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Self-Assembled pH-Responsive Polymeric Micelles for Highly Efficient, Non-Cytotoxic Delivery of Doxorubicin Chemotherapy to Inhibit Macrophage Activation: In Vitro Investigation Zhi-Sheng Liao, Shan-You Huang, Jyun-Jie Huang, Jem-Kun Chen, Ai-Wei Lee, Juin-Yih Lai, Duu-Jong Lee, and Chih-Chia Cheng Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00380 • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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Self-Assembled pH-Responsive Polymeric Micelles for Highly Efficient, Non-Cytotoxic Delivery of Doxorubicin Chemotherapy to Inhibit Macrophage Activation: In Vitro Investigation Zhi-Sheng Liao,a Shan-You Huang,a Jyun-Jie Huang,a Jem-Kun Chen,b Ai-Wei Lee,c Juin-Yih Lai,aef Duu-Jong Lee,def and Chih-Chia Chenga* a. Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Taipei 10607, Taiwan. E-mail: [email protected] b. Department of Materials Science and Engineering, 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, Taipei 10617, Taiwan. e. Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan. f. R&D Center for Membrane Technology, Chung Yuan Christian University, Chungli, Taoyuan 32043, Taiwan. KEYWORDS. Drug delivery; Macrophages; Self-assembly; pH-responsive micelles; Multifunctional water-soluble polyurethanes.

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ABSTRACT. Self-assembled pH-responsive polymeric micelles, a combination of hydrophilic poly(ethylene glycol) segments and hydrogen bonding interactions within a biocompatible polyurethane substrate, can spontaneously self-assemble into highly controlled, nanosized micelles in aqueous solution. These newly-developed micelles exhibit excellent pH-responsive behavior and biocompatibility, highly controlled drug (doxorubicin; DOX) release behavior and high drug encapsulation stability in different aqueous environments, making the micelles highly attractive potential candidates for safer, more effective drug delivery in applications such as cancer chemotherapy. In addition, in vitro cell studies revealed the drug-loaded micelles possessed excellent drug entrapment stability and low cytotoxicity towards macrophages under normal physiological conditions (pH 7.4, 37 °C). When the pH of the culture media was reduced to 6.0 to mimic the acidic tumor microenvironment, the drug-loaded micelles triggered rapid release of DOX within the cells, which induced potent anti-proliferative and cytotoxic effects in vitro. Importantly, fluorescent imaging and flow cytometric analyses confirmed the DOX-loaded micelles were efficiently delivered into the cytoplasm of the cells via endocytosis, then subsequently gradually translocated into the nucleus. Therefore, these multifunctional micelles could serve as delivery vehicles for precise, effective, controlled drug release to prevent accumulation and activation of tumor-promoting tumor-associated macrophages in cancer tissues. Thus, this unique system may offer a potential route towards the practical realization of next-generation pH-responsive therapeutic delivery systems.


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INTRODUCTION

Macrophages are phagocytic cells responsible for inducing and activating an immune response in response to the presence of pathogens. Most macrophages have high levels of plasticity and their function is regulated by post-translational modifications that occur in response to environmental cues, resulting generation of subpopulations of macrophages that possess varied functions.1,2 However, large numbers macrophages accumulate around tumor tissues, and can polarize into the tumor-suppressive M1 or tumor-promoting M2 phenotypes of tumor-associated macrophages (TAMs).3-5 Although classically activated M1-like TAMs suppress tumor progression, the majority of TAMs in the tumor stroma have an alternatively activated M2-like phenotype, which is associated with increased tumor cell proliferation, tumor progression and a poorer prognosis in various cancers.6-8 The molecular mechanisms and factors that regulate M1 and M2 macrophage polarization are still not completely understood.9,10 In order to inhibit or prevent induction and accumulation of TAMs with the M2-like phenotype around tumors and provide a practical solution, it would be highly desirable to construct a multifunctional nanocarrier system that effectively controls the delivery of anticancer drugs under physiological conditions and achieves triggered drug release at the precise target sites where M2 macrophages and cancer cells are located in the tumor microenvironment. Such a strategy could possibly reduce the infiltration of tumor-promoting M2 macrophages into tumor tissues, and may represent a highly effective and safer chemotherapeutic approach for cancer. With respect to the features required to solve this issue, stimuli-responsive polymeric micelles (SRPMs) have recently attracted much attention as a platform for development of controlledrelease drug delivery systems. SRPMs have the ability to provide a wide range of desired drug release profiles and enable tuning of the drug release rate in response to changes in the


