell cytoplasm, activating the expressed KR. Once activated, KR triggers the generation of reactive oxygen species (ROS). We prepared cadmium selenide (CdSe)/zinc sulphide (ZnS) QDots and evaluated their optical properties. Subsequently, we performed morphology studies, elemental analysis, thermogravimetric analysis (TGA), and measurements of particle size and surface charge of prepared QDots encapsulated in PHEA-g-PEG-bPEI (PPP-QDot). Cellular uptake of PPP-QDot and PPP-QDot/KR nanoparticles was confirmed using confocal microscopy, and the cellular toxicity and transfection efficiency associated with uptake of PPP-QDot/KR nanoparticles were analyzed. KR expression in normal cells and cancer cells was confirmed using confocal microscopy and Western blotting. Cellular morphologies before and after intracellular activation of KR were observed using phase contrast, fluorescence, and confocal microscopy. Cell fate after exposure to blue light-emitting diode lighting was determined using apoptosis staining and a cell proliferation assay, confirming a suppression in proliferation and a reduction in metabolic activity. We determined that ROS generation contributed to cellular damage after treatment with PPP-QDot/KR nanoparticles and blue light exposure. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Polymeric micelles have various advantages over other carriers for biomedical applications [1]. The core of a polymeric micelle has a hydrophobic nature that is compatible with hydrophobic substances, such as hydrophobic drugs, superparamagnetic iron oxide nanoparticles, and quantum dots (QDots). The surface corona of a polymeric micelle is hydrophilic as a result of conjugating with commonly utilized polymers such as polyethylene glycol (PEG) [2]. The hydrophilic surface of a polymeric micelle naturally gives a surface-smoothing effect, reducing its interaction with serum proteins, enhancing bio-compatibility, and lengthening the blood circulation time when administered in vivo [3,4].

∗ Corresponding author at: Department of Biomedical Science, Chonnam National University Medical School, 5, Hak-1-dong, Gwangju 501-746, South Korea. Tel.: +82 61 379 8481; fax: +82 61 379 8455. E-mail address: [email protected] (I.-K. Park). 0927-7765/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2014.01.001

When the surface of a polymeric micelle is designed with cationic polymers such as polyethylenimine (PEI) along with PEG, the polymeric micelle will acquire a positive charge from the protonated amine groups of PEI [5,6]. PEI complexes well with negatively charged plasmid DNA, which can be delivered efficiently inside cells and discharged in the cytoplasm as a result of the proton sponge effect. The use of QDots for cell tracking is an established practice [7]. QDots can absorb and emit light even when they are inside cells [8,9]. Compared to dyes, which can quickly degrade, QDots are more stable and persistent and avoid the quenching effect [10]. The delivery of a therapeutic gene that can be specifically activated by an external light source has many advantages for normal cells, because they are less likely to be disturbed when the external light absorbent polymer with QDot does not reach them. Killer Red (KR) is a genetically encoded photosensitizer that can be selectively activated by green light. Its activation results in the generation of reactive oxygen species (ROS) that observably damage the cell and trigger cell death [11–13]. KR is capable of

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(TGA) analysis, and measured the particle size and surface charge of the prepared PPP-QDots. Uptake of the PPP-QDot and PPP-QDot/plasmid nanoparticles was confirmed using confocal microscopy. The cellular toxicity and transfection efficiency following uptake of PPP-QDot/plasmid nanoparticles were analyzed. KR expression in normal cells and in cancer cells after PPP-QDot/KR nanoparticles treatment was confirmed using confocal microscopy and Western blotting. Cellular morphologies before and after blue light exposure were observed using phase contrast, fluorescence, and confocal microscopy. Cell fate after blue light exposure was tested using a fluorescent apoptosis marker and a proliferation assay. We confirmed that ROS generation was responsible for the cellular damage in MCF-7 breast adenocarcinoma cells treated with PPP-QDot/KR nanoparticles and irradiated with blue light. 2. Materials and methods 2.1. Materials

Scheme 1. Preparation of PPP-QDots and the mechanism of photoinduced cellular damage after delivery and expression of KR plasmid by PPP-QDots.

