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Highly Effective Photocontrollable Drug Delivery Systems Based on Ultrasensitive Light-Responsive Self-Assembled Polymeric Micelles: An In Vitro Therapeutic Evaluation Chih-Chia Cheng, Jyun-Jie Huang, Ai-Wei Lee, Shan-You Huang, Chien-Yu Huang, and Juin-Yih Lai ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00146 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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ACS Applied Bio Materials

Highly Effective Photocontrollable Drug Delivery Systems Based on Ultrasensitive LightResponsive Self-Assembled Polymeric Micelles: An In Vitro Therapeutic Evaluation Chih-Chia Cheng,ab* Jyun-Jie Huang,a Ai-Wei Lee,c Shan-You Huang,a Chien-Yu Huang,e and Juin-Yih Laiabdf a. Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Taipei 10607, Taiwan. E-mail: [email protected] b. Advanced Membrane Materials Research Center, National Taiwan University of Science and Technology, Taipei 10607, Taiwan. c. Department of Anatomy and Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan. d. Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, 10607, Taiwan. e. Graduate Institute of Cancer Biology and Drug Discovery, Graduate Institute of Clinical Medicine and Department of Surgery, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan. f. R&D Center for Membrane Technology, Chung Yuan Christian University, Chungli, Taoyuan 32043, Taiwan. * Corresponding Author

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KEYWORDS. Controlled drug delivery; Polymer assembly; Photoresponsive micelles; Realtime cytotoxicity assays; Water-soluble functional block copolymer.

ABSTRACT. An ultrasensitive light-responsive block copolymer, a combination of multi-armed poly(ethylene glycol)-b-poly(caprolactone) polymer as a water-soluble element and maleimideanthracene linkers as a photosensitive group, was successfully synthesized and rapidly selfassembles to form spherical micellar nanoparticles in aqueous media and phosphate-buffered saline. Their unique characteristics, such as extremely low critical micelle concentration, desirable micellar stability, well-controlled light-responsiveness, tailorable drug-loading content and ultrasensitive light-induced drug release, make these micelles potential candidates for development of a more effective, safer drug delivery platform for cancer treatment. In vitro studies revealed the drug-loaded micelles exhibited high structural stability in serum-containing media and very low toxicity towards normal and cancer cells under physiological conditions. Irradiation of cancer cells incubated with the drug-loaded micelles with ultraviolet light at 254 nm for only 10 seconds triggered rapid and complete release of the drug in the intracellular environment, and induced strong antiproliferative/cytotoxic activity. Importantly, real-time cytotoxic assays and ﬂuorescence imaging analysis further demonstrated the drug-loaded micelles were rapidly taken up into the cytosol or nuclei of the cells and subsequent ultraviolet exposure induced drug release and apoptotic cell death. Given their simplicity of design, high reliability and performance, this new light-sensitive micelle may provide a promising route for developing a multifunctional therapeutic nanocarrier system.


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

Introduction

Over the last few decades, amphiphilic block copolymers (ABCs) that spontaneously selfassemble in an aqueous environment into spherical micellar nanoparticles with a hydrophobic segment and hydrophilic segment have an essential role in medicine as nanocarriers to enhance the effectiveness of chemotherapy.1-6 The ability to control the self-assembly of ABCs in aqueous media provides versatility and adaptability to tune the formation of various micellar nanostructures, including core-shell nanospheres, nanocapsules, hollow spheres and vesicular structures.7-10 These strategies have enabled the design of effective drug delivery systems that offer stable transport of anticancer drugs and also improve therapeutic safety and minimize side effects through the enhanced permeability and retention (EPR) effect.11-13 In general, fabrication of biodegradable/biocompatible ABC-based micelles has recently attracted the notice of researchers from different medical specialties due to the potential of polymeric micelles to enhance the safety and effectiveness of chemotherapy in clinical trials.9,14-16 For example, the poly(ethylene

glycol)-block-poly(ε-caprolactone)

