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Controlling Endosomal Escape Using pH-Responsive Nanoparticles with Tunable Disassembly Nachnicha Kongkatigumjorn,† Samuel A. Smith,† Moore Chen,‡ Katie Fang,† Shenglin Yang,† Elizabeth R. Gillies,§,∥ Angus P. R. Johnston,*,‡ and Georgina K. Such*,† †

School of Chemistry, The University of Melbourne, Parkville, Victoria 3010, Australia Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia § Department of Chemistry and Centre for Advanced Materials and Biomaterials Research, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 5B7, Canada ∥ Department of Chemical and Biochemical Engineering, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 5B9, Canada

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ABSTRACT: Endosomal escape is a bottleneck in the efficient delivery of therapeutics using nanoparticles; therefore understanding how this property can be optimized is important for achieving better therapeutic outcomes. It has been demonstrated that pH-responsive nanoparticles (pHlexi nanoparticles) have potential to achieve effective escape from the endosomal compartments of the cell. In this paper a library of five pHlexi particles with tunable disassembly pH were synthesized by combining poly(ethylene glycol)-b-poly(2(diethylamino)ethyl methacrylate) (PEG-b-PDEAEMA) with random copolymers of 2-(diethylamino)ethyl methacrylate and 2(diisopropylamino)ethyl methacrylate. A series of cellular studies were conducted to investigate the effect of particle composition on in vitro behavior. Endosomal escape was probed using a calcein escape assay in NIH/3T3 fibroblast cells, demonstrating endosomal escape increased with increasing particle concentration. Interestingly, it was shown that endosomal escape was most efficient with particles that disassemble at high (pH 7.2) or low (pH 4.9) pH, with particles that disassemble between pH 5.8 and 6.6 inducing decreased levels of endosomal escape. This change in endosomal escape behavior suggests particles can induce escape by different pathways. The results show that tuning the core component of pHlexi particles can improve the effectiveness of endosomal escape capabilities and thus their ability to act as effective delivery systems. KEYWORDS: endosomal escape, nanoparticle, pH-responsive, delivery, stimuli-responsive

1. INTRODUCTION Advances in nanoparticle technology have led to new opportunities in the use of nanoparticles as therapeutic delivery systems. To be effective for delivery applications nanoparticles need a range of characteristics such as the ability to target specific cells as well as controlled release within a specific region of the cell/tissue.1−3 Significant interest in this field has led to the development of a variety of nanoparticles with different architectures and compositions, and with the ability to respond intelligently to a range of biologically relevant stimuli.3−5 It has been well-demonstrated that nanoparticles typically internalize into cells via endocytosis, leading to entrapment inside acidic compartments called endosomes/lysosomes. This is not the site of action for the majority of therapeutics; thus escape from this compartment, termed endosomal escape, is integral to improving therapeutic efficacy.6−8 A number of nanoparticles have been reported that demonstrate endosomal escape capabilities;9,10 however there © XXXX American Chemical Society

are still many questions around the mechanisms of endosomal escape and how it can be enhanced. In this paper we design a library of pH-responsive nanoparticles with tunable disassembly pH and demonstrate the compositional variation has significant impact on endosomal escape behavior. For the majority of applications the therapeutic cargo needs to be released inside the cell, which relies on engineering nanoparticles to release their cargo in response to a biological stimulus. Many stimuli have been investigated to achieve this;11 however release in response to a drop in pH has generated particular research interest. This is due to the inherent decrease in pH that occurs during the internalization process from the bloodstream (pH 7.4) into the early endosome (pH 6.0−6.5) and then to the late endosome/ Received: March 1, 2018 Accepted: June 5, 2018 Published: June 5, 2018 A

DOI: 10.1021/acsanm.8b00338 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials

Scheme 1. Synthesis of the Library of pHlexi Nanoparticles Based on Poly(ethylene glycol)-b-poly(2-(diethylamino)ethyl methacrylate) (PEG-b-PDEAEMA) with Random Copolymers of 2-(Diethylamino)ethyl Methacrylate and 2(Diisopropylamino)ethyl Methacrylate