Stannous octate, trioctylphosphine oxide (TOPO), trioctylphosphine (TOP), cadmium oxide, cadmium chloride (technical grade), tellurium powder (−200 mesh), selenium powder, n-tetradecylphosphonic acid (TDPA), 1-octadecene (ODE), trioctylamine (TOA), dichloromethane (DCM), and hexadecylamine (HDA) were purchased from Sigma Aldrich (St. Louis, MO, USA). Dimethylcadmium (CdMe2 ) and diethylzinc (ZnEt2 ) were purchased from Fluka (Seelze, Germany). 2.2. Synthesis of CdSe/ZnS QDots

both singlet-oxygen and superoxide-ion generation upon irradiation with green light [14,15]. However, this process only affects cells that express KR proteins. If normal cells are prevented from uptaking the KR-encoding therapeutic plasmids, they will remain intact and healthy. Therefore, delivery of the therapeutic plasmid via polymeric micelles with multifunctional capacity can be an effective cancer treatment strategy [16]. Surface polymers can be conjugated with peptides, aptamers, or antibodies to enable specific targeting [17–20]. Imaging of polymeric micelles is performed via encapsulation of magnetic nanoparticles for magnetic resonance imaging (MRI) detection or QDots for optical detection [21,22]. Alternatively, hydrophobic drugs or contrast agents can be encapsulated in the core of the polymeric micelle [23]. The utility of the optically activatable gene is that it can be induced from outside the cell. Its multifunctionality should lead to more developments regarding this kind of cancer treatment. The present study is novel because it employs a genetically encoded photosensitizer, KR, that is delivered with polymeric micelle-encapsulated QDots and that is activated intracellularly by QDots encapsulated in the same carrier. Previously, KR plasmid DNA without any carrier was delivered and activated directly with green light [15]. In this study, the KR plasmid was delivered with polymeric micelle-encapsulated QDots, and KR protein was activated locally with the same carrier. Specifically, PHEA-g-PEG-bPEI (PPP)-QDots were prepared to deliver the optically activatable KR plasmid to cancer cells. Upon plasmid delivery and protein expression, KR can be activated by blue light, which is absorbed by the CdSe/ZnS QDots encapsulated inside the polymeric micelle and emitted locally in the green wavelength within the cytoplasm of the cells. The activated KR protein triggers ROS generation, and as ROS increase intracellularly, they induce morphological changes that lead to cellular damage and apoptosis (Scheme 1). The CdSe/ZnS-QDot was prepared and characterized and its optical properties were studied. We examined the morphology, performed elemental analysis and thermogravimetric

CdSe/ZnS nanocrystals were synthesized as follows. A mixture of CdO (0.026 g, 0.20 mmol) and lauric acid (0.260 g, 1.3 mmol) was heated to 180 ◦ C to obtain a clear solution, then the mixture was cooled to room temperature. TOPO and hexadecylamine (3 g of each) were added to the flask, and the mixture was heated to 300 ◦ C. The selenium precursor (0.316 g Se powder dissolved in 4 mL TOP) then was swiftly injected into a reaction flask. A mixture containing 100 ␮L (TMS)2 S and 800 ␮L Zn(Et)2 premixed in 2.0 mL TOP was injected into the solution at a rate of 0.1 mL/3 min at 160 ◦ C. The mixture was reacted at 150 ◦ C with stirring for 1 h. The solution then was cooled to room temperature, and the resulting CdSe/ZnS QDots were washed numerous times with chloroform and methanol. 2.3. Preparation and characterization of PPP-QDot polymeric micelles We previously described the synthesis of poly(2-hydroxyethyl aspartamide) grafted PEG and bPEI (PPP) polymeric micelles in detail [24,25]. PPP was loaded with hydrophobic CdSe/ZnS QDots with a uniform morphology to prepare the PPP-QDot polymeric micelles. The PPP and QDots were dissolved in DMSO and dialyzed against water. The samples then were lyophilized and dissolved in water for further characterization. Transmission electron microscopy (TEM) was used to evaluate the morphology of the PPP-QDot micelles (JEM-2000 FXII, JEOL, Japan), elemental analysis of the micelles was conducted using an ELS-8000 (Otsuka Electronics, Japan) to measure the hydrodynamic size and zeta potential, and TGA analysis (Mettler-Toledo, SDT851, Columbus, OH, USA) was performed to determine the chemical composition. 2.4. Preparation of PPP-QDot/plasmid DNA nanoparticles The plasmid DNA binding ability of the PPP-QDots was evaluated by electrophoresis through a 1.2% agarose gel containing ethidium bromide intercalating dye. The nitrogen-to-phosphate (N/P) molar