(PEG-b-PCL)

copolymer,

a

highly

biodegradable and biocompatible functional biomaterial, has already been recognized by the U.S. Food and Drug Administration for clinical use.17-19 Thus, PEG-b-PCL and its derivatives have been widely used in various biomedical application..19,20 However, the use of PEG-b-PCL copolymers as nanoparticle drug delivery systems still presents several serious limitations. With respect to PEG-b-PCL polymeric micelle-based drug delivery systems, restrictions such as a slow hydrolytic degradation of the PCL block in the aqueous environment and the lack of responsiveness of drug-encapsulated micelles to specific environmental triggers in the tumor microenvironment result in insufficient drug-induced cytotoxicity in cancer cells and limit the efficacy of chemotherapy.21-26 The challenge still needs to overcome these obstacles: design and


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exploitation of an externally stable, environmental-stimuli-responsive PEG-b-PCL system with improved micellar drug encapsulation capability that exhibits highly effective stimuli-responsive drug release in the extracellular conditions of the tumor microenvironment, may represent a promising therapeutic route for cancer treatment. Development of stimuli-responsive systems in recent years has provided an effective strategy to resolve the issues described above. Environmental stimuli-responsive systems have gained significant research attention as potential polymeric nanocarriers for the encapsulation and delivery of active anticancer agents, due to their ability to accommodate drugs with a wide range of characteristics and allow well-controlled drug release in response to changes in both the external environment and internal physiologic environment.27-30 Among the various environmental stimuli that affect the efficiency of drug delivery from associative ABC systems, temperature,31 pH,32 gas,33 redox34 and light are the most widely used and well-studied; light stimuli have recently received increasing attention in the research community.35-38 Lightresponsive polymeric micelles (LRPMs) can be rapidly activated by short-term light exposure to rapidly release the encapsulated drug, thus light has attracted enormous research interest as an effective stimulus for a broad range of biotechnical and biomedical applications from drug delivery to photothermal/photodynamic therapy.39 Although many LRPMs possessing unique physical properties have been designed for cancer treatment,36,37,39 they do not achieve sufficiently specific delivery and release of the caged anticancer drug in the tumor interstitium. and the drug-loaded micelles appear to lack long-term stability in serum-containing biological media. These issues and lead to premature drug leakage and accumulation of the drug in nonspecific tissues, thus significantly increasing the risk of chemotherapy side-effects.17,40-43 Thus, LRPMs with improved micellar stability in blood and whose uptake by cells of


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reticuloendothelial system has effectively promoted development of nanocarrier-based platforms for safer and more effective biomedical applications. We recently successfully demonstrated that incorporation of stimuli-sensitive molecular groups (SSMGs) into functional PCL possesses excellent stimuli-responsiveness and mechanical performance in combination with substantially improved thermal stability and self-healing ability.44-47 When SSMGs were incorporated into a PEG/PCL blend system, the resulting polymers directly self-assembled in water into spherical micellar structures suitable for drug delivery applications; these micelles had a high degree of drug-loading capacity and exhibited stable drug entrapment and modulated drug release in response to changes in their environment, such as pH and temperature.48 Based on our findings from previous studies on SSMGs that illustrated the pivotal role of the PEG and PCL polymers in establishment of stimulus-responsive connections, we investigated whether introducing highly light-sensitive molecular groups (maleimide-anthracene linkers) into the segment junctions of multi-armed PEG-functionalized PEG-b-PCL polymers would significantly influence their ability to form an ultrasensitive lightresponsive LRPM. As hypothesized, this strategy generated micelles with unique amphiphilic characteristics, high micellar stability, rapid light-responsiveness, tailorable drug-loading content and controlled drug release profiles, with promising potential as a multifunctional nanocarrier for the development of advanced drug-delivery systems (Scheme 1). The drug-loaded micelles showed extremely stable in serum-rich media over long periods of time, and rapid and complete drug release after exposure to ultraviolet (UV) light for only 10 seconds; these micelles increased the cytotoxicity of the encapsulated drug towards human oral cancer cells in vitro. More importantly, real-time cytotoxic assays and fluorescence imaging indicated the drug-loaded micelles were rapidly and efficiently taken up by cells under physiological conditions and