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surrounding environment, such as salinity, temperature, oxygen, light and pH levels.11-20 Among the different external environmental stimuli present in biological systems, a change in pH as an internal stimulus is particularly crucial for the controlled release of anticancer drugs in tumor tissues or within intracellular endosomal/lysosomal compartments. Compared to the constant extracellular and intracellular pH values of blood and healthy tissues (pH 7.4 and 7.2, respectively), the extracellular pH values of tumor tissues range from 6.0 to 7.2.21-23 Furthermore, after endocytosis of extracellular material by host tumor cells, the intracellular pH of the endosomal compartments can drop substantially, to as low as 6.5 in early endosomes, 5.0– 6.0 in late endosomes and 4.0–5.0 in lysosomes.24-27 Thus, pH stimuli-responsive polymeric micelles have the potential to enhance the delivery of drugs to tumor cells and rapidly release encapsulated drug in response to the slightly acidic pH of the tumor microenvironment. Although numerous pH-sensitive polymeric micellar systems for loading and releasing anticancer drugs in vitro and in vivo have been reported,28-34 these systems do not overcome the serious side-effects of chemotherapy and may have limited therapeutic efficacy. For instance, previously reported pH-responsive polymeric micelles did not meet their primary objective to achieve efficient release of the entrapped drug at tumor sites or provide long-term drug entrapment stability in serum-rich biological environments.35-38 Thus, SRPMs that can circulate for longer in the blood by limiting their uptake by the reticuloendothelial system urgently need to be developed to improve the safety and effectiveness of chemotherapeutic treatments for cancer and further guide the development of SRPM-based platforms for drug delivery applications. Previous studies from our laboratory have successfully demonstrated that hydrogen-bonded functional polymers (HBFPs) possess unique physical properties and self-assembly behavior,39-43 which substantially enhanced the optoelectronic performance of the resulting hole-conductor


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devices.44 Due to the presence of hydrogen bonding interactions within the polymer matrix, water-soluble HBFPs can spontaneously self-assemble in aqueous solution into complex hierarchical micelles that are suitable for drug delivery applications. These micelles possess excellent micellar stability and high drug loading capacity and undergo substantially enhanced cellular uptake, thus reducing the concentrations of drug required in vitro.45-48 Based on these findings and the critical role of the hydrogen bonding moieties in the polymer backbone, we investigated whether introduction of hydrophilic polyethylene glycol (PEG) into the side-groups of the hydrophobic hydrogen-bonded polyurethane (PU) backbone would significantly affect its structural features, self-assembly and hydrated ability to form an ultrasensitive pH-responsive polymeric micelle (PU-PEG) that effectively encapsulates the anticancer drug doxorubicin (DOX) within the micellar interior in aqueous solution, as presented in Scheme 1. Importantly, the micelle-encapsulated DOX remained highly stable under normal physiological conditions in serum-containing media over the long-term, was sensitively released at a mildly acidic pH of 6.0 and effectively reduced the viability of RAW 264.7 cells in vitro. In turn, the efficient cellular uptake and efficient penetration of the drug nanocarriers into the cells activated cellular apoptotic pathways, which indicate PU-PEG may serve as an efficient delivery vehicle for chemotherapeutic treatments and could effectively prevent the ability of M2-like polarized TAMs to promote tumor growth. To the best of our knowledge, this is the first example of an ultrasensitive pH-responsive PU-based micelle that can effectively deliver its payload into the desired cells, which could potentially reduce the side-effects of DOX in healthy tissues, manipulate drug release profiles and achieve enhanced cellular uptake to improve the efficacy of chemotherapy.


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Scheme 1. Graphical illustration of drug loading, drug release and chemotherapeutic treatment by pH-responsive PU-PEG micelles.

EXPERIMENTAL SECTION

1. Materials. All chemicals, reagents and organic solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) at the highest purity available. Tetrahydrofuran (THF) and toluene were distilled under argon over calcium hydride before use. 2. Characterization. Proton nuclear magnetic resonance (1H-NMR). 1H-NMR spectra were measured at 20 °C on a Varian Inova-400 MHz magnetic resonance spectrometer (Palo Alto, CA, USA). Samples (15 mg) were dissolved in approximately 0.8 mL of deuterated chloroform (CDCl3). Size Exclusion Chromatography (SEC). SEC elution profiles were measured using a Waters GPC system (Waters, MA, USA). THF was used as the eluent at a flow rate of 1.0


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mL/min at 40 °C with calibration against polystyrene standards (Polymer Standards Service, Silver Spring, MD, USA). Dynamic light scattering (DLS) and zeta potential measurements. The average particle size and zeta potentials of the aqueous samples were measured using a Nano Brook 90 Plus PALS (Brookhaven Instruments Corp., Holtsville, NY, USA). Temperaturedependent DLS measurements were carried out at temperatures from 25 to 50 °C, and incubated at each temperature for at least 30 min before each measurement. Photoluminescence (PL) and Ultraviolet-Visible (UV-Vis) Spectra. PL and UV-Vis spectra were recorded using a Hitachi F4500 luminescence spectrometer and Jasco V-730 Spectrophotometer (Hitachi, Tokyo, Japan), respectively. Determination of Critical Micelle Concentration (CMC). CMC values were assessed using pyrene as an extrinsic fluorescence probe, following procedures described previously.51,52 Transmission Electron Microscopy (TEM). Digital TEM images were captured at 25 °C using a JEOL 2000 FX2 (Jeol USA, Inc., Peabody, MA, USA). Aqueous samples were dropped onto the surface of copper grid with a carbon film, then the water was evaporated slowly at ambient temperature for 2 days. Scanning Electron Microscopy (SEM). Digital SEM images were captured using a JEOL JSM-6500F system (JEOL, Tokyo, Japan). Diluted samples in deionized water were spin-coated onto silicon wafers then dried in a vacuum oven at 25 °C for 6 h. Atomic Force Microscopy (AFM). The topography of DOX-loaded micelles was measured using an atomic force microscope (NX10, AFM Park systems, Suwon, South Korea). AFM samples were prepared following the same procedure as for SEM.