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ratio was varied by adding predetermined PPP-QDot concentrations to a fixed amount of the plasmid DNA. The PPP-QDot and plasmid DNA solutions were mixed at N/P ratios from 1 to 50 and vortexed briefly. The complexes were kept at room temperature for 30 min for complete complexation before being loaded into an agarose gel. Electrophoresis was carried out at 100 V for 50 min, and DNA retention was visualized under ultraviolet (UV) illumination. 2.5. Transgene delivery analysis of PPP-QDot Adherent MCF-7 cells (breast adenocarcinoma cells from a BALB/c mouse) were cultured at 37 ◦ C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) (WelGENE, Korea) supplemented with 1% penicillin–streptomycin (WelGENE) and 10% fetal bovine serum (FBS; WelGENE). Cells were seeded at a density of 5 × 104 cells per well in 24-well plates. After overnight culture, adherent cells were washed twice with PBS (Biosesang, Korea) and then were incubated with the PPP-QDot/luciferase plasmid (pLuc) DNA complex solution for 4 h. Transfection results were obtained 48 h post-transfection by measuring the extent of transgene expression with a Luciferase Assay System (Promega, WI, USA). A microplate luminometer (Microlumat Plus LB96V, Berthold Technologies, Germany) was set for a 3-s delay with signal integration for 10 s. Luciferase activity was normalized to the amount of total protein in the sample using a BCA assay (Intron Biotechnol Co., Korea). A calibration curve with bovine serum albumin standards was used to measure protein concentrations. Transfection efficiency was expressed as relative light units (RLU) per mg of cell protein. 2.6. Cytotoxicity of PPP-QDot/plasmid DNA nanoparticles MCF-7 cells were seeded in 96-well tissue culture plates at a density of 1 × 104 cells per well in DMEM containing 10% FBS for 24 h. The cytotoxicity of PPP-QDot/plasmid DNA was evaluated by determining cell viability after 4 h of incubation in serumfree DMEM followed by 24 h in DMEM containing 10% FBS under the same conditions as for transgene expression. The cytotoxicity of PPP-QDot/plasmid DNA nanoparticles was examined at N/P ratios of 10–50 by varying the polymer concentration. The number

of viable cells was determined by measuring their mitochondrial reductase activities using the tetrazolium-based colorimetric MTS assay (Promega). 2.7. Cellular uptake of PPP-QDot and PPP-QDot/plasmid DNA nanoparticles MCF-7 cells were seeded in 8-well chambered slides at a density of 1 × 104 cells per well in DMEM medium containing 10% FBS for 24 h. The cells were treated with PPP-QDot for 4 h in Optimem (Gibco Life Technologies, NY, USA), and then cultures were replaced with DMEM medium containing 10% FBS. The cells were stained with WGA-A555 red membrane stain according to the manufacturer’s protocol (Invitrogen, NY, USA) and with DAPI blue nuclear stain (Invitrogen). The slides were visualized under confocal microscopy. Plasmid DNA was labeled with BOBO-3 (Invitrogen) dimeric cyanine nucleic acid stain to prepare the PPP-QDot/plasmid DNA. MCF-7 cells were cultured, treated as described above, fixed with formaldehyde, and stained with DAPI (Invitrogen). 2.8. Detection of KR protein expression MCF-7 cells were seeded in 8-well chambered slides at a density of 1 × 104 cells per well in DMEM medium containing 10% FBS for 24 h. The cells were treated with PPP-QDot/KR nanoparticles for 4–6 h in Optimem (Gibco Life Technologies), and cultures then were replaced with DMEM medium containing 10% FBS. The control NIH/3T3 fibroblasts were cultured under conditions similar to the MCF-7 cells. After 24 h, the chambered slides were stained with DAPI and observed under confocal microscopy. Total proteins were isolated, and Western blotting was conducted using a KR-specific antibody (Evrogen, USA). 2.9. Intracellular activation mediated by PPP-QDots MCF-7 cells were seeded in 8-well chambered slides at a density of 1 × 104 cells per well in DMEM medium containing 10% FBS for 24 h. The cells were treated with PPP-QDot/KR nanoparticles for 4–6 h in Optimem (Gibco Life Technologies), and then cultures were replaced with DMEM medium containing 10% FBS. After 24 h, the

Fig. 1. Characterization of green fluorescence and QDot-encapsulated polymeric micelles. TEM image and excitation/emission wavelengths of (a) CdSe/Zns QDots and (b) PPP-QDots.