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accumulate within the nucleus. Incubation of cells with the drug-loaded micelles for different periods of time followed by short-term exposure to UV radiation effectively suppressed the proliferation and viability of cancer cells lines in vitro, indicating this distinctive class of LRPM may act as highly efficient drug-delivery vehicle. Although UV light may not be able to reach deeper layers of human skin, the reaction for the destruction of maleimide-anthracene linkers can be conducted throughout a broad range of UV region, which may meet the requirements for chemotherapy and targeted treatments.49 To the best of our knowledge, this is the first reported development of light-responsive block copolymer micelles with tunable micellar characteristics that achieve the goal of highly efficient light-triggered drug delivery. These micelles could potentially reduce the toxicity of anticancer drugs in normal tissues, and enable enhanced cellular drug uptake and precisely intracellular release of drugs to enhance the effectiveness of cytotoxic chemotherapy. Thus, this work indicates these newly-developed polymeric micelles with lightresponsive drug release performance could improve the safety and efficacy of chemotherapy.


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Scheme 1: Structural and graphical representations of controlled drug loading and release by light-sensitive 3PEG-PCL micelles.

 Experimental section The experimental procedures, spectroscopy, analytical techniques, monitoring instrument and equipment used in this study are described in detail in Electronic Supplementary Information.



Results and Discussion


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Our general strategy of producing light-responsive PEG-b-PCL involved an innovative, efficient synthetic route via a simple three-step reaction, as presented in Schemes S1 and S2 (see Materials and Methods Section of Electronic Supplementary Material for further detail). We prepared well-controlled mono- and three-armed anthracene end-capped PCLs (1An-PCL and 3An-PCL) via a combination of ring-opening polymerization and a copper-catalyzed azidealkyne cycloaddition reaction. Subsequent functionalization via a Michael addition reaction with maleimide end-capped PEG (maleimide-PEG; average molecular weight = 5000, ca. 114 repeat units) was employed to produce two different multi-armed PEG-functionalized PEG-b-PCL polymers, the chemical structures of which are presented in Schemes 1 and S2. PEG-b-PCLs containing two different arm numbers of PEG blocks (mono- and tri-arm PEGs), hereafter termed 1PEG-PCL and 3PEG-PCL, respectively, were purified by dialysis. These polymers were recovered at high yields (> 85%) and the resulting products possessed the desired structures and had high molecular weights (Mw) and narrow polydispersity indexes (PDIs), as confirmed by proton nuclear magnetic resonance spectroscopy (1H-NMR; Figures S1-S4) and gel-permeation chromatography (GPC; Figures S5 and S6). Interestingly, water-solubility tests showed introduction of the PEG segments into the block copolymer structures substantially improved hydrophilicity and led to very high water solubility, even at concentrations above 40 mg/mL. This feature may probably be attributed to the hydrophobic-hydrophilic balance that promotes the formation of self-assembled hierarchical structures in aqueous environments. To understand the influence of phase separation between the hydrophobic PCL and hydrophilic PEG segments on the water solubility-structure relationships in the PEG-b-PCL system, we determined the critical micelle concentrations (CMCs) of 1PEG-PCL and 3PEG-PCL in water via a fluorescence and ultraviolet-visible technique using hydrophobic pyrene as a fluorescent probe.50 As shown in


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Figure 1a, the 3PEG-PCL polymer had an extremely low CMC value of 4.2 x 10-6 mg/mL, significantly lower than that of 1PEG-PCL (approximately 5.5 x 10-6 mg/mL). In contrast, a lack of CMC characteristics were measured over a wide concentration range for maleimide-PEG, suggesting that increasing the PEG content of PEG-b-PCL micelles significantly improved their structural stability in water. This observation also demonstrates more branched PEGs effectively contain distinct hydrophilic and hydrophobic regions, which enables further self-assembly into stable, spherical micelles in aqueous environments. To confirm the formation of micelles in water, dynamic light scattering (DLS), atomic force microscopy (AFM) and scanning electron microscopy (SEM) measurements were carried out at 25 °C. When the polymer concentrations were above the CMC (1.0 mg/mL in water), 1PEGPCL and 3PEG-PCL exhibited mean particle sizes of 150.2 ± 18.3 nm and 34.5 ± 10.5 nm, respectively, suggesting the amphiphilic PEG-b-PCL polymers composed of hydrophilic and hydrophobic blocks spontaneously formed nanosized micelles in aqueous media, and a higher branching density and PEG fraction within the block copolymer structure enhanced hydrophilicity, leading to smaller, more tightly packed micelles (Figure 1b). SEM and AFM validated the DLS results, and confirmed the 3PEG-PCL polymer formed spherical micelles with a spherical micellar size in the range of 30–50 nm (Figure S7). These experiments also revealed the 3PEG-PCL micelles possess a highly stable morphology due to the well-controlled hydrophilic properties of the tailored polymer structure, which results in formation of lowdimensional spherical nanoparticles. To gather further insight into the stability of the micelles in aqueous physiological environments, kinetic DLS assays of 1PEG-PCL and 3PEG-PCL were performed at 25 °C in the presence of biological media containing 66 v% deionized water and 34 v% fetal bovine serum (FBS), which functions as an effective nanoparticle-destabilizing