3. Synthesis of PEG-functionalized polyurethane (PU-PEG). PU-PEGs were prepared by a combination of condensation polymerization and copper-catalyzed click reaction. All chemical synthetic procedures in this work are described in Supplementary Information.


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4. Preparation of DOX-loaded PU-PEG micelles. PU-PEG4T (or PU-PEG2T) was used to directly encapsulate DOX via a simple dialysis method. Different amounts of DOX (ranging from 0.2 to 1.0 mg) were mixed with 1 mg of PU-PEG4T and 0.2 mL of triethylamine in 10 mL dimethylformamide (DMF) with gentle stirring at 40 °C for 60 min. The mixed solution was dialyzed against deionized water in dialysis membrane (MWCO 6000-8000 Da) to remove unloaded free DOX and organic solvents for 2 days at 25 °C. Loading of DOX into the micelles was directly assessed by UV-Vis spectroscopy at λ = 486 nm for DOX, and then the micelles were lyophilized to determine total DOX loading. The loading content of DOX was calculated according to the following equation:

DOX loading content (wt%) = WDOX/ Wtotal × 100%

where WDOX is the weight of DOX loaded into polymeric micelles and Wtotal, the weight of DOX-loaded polymeric micelles after dialysis and lyophilization.

5. In vitro DOX release assay. DOX release from micelles was studied using a dialysis method in phosphate buffer saline (PBS) at different pH values at 37 °C. PBS for different pH values (7.4, 6.0 and 4.0) were purchased from Sigma-Aldrich. After that, the DOX-loaded micelle solution was dialyzed against large volumes of PBS for 1 day at 37 °C under three different pH conditions to monitor real-time release of DOX from pH-responsive micelles. The cumulative DOX release was assessed using UV-Vis spectroscopy by comparison with a calibration curve of free DOX in DMF determined under the same experimental conditions. The rates of DOX release from the micelles at three different pH values (4.0, 6.0 and 7.4) were measured and plotted against time, as shown in Figure 3c.


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6. Long-term stability evaluations of blank and DOX-loaded micelles. Fetal bovine serum (FBS) is a biological micelle-destabilizing agent commonly used to assess the kinetic stability of micelles in aqueous environments. Detailed procedures have been previously described in detail. 36,37

7. Cell culture. RAW 254.7 cells (American Type Culture Collection; ATCC, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma Chemical Company, St. Louis, MO, USA) containing 10% FBS and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified atmosphere containing 5% CO2 at 37 °C. For experiments, the cells were trypsinized and resuspended in PBS containing 0.1% Trypan Blue (Thermo Fisher, Waltham, MA, USA) and counted using a hemocytometer and a microscope.

8. In vitro cytotoxicity studies. The in vitro cytotoxicity of free DOX, blank and DOX-loaded micelles against RAW 254.7 cells were assayed using a standard methyl thiazolyl tetrazolium (MTT) assay, as previously described.47,48 Briefly, the in vitro cytotoxicity of sample solutions against RAW 254.7 cells was evaluated using the MTT assay. RAW 254.7 cells were seeded in a 96-well plates at a density of 1 × 104 cells per well in 100 µL of medium and then incubated with sample solutions at the designed concentrations (0.01-100 µg/mL) for 24 h at 37 °C. Then, 20 µL of MTT solution (5 mg/mL) in PBS was added to each well, incubated for 4 h, the medium containing unreacted dye was removed carefully. Finally, the blue formazan crystals were dissolved in 150 µl of solution of dimethyl sulfoxide, and absorbance was measured using a microplate reader (BioTek, ELx800, Winooski, VT, USA) at a wavelength of 570 nm.

9. Analysis of cellular uptake by confocal laser scanning microscopy. RAW 264.7 cells were seeded into 6-well plates at a density of approximately 1 × 105 cells per well, cultured for 24 h,


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free DOX or DOX-loaded micelles were added, then the cells were incubated at 37 °C at various pH values for different time periods (4, 8, 24 h). Cells were fixed in 4% formaldehyde at ambient temperature for 10 min, washed several times with PBS (pH 7.4), stained with 4′,6-diamidino-2phenylindole (DAPI; Sigma-Aldrich) and examined by CLSM (iRiS™ Digital Cell Imaging System, Logos Bio Systems, Korea). The CLSM images are presented in Figures 4 and S11.

