Tempo-spatial Activation of Sequential Quadruple Stimuli for High

Feb 15, 2017 - Department of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Ko...
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Tempo-spatial Activation of Sequential Quadruple Stimuli for High Gene Expression of Polymeric Gene Nanocomplexes Hana Cho,† Young-Woo Cho,‡ Sun-Woong Kang,‡ Mi-Kyoung Kwak,† Kang Moo Huh,§ You Han Bae,¶,& and Han Chang Kang*,† †

Department of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Bucheon-si, Gyeonggi-do 14662, Republic of Korea ‡ Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, 141 Gajeong-ro, Yuseong-gu, Daejeon, 34114, Republic of Korea § Department of Polymer Science and Engineering, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of Korea ¶ Department of Pharmaceutics and Pharmaceutical Chemistry, The University of Utah, 30 S 2000 E, Rm 2972, Salt Lake City, Utah 84112, United States & Utah-Inha Drug Delivery Systems (DDS) and Advanced Therapeutics Research Center, 9 Songdomirae-ro, Yeonsu-gu, Incheon 21988, Republic of Korea S Supporting Information *

ABSTRACT: The clinical application of intracellular gene delivery via nanosized carriers is hindered by intracellular multistep barriers that limit high levels of gene expression. To solve these issues, four different intracellular or external stimuli that can efficiently activate a gene carrier, a gene, or a photosensitizer (pheophorbide A [PhA]) were assessed in this study. The designed nanosized polymeric gene complexes were composed of PhA-loaded thiol-degradable polycation (PhA@RPC) and cytomegalovirus (CMV) promoter-equipped pDNA. After cellular internalization of the resulting PhA@ RPC/pDNA complexes, the complexes escaped endosomal sequestration, owing to the endosomal pH-induced endosomolytic activity of RPC in PhA@RPC. Subsequently, intracellular thiol-mediated polycation degradation triggered the release of PhA and pDNA from the complexes. Late exposure to light (for example, 12 h post-treatment) activated the released PhA and resulted in the production of reactive oxygen species (ROS). Intracellular ROS successively activated NF-κB, which then reactivated the CMV promoter in the pDNA. These sequential, stimuli-responsive chemical and biological reactions resulted in high gene expression. In particular, the time-point of light exposure was very significant to tune efficient gene expression as well as negligible cytotoxicity: early light treatment induced photochemical internalization but high cytotoxicity, whereas late light treatment influenced the reactivation of silent pDNA via PhA-generated ROS and activation of NF-κB. In conclusion, the quadruple triggers, such as pH, thiol, light, and ROS, successively influenced a gene carrier (RPC), a photosensitizer, and a genetic therapeutic, and the tempo-spatial activation of the designed quadruple stimuli-activatable nanosized gene complexes could be potential in gene delivery applications. KEYWORDS: CMV promoter, photosensitizer, polymeric gene delivery, stimuli-responsive gene delivery, reactive oxygen species

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pDNA and then intracellularly deliver pDNA.3,4 We sought to determine how a subset of these gene carriers, polymer-based gene complexes, express high transfection efficiencies and maintain desirable characteristics (e.g., customized design, low cytotoxicity, low immunogenicity, no tumorigenicity, no loading limitations in the size and dose of pDNA, and ease

INTRODUCTION Over the past few decades, various gene delivery and therapeutic strategies have been investigated in hopes of reaching a consensus regarding clinical treatments for various disorders, such as cancers, aging, diabetes, and neurodegenerative and cardiovascular diseases.1,2 The use of plasmid DNA (pDNA) in gene delivery is an attractive option. However, pDNA is easily degraded by serum nucleases, and its cellular internalization is strongly limited by its hydrophilicity and negative charge.3,4 To overcome these issues, viral, nonviral, and hybrid vectors have been applied to protect (encapsulate) © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

November 23, 2016 January 18, 2017 February 15, 2017 February 15, 2017 DOI: 10.1021/acs.molpharmaceut.6b01065 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 1. (a) Research design concepts of quadruple stimuli (pH, thiol, light, and ROS)-triggered polymeric gene nanocomplexes performed with LE12 and (b) cytotoxic photochemical internalization induced by either LE02 or LE04.

endocytosis, a cationic vector surface and light irradiation may allow for direct penetration of the plasma membrane.6,8,9 Second, after the cellular entry of the vectors via endocytic pathways, the endocytosed vectors are mostly sequestered into the endolysosomes, thus resulting in low transfection efficiency.3,4,10 To improve transgene expression, endolysosomal vector escape may be induced by destabilizing the endolysosomal membrane through various mechanisms (e.g., osmotic shock,11−14 fusogenicity,13,14 light stimulation,8,9 and combinations thereof13,14). Third, the gene carriers released from the endolysosomes travel to subcellular targets (e.g., the nucleus and mitochondria).15,16 The introduction of certain components from either motor assemblies17 or organelletranslocating complexes,18,19 which use the cytoskeleton network, targets the polymeric vectors to the subcellular organelles of interest. Fourth, the gene carriers release the

of manufacture) while ameliorating the shortcomings of polymeric vectors (e.g., low transfection efficiency and low cellular and subcellular delivery efficiencies). In general, the aforementioned drawbacks of polymeric gene delivery are frequently caused by various membrane barriers (e.g., plasma, endosomal, and nuclear membranes), various transport hurdles (e.g., cell targetability, subcellular targetability, and cytosolic transport), and inappropriate gene release from the vectors.3 First, the intracellular delivery of polymeric vectors can be improved by introducing specific ligands onto the gene carriers, thereby improving the vector targeting of the corresponding receptor-expressing cells of interest for rapid internalization via receptor-mediated endocytosis.5 Vector surfaces that are hydrophobic, positively charged, or both can induce rapid endocytosis of the vectors via adsorptive endocytosis.6,7 In addition, because some vectors bypass B

DOI: 10.1021/acs.molpharmaceut.6b01065 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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tively, and light irradiation can cause the photochemical internalization (PCI) of polyplexes via membrane destabilization. Specifically, we expected light-triggered PhA to have different biological activities depending on the time-point of light exposure (LE), PCI-mediated cellular entry of polyplexes at an early step, PCI-mediated endosomal escape of polyplexes at an intermediate time-point, and the production of ROS at a later stage. Furthermore, the fourth trigger, light-induced ROS, can reactivate an inactive cytomegalovirus (CMV) promoter in pDNA via the NF-κB pathway, thus resulting in high gene expression. In general, most stimuli-responsive gene delivery systems are composed of responsive-gene carriers and nonresponsive genes. However, in the designed PhA@RPC/pDNA complexes, both essential components are stimuli-sensitive; the PhA@RPC gene carrier responds to pH, thiol, and light, and the genes with CMV promoters are stimulated by ROSactivated NF-κB. Specifically, in contrast to intracellular pH and thiol, the time-point and strength of LE (external stimuli) can be tuned and can control ROS levels and, subsequently, NF-κB levels. Therefore, this study investigated the effects of the timepoint of LE (i.e., 2 h for a quick cellular entry, 4 h for an endosomal escape, and 12 h for an NF-κB-triggered CMV activation) on the transfection of quadruple stimuli-activatable PhA@RPC/pDNA complexes in terms of cytotoxicity, gene transfection, cellular uptake, and nuclear uptake.