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Fig. 2. Optical and chemical characterization of QDot-encapsulated polymeric micelles. (a) Confocal microscopic image of PPP-QDots showing green emissions from the QDots encapsulated within the PPP polymeric micelle shell. (b) The energy-dispersive spectrum of prepared PPP-QDots revealed that Cd, Se, Z, S, and C were present. TGA analysis of (c) the prepared PPP-micelle and (d) PPP-QDots. Comparing the free PPP polymer and the QDot-encapsulated PPP polymer, about 20–25 wt% of QDots was encapsulated in the PPP.

chambered slides were exposed to blue light-emitting diode (LED) lighting (SH-FTL-395, SH system, Korea) for 40 min and incubated for 1 h in a 5% CO2 incubator. The cells then were stained with DAPI and WGA-A488 green membrane staining according to the manufacturer’s protocols (Invitrogen). The slides were visualized under phase contrast, fluorescence, and confocal microscopy. As a control experiment, MCF-7 cells were treated with PPP-QDots alone and then were exposed to blue light for 40 min. Cells then were incubated for 1 h in a 5% CO2 incubator. Finally, the cells were fixed and stained with DAPI and WGA-A555 red for morphology analysis using confocal microscopy. 2.10. Apoptosis assay of cellular fate MCF-7 cells were seeded in 8-well chambered slides at a density of 1 × 104 cells per well in DMEM medium containing 10% FBS for 24 h. The cells were treated with PPP-QDot/KR nanoparticles for 4–6 h in Optimem (Gibco Life Technologies), and then cultures were replaced with DMEM medium containing 10% FBS. After 24 h, the chambered slides were exposed to blue light for 40 min and then were incubated for 1 h in a 5% CO2 incubator. The cells were stained with propidium iodide (PI) and were counterstained with DAPI for apoptosis detection. The cells also were treated with appoflamma PS series apoptosis membrane markers according to the manufacturer’s protocol (BioActs, Incheon, Korea).

24 h in DMEM containing 10% FBS under the same conditions as for transgene expression. Tissue culture plates then were exposed to blue light for 40 min and incubated for 1 h in a 5% CO2 incubator. We determined the number of proliferating cells by the MTS assay (Promega). 2.12. Detection of ROS in cells treated with PPP-QDot/KR nanoparticles and blue light MCF-7 cells were seeded in 96-well tissue culture plates at a density of 1 × 104 cells per well in DMEM containing 10% FBS for 24 h. The cells then were treated with PPP-QDot/KR nanoparticles and PPP-QDots at various N/P ratios for 24 h. Subsequently, the cells were exposed to blue light for 40 min and were incubated for 1 h in a 5% CO2 incubator. ROS generation then was measured using an OxiSelect Intracellular ROS Assay kit according to the manufacturer’s protocols (Cell Biolabs, San Diego, CA, USA). 2.13. Statistical analysis Quantitative data are expressed as means ± SD. The means were compared using an independent samples t-test. p values less than 0.05 were considered statistically significant. 3. Results

2.11. Proliferation assay following intracellular KR activation

3.1. CdSe/ZnS characterization

MCF-7 cells were seeded in 96-well tissue culture plates at a density of 1 × 104 cells per well in DMEM containing 10% FBS for 24 h. The proliferation of MCF-7 cells after treatment with PPPQDot/KR nanoparticles was evaluated by MTS assay. After 4 h of incubation in serum-free DMEM, MCF-7 cells were incubated for

TEM imaging of bare CdSe/ZnS QDots indicated a round morphology with a diameter of approximately 5 nm. The excitation and emission wavelengths of the CdSe/ZnS QDots were approximately 340 nm and 520 nm, respectively (Fig. 1a). The QDots exhibited a broad excitation range and a narrow emission range, which helped