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agent.40,51,52 As shown in Figure 1c and 1d, all observations in the absence of FBS agreed well with the DLS size results. After 24 hours of monitoring in the presence of FBS, the size and shape distributions of 3PEG-PCL micelles remained almost unchanged, indicating that increased PEG branching protects the hydrophobic PCL segments within the inner part of the micelle, thus conferring the structural stability required to resist prolonged FBS treatment. Conversely, the size and shape distribution of 1PEG-PCL micelles changed significantly during long-term treatment with FBS, even though mono-armed PEG segment was stably present in the hydrophilic segment of the block copolymer architecture. This implies that both the volume fraction and geometric conformation of the hydrophilic segment play critical roles to modulate and stabilize micellar structure. Multi-armed or branched PEG segment in the micellar outer shell maintain its overall spherical integrity, and FBS can barely diffuse into the internal region of 3PEG-PCL micelles to disrupt the hydrophobic interactions between the aggregated PCL segments. These results also demonstrated that the 3PEG-PCL system is sufficiently stable under physiological conditions, making this material potential suitable for a broad range of medical applications related to nanotechnology.


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Figure 1: (a) CMC determination and (b) DLS analyses of 1PEG-PCL and 3PEG-PCL micelles in water at 25 °C. Particle size distributions measured at 25 °C for (c) 3PEG-PCL and (d) 1PEGPCL micelles over time after incorporation of FBS into the polymer solution. (c, d) The insets show the particle sizes of 1PEG-PCL and 3PEG-PCL micelles in aqueous solution over time after incorporation of FBS into the polymer solution. To further evaluate the environmentally responsive behavior of 3PEG-PCL micelles in aqueous medium, environment-dependent DLS studies were carried out by detecting the particle size changes of the sample solutions in situ as variations of pH (7.4 and 6.0), temperature (25–55 °C) and UV light exposure (254 nm; 50-70 mW/cm2). As shown in Figures S8 and S9, the average particle diameter and intensity autocorrelation function (G) of 3PEG-PCL micelles in


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aqueous solution did not change significantly when the environmental pH was decreased from 7.4 to 6.0 and maintained for 24 h or the environmental temperature was stepwise increased to 55 °C, suggesting the micelles do not undergo dramatic structural transitions in response to changes in pH and temperature under a wide range of physiological conditions. However, the particle size distribution and fluorescence intensity of the micelles significantly changed after exposure to UV light for varying periods of time. The photoluminescence (PL) spectra in Figure S10 demonstrated the rapid, well ‐ controlled photo-cleaving kinetics: the fluorescence intensity of 3PEG-PCL solution at 419 nm abruptly decreased by up to 25% after 10 seconds of UV irradiation and by up to 78% after 60 seconds irradiation, indicating exposure to UV light rapidly disrupted the maleimide-anthracene cycloadduct structures within the interior of the micelles, which promoted disassembly of the micelle structures and decreased the fluorescence intensity. After UV irradiation for 60 seconds, the G curve of 3PEG-PCL micelles gradually shifted to higher delay times (τ) as increasing measuring time, suggesting that larger hydrophobic clusters are formed in aqueous solution. In other words, irradiation cleaved the 3PEG-PCL polymers back to their original homopolymers, leading to degradation of the micelles and formation of hydrophobic PCL aggregates in aqueous solution. Thus, these features indicate ultrasensitive light-responsive 3PEG-PCL micelles possess high potential for improving the release of drug payloads in response to external UV irradiation. Next, we further assessed the drug-loading capacity and stability of 3PEG-PCL micelles incorporating anticancer drug doxorubicin (DOX). After DOX was encapsulated by 3PEG-PCL, the average micelle size increased progressively from 159 ± 3 nm to 272 ± 18 nm in a monodisperse distribution (PDI > 0.04) as the loaded DOX content increased from 4.3 to 11.1% (Figure S11a and Table S1), indicating micellar expansion can provide a loading space to