10. Detection of apoptosis by flow cytometry after Annexin V/PI staining. RAW 264.7 cells were seeded into 6-well plates at an initial density of 1 × 105 per well in DMEM media for 24 h, then incubated with blank or DOX-loaded micelles at 37 °C at various pH values for different time periods (4, 8, 24 h). Cells were washed and resuspended in PBS. The cells were harvested, washed twice with cold PBS, stained using Annexin V/PI,57-59 and examined by flow cytometry analysis (FACSAriaTM III, BD Biosciences, San Jose, CA, USA).

11. Statistical analysis. All in vitro experiments were repeated at least five times to confirm reproducibility, values are presented at the mean plus or minus (±) the standard deviation of three independent experiments. Statistical significance (P < 0.05) was assessed using the Student’s ttest.

RESULTS AND DISCUSSION Novel stimuli-responsive water-soluble polymers, PEG-grafted PUs (PU-PEG), were

developed via a simple two-step reaction, as illustrated by the chemical synthesis procedures shown in Scheme S1 (see Supporting Materials and Methods for further details). First, condensation polymerization of 4,4'-methylenediphenyl diisocyanate (MDI) and azide-endcapped oligomeric polypropylene glycol (PPG-N3; average molecular weight [Mw] of 640 g/mol


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or approximately 10 repeat units)49 was performed using dibutyltin dilaurate as catalyst at 50 °C. The pendant azide-functionalized PU was recovered at high yield (84%), with the resulting polymeric product exhibiting a Mw of ca. 14,200 g/mol, polydispersity index (PDI) of 1.3 and approximately 8.0 % of the azide groups linked in the side chains of the polymer, as identified by proton nuclear magnetic resonance and size exclusion chromatography (Figures S1-S5). Subsequently, azide-grafted PU-N3 was further functionalized with different molecular weights of monopropargyl terminated PEG50 via the copper catalyzed click reaction to generate high quality PU-PEG polymers (yields, ≥ 90%), the chemical structure of which are presented in Scheme 1. After purification by dialysis, PU-PEG containing different molecular weights of pendant PEG chains (PEG 2000 and PEG 4000) could be obtained, hereafter termed PU-PEG2T and PU-PEG4T, respectively. Interestingly, these samples dissolved easily in aqueous solutions, even at concentrations up to 30 mg/mL. This property can be attributed to the presence of the PEG segments, which enhance the aqueous solubility and dissolution of PU-PEG. To further explore the influence of phase separation between hydrophilic PEG and the hydrophobic PU backbone on solubility, we performed dynamic light scattering (DLS) measurements and transmission and scanning electron microscopy (TEM and SEM) at 25 °C. At sample concentrations of 1.0 mg/mL in aqueous solution, PU-PEG2T and PU-PEG4T displayed average particle sizes of 58 ± 17 nm (PDI = 0.086) and 25 ± 11 nm (PDI = 0.194), respectively, implying introduction of the hydrophilic PEG grafts promoted formation of the nano-sized micelles, and a higher hydrophilic PEG fraction of PU-PEG4T enhanced the hydrophilicity of the micelles, leading to tight packing of particles with a smaller diameter (Figure 1a). TEM and SEM were performed to validate the DLS experiments, and confirmed the PU-PEG4T micelles have a small diameter of 20–30 nm (Figure 1b-d). In addition, these observations also imply the


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PU-PEG micelles possess stable morphological features due to the hydrogen bonding network within the hydrophobic PU backbone, resulting in low-dimensional self-assembled polymer nanostructures. Next, the critical micelle concentration (CMC) values of PU-PEG micelles formed in aqueous solution were determined by fluorescence methods using pyrene as a hydrophobic probe.51,52 As shown in Figure S6, PU-PEG4T polymer exhibited a markedly lower CMC value (approximately 2 x 10-6 mg/mL) than the PU-PEG2T system (approximately 8 x 10-6 mg/mL), suggesting micelle stability significantly improves as PEG content and pendent chain length increases. This evidence also demonstrates the long hydrophilic length of the PEG chains creates distinct hydrophilic and hydrophobic areas that enable spontaneous self-assembly in aqueous solution and induce the formation of smaller, more stable self-assembled micelles.


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Figure 1. (a) DLS analyses of 1.0 mg/mL aqueous solutions of PU-PEG2T and PU-PEG4T at 25 °C. (b) TEM and (c, d) SEM images of PU-PEG4T micelles.

Figure 2. Correlation functions and particle size distributions measured at 90° for (a) PUPEG2T and (b) PU-PEG4T micelles over time after addition of FBS. DLS analysis of 1.0 mg/mL aqueous solutions of PU-PEG4T at various (c) temperatures and (d) pH values. (c) The inset shows the particle size of PU-PEG4T in aqueous solution at various temperatures.