exogenous pDNA to produce the corresponding protein in the intracellular space but not the extracellular space.20,21 In general, the release of pDNA from electroattraction-driven polymer/gene nanocomplexes is induced by counter-charged molecules (e.g., polyanions such as negatively charged membrane components, intracellular RNA, nuclear DNA, and mitochondrial DNA).22−25 The decomplexation rates (i.e., the pDNA release rates)25,26 are strongly dependent on the complexation strength between exogenous pDNA and polymers. Gene complexes that are too loosely bound can easily release pDNA into the blood or on the plasma membrane through interactions with negatively charged serum proteins or negatively charged membrane components, respectively, whereas gene complexes that are too tightly bound cannot release pDNA and result in expression of the encoded protein.25,27 Thus, the interactions between polymeric gene vectors and pDNA should interact tightly enough (e.g., stable colloids) to protect the gene in extracellular environments but loosely enough (e.g., unstable colloids) to release the gene into intracellular environments. Site-specific control of the complexation strength can be achieved by using intracellular stimuli, such as acidic pHs in the endosomes,28 reduced glutathione (GSH) levels in the cytosol, mitochondria, and nucleus,26,29,30 or cytosolic reactive oxygen species (ROS),31 to cleave long polymer chains into short blocks. In addition, light irradiation as an external stimulus influences polymeric degradation and facilitates pDNA release.9 Though overcoming all of these hurdles is very important for high transfection efficiency, only a few strategies have been applied in most studies because of the difficulty of applying all of these solutions to polymeric vectors. Specifically, many researchers have achieved high transfection efficiencies through high cytosolic levels of pDNA. In our previous studies, we have synthesized high molecular weight (HMW) bioreducible poly(ethylenimine)s (RPCbPEI0.8 kDa; further abbreviated as RPC) from low molecular weight (LMW), 0.8 kDa poly(ethylenimine)s (bPEI0.8 kDa).26 The resulting RPCs have been complexed with various HMW drugs (i.e., pDNA26 and proteins32) and have successfully demonstrated acid pH-triggered, bPEI-induced endosomal escape of the complexes and the rapid, GSH-triggered intracellular release of payloads from the complexes. RPC also has a capacity for physical loading of hydrophobic drugs (e.g., pheophorbide a [PhA] and Sudan III); the resulting PhAloaded RPC (RPC-bPEI0.8 kDa-PhA; further abbreviated as PhA@RPC) induces light irradiation-dependent cytotoxicity.33 Thus, in the present study, quadruple stimuli (i.e., pH, GSH, light, and ROS)-activatable polymeric gene complexes with PhA@RPC were used to achieve a high transfection efficiency (Figure 1). HMW RPC in PhA@RPC can form tight complexes with pDNA, thus resulting in the formation of nanosized PhA@RPC/pDNA complexes that can enter the cell via endocytosis. The subsequent first trigger (i.e., the endosomal acidic pHs) induces the bPEI-mediated endosomal release of the complexes. In the cytosol, the second trigger (i.e., the cytosolic or nuclear GSH) cleaves the disulfide bonds in the HMW RPC into LMW bPEI, thus leading to the cytosolic or nuclear dissociation of the complexes for pDNA release. During the destruction of PhA@RPC, both pDNA and PhA are released. The third trigger (i.e., light) generates ROS in the presence of cytosolic oxygen. If some PhA is passively released from the PhA@RPC in the extracellular medium or the endolysosomes, the released PhA can be entrapped in the plasma membrane or the endolysosomal membrane, respec-

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EXPERIMENTAL SECTION Materials. Two branched polyethylenimine (bPEI) molecules with an Mw of 0.8 kDa (Mn 0.6 kDa) and 25 kDa (Mn 10 kDa) (abbreviated as bPEI0.8 kDa and bPEI25 kDa, respectively) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), RPMI1640 medium, Ca2+-free and Mg2+-free Dulbecco’s phosphate buffered saline (DPBS), 2-iminothiolane, L-cysteine hydrochloride monohydrate, dimethyl sulfoxide (DMSO), 4-(2hydroxy-ethyl)-1-piperazine (HEPES), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), D-glucose, sodium bicarbonate, fetal bovine serum (FBS), penicillin− streptomycin antibiotics, trypsin−EDTA solution, hydrogen peroxide (H2O2), ethidium bromide (EtBr), TBE buffer, and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pheophorbide A (PhA) and Calbiochem 2′,7′-dichlorofluorescein diacetate (DCFDA) were obtained from Frontier Scientific Inc. (Logan, UT, USA) and Merck Millipore (San Diego, CA, USA), respectively. A plasmid (pDNA or pLuc) with a firefly luciferase gene and a CMV promoter (pCMV-Luc) was purchased from Aldevron, Inc. (Fargo, ND, USA). The CMV promoter in the pCMV-Luc was replaced with an SV40 promoter to construct a pDNA with a firefly luciferase gene and an SV40 promoter (pSV40-Luc), and the gene modification was performed by B2Bio, Inc. (Seoul, Republic of Korea). The phosphorylated NF-κB p-p105 and antitubulin antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Pierce BCA protein assay kit, antirabbit IgG antibodies, Pierce ECL Western Blotting Substrate, and YOYO-1 iodide were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The luciferase assay kit and the μ-slide 8-well were bought from Promega Corporation (Madison, WI, USA) and Ibidi GmbH (Martinsried, Germany), respectively. A bPEI0.8 kDa-based reducible polycation (RPC-bPEI0.8 kDa or RPC) was synthesized and characterized as previously reported,34 and its molecular weight, as measured by viscosity, was 20 kDa. PhA-loaded RPC C

DOI: 10.1021/acs.molpharmaceut.6b01065 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics (PhA@RPC) was also prepared as a previously reported,33 and the weight content of PhA in PhA@RPC was 4.1% (w/w). Cells and Cell Culture. MDA-MB-231 cells (human breast cancer cell line) and HeLa cells (human cervical cancer cell line) were used at 37 °C in humidified air containing 5% CO2. MDA-MB-231 cells were cultured in DMEM, and HeLa cells were cultured in RPMI1640; these media were supplemented with D-glucose (4.5 and 2 g/L, respectively), 10% (v/v) FBS, and 1% (v/v) antibiotics. Preparation and Characterization of PhA@RPC/pDNA Complexes. To form the polymer-based pDNA complexes (i.e., polyplexes), polycation solutions (e.g., RPC, PhA@RPC) of various concentrations and a pDNA solution (0.1 mg/mL) were separately prepared in HEPES buffer (pH 7.4, 20 mM), and the two solutions with an equal volume (i.e., 10 μL) were mixed, vortexed, and then incubated at room temperature (RT) for 30 min to form polyplexes (e.g., RPC/pDNA, PhA@RPC/ pDNA). The polyplex solution was used with or without dilution with HEPES buffer (20 mM, pH 7.4) for further studies. The complexation ratio of the designed polyplexes was expressed by using a weight ratio (WR) of a weight of polymer (e.g., RPC, PhA@RPC, etc.) to a weight of pDNA as in the following equation: weight ratio (WR) =