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accommodate DOX within the micellar core to obtain a high DOX-loading capacity. The maximum DOX-loading content was ca. 11.1% with a zeta potential value of 30.5 ± 1.8 mV, suggesting the final DOX loading content of the micelles can be efficiently tailored by simply varying the blend composition. In further verification of the size and shape of the DOX-loaded micelles, AFM and SEM images (Figure S11b-d) demonstrated 3PEG-PCL micelles containing 4.5% DOX were large spherical particles with diameters of 100-160 nm, which is in agreement with the DLS data (Figure S11a). These results further indicated DOX could be successfully loaded into the hydrophobic PCL segment of the polymeric micelles, thus conferring preciselytailorable drug loading content. The structural stability of DOX-loaded 1PEG-PCL and 3PEGPCL micelles in the presence of FBS-containing medium was directly assessed via DLS at 25 °C (Figure 2a).40,51,52 After 24 h of monitoring, the incorporation of a high concentration of FBS in the aqueous media did not remarkably affect the shape and size distribution of DOX-loaded 3PEG-PCL micelles, suggesting high structural stability. Conversely, large aggregate peaks centered at 1700 nm gradually formed in the DOX-loaded 1PEG-PCL micellular solution as monitoring time increased, possibly attributed to the release of hydrophobic DOX from the micelles, thus resulting in the formation of large DOX aggregates.43 In other words, the lower the fraction of PEG segments, the lower the DOX-entrapment stability of the micelle; lower stability caused in sustained release behavior of the drug and formation of large hydrophobic DOX clusters. These observations suggest that the hydrophilicity and branch geometry of the PEG segments in the outer shell of the micelles play vital roles in long-term stabilization of micellar structure during prolonged FBS treatment; a high-volume fraction and increased branch structure within the PEG segments maintain the integrity of micellar conformation. FBS could barely diffuse into the 3PEG-PCL micelles and thus had limited ability to disturb the hydrophobic


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interactions between DOX and PCL segments; such high stability under physiological conditions is a feature that is highly desirable in conventional block copolymer-based drug delivery systems. Hence, these results inspired us to further explore the pH/light-triggered drug-release profiles and in vitro cytotoxicity of DOX-loaded 3PEG-PCL in phosphate buffered saline (PBS).

Figure 2: (a) DLS results measured at 25 °C for DOX-loaded 1PEG-PCL and 3PEG-PCL micelles over time after incorporation of FBS into the polymer solution. (b) Drug release profiles for DOX-loaded 3PEG-PCL micelles in PBS at 37 °C with different pH conditions (pH 6.0 and 7.4). (c) UV light-responsive DOX release profiles for 3PEG-PCL micelles in PBS at 37 °C in response to different durations of UV exposure. The inset graph shows the destructive bondscission reaction that occurs within 3PEG-PCL micelles after UV irradiation. We subsequently assessed the effects of UV radiation and pH on drug release behavior. As presented in Figure 2b, DOX-loaded 3PEG-PCL micelles in PBS exhibited a slow sustainable release under physiological conditions (pH 7.4 and 37 °C) with only 22.1 ± 1.6% of the drug