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Fig. 3. Physicochemical characterization and in vitro studies of QDot-encapsulated polymeric micelles complexed with plasmid DNA. QDots encapsulated within polymeric micelles were taken up by MCF-7 cells. (a) Confocal microscopic image of PPP-QDot endocytosed by MCF-7 cells. (a, left side) Blue, DAPI nuclear stain; red, WGA-A555 membrane stain; green, PPP-QDot emission. (a, right side) MCF-7 uptake of plasmid DNA complexed with PPP-QDots. Red, BOBO-3 plasmid DNA stain; green, PPP-QDots; blue, DAPI. (b and c) The particle size and surface charge of PPP-QDot/plasmid nanoparticles were analyzed by dynamic light scattering and zeta potential measurement, respectively. (d) The transfection efficiency of PPP-QDots was measured by luciferase assay. (e) Cell viability after treatment with PPP-QDot/plasmid nanoparticles was analyzed by MTS assay. p ≤ 0.001 is represented as #.

to establish the optical properties required for labeling and other uses. 3.2. Physicochemical characterization of PPP-QDots PPP-QDots were prepared by the dialysis method. The PPP polymer has a hydrophobic core that interacts with and encapsulates the hydrophobic QDots, forming polymeric micelles with hydrophilic head groups and cationic polymers on the surface. PPP-QDots were analyzed by TEM, and the image confirmed the rounded morphology of the polymeric micelles (Fig. 1b). Elemental analysis indicated that the PPP-QDots were composed of cadmium (Cd), selenide (Se), zinc (Z), sulphide (S), oxygen (O), and carbon

(C) (Fig. 2b). The estimated composition of the QDots encapsulated in the polymeric micelle was 20–25 wt%. The polymer formed the shell, and the QDots were encapsulated in the core. Heating to 500 ◦ C could thermally decompose the polymer without disrupting the integrity of the QDots. The percentage of QDots remaining after thermal decomposition could be calculated by comparing the PPP polymer and PPP-QDots (Fig. 2c). The polymer alone had free functional groups that were highly sensitive to the temperature change. In contrast, the PPP-QDots was in the micelle form and had a metal ion in its core that was closely associated with the hydrophobic portion of the encapsulating PPP. This protected the coated polymer from thermal decomposition and was responsible for the variation in the thermal decomposition peaks. Moreover,

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Fig. 4. In vitro KR gene expression in normal and cancer cells. Confocal microscopy images of the KR-encoding plasmid delivered by PPP-QDots in (a and b) NIH3T3 and (c and d) MCF-7 cells. (e) Confirmation of KR expression after delivery by PPP-QDots was achieved by Western blotting. Note: b and d are the Z-stack images of the respective figures.

Matusinovic et al. reported that free radical absorption by QDots during the thermal decomposition of poly(methyl methacrylate) retards polymer mobility, resulting in a shift to higher temperatures in the composites [26]. 3.3. Optical properties of PPP-QDots We measured the optical characteristics of PPP-QDots to confirm whether encapsulation in the polymeric micelle had altered the optical properties of CdSe/ZnS. Excitation and emission characteristics were retained after encapsulation within the polymeric micelle, as evident from spectrofluorometric analysis (Fig. 1a and b). Confocal microscopy of PPP-QDots indicated green emission from the polymeric micelles (Supplementary Figs. 1a and 2a). The green emission was derived from the CdSe/ZnS QDots encapsulated inside the polymeric micelles and not from the polymeric micelles themselves, as they have no light emission properties. 3.4. PPP-QDot/KR complexation Complexation of the KR plasmid with the PPP-QDot carrier was analyzed using a gel retardation assay. N/P ratios of 15, 20, and

30 complexed well with the plasmid DNA, and their movement was retarded in the wells compared to the control and to carriers with lower N/P ratios (Supplementary Fig. 1b). The sizes of the PPPQDots ranged from 200 to 250 nm as measured by dynamic light scattering. The surface charge of the PPP-QDot was approximately +10–18 mV as determined by zeta potential measurement (Fig. 3b and c). The polymeric micelle carried a positive surface charge as indicated by the appearance of bPEI at the surface, irrespective of PEG in the same region. 3.5. Cellular uptake of PPP-QDots and PPP-QDot/KR plasmid DNA nanoparticles Uptake of the PPP-QDots can be visualized by confocal microscopy without any dye conjugation, because QDots within the polymeric micelles emit green light. MCF-7 cells were incubated with PPP-QDots, nuclei were DAPI-stained, and membranes were stained with WGA-A555 red. Cellular uptake was evident from the green color emitted from the cell cytoplasm (Fig. 3a, left). Plasmid DNA was labeled with BOBO-3 dye and complexed with PPP-QDots. MCF-7 cells took up the complex as evidenced by the release of plasmid DNA into the cytoplasm (Fig. 3a, right).