performed after changing the medium. LE was carried out by using a 670 nm light with 1.7 J/cm2 illumination for 63 s at predetermined time-points (i.e., 2, 4, or 12 h). At 24 h posttreatment, MTT solution (10 μL of 5 mg/mL) was added to the culture medium (0.1 mL) of the polycation-treated or PhAtreated cells, and the cells were incubated for an additional 4 h. After the MTT-containing medium was discarded, DMSO (100 μL/well) was added into the well to dissolve the formazan crystals produced by live cells, and then the absorbance at 570 nm was measured with a SpectraMax M5 multiplate reader (Molecular Devices, Sunnyvale, CA, USA). Second, the cytotoxicities of the polyplexes were tested by seeding MDA-MB-231 and HeLa cells at a density of 5 × 104 cells/well into the wells of a clear 24-well plate with a clear bottom and incubating the cells in serum-containing culture media for 24 h. One hour before the polyplex treatment, the culture medium was replaced with serum-free transfection medium (0.5 mL). After the addition of polyplexes (0.5 μg pDNA in 10 μL) with various WRs into the cells, the polyplextreated cells were incubated for an additional 4 h. At 4 h posttreatment, the serum-free medium was replaced with a fresh serum-containing culture medium, and the polyplex-treated cells were further incubated for 20 h. To determine the darktoxicities, LE was not applied, whereas LE02, LE04, LE06, LE10, or LE12 were applied to determine the phototoxicities. The strength and duration of LE with a 670 nm light were 8.67 mW and 63 s, respectively. At 24 h post-treatment, MTT solution (50 μL of 5 mg/mL) was added into the cellincubating medium (0.5 mL), and the cells were incubated for an additional 4 h. The MTT-containing medium was removed, and then DMSO (500 μL/well) was used to dissolve the formazan crystals to measure the absorbance at 570 nm. Specifically, LE04 was performed after changing the medium. In Vitro Transfection Efficiency of Polyplexes. MDAMB-231 and HeLa cells (1 × 105 cells/well) were seeded into the wells of a 12-well plate and then incubated in serumcontaining culture medium for 24 h. One hour prior to transfection, the culture medium was replaced with serum-free transfection medium (1 mL). After treatment of the cells with polyplexes (1 μg of pDNA in 20 μL), the cells were incubated for 4 h. Then, the transfection medium was replaced with fresh serum-containing culture medium, and the polyplex-transfected cells were cultured for an additional 20 h. For the transfections under dark conditions, LE was not applied, whereas LE02, LE04, and LE12 were applied for light-triggered transfection. The strength and duration of LE with a 670 nm light were 8.67 mW and 63 s, respectively. Specifically, LE04 was carried out after the medium was changed. After the transfection, the cells were rinsed with DPBS and then lysed with a lysis buffer according to the manufacturer’s instructions. The relative luminescence units (RLUs) of luciferase and the total cellular protein levels were measured using a luciferase assay kit and a BCA protein assay kit, respectively. Cellular Uptake and Nuclear Uptake of Polyplexes. To determine the cellular uptake and nuclear uptake of the RPC/ pDNA and PhA@RPC/pDNA complexes, similar experiments to those used in the transfection studies were used. Instead of pDNA, the YOYO-1-intercalated pDNA was used and prepared by mixture of YOYO-1 with pDNA at a ratio of the nucleotide in the pDNA:YOYO-1 = 10:1 (mol/mol), and the cells were treated with the resulting YOYO-1-containing polyplexes. In contrast to the transfection studies, the uptake studies included an additional incubation for 30 min after LE02, LE04, or LE12.

weight of polymer weight of pDNA

The particle sizes and zeta-potentials of the RPC/pDNA and PhA@RPC/pDNA complexes in HEPES buffer (20 mM, pH 7.4) were monitored with a zeta-potential and particle size analyzer (ELS-Z; Photal Otsuka Electronics, Osaka, Japan) with a wavelength of 677 nm and a constant angle of 90° at RT. The concentration of pDNA in the polyplex solution (1 mL) was 5 μg/mL. In addition, the sizes of RPC/pDNA and PhA@RPC/ pDNA complexes (WR 2) were evaluated by transmission electron microscope (TEM; JEM-1010, JEOL USA Inc.) at 60 kV after the polyplexes were dropped onto a cooper grid and then subsequently dried at RT for 5−10 min. To evaluate whether pDNA was complexed with a polycation or was exposed on the surfaces of polyplexes, the polyplexes were subjected to gel electrophoresis. The polyplexes were loaded into a well in a 0.8 wt/vol % agarose gel (100 mL) with EtBr (75 ng/mL), and a constant voltage (100 V) was then applied to the polyplex-loaded gel in 0.5×TBE buffer for 60 min. Uncomplexed pDNA or exposed pDNA in the polyplexes was detected by a Gel Doc EZ imager (Bio-Rad laboratories, Hercules, CA, USA) with a UV tray. Dark-Toxicity and Phototoxicity of Polycations and Polyplexes. The dark-toxicity and phototoxicity of polycations (i.e., RPC, PhA@RPC), polyplexes (i.e., RPC/pDNA, PhA@ RPC/pDNA), or free PhA were examined with an MTT-based cell viability assay in the absence or presence of LE.33 First, for the cytotoxicities of the polycations or free PhA, MDA-MB-231 cells with a density of 5 × 103 cells/well were seeded in each well of a clear 96-well plate with a clear bottom and incubated in a serum-containing culture medium for 24 h. After treatment of the cells with either polycations or free PhA at various concentrations, the cells were incubated for an additional 24 h. The dark-toxicity tests were performed in the dark, whereas the phototoxicities were obtained after LE at 2, 4, or 12 h posttreatment (further abbreviated as LE02, LE04, or LE12, respectively). At 12 h post-treatment, the culture medium was replaced with fresh medium, especially, LE12 was D

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Figure 2. Dark-toxicity and phototoxicity of (a) free PhA and (b) PhA@RPC in MDA-MB-231 cells at 24 h post-treatment. After the cells were treated with the polymer or photosensitizer, light exposure at 2, 4, and 12 h (LE02, LE04, and LE12, respectively) was accomplished with a 670 nm laser (8.67 mW) for 63 s. The data are expressed as the means ± standard errors (SE) (n = 6).

incubated with DCFDA (20 μM)-containing culture medium for 30 min at 37 °C. For controls, NAC (5 mM) and H2O2 (0.3 mM) were added to the cells at 1 h before LE and at 1 h before imaging by confocal microscopy, respectively. After the cells were rinsed with DPBS, the intracellular ROS fluorescence of the cells was monitored by using a LSM 710 confocal laser scanning microscope (ZEISS, Oberkochen, Germany) equipped with an excitation laser (488 nm for diode) and variable band-pass emission filters in the dark. The obtained images were analyzed using the ImageJ software program (1.47v, National Institute of Health, MD, USA). Detection of NF-κB Activation in Polyplex-Transfected Cells. After the MDA-MB-231 cells (5 × 105 cells/ dish) were seeded in a 60 mm cell culture dish, the cells were cultured, treated with polyplexes, and then exposed to a 670 nm, 8.67 mW light for 63 s according to the aforementioned intracellular ROS assay. The cells were incubated for 1 h after light irradiation, washed twice with ice-cold DPBS, harvested from the plate with a cell scraper, and then pelleted by centrifugation at 13,000 rpm for 5 min. The cell pellet was lysed with an ice-cold protein extraction solution, and the lysate was further incubated at 4 °C for 30 min to obtain soluble proteins. After insoluble debris was removed by centrifugation at 13,000 rpm for 10 min, the final soluble protein concentration was determined with a BCA protein assay. Then, 20 μg of protein in the lysate was separated on a 10% SDS-polyacrylamide (PAGE) gel and transferred onto a nitrocellulose membrane. After being blocked with EzBlock BSA at RT for 1 h, the membrane was incubated overnight at 4 °C with primary antibodies to specifically detect the phosphorylated NF-κB p-p105 as a target and tubulin as a control. The membrane was then incubated with HRP-conjugated antirabbit IgG antibodies at RT for 1 h, rinsed three times with a TBST buffer, and developed using a Pierce ECL Western Blotting Substrate.