lysosome (pH 4.5−5.5).12−16 To design pH-responsive materials two strategies are commonly used: first, the incorporation of pH-responsive linkages within the material or second, the use of building blocks which are pH-responsive. Charge-shifting polymers are an example of the latter class of material. They switch from uncharged and hydrophobic above their pKa to charged and hydrophilic when below their pKa.17 Poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) is a well-known charge-shifting polymer, which has a pKa value of ∼7. This transition can be exploited for intracellular release, as the pH transition from the extracellular environment to endosomes falls within this range.18−21 This pH transition can also be tuned by the substituents present on the amine group. When applied to nanoparticles it has been demonstrated this hydrophobic to hydrophilic transition can induce efficient nanoparticle disassembly and release of a therapeutic payload.22 Zhou et al. have designed a series of pH-responsive micellar nanoparticles using poly(ethylene oxide) block copolymers with different charge-shifting components to tune the pH transition of the nanoparticle. These materials were used as extracellular and intracellular sensors by conjugating tetramethyl rhodamine (TMR) to the polymer and using the color change as a signal of nanoparticle disassembly. 23,24 This group also reported poly(2(dimethylamino)ethyl methacrylate)-block-poly(2(diisopropylamino)ethyl methacrylate) (PDMAEMA-bPDPAEMA) micellar nanoparticles conjugated with siRNA. They demonstrated little siRNA release with the nanoparticles alone but achieved good release when they added a drug known to promote membrane permeability, amphotericin B.25 Stayton and co-workers have also performed a number of studies in this area demonstrating pH dependent membrane interaction leads to improved delivery of peptide antigens.26 A number of groups have investigated PDEAEMA and PDPAEMA micelles for therapeutic delivery, but controlling

the endosomal escape by tuning the ratio of PDEAEMA and PDPAEMA has not been studied. Recently, we demonstrated a pH-responsive nanoparticle system composed of a poly(2-diethylamino)ethyl methacrylate)-b-poly(ethylene glycol) (PDEAEMA-b-PEG) block copolymer and a PDEAEMA homopolymer.27 The pHresponsive particles, termed pHlexi particles, were taken up by 3T3 fibroblast cells and were able to induce endosomal escape due to the pH transition of the PDEAEMA components.28 In addition, it was demonstrated molecular weight of the homopolymer allowed tuning of the endosomal escape capabilities. Herein, we report the synthesis of a library of pHlexi nanoparticles with tunable disassembly pH from 7.2 to 4.9 (Scheme 1). We demonstrate this compositional variation has important implications on both material properties and their cellular behavior. The particles were synthesized by combining a PEG-b-PDEAEMA block copolymer with a random copolymer comprising PDEAEMA and PDPAEMA at mol ratios of 1:0, 3:1, 1:1, 1:3, and 0:1, respectively. Endosomal escape was investigated using the calcein assay in NIH/3T3 fibroblast cells at particle concentrations of 0.5 × 109, 1.5 × 109, 5 × 109, and 15 × 109 particles/mL. It was shown particle concentration was an important determinant of endosomal escape. In addition, an interesting trend of endosomal escape was observed across the particle library. Highest escape was seen with the nanoparticles that had the extreme disassembly pHs, e.g., PDEAEMA (pH 7.2) and PDPAEMA (pH 4.9); endosomal escape then decreased down to a minimum for the 1:1 composition. The decrease in endosomal escape in the particles with the intermediate particle disassembly pH suggests two different mechanisms are playing a role in endosomal escape. Highest association was observed for the PDEAEMA and PDEAEMA-b-PDPAEMA (1:1) particles. This study indicates that changes in composition can play a significant role in cell behavior. B

DOI: 10.1021/acsanm.8b00338 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials