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Fig. 5. Intracellular activation after KR gene expression. Confocal microscopy images of MCF-7 cells after blue light exposure to control cells alone, bPEI/KR nanoparticles (N/P 10), PPP-QDot (N/P 40)/KR nanoparticles. Blue, DAPI nuclear stain; green, WGA-A488 membrane stain; red, KR gene expression; combined image.

The polymersome exhibited a PEI moiety grafted over nearly 45% of the polymer backbone and exposed outside. The plasmid DNA was bound predominantly to the PEI exposed outside. We incorporated PEI specifically for plasmid complexation, and its escape from the endosome via the proton sponge effect is an established phenomenon. 3.6. Biocompatibility of PPP-QDot/plasmid DNA nanoparticles Following uptake of PPP-QDots, we investigated whether potentially toxic effects would result by performing an MTS assay of cell viability. Control PEI and the PPP-QDot equivalents of the N/P 20, 30, and 40 were incubated with MCF-7 cells for 24 h, and their toxicities were measured. The PPP-QDot carrier showed no toxicity in comparison to the control bPEI carrier (Fig. 3e). bPEI alone is associated with some toxicity due to its high protonation. In contrast, PPP-QDots have PEG at the surface along with bPEI, which increases the hydrophilic nature outside the carrier, making the carrier more cyto-friendly than bare PEI. 3.7. Transfection efficiency of PPP-QDots The transfection efficiency of PPP-QDots was analyzed because the carrier was to be utilized for the delivery of the plasmid DNA. PPP-QDots were complexed with a luciferase plasmid and were delivered to MCF-7 cells. Luciferase expression then was detected with a luminometer. The N/P ratio enables a determination of the concentration at which complexation occurs. In general, as the N/P

ratio increases, the positive charge of the complex increases. This facilitates interaction with the negatively charged cell membrane, thereby increasing the transfection efficiency. As the plasmid concentration is increased, the chances of transfecting larger numbers of cells increases, and the transgene integration probability also increases. As the hydrophobic block length increases, the size of the micelle increases. This hinders cellular uptake, and in vivo, sizebased particle retention will prevent the micelles from reaching the region of interest [27]. The transfection efficiency of PPP-QDots was comparable to that of control bPEI and lipofectamine, a commercially available transfection reagent (Fig. 3d).

3.8. PPP-QDot-mediated KR expression The transfection efficiency of PPP-QDots carrying the luciferase plasmid was confirmed to be sufficient to deliver a loaded plasmid for subsequent protein expression. We next used the carrier for the delivery of plasmid-encoded KR protein. After treatment with PPPQDot/KR nanoparticles, we probed for KR protein expression using confocal microscopy. The KR expression patterns in normal NIH3T3 cells and in MCF-7 cancer cells were different. Specifically, the pattern of KR expression in normal cells was uniform and in cancer cells was punctuated. When KR plasmid was delivered via control bPEI, fewer cells expressed KR compared with when PPP-QDots were used as the carrier (Supplementary Fig. 2a). In the image, nuclei were labeled with DAPI blue, and expressed therapeutic KR protein is labeled in red (Fig. 4a–d).

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Fig. 6. Detailed analysis of intracellular activation after KR gene expression. Confocal microscopy images of MCF-7 cells after blue light exposure. Blue, DAPI nuclear stain; green, WGA-A488 membrane stain; red, KR gene expression; combined image. Yellow arrows indicate regions of higher KR expression and enhanced cell shrinkage. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)