That is, the cellular uptake and nuclear uptake with LE02, LE04, or LE12 were evaluated for 2.5, 4.5, or 12.5 h, respectively. For LE, a 670 nm, 8.67 mW light was used to irradiate the cells for 63 s. Specifically, the studies for LE02 were carried out in serum-free medium, whereas LE04 was conducted after replacement of the serum-free medium with the serum-containing medium. The cellular uptake of polyplexes was assayed by detaching the polyplex-transfected cells with a trypsin-EDTA solution, pelleting the cells, resuspending them in DPBS (0.3 mL), and then fixing them with a 4 w/v% formalin solution. After resuspension of the cells in DPBS, YOYO-1 fluorescence in the polyplex-transfected cells was measured by using a FACScanto II flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) equipped with a primary argon laser (488 nm) and a fluorescence detector (530 ± 15 nm). After detection of 1 × 104 polyplex-transfected cells, the cellular YOYO-1 fluorescence was determined in the cells distributed in a gated population of live cells. The nuclear uptake of polyplexes was determined by rinsing the polyplex-transfected cells twice with DPBS and detaching them. Then, the nuclei were prepared and isolated by using the Nuclei PURE Prep nuclei isolation kit according to the manufacturer’s protocol. Nuclear YOYO-1 fluorescence was measured by using a FACScanto II flow cytometer with a primary argon laser (488 nm) and a fluorescence detector (530 ± 15 nm). After detection of 1 × 104 nuclei isolated from the polyplex-transfected cells, the nuclear YOYO-1 fluorescence of the nuclei located in a gated region for a single nucleus was used. Intracellular ROS Levels in Polyplex-Transfected Cells. MDA-MB-231 cells were seeded in a μ-slide 8 well (1 × 104 cells/well) and incubated in serum-containing culture medium for 24 h. One hour prior to the polyplex treatment, the culture medium was replaced with serum-free transfection medium. The cells were transfected with RPC/pDNA (WR 2) or PhA@ RPC/pDNA (WR 2) complexes and then cultured for 12 h. During the procedure, the serum-free transfection medium was replaced with fresh serum-containing culture medium at 4 h post-transfection. At 12 h post-treatment (LE12), a 670 nm, 8.67 mW light was used to irradiate the polyplex-transfected cells for 63 s. Then, the cells were washed with DPBS and

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RESULTS AND DISCUSSION Phototoxicity Characteristics of the PhA@RPC Polymer. In general, carrier materials for drug delivery must have either negligible or minimal cytotoxicity. Thus, the PhA@RPC polymer and RPC (MW 20 kDa) polymer used in this study were designed to exhibit reasonable cytotoxicity during gene delivery. According to our previous reports, HMW RPC E

DOI: 10.1021/acs.molpharmaceut.6b01065 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 3. (a) Particle sizes and zeta-potentials and (b) gene-condensational characteristics of PhA@RPC/pDNA and RPC/pDNA complexes. The data in (a) are expressed as the means ± standard deviation (SD) (n = 5), whereas the electrophoresis image in (b) depicts a representative result.

carrier toxicity. Fifth, the phototoxicity differences between PhA@RPC and free PhA decreased over longer incubation times; PhA@RPC had 22.6-fold, 22.2-fold, and 5.4-fold lower phototoxicities than free PhA at LE02, LE04, and LE12, respectively. These results indicate that the time-point of LE might induce PhA-mediated phototoxicity via different mechanisms. Specifically, the phototoxicity differences between PhA@ RPC and free PhA at different time-points of LE were strongly affected by the rates and mechanisms of their cellular internalization as well as their extracellular or intracellular locations. Without light irradiation, it was expected that hydrophobic PhA would be internalized by either direct penetration through the plasma membrane, hydrophobicitymediated adsorptive endocytosis, or both. In contrast, PhA@ RPC can utilize positive charge-mediated adsorptive endocytosis for its intracellular entry. Although it is difficult to say which of these mechanisms is faster or slower at internalizing free PhA and PhA@RPC, hydrophobic PhA may have had faster internalization rates than PhA@RPC because negatively charged serum proteins interfered with the cellular internalization of positively charged PhA@RPC. Specifically, the faster cellular uptake rates of free PhA compared with PhA@RPC were supported by our previous results showing that the cellular uptake of free PhA is 7−9-fold greater than that of PhA@RPC at 4 h post-treatment in three cell lines (i.e., MCF7, MCF7/ADR-RES, and HeLa cells).33 In addition, in contrast to the almost identical phototoxicities of free PhA at different time-points of LE, different phototoxicities of PhA@RPC at different time-points of LE may have been influenced by the amount of PhA released from PhA@RPC. From the expected cellular internalization rates of PhA@RPC, it was inferred that the major population of PhA@RPC was distributed in the extracellular medium at 2 h post-treatment, in the endolysosomes at 4 h post-treatment, and in the cytosol at 12 h posttreatment. The shortage of the extracellular and endolysosomal thiols did not permit the release of PhA from PhA@RPC, thus resulting in low photoactivity. However, cytosolic PhA@RPC was able to release PhA via the cytosolic thiol-triggered degradation of RPC, thus resulting in LE12-activated PhA photoactivity. These different extracellular or intracellular locations of PhA@RPC may have influenced the phototoxicity differences between PhA@RPC and free PhA, which decreased from approximately 22-fold to 5-fold at later LE time-points.

polymers exhibit either negligible cytotoxicity or less cytotoxicity than HMW bPEI (e.g., MW 25 kDa) because intracellular thiols (e.g., GSH) trigger the degradation of HMW RPC to LMW bPEI0.8 kDa derivatives via disulfide cleavage.26,32 Moreover, when light irradiation is provided at 12 h posttreatment, PhA@RPC induces light-triggered cytotoxicity in the thiol-rich environments because intracellular thiols cause the release of the PhA from the polymers into the cytosol.33 Though LE at different time-points may induce different PhAmediated biological actions during polymeric transfection, thereby resulting in different phototoxicity phenomena, the time-point effects of LE on the carrier cytotoxicity of PhA@ RPC were not investigated in our previous studies. To understand this issue, the toxicities in PhA@RPC, RPC, or PhA-exposed MDA-MB-231 cells were examined at 1 d posttreatment with or without LE. RPCs at a concentration of ≤36 μg/mL induced death in less than 20% of the cells regardless of the presence/absence of light and the time-point of LE, whereas the highest RPC concentration (72 μg/mL) tested killed approximately 45−60% of the cells; notably, the killing activities were not strongly dependent on the LE time-point (Figure S1). For free PhA, the photosensitizer did not cause any cell-killing effects in the dark but killed approximately 50% of the cells at [PhA] = 0.036−0.056 μg/mL with LE (Figure 2a). In the case of PhA@RPC, the dark-toxicity IC50 value was [PhA] ≈ 2 μg/mL, whereas PhA@RPC exhibited greater phototoxicity when LE was performed after a longer incubation time (the phototoxicity IC50 values: 1.13 μg/mL for LE02, 0.80 μg/mL for LE04, and 0.30 μg/mL for LE12) (Figure 2b). On the basis of these results, the toxicity characteristics of PhA@ RPC could be inferred as follows. First, the dark-toxicity of PhA@RPC may have resulted not from PhA but instead from RPC because [PhA] ≈ 2 μg/mL in PhA@RPC corresponds to [PhA@RPC] ≈ 49 μg/mL, and the dark-toxicity IC50 values of RPC were between 36 and 72 μg/mL. Second, unlike the phototoxicity of free PhA, the phototoxicity of PhA@RPC was strongly influenced by the time-point of LE. Third, the lower phototoxicity IC50 values of PhA@RPC relative to its darktoxicity IC50 value indicate that the PhA@RPC phototoxicity was strongly mediated by the LE-triggered PhA. Fourth, in particular, LE12 might be more beneficial for polymer-based gene delivery than LE02 and LE04 because the former condition could use less PhA@RPC, resulting in its negligible F