Long-term kinetic experiments were performed using DLS in the presence of a high concentration of fetal bovine serum (FBS; 33%, v/v; deionized water/FBS), which acts as a


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biological micelle-destabilizing agent in aqueous media.36,37 As shown in Figure 2a and 2b, the presence of FBS in the PU-PEG4T micelle solutions did not affect particle size and size distribution after 24 h of monitoring at 25 °C, indicating the increased PEG segment chain length protects the hydrogen-bonded network of the PU backbone within the micelle interior, thus forming a stable micellar structure that is resistant to long-term treatment with high concentrations of FBS. Conversely, the size distribution of the PU-PEG2T micelles substantially increased during prolonged FBS treatment, even though shorter PEG chains are present in the polymer side chain. This suggests that both the hydrophilic character and weak hydrogen-bonded network play critical roles in stabilizing and organizing the micellar structure. While longer PEG chains in the outer shell of the micelle preserved the integrity of the spherical structure, FBS hardly disrupted the hydrogen bonding network of the hydrophobic PU backbone within the PUPEG4T micelles. To further investigate the environmental-responsive micellization behavior of PU-PEG4T in aqueous solution, temperature and pH-dependent DLS experiments were performed by monitoring the particle size distribution of the polymer solutions as function of temperature and pH from 25–50 °C and pH 4–7. As shown in Figure 2c, the average particle diameter and size distribution of PU-PEG4T in aqueous solution remained almost unchanged when the temperature of solution gradually increased from 25 °C to 50 °C, indicating the micelles did not undergo a temperature-induced structural transition over the normal physiological temperature range. Conversely, the micelles exhibited an almost linear decrease in particle diameter as the pH of the aqueous solution decreased. The pH-dependent DLS data shown in Figure 2d confirms PU-PEG4T micelles had a very high stability in aqueous solution at pH 7.0 and 25 °C as the diameter of the micelles remained practically unchanged over 48 h. When the pH was decreased to 6.0 (or 4.0) and the temperature remained at 37 °C, micellar size


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decreased gradually over time, from 20 to 8 nm, implying that the protons in the mildly acidic medium rapidly disrupted the hydrogen bonding network within the inner region of the micelles, thus decreasing the micelle size and increasing the hydrophilicity of PU-PEG4T. Thus, the combined features of excellent micellar stability and sensitive pH-responsive behavior indicate the PU-PEG4T micelles have significant potential to improve the control of drug delivery and drug release in response to a change in the pH of the external environment. Next, we further investigated the drug encapsulation and in vitro drug release behavior of the PU-PEG micelles using the hydrophobic anticancer agent DOX. Due to its poor aqueous solubility,

DOX

hydrochloride

was

initially

dissolved

in

a

dimethylformamide

(DMF)/triethylamine cosolvent mixture and then mixed with DMF solutions containing different proportions of PU-PEG. The solutions were carefully transferred into dialysis membrane and dialyzed against deionized water for at least 2 days to remove organic solvents and nonencapsulated DOX. The average micelle size of the DOX-loaded PU-PEG4T micelles increased gradually from 99 ± 18 nm (PDI = 0.033) to 291 ± 23 nm (PDI = 0.006) as the encapsulated DOX content increased (Figure S7a), implying that a significant increase in micelle size was required to accommodate DOX in the interior of the micelle to achieve a high DOX encapsulation efficiency. In addition, the DOX-loading content of the PU-PEG4T micelles reached a maximum at 9.1 ± 0.5% with a zeta potential of -22.6 mV (Table S1), indicating the DOX loading content could be easily tailored to achieve the desired micelle properties. In further confirmation of the morphology of the DOX-loaded micelles, atomic force microscopy (AFM) and SEM images confirmed that PU-PEG4T micelles containing 8% DOX formed large spherical structures with a mean diameter of 250 nm, as shown in Figure S7b and S7c. TEM demonstrated clear core-shell structures, with the cores appearing bright due to the presence of


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DOX (inset of Figure S7d), revealing DOX was physically entrapped in the hydrophobic interior of the micelles and may possibly promote the formation of some specific intermolecular interactions between the DOX molecules and aromatic groups of the PU backbone. These observations were consistent with the DLS (Figure S7a), and further confirmed the hydrophobic PU segments within the micelles enhance the encapsulation of DOX within the core of the micelles, and thus confer tunable drug-loading capacity. To explore the long-term circulation ability of DOX-loaded micelles in vivo using an in vitro assay, the stability of DOX-loaded PU-PEG2T and PU-PEG4T micelles in FBS-containing media were evaluated by DLS at 25 °C (Figure 3a and 3b).36,37 The intensity autocorrelation function (G) of DOX-loaded PU-PEG4T micelles did not change significantly over 24 h in the presence of high concentrations of FBS, indicating an extremely high drug-entrapment stability. Conversely, DOX-loaded PU-PEG2T micelles gradually formed a second relaxation mode within high delay times (τ) ranging from 104 to 106 µs as monitoring time increased, indicating the formation of larger particles. In other words, the presence of FBS in the aqueous media rapidly disrupted the spherical conformation of the PU-PEG2T micelles, resulting in a burst of hydrophobic DOX release and formation of large DOX aggregates. PU-PEG4T had substantially higher long-term micellar and DOX-entrapped stabilities due to the balanced hydrophilic and hydrophobic interactions between PU and the PEG segments, a feature that is extremely rare in traditional polymeric drug carrier systems. These unique characteristics prompted us to further investigate the in vitro drug-release behavior and cytotoxicity effect of DOX-loaded PU-PEG4T micelles in PBS solution.