3.9. Confirmation of PPP-QDot-mediated KR expression by Western blotting Proteins were isolated from KR-expressing cells, and KR was detected by Western blotting. No KR-reactive bands were visible from control cell protein samples, and very few bands were observed from bPEI/KR-treated cells. In contrast, bands were obvious in the PPP-QDot (N/P 40)/KR nanoparticle-treated cells (Fig. 4e and Supplementary Fig. 2c). 3.10. Intracellular activation of KR-expressing cells by PPP-QDot nanomicelles Once intracellular KR expression was confirmed, the cells were activated with blue light. Cells then were stained with WGAA488 green membrane stain and DAPI blue nuclear stain to detect changes in the morphology of the treated cells compared with normal cells. Control cells lacking PPP-QDot/KR nanoparticles showed no morphological changes. When cells were treated with bPEI (N/P 10)/KR, only a few cells exhibited mild KR expression, and cell damage was not observed. In contrast, PPP-QDot/KR nanoparticletreated cells shrunk and moved apart from each other, altering their typical propensity toward clumping. The rounded morphology may precede further damage (Supplementary Fig. 2b and Fig. 5). The morphological effects of KR expression and activation following delivery by PPP-QDot/KR nanoparticles were obvious (Fig. 6,

yellow arrow). As a control, MCF-7 cells were treated with PPPQDots alone and then exposed to blue light. The cells showed no morphological abnormalities following this treatment (Supplementary Fig. 3a).

3.11. Intracellular activation-mediated apoptosis detection We next investigated whether morphological changes in KRexpressing and -activated cells would lead to apoptosis. Following PPP-QDot/KR nanoparticle treatment and blue light exposure, we stained cells with PI for apoptosis detection. PI accumulation was detected in nuclei, indicating apoptosis (Fig. 7b). Conversely, PPP-QDot-treated and blue light-exposed cells lacked nuclear PI accumulation, indicating cellular survival (Supplementary Fig. 3b).

3.12. Detection of intracellular KR activation and proliferation suppression Cellular proliferation after exposure to PPP-QDot/KR nanoparticles was measured using the MTS assay. One hour after exposure, cells exhibited a 50% reduction in activity when compared to cells treated with the control carrier (Fig. 7a, right). Cells not exposed to blue light showed no reduction in proliferation (Fig. 7a, left).

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Fig. 7. Cell damage after PPP-QDot-mediated KR delivery and intracellular activation. The MTS assay was used to measure MCF-7 cell proliferation (a, left) before blue light exposure and (a, right) 1 h after blue light exposure. Confocal microscopy images of PPP-QDot/KR nanoparticle-treated cells after blue light exposure. (b) Apoptosis was detected with PI staining (red, in nucleus); blue, DAPI nuclear stain. (c) ROS production in PPP-QDot- and PPP-QDot/KR nanoparticle-treated cells after blue light exposure was measured using a ROS assay. PPP-QDot/KR nanoparticles at different N/P ratios (N/P 20, 30, and 40) and varied amounts of PPP-QDots (PQ) equivalent to respective N/P ratios (∼PQ 20, 30, and 40) were compared. The carrier, bPEI (N/P 10), and cells alone were assayed as controls. p ≤ 0.001 is represented as #.

3.13. ROS detection after PPP-QDot/KR nanoparticle treatment and blue light exposure The amount of ROS produced after PPP-QDot/KR nanoparticletreated MCF-7 cells were exposed to blue light was quantified using the DCFH-DA assay. We detected a significant increase in ROS generation, especially at an N/P ratio of 40, when compared to cells treated with the bPEI control carrier and to control cells alone (Fig. 7d). 4. Discussion KR activation results in the production of free radicals and hydrogen peroxide, supporting a Type I photosensitization mechanism. The phototoxicity of KR is due to protein cross-linking through radical reactions. Type I sensitization assumes the formation of radicals via electron transfer (reductive or oxidative), involving the triplet state of the photosensitizer [14]. The combination of protein-mediated associations and photoinduced cell death raises the possibility of a photodynamic therapy using a biogenerated agent rather than a synthetic external agent. We describe the application of QDot-containing polymeric micelles for the delivery of a KR-encoding plasmid and intracellular activation of the same carrier with blue light following KR gene expression. QDots were encapsulated within the PPP polymeric micelle by dialysis. PPP-QDots consisted of a PPP shell and QDots aggregated with C18 alkyl chains as the core. We confirmed that PPP-QDots retained the characteristics of the QDots after encapsulation. The