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Figure 4. Time-point effects of light exposure and WR effects of polyplexes on (a,c) transfection efficiency and (b,d) cell viability of the PhA@RPC/ pDNA and RPC/pDNA complexes in MDA-MB-231 cells at 24 h post-treatment. For the experiments in (a) and (b), dark (no light), LE02, LE04, and LE12 were applied after the cells were treated with the polyplexes (WR 2). For the experiments in (c) and (d), various WR polyplexes were evaluated under either dark conditions or LE12. The data are expressed as the means ± SE (n = 4−23).

Moreover, this result may explain why LE12 caused approximately 4.4-fold and 2.7-fold more phototoxicity in PhA@RPC than LE02 and LE04, respectively. It was expected that the time-point of LE would tune the levels of ROS production as well as different light-triggered actions (e.g., the destabilization of the plasma membrane, the endosomal membrane, and the nuclear membrane, and the ROS-triggered NF-κB activation). Furthermore, different time-points of LE probably influenced the transfection efficiencies of PhA@RPCbased gene complexes. Physicochemical and Gene-Condensational Characteristics of PhA@RPC/pDNA Complexes. To confirm the potential of PhA@RPC as a gene carrier, its pDNA complexes were first constructed, and particle formation was then confirmed on the basis of gene condensation, sizes, and zetapotentials. First, when PhA@RPC was complexed with pDNA at weight ratio (WR) of 1, the sizes of the PhA@RPC/pDNA complexes were approximately 500 nm in diameter. At WR ≥ 2, the PhA@RPC/pDNA complexes were below 80 nm in diameter (Figure 3a). The WR-dependent size pattern of the PhA@RPC/pDNA complexes was similar to that of the RPC/ pDNA complexes used as the control polyplexes. In addition, as

shown in Figure S2, TEM images represented that the sizes of RPC/pDNA and PhA@RPC/pDNA complexes (WR 2) were about 30 nm in diameter because the dried sizes of polyplexes in a TEM image are generally smaller than the hydrodynamic size of polyplexes in a DLS method. Second, the zeta-potentials of the PhA@RPC/pDNA complexes were approximately 20 mV at 1 ≤ WR ≤ 2 and reached approximately 30 mV at 5 ≤ WR ≤ 10 (Figure 3a). Although the RPC/pDNA complexes had slightly different patterns in zeta-potentials, their zeta-potentials were within approximately 15−25 mV of the same WR range. Third, gene condensation with PhA@RPC was evaluated with a gel retardation assay. The experiment was performed with polyplexes of 2 ≤ WR ≤ 10 because the polyplexes of WR 1 were not reasonably sized for in vivo applications. No EtBr fluorescence was detected from the PhA@RPC/pDNA complexes prepared at 2 ≤ WR ≤ 10 (Figure 3b). The results indicate that in aqueous solution, the tested PhA@RPC/pDNA complexes did not contain any uncomplexed pDNA or any exposed pDNA on their nanoparticular surfaces, thus suggesting that PhA@RPC was completely condensed and G

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transfection efficiency regardless of the absence or presence of PhA. However, it is unclear why light irradiation enhances the polymeric transfection efficiency. The polymeric transfection of the PhA@RPC/pDNA complex-transfected cells and the RPC/ pDNA complex and PhA-cotreated cells suggested that light treatment may differentially influence the polymeric transfection processes depending on the time-point of LE; in particular, the transfection effects at LE02 and LE04 may be markedly different from those at LE12. Although the lightinduced transfection results of the PhA@RPC/pDNA complexes are not clearly explained, we expect that enhanced cellular uptake, endosomal release, or nuclear uptake induced by PCI (photochemical internalization) and the reactivation of the CMV promoter in the pDNA via ROS-mediated NF-κB activation may mediate the transfection enhancement. If a polyplex exhibits high cytotoxicity, the polyplex cannot be applied in clinical gene therapies, regardless of whether the transfection efficiency is high. Thus, the dark-toxicity and phototoxicity of the polyplexes to the transfected cells were assessed without and with LE, respectively (Figure 4b). As expected, both the presence/absence and time-point of LE did not significantly affect the viability of the RPC/pDNA complextransfected cells (which was above 80%). However, although the dark treatment (no LE) and LE12 maintained the viability of the cells cotreated with both the RPC/pDNA complexes and free PhA (5 ng/mL) to above 80%, 50−55% of the cells were killed by LE02 and LE04. Interestingly, similar LE time-pointdependent cytotoxicity patterns after the cotreatment described above were observed in the PhA@RPC/pDNA complextransfected cells. The cell viabilities of the PhA@RPC/pDNA complex-transfected cells were above 80% when either LE12 or no LE was applied, whereas 40−50% of the transfected cells died by LE02 and LE04. The phototoxicity results of the cotreatment system at LE02 and LE04 indicate that LE at these time-points caused PhA (5 ng/mL)-mediated necrosis of the transfected cells via phospholipid-stimulated membrane rupture in which the ROS produced by the light-activated PhA oxidize the membrane phospholipids, and the oxidized phospholipids damage membrane stability.8 The phototoxicity results of the PhA@RPC/pDNA complex-transfected cells at LE02 and LE04 also suggested that unexpectedly a small amount of free PhA (82 ng/mL) may have been released from PhA@RPC/pDNA complexes via passive diffusion, and the subsequent release of PhA may have damaged the membrane phospholipids via lightinduced ROS. However, the negligible phototoxicities at LE12 indicate that the amount of free PhA or the release of PhA into the cytosol may have generated ROS but at levels insufficient to kill the transfected cells. Additional phototoxicity tests of PhA@ RPC/pDNA complexes at LE06 or LE10 supported that late LE could not damage on cell viability (Figure S3). Importantly, when PhA was present, regardless of its location inside or outside of the polyplexes, the early light irradiation (e.g., LE02 and LE04) caused lethal damage to the cells; this result was in marked contrast to the negligible cytotoxicity observed at late light irradiation (e.g., LE12). The phototoxicity results at different time-points of LE suggested that unlike LE12, LE02, and LE04 may not be acceptable conditions for transfection studies. For higher WR polyplexes (i.e., WR 5 and WR 10), additional transfection and cytotoxicity studies were performed at LE12 in MDA-MB-231 cells and HeLa cells. In MDA-MB231 cells, the transfection efficiencies of the RPC/pDNA complexes increased at greater WRs in the dark (Figure 4c).