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Figure 3. Correlation functions measured at 90° for (a) DOX-loaded PU-PEG2T and (b) DOXloaded PU-PEG4T micelles over time after addition of FBS. (c) In vitro drug release-time profiles for DOX-loaded PU-PEG4T micelles in PBS buffer at 37 °C under different pH conditions. (d) Cell viability of RAW 264.7 cells treated with different concentrations of free DOX or DOX-loaded PU-PEG4T micelles under different environmental conditions for 24 h.

Next, we examined the effects of pH on the in vitro drug-release profiles. As shown in Figure 3c, DOX-loaded PU-PEG4T micelles (DOX loading content = 9.1 %) in PBS demonstrated slow and restrained release of DOX at pH 7.4 and 37 °C with only 38 ± 5.4 % of DOX released after 48 h, much lower than at either pH 6.0 (71 ± 4.8 %) or pH 4.0 (91 ± 2.8 %) at 37 °C. This


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suggests the specific intermolecular interactions between the DOX molecules and aromatic groups of the PU backbone rapidly dissociated under low pH or acidic conditions, which efficiently manipulated/tuned the properties of the DOX-loaded micelles and endowed specific, sensitive pH-responsive ability in the physiological conditions of the tumor environment. Interestingly, when the pH of the DOX-loaded micelle PBS solutions was gradually decreased to mildly acidic conditions, the cumulative amounts of DOX released after the initial 10 h increased to 60% and 80% at pH 6.0 and 4.0, respectively, followed subsequently by more sustained, slower release of DOX over the next 38 h. This demonstrates that the well-controlled pHresponsive drug release of PU-PEG4T micelles could reasonably be expected to meet the rising demand for a safe, controllable system for delivering DOX to tumor cells to reduce the sideeffects of treatment in healthy cells while maintaining the efficacy of chemotherapy in tumor cells. Since pH is lower in the tumor microenvironment than the surrounding normal tissues,21-27 DOX-loaded PU-PEG4T micelles could be rapidly and precisely triggered to release DOX in the slightly lower pH of the tumor microenvironment. In order to further prove the contribution of the acidic pH environment to DOX release, DOX-loaded PU-PEG4T micelles in PBS at pH 4.0 and 37 °C were observed by DLS, as shown in Figure S8. After 8 h continuous monitoring at 37 °C, DLS analysis revealed two clearly distinct peaks appeared at 320 nm and 850 nm. The first peak at 320 nm can be attributed to the original DOX-loaded micelles from comparison with the DLS data in Figure S7a. In addition, the second broad peak centered at 850 nm may be possibly attributed to gradual release of DOX into the surrounding media, causing formation of abnormally large DOX aggregates due to the poor solubility of DOX in aqueous medium (Figure S9). This observation further clearly demonstrates that PU-PEG4T represents a sensitive, pHresponsive nanocarrier with significant potential for efficient and safe drug delivery that is


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suitable for application in cancer therapeutics, as the system can be adapted to specific parameters, stably deliver drug payloads and rapidly release the encapsulated drug in mildly acidic microenvironments. Based on these findings, we confidently hypothesize the DOX-loaded PEG-PU-PEG4T micelles would significantly improve DOX uptake in macrophages and reduce infiltration of TAMs to promote anti-tumor immunity. The cytotoxicity of pristine nanocarriers, DOX-loaded PU-PEG4T micelles and free DOX toward RAW 264.7 cells were assayed in vitro using the MTT method. After incubation for 24 h at 37 °C, the pristine nanocarriers did not exert any cytotoxic effects in RAW 264.7 cells at concentrations ranging from 10 µg/mL to 500 µg/mL (Figure S10), indicating these nanocarriers have very low toxicity towards macrophages. Furthermore, DOX-loaded PU-PEF4T micelles containing 9.1% DOX did not significantly reduce cell viability at pH 7.4 compared to blank micelles (Figure 3d), suggesting DOX did not appreciably extravasate from the micelles and accumulate in RAW 264.7 cells, demonstrating DOX-loaded PU-PEG4T micelles exhibit excellent drug-entrapped stability under normal physiological conditions. When the pH was decreased from 7.4, the DOX released from the PUPEG4T micelles at pH 6.0 and pH 4.0 exerted a cytotoxic effect, with half maximal inhibitory concentration (IC50) values of 52 ± 3.2 µg/mL and 23 ± 3.8 µg/mL, respectively. This suggests the DOX-loaded PU-PEG4T micelles enhanced the cellular uptake of DOX to elicit the desired pharmacological response in the cells. Compared to the IC50 value of free DOX (14 ± 4.5 µg/mL), the significantly higher IC50 values of the DOX-loaded micelles may be attributed to the time required for DOX to be released from the micelles and subsequently delayed nuclear uptake of DOX in RAW 264.7 cells. On further increasing the concentration of DOX-loaded micelles to 100 µg/mL, the IC50 in RAW 264.7 cells incubated in pH 6.0 and pH 4.0 media