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released after 48 h, suggesting high structural stability of DOX-loaded 3PEG-PCL micelles. Even when the environmental acidity was decreased to pH 6.0 and the temperature maintained at 37 °C, only 29.8 ± 6.0% of the DOX was released within the initial 12 h with cumulative release of 45.9 ± 6.8% after 48 h, indicating that the weakly acid environment only had a limited influence on drug release behavior, even over an extended time period. This further confirmed pH does not trigger drug release from DOX-loaded 3PEG-PCL micelles under weakly acidic conditions.21-26 Conversely, DOX-loaded micelles in PBS exhibited excellent controlled drug release characteristics when exposed to UV light for different periods of time. The micelles rapidly and completely released the DOX after only 10 seconds exposure to UV light in PBS solution at pH 7.4 and 37 °C (Figure 2c). This effect could be attributed to disruption of the maleimide-anthracene cycloadduct structures by UV light followed by rapid reversion back to the original precursor materials (as shown in the inset of Figure 2c), leading to complete release of the encapsulated drug within a few seconds. Thus, a combination of the above-mentioned features (excellent micellar stability and ultrasensitive light-triggered controlled drug release) indicates 3PEG-PCL micelles exhibit high potential for controlling the drug release behavior in response to changes in the presence of external light stimuli. Furthermore, we explored the cytotoxic effects of pristine 3PEG-PCL and irradiated 3PEG-PCL micelles towards murine macrophage (RAW 264.7), Meng-1 oral epidermoid carcinoma (OECM-1) and human oral squamous cell carcinoma (SAS) cell lines using a conventional methylthiazolyldiphenyltetrazolium bromide (MTT) assay. Cells were incubated with 3PEG-PCL-containing medium at pH 7.4 and 37 °C for 24 h; no cytotoxic effects were noted in RAW 264.7, OECM-1 or SAS cells over a wide concentration ranging from 0.1 to 100 µg/mL (Figures S12-S15), indicating these polymeric materials and precursors have low toxicity towards normal and cancer cell lines.


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These observations also suggest the irradiation process did not cause any adverse toxic effects towards each cell line (Figure S15 and Table S2). Consequently, we expect DOX-loaded 3PEGPCL micelles would exhibit high cellular uptake rates and could enhance chemotherapeutic efficacy.

Figure 3: (a) Viability analysis of SAS cells treated with free DOX or DOX-loaded 3PEG-PCL micelles determined by MTT assay after incubation for 24 h at 37 °C and pH 7.4. (b) Confocal microscopic images of SAS cells incubated with 50.0 µg/mL of UV-irradiated DOX-loaded 3PEG-PCL micelles (absolute amount of DOX = 5.6 µg; DOX loading content = 11.1 %) for 4, 12 or 24 h at pH 7.4 and 37 °C. SAS cells were stained with the fluorescent nuclear marker DAPI (blue). The DOX conjugate emits red fluorescence at wavelengths ranging from 550 nm to 600 nm (λex = 480 nm). Upper panels, bright field images; middle panels, fluorescence images; lower panels, merged fluorescence images. Scale bars in the lower right corner of each image are


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50 μm. (c) In vitro cytotoxicity profiles determined using the real-time xCELLigence impedance analyzer. Cell proliferation is expressed as cell index (CI) recorded over time. SAS cells were cultured for 22 h. Then, DOX-loaded 3PEG-PCL micelles were added and incubated for another 4 or 12 h before UV irradiation. The concentration and DOX-loading content of 3PEG-PCL micelles were 50.0 µg/mL and 11.1%, respectively. To further investigate the therapeutic effects of this newly-developed photosensitive micelle on human cancer cells, the in vitro cytotoxicity of DOX-loaded 3PEG-PCL micelles and free DOX towards SAS cells were compared using the sulforhodamine-B (SRB) assay. After 24 h of co-culture, free DOX and DOX-loaded 3PEG-PCL micelles exhibited IC50 values (halfmaximal inhibitory concentration) of 4.17 µg/mL and 13.86 µg/mL, respectively (Figure 3a). This result implies that the DOX-loaded nanocarriers entered the cells and exerted the desired pharmacologic effects. Further, compared with free DOX, the higher in vitro IC50 values of DOX-loaded micelles possibly caused by physical entrapment of drugs in the micelle cores, which can significantly affect the release of DOX from the micelles into the cancer cells by diffusion and thus delayed nuclear uptake.43,46 The in vitro MTT assay confirmed similar trends in the cytotoxicity profiles of DOX-loaded 3PEG-PCL micelles as the SRB assay (Figure S16), further confirming that DOX-loaded micelles exhibit dose-dependent cellular toxicity. In order to gain deeper insight into the mechanisms of intracellular DOX-release behavior and cellular uptake, DOX-loaded 3PEG-PCL micelles or free DOX were directly incubated with SAS cells at 37 °C and pH 7.4 for 4, 12 or 24 h, then the cells were stained with 4,6-diamidino-2phenylindole (DAPI) and observed using confocal laser scanning microscopy (CLSM). Based on the results of the SRB cytotoxicity assay (Figure 3a), the concentration of 50.0 µg/mL DOXloaded 3PEG-PCL micelles (or free DOX) in PBS was chosen, which led to the appropriate