shielded pDNA. Similarly, the control polyplexes also showed complete gene condensation. Light-Induced Transfection and Phototoxicity Characteristics of the PhA@RPC/pDNA Complexes. To understand whether the LE time-point influenced the polymeric transfection process, the light-induced transfection efficiencies of the PhA@RPC/pDNA complexes were examined. Specially, the PhA@RPC/pDNA (WR 2) complex instead of a system with a higher WR was used as the starter polyplex. For the control polyplex, the RPC/pDNA (WR 2) complex was applied and cotreated with free PhA. The first tested dose of free PhA was 82 ng/mL because of the amount of PhA loaded in the PhA@RPC/pDNA complexes (i.e., approximately 41 ng of PhA in 1 μg of PhA@RPC). However, this dose killed almost all of the RPC/pDNA complex-transfected cells in the dark. Thus, the dose of free PhA was reduced to 5 ng/mL to maintain the viability of transfected cells. After cells were treated with polyplexes alone or with free PhA, the expression levels (i.e., transfection efficiencies) of the model luciferase gene under control of the CMV promoter (i.e., pCMV-Luc) in the transfected cells were investigated at 1 d post-transfection. Although the RPCs did not contain any PhA molecules, the RPC/pDNA complex-transfected cells showed enhanced LE-mediated transfection; LE02, LE04, and LE12 demonstrated 5.8-fold, 2.1-fold, and 2.6-fold higher transfection efficiencies compared with the treatment without LE (dark) (Figure 4a). When both RPC/pDNA complexes and free PhA (5 ng/mL) were applied to the cells, the LE02, LE04, and LE12 transfected cells exhibited 44.1-fold, 77.8-fold, and 1.1-fold higher transfection efficiencies than the cells with no LE (Figure 4a). As expected, the transfection efficiencies of the two systems (i.e., a single treatment of RPC/pDNA complexes and cotreatment with both RPC/pDNA complexes and free PhA) were not significantly different in the dark. However, the cotreatment at LE02 and LE04 induced 10.5-fold and 51.5-fold higher transgene expression, respectively, compared with a single treatment with RPC/pDNA complexes. Interestingly, the transfection efficiencies of the cotreatment at LE12 were 1.7fold lower than that of the single treatment. In the case of the PhA@RPC/pDNA complexes, the LE time-point caused unexpected transfection efficiencies on the basis of the transfection results of RPC/pDNA complexes with or without free PhA. Although no LE was applied, the PhA@ RPC/pDNA complexes generated 20.3-fold and 14.8-fold higher gene expression than that obtained with the RPC/ pDNA complexes without and with free PhA (5 ng/mL), respectively (Figure 4a). Moreover, the transfection efficiency of PhA@RPC/pDNA complexes at LE12 was 11.5-fold higher than the transfection efficiency in the dark. By contrast, LE02 and LE04 induced only 1.3-fold and 2.3-fold transfection enhancement, respectively, compared with the no LE conditions. Interestingly, the PhA@RPC-based polyplexes showed interesting transfection enhancements compared with the RPC-based polyplexes (20.3-fold at no LE, 4.4-fold at LE02, 22.5-fold at LE04, and 91.2-fold at LE12). The PhA@RPC/ pDNA complexes also produced approximately 2.3-fold lower gene expression levels than those obtained through cotreatment with RPC/pDNA complexes and free PhA at LE02 and LE04, whereas the PhA@RPC/pDNA complexes resulted in a 157fold higher transfection efficiency than that obtained after cotreatment at LE12. These transfection results (specifically those of the RPC/ pDNA complexes) indicate that light irradiation enhances the H

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Figure 5. Time-point effects of light exposure on the (a) cellular uptake and (b) nuclear uptake of PhA@RPC/pDNA and RPC/pDNA (WR 2) complexes in MDA-MB-231 cells after an additional 30 min of incubation after LE02, LE04, or LE12. The FACS histograms were obtained from representative results.

However, in contrast to the 2.6-fold higher transfection efficiency of the RPC/pDNA complexes (WR 2) at LE12 relative to the absence of LE, LE12 did not affect the transfection enhancement of the WR 5 and WR 10 complexes.

In the case of the PhA@RPC/pDNA complexes, the transfection efficiencies were increased to WR 5 and then dropped at WR 10 in the dark; that is, WR 5 and WR 10 produced 36.8-fold and 1.1-fold higher transfection efficiencies, I

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incubation for 30 min was used to achieve at least lightmediated cellular internalization. In particular, the fluorescent intensity of YOYO-1-intercalated pDNA in the polyplextransfected cells was determined from a gated population regarded as live cells after the transfected cells were rinsed with DPBS and detached by trypsin-EDTA solution. A subset of this population of detached cells (1 × 104) was counted to evaluate the amount of polyplex-delivered pDNA that was internalized into the cells and, after live cells were distinguished from dead cells, the number of surviving cells. First, when MDA-MB-231 cells were treated with the RPC/ pDNA complexes, the cellular uptake was almost identical regardless of the absence/presence of light irradiation or the LE time-point (Figure 5a). These results indicate that lighttriggered transfection enhancement may have resulted from a lack of PCI from the polyplexes or other reasons because the polyplexes did not contain PhA and light could not alter the polyplex size or surface charge. Second, without light irradiation, the cellular uptake of the PhA@RPC/pDNA complexes substantially increased relative to that of the RPC/ pDNA complexes at both 2.5 and 4.5 h post-transfection, whereas the PhA@RPC/pDNA complexes showed a slightly higher cellular uptake relative to the RPC/pDNA complexes at 12.5 h post-transfection (Figure 5a). In the dark, the cellular uptake differences between the PhA@RPC/pDNA and RPC/ pDNA complexes may have been influenced by the PhAmediated increase in hydrophobicity of the PhA@RPC/pDNA complexes compared with the RPC/pDNA complexes because these two polyplexes had similar sizes and zeta-potentials. In addition, the PhA@RPC/pDNA complexes had both hydrophobic and cationic surfaces that may have permitted faster cellular internalization than that resulting from cationic polyplexes alone. Although additional effectors may exist, the improved hydrophobicity of polyplexes via the encapsulation of PhA probably influenced the higher transfection efficiencies of the PhA@RPC/pDNA complexes compared with the RPC/ pDNA complexes in the dark. Interestingly, the LE time-point resulted in different cellular uptakes of the PhA@RPC/pDNA complexes. During the polymeric transfection of MDA-MB-231 cells with the PhA@ RPC/pDNA complexes, the LE02 and LE04 treatments significantly increased the fluorescence intensities of the YOYO-1-intercalated pDNA delivered by the PhA@RPC/ pDNA complexes when compared with the absence of LE (i.e., in the dark) at 2.5 and 4.5 h post-transfection, respectively (Figure 5a). The improved cellular uptake of the PhA@RPC/ pDNA complexes at LE02 and LE04 may have been caused by their hydrophobicities as well as the PCI effects of the PhA released from the complexes. Specifically, after LE02 and LE04 were applied, the large reduction in the number of fluorescent PhA@RPC/pDNA complex-transfected cells indicates that LE02 decreased the viability of PhA@RPC/pDNA complextransfected cells and that LE04 severely affected cell death (Figure 5a). It was expected that a major contributor to the LE02 and LE04-mediated phototoxicity and transfection results would be the PhA-initiated PCI effect, which resulted in the improved cellular uptake of the PhA@RPC/pDNA complexes and, consequently, the significant phototoxicities mediated by damaged phospholipids in the plasma and endosomal membranes. This finding may also explain the high transfection efficiencies because dying cells show a greater cellular uptake of the complexes. In addition, the hydrophobic character of the polyplexes would have been a minor contributor to their