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reduced to 37 ± 3.2 µg/mL and 16 ± 3.9 µg/mL, respectively. These results prove the DOXloaded micelles exert a concentration-dependent, positive, linear cytotoxic effect towards RAW 264.7 cells.

Figure 4. CLSM images of RAW 264.7 cells incubated with free DOX or DOX-loaded PUPEG4T micelles (DOX loading = 9.1%) for 4, 8 or 24 h at 37 °C under different pH conditions (pH 6.0 and 7.4). RAW 264.7 cells were stained with DAPI as a nuclear stain (blue fluorescence). DOX emits characteristic red fluorescence at 550−600 nm when excited at 480 nm. The upper and middle panels show the bright-field and fluorescence images, respectively;


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the lower panel is the merged image of both fluorescence channels. Scale bar = 20 µm for all images. To gain further understanding of mechanisms relevant to cellular uptake and intracellular release behavior, free DOX or DOX-loaded PU-PEG4T micelles were incubated with RAW 264.7 cells at various pH (pH 4.0, 6.0, and 7.4) values at 37 °C for 4, 8 or 24 h, then the cells were observed by confocal laser scanning microscopy (CLSM). In Figure 4, the red domains indicate the characteristic fluorescence of DOX, while the blue domains represent nuclear staining by 4',6-diamidino-2-phenylindole (DAPI). After only 4 h of incubation with free DOX at pH 7.4, formation of pink domains was observed in the nuclei (in the merged images), indicating free DOX was rapidly transported by passive diffusion into the cells.53,54 In contrast, in cells incubated with the DOX-loaded PU-PEG4T micelles for 4 h at pH 7.4, the red DOX fluorescence was strongly confined within the micelle interior and did not colocalize to the cell surface (Figure S11). Even after extended incubation periods of up to 24 h, only a small fraction of weak pink fluorescence was dispersed on the outer surface of the cells. These observations indicate PU-PEG4T micelles function as efficient carriers for delivering DOX across the plasma membrane, and can enhance the stability of DOX encapsulation and retention in the cell under physiological conditions compared to free DOX.55,56 When the pH was decreased to 6.0 (Figure 4), the DOX fluorescence of the DOX-loaded PU-PEG4T micelles was mainly located on the surface of the intracellular matrix, indicating DOX was gradually released from the micelles under slightly acidic conditions after 4 h, leading to diffusion of DOX within the intracellular space. After further incubation for 24 h, significant accumulation of pink fluorescence was observed in the nucleus. These results reveal the DOX-loaded micelles were rapidly delivered into the cytoplasm of the RAW 264.7 cells and the sequentially released DOX was effectively


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translocated into the cell nuclei via a combination of cellular uptake via nonspecific endocytosis and intracellular pH-triggered drug release.

Figure 5. Comparative flow cytometric dot plot quadrant charts: Annexin V/PI analysis of RAW 264.7 cells after incubation with (a) control media, (b) blank PU-PEG4T micelles and (c-h) DOX-loaded PU-PEG4T (DOX loading = 9.1%) for 4, 8 or 24 h at 37 °C at pH 6.0 or 7.4. (i) The chart quadrants from the lower left to the upper left (anti-clockwise) represent live, early apoptotic, late apoptotic, and necrotic cells, respectively. The proportions of cells inside each quadrant are indicated within the dot-plot charts.


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In order to identify the endocytosis pathways for micelles and assess activation of cellular apoptotic processes, flow cytometry analysis was performed by Annexin V-Alexa Fluor488 (Annexin V)/propidium iodide (PI) double staining of RAW 264.7 cells exposed to DOX-loaded PU-PEG4T micelles (containing 9.1% DOX) at various pH conditions. Annexin V was used to detect the presence of phosphatidylserine expression on early apoptotic cells, while PI was used to label intracellular DNA, which is released after the integrity of the plasma membrane has been compromised in late apoptotic cells.57,58 Combined Annexin V and PI staining enables discrimination of early apoptotic cells (Annexin V+, PI-), late apoptotic cells (Annexin V+, PI+), necrotic cells (Annexin V-, PI+), and viable cells (Annexin V-, PI-),59 the proportions of which can be easily quantified by flow cytometry (Figure 5i). When incubated with DOX-loaded micelles for up to 24 h at pH 7.4 and 37 °C, the percentage of viable RAW 264.2 cells remained quite high (approximately 90%; Figure 5e), indicating the extremely stable micellar properties under normal physiological conditions effectively prevented premature leakage of DOX before reaching the target tissue or cells. Even when the pH was decreased to 6.0 and the temperature remained at 37 °C, a similar a high cell viability of 90.4% was observed after the initial 8 h period (Figure 5g). This implies that the DOX loaded within the micelles can be quickly transported across the plasma membrane via endocytosis, and subsequently, the acidic media gradually diffuses into the inner core of the micelles in the cells. Surprisingly, on further extending the incubation time up to 24 h, the relative number of apoptotic cells increased compared to the number of apoptotic cells at the beginning of incubation. As shown in Figure 5h, the quantity of early apoptotic RAW 264.7 cells reached 59% and the percentage of late apoptotic cells increased to 7.4%; necrotic cells were not detected. Even though a further