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compromise between DOX dose and cell viability for further CLSM studies. In Figure S17, the red areas indicate DOX fluorescence while the blue areas represent DAPI-stained cell nuclei. After 8 h incubation with free DOX, formation of pink spots was seen in the merged image, suggesting free DOX was rapidly internalized by cells through passive diffusion.53,54 In contrast, pink-fluorescent color was randomly distributed on the outside surface of cells incubated with the DOX-loaded 3PEG-PCL micelles, even after 24 h (Figure S18). These results illustrate 3PEG-PCL micelles function as a stable nanocarrier to efficiently penetrate the plasma membrane of cells and enhance structural stability of physically entrapped DOX during cellular internalization.55,56 Surprisingly, when cells incubated with DOX-loaded 3PEG-PCL micelles were exposed to UV irradiation, the location of drug release in the cells could be easily manipulated by altering the duration of co-incubation prior to irradiation, and the resulting CLSM images also revealed different cellular uptake efficiencies (Figure 3b). After incubating DOX-loaded micelles with cells for 4 h and then exposed to UV irradiation for 10 seconds, the red fluorescent color from DOX was mainly distributed on the cell surface, suggesting DOXloaded micelles slowly diffused across the cell membrane over 4 h, and UV irradiation led to release of DOX and disassembly of the micellar structure within the cytoplasm. As the time of drug incubation was prolonged to 12 h or 24 h before the cells were exposed to UV irradiation of 254 nm for 10 seconds, the pink fluorescence of DOX-loaded micelles gradually transferred from the cell membrane to the nucleus, in stark contrast to non-irradiated cells incubated with free drug and drug-loaded micelles (Figures S17 and S18). These observations demonstrate the DOX-loaded 3PEG-PCL micelles were progressively transported from the cytoplasm into the nucleus. Furthermore, programmed, controlled release of DOX within specific sites of the cell could be achieved via a combination of the above-mentioned features (time-dependent cellular


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uptake and ultrasensitive light-triggered intracellular drug release); these observations are fully consistent with the results of the in vitro cellular cytotoxicity studies (Figures 3a and S16). Overall, these data further prove that 3PEG-PCL represents a well-controlled light-responsive micelle for safe, precise and highly efficient drug delivery. These micelles may have significant potential for and impact on cancer treatment, as the system is extremely sensitive to UV light, could be easily adapted for specific treatment of superficial basal cell carcinomas of the skin and oral cavity, and the cellular uptake and cytotoxicity properties can be precisely tuned to achieve the desired chemotherapeutic efficacy. To further validate the extent of cellular uptake and importance of UV treatment on the cytotoxic effects of the drug-load micelles towards cancer cells, the xCELLigence real-time cell analyzer was used to monitor the viability of SAS cells exposed to DOX-loaded 3PEG-PCL micelles after different periods of UV exposure. After undisturbed culture at 37 °C for 22 h, DOX-loaded 3PEG-PCL micelles (DOX loading content = 11.1 %; absolute amount of DOX = 5.6 µg) were added to the cells at a concentration of 50.0 µg/mL. As presented in Figure 3c, the impedance and cell index (CI) values of the cells substantially increased during further coincubation (for 24 h) with DOX-loaded micelles, indicating DOX remained stably entrapped within the interior of the micelles and did not show significant cytotoxic effects against SAS cells under physiological conditions in vitro, further indicating that the 3PEG-PCL micelles did not prematurely leak DOX before entering the tumor sites or in response to any internal and external stimuli. Conversely, as DOX-loaded micelles were co-incubated with SAS cells for 4 h and then exposed to UV irradiation for 10 seconds, the release of DOX led to 30% cell death within the first 10 h and the CI value rapidly decreased to 18% after 20 h. This implies that after 4 h of co-incubation with cells, the DOX carried by micelles was only transported to the cell