respectively, compared with that produced by WR 2. Specifically, in the dark, the PhA@RPC/pDNA complexes caused 20.3-fold and 146.3-fold higher transfection efficiencies at WR 2 and WR 5, respectively, compared with that produced by the RPC/pDNA complexes, whereas these two polyplexes had approximately the same transfection efficiencies at LE12. The LE12-triggered transfection efficiencies of PhA@RPC/ pDNA complexes were also 11.5-fold (at WR 2), 2.6-fold (at WR 5), and 3.0-fold (at WR 10) higher than those in the dark and 91.2-fold (at WR 2), 470.4-fold (at WR 5), and 3.1-fold (at WR 10) higher than the transfection efficiencies of the RPC/ pDNA complexes. The cytotoxicities of the RPC/pDNA complexes were not significantly affected by the WR ranges tested or by light irradiation at 12 h post-treatment (Figure 4d). However, although the absence/presence of light did not induce cytotoxicity in the PhA@RPC/pDNA complex-transfected cells, their light-induced cell viabilities at LE12 were slightly decreased from approximately 85−90% at WR 2 to 55−60% at WR 10. The effects of LE on the transfection efficiency and cytotoxicity were also investigated in HeLa cells. Similarly to the transfection results in MDA-MB-231 cells, LE12 caused very slight increases or no change in the transfection efficiencies of the RPC/pDNA complexes (i.e., 1.7-fold at WR 2, 1.3-fold at WR 5, and 1.0-fold at WR 10 compared with the transfection efficiencies of the complexes in the dark) (Figure S4a). Moreover, the PhA@RPC/pDNA and RPC/pDNA complexes showed similar WR-dependent transfection patterns in the dark: the PhA@RPC/pDNA (WR 5) complexes presented a transfection efficiency that was 291-fold greater than those of RPC/pDNA (WR 5) complexes, but the transfection efficiencies of the two polyplexes did not differ at WR 2 and WR 10. Interestingly, LE12 enhanced the transfection efficiencies of the PhA@RPC/pDNA complexes by as much as 244.9-fold at WR 2, 2.5-fold at WR 5, and 3.0-fold at WR 10 compared with the transfection efficiency without LE. The LE12-mediated transfection enhancement of the PhA@RPC/ pDNA complexes was 160.3-fold (at WR 2), 572.2-fold (at WR 5), and 3.1-fold (at WR 10) higher than the transfection efficiency of the RPC/pDNA complexes. The viabilities of the polyplex-transfected HeLa cells were above 90% for the tested WRs (Figure S4b). From the light-induced transfection and cytotoxicity results, we found that (1) light irradiation enhanced the polyplex transfection efficiency despite the deficiency in PhA; (2) the PhA@RPC/pDNA complexes induced higher transfection efficiencies than those obtained with the RPC/pDNA complexes in the dark; (3) both LE02 and LE04 caused severe phototoxicity and light-induced transfection enhancements in the PhA@RPC/pDNA complexes; and (4) LE12 induced both negligible phototoxicity and light-induced transfection enhancement in the PhA@RPC/pDNA complexes. Photoinduced Cellular and Nuclear Internalization of the PhA@RPC/pDNA Complexes. In general, it is known that the size, zeta-potentials, and hydrophilicity/hydrophobicity of polyplexes influence directly their cellular uptake and as a result, their transfection efficiency and cytotoxicity.35 Thus, to understand these findings in transfection and viability studies, the cellular uptake of the polyplexes (i.e., PhA@RPC/pDNA and RPC/pDNA complexes) was examined at predetermined time-points by using the pDNA-intercalating YOYO-1 fluorescent dye at WR 2. After light irradiation, an additional J

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Figure 6. (a) Intracellular ROS levels, (b) activated NF-κB p-p105 levels, and (c) gene promoter (CMV versus SV40)-dependent transfection efficiency in PhA@RPC/pDNA complex-transfected and RPC/pDNA complex-transfected MDA-MB-231 cells at 13 h post-transfection (i.e., an additional 1 h of incubation) (a,b) or 24 h post-transfection (i.e., an additional 12 h of incubation) (c) after treatment with either no LE or LE12. The data in (c) are expressed as the means ± SE (n = 9−20).

the nuclear uptake of polyplexes was remarkably lower in the light-exposed cells than in the cells without light (Figure 5b). These results indicate that the nuclear uptake of polyplexes was not influenced by either the PhA-mediated PCI effects or the PhA-mediated hydrophobic surface character of the polyplexes. Specifically, a lack of PCI effects in the nuclear uptake of polyplexes in the presence of light might be supported by the preferred mitochondrial localization of PhA.33,36−38 In spite of the slightly lower nuclear uptake of polyplexes in under LE relative to those in the dark, the LE-mediated transfection enhancements suggested that other PhA-induced effectors may exist. Transfection Enhancement of PhA@RPC/pDNA Complexes via ROS-Triggered NF-κB Activation. We considered ROS to be a potential effector for LE (specifically, LE12)mediated transfection enhancement in PhA@RPC/pDNA complex-transfected cells because PhA@RPC complexes were more hydrophobic than RPC and because ROS could be produced by polycations and PhA in the dark. ROS have proven to be a double-edged sword: though high ROS levels usually cause mitochondrial dysfunction and cell death,39 at certain concentrations, ROS improve cell survival rates and activate various signaling pathways, such as NF-κB. Interestingly, it has been shown that the NF-κB activated by ROS translocates from the cytosol into the nucleus and then the nuclear NF-κB reactivates a silent CMV promoter,40−43 thus resulting in the resumed gene expression of pDNA equipped with a CMV promoter. This phenomenon has been demonstrated in our previous islet transfection study, in

improved cellular uptake at LE02 and LE04. However, unlike both the enhanced cellular uptake and the lethal phototoxicities of the PhA@RPC/pDNA complexes at LE02 and LE04, the fluorescent YOYO-1 intensities of the polyplexes in the transfected cells at LE12 were slightly higher than those without LE, and the cells showed negligible phototoxicity because the light irradiation did not induce any changes in the number of gated live cells (Figure 5a). In the case of transfection with the PhA@RPC/pDNA complexes at LE12, an effector for their improved transfection efficiencies may have been the hydrophobicity induced by loading PhA into the polyplexes because the negligible phototoxicity may have negated the role of PhA-mediated PCI effects as a major effector. Although high cellular uptake of pDNA generally results in high transfection efficiencies, the nuclear uptake of pDNA is more strongly correlated than its cellular uptake with the estimated transfection efficiency. Thus, the nuclear uptake of YOYO-1-intercalated pDNA delivered by polyplexes was investigated after isolation of the nuclei from polyplextransfected MDA-MB-231 cells. First, the nuclear uptake of PhA@RPC/pDNA complexes was higher than that of the RPC/pDNA complexes, regardless of the absence or presence of LE or the LE time-point (Figure 5b). The results indicate that the high cellular uptake of polyplexes resulted in a high nuclear uptake. Moreover, although the higher nuclear uptake of polyplexes in the light-exposed cells was expected compared with that in the light-unexposed cells, LE caused the unexpected nuclear uptake of polyplexes. That is, interestingly, K