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decrease in pH to 4.0 accelerated induction of apoptotic processes, the proportion of late apoptotic cells remained almost unchanged (1.6% increase) after 24 h incubation and the majority of apoptotic cells were still early apoptotic cells (up to 87.8% of cells overall; Figure S12). Therefore, the flow cytometry demonstrates that the DOX-loaded micelles effectively penetrated into cells, and the consequent intracellular release of DOX from the micelles at pH 6.0 directly induced apoptotic cell death in RAW 264.7 cells. This finding indicates this system could significantly reduce the side-effects of chemotherapy in healthy organs. Collectively, the findings of this study clearly show that ultrasensitive pH-responsive PU-PEG4T nanocarriers may not only markedly enhance the therapeutic efficiency of anticancer drugs, but remain highly stable under normal physiological conditions and could potentially facilitate precise drug release to inhibit the infiltration and tumor-promoting effect of TAMs in tumor tissues.

CONCLUSION In summary, we successfully synthesized and developed an ultrasensitive pH-responsive

polymeric micelle, PU-PEG, based on the presence of a hydrophobic PU backbone and hydrophilic PEG side chains. In aqueous environments, PU-PEG can rapidly organize into remarkably uniform, nanosized micelles that possess a number of unique physical properties, including extremely low CMC and cytotoxicity, excellent pH-responsive behavior and micellar stability, as well as tunable drug-loading capacity and high drug-encapsulated stability in the presence of serum. The combination of these characteristics in a multifunctional polymer-based micelle is highly desirable, yet exceedingly rare in traditional polymeric drug carriers. In addition, in vitro experiments demonstrated the DOX-loaded PU-PEG micelles exhibited good drug-entrapment stability and low cytotoxicity towards RAW 264.7 cells under normal


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physiological conditions (pH 7.4 and 37 °C). When the pH was decreased to pH 6.0, DOX was rapidly released from the micelles as a result of disruption of the intermolecular interactions between DOX and the aromatic groups of the PU backbone, and the released DOX exerted potent anti-proliferative and cytotoxic effects in vitro as the micelles were safely and efficiently delivered DOX into the cell nuclei. Importantly, CLSM and flow cytometric analyses further confirmed the DOX-loaded micelles were efficiently taken up by RAW 264.7 cells via endocytosis, firstly into the cytoplasm, and then gradually transferred to the nucleus. Switching the pH of the media to 6.0, to mimic the acidic tumor microenvironment, resulted in release of the drug and apoptosis of the macrophages, which may halt tumor progression in vivo. These encouraging results strongly suggest that PU-PEG could be employed to dramatically improve the effectiveness of chemotherapy in the low pH tumor microenvironment, while substantially reducing the toxic effects associated with premature drug release in healthy cells under normal physiological conditions. Thus, this work not only provides a potential route towards the development of multifunctional pH-sensitive micelles, but may also help to enhance the safety of anticancer drugs, increase chemotherapeutic efficacy and potentially offers an exciting route to inhibit the accumulation and tumor-promoting effects of TAMs in cancer tissues.


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ASSOCIATED CONTENT Supporting Information. Synthetic procedures, Structural characterizations and analytical data are provided in Supplementary Information. This material is available free of charge on the ACS Publications website at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] Present Addresses 43 Keelung Rd., Sec. 4, Da'an Dist., Taipei City 10607, Taiwan. Author Contributions C.-C. Cheng devised the project, planned the experiments, supervised the work and wrote the manuscript. Z.-S. Liao, S.-Y. Huang and J.-J. Huang performed all experiments. All authors discussed the results and provided constructive comments to the final manuscript. Funding Sources This study was supported financially by the Ministry of Science and Technology, Taiwan (contract no. MOST 105-2628-E-011-006-MY2). Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT This study was supported financially by the Ministry of Science and Technology, Taiwan (contract no. MOST 105-2628-E-011-006-MY2).

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Table of Contents.

A new ultrasensitive pH-responsive polymeric micelle possessing high drug-entrapped stability and excellent chemotherapeutic efficiency in vitro, potentially suitable for effective, safe release of anticancer drugs to prevent accumulation of tumor-associated macrophages within the tumor microenvironment.


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