excitation and emission characteristics from the PPP-QDots indicated the optical properties of the CdSe encapsulated in the polymeric micelle (Fig. 1). Elemental analysis of the PPP-QDots revealed the presence of Cd, Se, Z, and S in the polymeric micelles as well as C, owing to carbon present in the polymers. TGA analysis indicated that QDots accounted for approximately 20 wt% of the polymeric micelles when compared to the free polymeric micelles (Fig. 2). CdSe/ZnS alone is toxic [28], but when covered by a polymeric shell, the toxicity is reduced. This is similar to PEI, which was one of the PPP surface components. When PEI is used as such, it is toxic [29,30] and a comparatively large amount of carrier complexed with plasmid DNA is needed to deliver a transgene. In this system, PEI is used along with hydrophilic PEG, which gives a smoothing effect to the carrier, protecting the cells from bare QDots and PEI (Fig. 3e). PPP-QDot/plasmid nanoparticles were approximately 250 nm in diameter, which is suitable for tumor accumulation by the enhanced permeability and retention effect (Fig. 3b). The surface charge of the PPP-QDot/plasmid nanoparticles was approximately +10 to +15, owing to the presence of PEI on the surface of the polymeric micelle (Fig. 3c). We delivered a KR-encoding plasmid in this study. KR is phototoxic upon irradiation with light of 540–580 nm due to the formation of ROS [14]. The KR protein expression patterns in cancer cells and normal cells were consistent with previous reports (Fig. 4a–d) [31]. Once KR expression was confirmed with Western blotting (Fig. 4e), the cells were exposed to blue light from an optical fiber source, and

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the fate of the cells was observed (Fig. 5). The cells were activated by an intracellular mechanism detailed in Supplementary Scheme 1. KR proteins become activated by green light from an external light source. We instead used blue light activation because QDots inside the polymeric micelles absorb blue light and emit green light locally and more effectively than direct activation with green light [32]. The cultured cells were directly exposed to a blue light source. KR activation was not affected substantially by light intensity in our in vitro study. In addition, prolonged exposure of the KR-expressing cells to the light source (>40 min) did not significantly affect KR activation. However, we believe that the wavelength and intensity of the light source will be critical factors when in vivo animal experiments are conducted because tissue penetration of the light is highly dependent on the wavelength and intensity of the source. Cells that underwent morphological changes were labeled red, due to the expression of KR in those cells. Cells not expressing KR retained their morphology even after exposure to blue light. Control cells and bPEI (N/P 10)/KR-treated cells remained intact after blue light exposure, but PPP-QDot/KR nanoparticle-treated cells became rounded off, shrunk, and lost their clumping feature as a result of KR activation (Figs. 5 and 6). QDots alone can emit ROS in cells after photoactivation. To rule out the possibility of QDot-mediated cellular damage, MCF-7 cells were treated with PPP-QDots alone and exposed to blue light. Confocal microscopy images clearly indicated that the cells were normal after this treatment and did not progress to apoptosis (Supplementary Fig. 3a and b). To quantitatively detect changes in cellular metabolic activity and proliferation, the MTS assay was performed. These results supported our confocal microscopic observations. When the concentration of delivered KR plasmid was elevated, cellular proliferation and metabolic activity were reduced drastically (Fig. 7a). The reduction in metabolic activity following intracellular activation of expressed KR led to apoptosis (Fig. 7b), and cells that underwent apoptosis were colocalized with KR expression. Changes in cellular morphology, reduced metabolic activity, and apoptosis all resulted from KR plasmid DNA delivery and intracellular activation of expressed KR by PPP-QDot nanomicelles in the cancer cells. PPP-QDot-mediated KR protein activation resulted in ROS generation, as confirmed through ROS assay (Fig. 7c). This ROS generation was the source of the cellular damage [33–35]. By targeting the uptake of PPP-QDot/KR nanoparticles to cancer cells, these cells can be selectively destroyed without affecting normal cells in terms of survival or proliferation. Our results suggest that translation of this treatment to in vivo models and clinical settings will result in the protection of normal cells from treatment side effects while inducing effective cancer cell death. 5. Conclusion In this work, we demonstrate that genetically encoded KR can be delivered and activated simultaneously by QDot-loaded polymeric micelles. Intracellular activation of expressed KR via PPP-QDots resulted in cellular damage, a reduction in metabolic activity, and apoptosis. Our results support that intracellular activation-based anticancer gene therapy is a promising approach in the fight against cancer. Acknowledgements This work was financially supported by the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A120899); the Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF), funded by the Ministry of

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