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transfection to a greater extent (approximately 233-fold in CMV polyplex-transfected cells versus 4.5-fold in SV40 polyplex-transfected cells) when LE12-triggered PhA@RPC/ pDNA complexes were compared with RPC/pDNA complexes in the dark. The different transfection enhancement of the MDA-MB-231 cells transfected with different promoter genes indicates that the ROS generation induced by both LE12 and PhA differentially affected the two promoters and that LE12 did not induce PhA-mediated PCI. Moreover, unlike the SV40 promoter, the CMV promoter may have been influenced by LE12, thus resulting in higher gene expression in the presence of PhA. With the activated NF-κB results (Figure 6b), the transfection results of the CMV-polyplexes further suggested that the ROS-triggered reactivation of NF-κB improved the gene expression of the delivered pDNA in cells. Most stimuli-responsive gene delivery systems have responded to one or two stimuli, and the use of three triggers is uncommon. However, the designed polymeric gene nanocomplexes were tempo-spatially and sequentially activated by four stimuli such as endosomal acidic pHs, intracellular thiols, external light, and intracellular ROS. Specifically, LE timepoints tuned either less toxic quadruple stimuli-activatable nanocomplex (at LE12) or highly toxic triple stimuli (i.e., light, pH, and thiol)-responsive nanocarriers (at LE02 or LE04). In addition, unlike other systems, all major components such as the gene carrying polymer (RPC), the photosensitizer (PhA), and the genetic therapeutic (pCMV-Luc) were responded to sequentially generating multistimuli, resulting in creating various chemical and biological solutions against intracellular multihurdles of low gene expression. The designed gene delivery systems and principles could be applied in various biomedical and pharmaceutical fields such as gene and drug delivery, gene design, diagnostics, and molecular imaging and also could be extensively utilized in cosmetics, agricultures, and food sciences.

which polyplex-transfected islets showed oscillating expression of the reporter protein despite a single treatment with the polyplex.44 Thus, the generation of ROS, the activation of NFκB, and the promoter-mediated effects were investigated during light-triggered gene transfection of the PhA@RPC/pDNA complexes. First, the intracellular ROS levels were monitored by using a fluorescent DCFDA dye. Treatment with NAC, a ROS scavenger, was also tested to confirm whether the detected green fluorescence was derived from ROS. Nontransfected cells showed very low ROS levels regardless of LE, whereas the treatment with the ROS trigger, H2O2, markedly generated intracellular ROS (Figure 6a). Despite our expectation of polycation-mediated ROS generation, the RPC/pDNA complex-transfected cells did not produce significant levels of intracellular ROS either in the dark or after light irradiation. These results may have been caused by the transformation of HMW RPC polymers to low-cytotoxic LMW bPEI derivatives via intracellular thiol-triggered biodegradation of the disulfide bonds in the RPC polymers. However, LE12 triggered PhA@ RPC/pDNA complex-transfected cells to produce a substantial level of intracellular ROS. Specifically, the quantitative analysis demonstrated that light-exposed PhA@RPC/pDNA complextransfected cells had approximately 3.2-fold higher intracellular ROS levels than either light-treated untransfected cells or lighttreated RPC/pDNA complex-transfected cells (Figure S5a). ROS-activated NF-κB signaling was also monitored by the detection of a phosphorylated IκB protein (p-p105). In general, the activity of NF-κB proteins is silenced by interactions with inhibitory IκB proteins, such as IκB family members and the precursor proteins, in the cytosol. The activation of NF-κB proteins proceeds on multiple steps: (1) intracellular ROS triggers the transformation of IκB kinases (IKK) to phosphorylated IKK; (2) the inhibitory IκB proteins are phosphorylated by the activated IKK; (3) the proteasomal degradation of IκB activates NF-κB subunits; and (4) the activated NF-κB subunits are translocated into the nucleus. Regardless of LE, a Western blot of nontransfected cells and RPC/pDNA complex-transfected cells showed very weak intensities of a phosphorylated precursor protein (p-p105), thus indicating expression of activated NF-κB proteins (Figure 6b). In the dark, the PhA@RPC/pDNA complex-transfected cells still exhibited a weak p-p105 signal. However, LE12 induced high levels of p-p105 in the PhA@RPC/pDNA complex-transfected cells, thus indicating high activity of NFκB. A quantitative analysis of p-p105 indicates that LE12 generated approximately 3.8-fold higher NF-κB activity than was generated in the absence of LE in PhA@RPC/pDNA complex-transfected cells (Figure S5b). The gene expression levels of two luciferase pDNAs with either a CMV promoter or an SV40 promoter were compared to determine whether the activated NF-κB would enhance gene expression via the reactivation of the CMV promoter in a luciferase reporter gene. In the RPC/pDNA complex-transfected cells, LE12 increased the luciferase expression of the pDNA with a CMV promoter by approximately 2.6-fold relative to the cells without LE, whereas the pDNA with an SV40 promoter produced a similar level of the corresponding protein regardless of LE (Figure 6c). Similarly, LE12 triggered approximately 11.5-fold and 4.5-fold higher levels of luciferase in the PhA@RPC/pCMV-Luc complex-transfected cells and PhA@RPC/pSV40-Luc complex-transfected cells, respectively. However, a combination of LE12 and PhA enhanced

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CONCLUSION In this study, we developed sequentially activatable polymeric gene complexes that responded to quadruple stimuli. For this purpose, a light-responsive photosensitizer, PhA, was physically loaded into a thiol-degradable and endosomolytic polycation, RPC, thus resulting in the formation of PhA@RPC. Then, the pDNA with the CMV promoter, which can be reactivated by NF-κB, was complexed with PhA@RPC, thus yielding PhA@ RPC/pDNA complexes. When we applied either LE02 or LE04 to the PhA@RPC/pDNA complex-transfected cells, lightinduced membrane disruption enhanced cellular uptake and endosomal release, but resulted in detrimental cytotoxicity. However, after the sequential activation of RPC-mediated proton buffering and intracellular biodegradation of PhA@ RPC/pDNA complexes in response to endosomal pH and intracellular thiol, respectively, light exposure (LE12) induced PhA-mediated ROS generation, NF-κB activation, and the reactivation of the CMV promoter, thus resulting in high transfection efficiency and negligible cytotoxicity of the PhA@ RPC/pDNA complexes. Our findings indicate that the light exposure time-point strongly influences the transfection efficiency and cytotoxicity of gene delivery systems. Moreover, with applying the late exposure of light, the PhA@RPC/pDNA complexes could realize tempo-spatial and sequential activation of consecutive quadruple stimuli (i.e., pH, thiol, light, and ROS), resulting in activating chemical or biological responses L

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Molecular Pharmaceutics

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and overcoming the multistep intracellular hurdles of nanosized gene delivery systems.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b01065. Dark-toxicity and phototoxicity of RPC in MDA-MB-231 cells, representative TEM images of polyplexes, timepoint effects of light exposure on cell viability of polyplexes in MDA-MB-231 cells, WR-dependent transfection efficiency and cell viability of polyplexes in HeLa cells, and light-triggered change in intracellular ROS levels and the activated NF-κB p-p105 levels in polyplextransfected MDA-MB-231 cells (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-2-2164-6533. Fax: +82-2-2164-4059. E-mail: [email protected]. ORCID

Kang Moo Huh: 0000-0002-2406-6659 Han Chang Kang: 0000-0003-0696-1155 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Research Foundation of Korea (NRF) grants funded by Ministry of Science, ICT, and Future Planning (NRF-2015R1A1A05001459). H.C. and H.C.K. were supported by BK21PLUS grant of NRF funded by Ministry of Education (22A20130012250).

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REFERENCES

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DOI: 10.1021/acs.molpharmaceut.6b01065 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.molpharmaceut.6b01065 Mol. Pharmaceutics XXXX, XXX, XXX−XXX