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surface, and subsequently, the released DOX lead to rapid necrotic-type cell lysis as the drug was not internalized into the nucleus after release from the micelles.57 Interestingly, on further prolonging the co-incubation period to 12 h and then exposing the cells to UV irradiation for 10 seconds, the CI value indicated the DOX-loaded 3PEG-PCL micelles significantly inhibited cell growth; this cytotoxic effect was maintained at a constant level and only started to diminish at 10 h after irradiation. Therefore, prolonging the co-incubation time from 4 h to 12 h significantly enhanced the cellular uptake of the DOX-loaded micelles by SAS cells and allowed the micelles to safely transport the loaded DOX into the cell nucleus. Subsequently, UV light-induced release of DOX allowed the drug to interact with the nuclear machinery and induce delayed apoptotic cell death.58 Therefore, the xCELLigence real-time cell analysis confirms that the DOX-loaded micelles have the ability to penetrate deep into the nuclei of SAS cells as the incubation time increased, and that subsequent UV irradiation triggered rapid release of DOX inside cells that induced apoptosis-like programmed cell death. Collectively, these findings clearly demonstrate that ultrasensitive light-responsive 3PEG-PCL micelles not only substantially enhance drug delivery to solid tumors, but also exhibit high structural stability under physiological environment and enable precisely controlled release of DOX within cell nuclei.



Conclusions

We successfully designed and synthesized an ultrasensitive light-responsive block copolymeric micelle, 3PEG-PCL, composed of a photosensitive maleimide-anthracene linker, hydrophilic PEG and hydrophobic PCL segments. 3PEG-PCL can rapidly self-assemble into spherical micellar nanoparticles in aqueous and PBS media that exhibits several unique amphiphilic characteristics, such as a low critical micellar concentration (below 10-5 mg/mL), high micellar


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stability, well-controlled light-responsive behavior, tailorable drug-loading and release performance. In vitro cellular studies confirmed the DOX-loaded 3PEG-PCL micelles showed high structural stability in serum-containing media and excellent biocompatibility towards normal and tumor cells. When irradiated with UV light for 10 seconds, the DOX-loaded micelles rapidly and completely released the drug within cells as a result of disruption of the maleimideanthracene cycloadduct linkers, and the released DOX exerted strong cytotoxic effects to inhibit cell proliferation and growth as the micelles had efficiently transported the DOX into cell nucleus. More importantly, a real-time cytotoxic assay and CLSM further demonstrated that DOX could be progressively transferred from the micelles into the cell interior and accumulate within the nucleus under normal physiological conditions, and subsequent UV radiation induced rapid release of the DOX and promoted apoptosis; this system may offer a potential route to suppress tumor growth and progression in vivo. The results obtained in the study further suggest that 3PEG-PCL micelles could enable safe drug delivery under normal physiological conditions and substantially enhanced the anti-tumor effect of chemotherapy in response to short-term UV treatment. Overall, this study offers a potentially effective pathway to the development of ultrasensitive light-responsive micelles and may help to greatly improve the overall efficiency and effectiveness of chemotherapy; this system could be potentially applied to provide a specific treatment for superficial basal cell carcinomas.

ASSOCIATED CONTENT Supporting Information


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Synthetic details, material characterizations, analytical methods and instrumentation are included in Electronic Supplemental Information.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] Present Addresses 43 Keelung Rd., Sec. 4, Da'an Dist., Taipei City 10607, Taiwan. Author Contributions Jyun-Jie Huang performed all experiments. Chih-Chia Cheng devised the study, designed the experiment and wrote the paper. All results presented in this paper were discussed and agreed by all authors and offered constructive suggestions or critiques to the final manuscript. Funding Sources Ministry of Science and Technology, Taiwan (contract no. MOST 105-2628-E-011-006-MY2 and MOST 107-2221-E-011-041-MY3). Notes The authors declare no competing financial interest. Acknowledgment


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This study was supported financially by the Ministry of Science and Technology (MOST), Taiwan, under contracts MOST 105-2628-E-011-006-MY2 and MOST 107-2221-E-011-041MY3.

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