controlled sample temperature and injection rate. Size and stability of particles were recorded on dynamic light scattering (DLS) (Horiba Scientific, SZ-100) at a fixed scattering angle of 90° and at 37 °C. Absorbance of the polymer solutions was recorded using an Agilent Technologies Cary 60 UV−vis spectrometer. 2.3. Synthesis of Poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA). Reversible addition−fragmentation chain transfer (RAFT) polymerization was used to synthesize PDEAEMA. The 2diethylaminoethyl methacrylate (DEAEMA) (2.0 g, 11 mmol), azobis(isobutyronitrile) (AIBN) (0.36 mg, 2.20 μmol), and 4cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (8.88 mg, 22 μmol) were prepared in 1,4-dioxane (2.0 g) to achieve a ratio of [monomer]/[CTA]/[initiator] = 500:10:1 and added to a Schlenk flask. The mixture was subjected to four freeze−thaw−pump cycles spanning 5, 10, 20, and 40 min, respectively, then backfilled with nitrogen. The degassed reaction mixture was stirred in an oil bath at 60 °C for 16 h. The reaction was then terminated by exposure of the reaction mixture to air. A portion of the crude PDEAEMA was precipitated in water for 24 h and then dried by vacuum filtration for another 24 h to yield PDEAEMA as a yellow tacky solid. The numberaverage molecular weight (Mn) (kDa) of PDEAEMA was determined by 1H NMR analysis to be 40 kDa (Table S1). This was done by comparing the peak from the RAFT agent at δ (ppm) of 1.25−1.2 ppm (CH3−C8H16−CH2−) to PDEAEMA polymer peak at δ of 4.1− 3.9 ppm (−COO−CH2−CH2−). 1H NMR (400 MHz, CDCl3): δ (ppm) 4.1−3.9 (−COO−CH2−CH2−), 2.7−2.6 (−CH2−CH2− N−), 2.6−2.5 (−N−CH2−CH3), 2.0−1.7 (backbone−C−CH2− C−), 1.25−1.2 (CH3−C8H16−CH2−), 1.15−0.95 (−N−CH2−CH3 backbone−CH2−C−CH3−), 0.95−0.8 (backbone−CH2−C−CH3−) (Figure S1). 2.4. Synthesis of Poly(2-(diisopropylamino)ethyl methacrylate) (PDPAEMA). PDPAEMA was synthesized via reversible addition−fragmentation chain transfer (RAFT). The 2-diisopropylaminoethyl methacrylate (6.40 g, 30 mmol), AIBN (0.98 mg, 6 μmol), and 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (24.22 mg, 60 μmol) were prepared in 1,4-dioxane (4.0 g) to achieve a ratio of [monomer]/[CTA]/[initiator] = 500:10:1 and added to a Schlenk flask. The mixture was subjected to four freeze−thaw−pump cycles and backfilled with nitrogen similar to PDEAEMA procedure. The mixture was polymerized in an oil bath at 60 °C for 16 h. Then, the reaction was terminated by exposure of the reaction mixture to air. A portion of crude PDPAEMA was precipitated in acetonitrile with 2 solvent exchanges. Rotary evaporation was used to remove all solvents from precipitation. The molecular weight was determined to be 48 kDa by comparing the peak from the RAFT agent at δ (ppm) of 1.25−1.2 ppm (CH3−C8H16−CH2−) to the PDPAEMA polymer peak at δ of 3.9−3.7 ppm (−COOH−CH2−CH2). 1H NMR (400 MHz, CDCl3): δ (ppm) 3.95−3.7 (−COO−CH2−CH2−), 3.0−2.85 (−CH2−CH2−N−), 2.7−2.5 (−N−CH−(CH3)2), 2.0−1.7 (backbone−C−CH2−C−), 1.25−1.2 (CH3−C8H16−CH2−), 1.15−0.85 (−N−CH−(CH3)2 and backbone−CH2−C−CH3−), 0.85−0.75 (backbone−CH2−C−CH3−) (Figure S2). 2.5. Synthesis of Poly(2-(diethylamio)ethyl methacrylateran-2-(diisopropylamino)ethyl methacrylate) (P(DEAEMA-rDPAEMA)). Three different P(DEAEMA-r-DPAEMA) were also synthesized via RAFT polymerization with different ratios of DEAEMA:DPAEMA monomer and were referred to as 3:1, 1:1, and 1:3 henceforth. All three polymers were prepared in 1,4-dioxane (4.0 g) to achieve the ratio of [monomer]/[CTA]/[initiator] = 250:10:1. The polymerization procedure was identical to those conducted for the polymers above. A typical polymerization procedure is described as follows: P(DEAEMA-r-DPAEMA) 3:1 was also synthesized with DEAEMA (4.40 g, 23.8 mmol), DPAEMA (1.60 g, 7.5 mmol), AIBN (0.98 mg, 6 μmol), and 4-cyano-4[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (24.22 mg, 60 μmol). After being cooled to room temperature, the crude mixture was terminated by exposure to the air. A portion of crude P(DEAEMA-r-DPAEMA) was precipitated similar to PDPAEMA procedure. Molecular weight (Mn) was determined by comparing the peak from the RAFT agent at δ (ppm) of 1.25−1.2 ppm (CH3−

Furthermore, pHlexi particles show potential benefits as smart carriers for future drug delivery research, as they combine low toxicity with pH-responsive disassembly and endosomal escape.

2. EXPERIMENTAL SECTION 2.1. Materials. The 4-cyano-5-[(dodecyl-sulfanylthiocarbony)sulfanyl] pentanoic acid (Sigma-Aldrich, 97%), poly(ethylene glycol)-4-cyano-4-(phenyl-carbonothioylthio)pentanoate (Sigma-Aldrich, 98%, PEG Mn = 2 kDa), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)4-methylmorpholinium chloride (DMTMM, ≥96.0%), cyanine-5 amine (Lumiprobe Life Science, 95%), and all solvents were used without modification. The water used in all materials experiments was water obtained from the reverse osmosis (RO) tap, whereas all cell experiments used high purity (Milli-Q) water of resistivity greater than 18.2 MΩ cm. To obtain 10 mM phosphate buffered saline (PBS), 1 L of 0.1 M PBS stock solution was prepared using sodium chloride (NaCl) (Sigma-Aldrich, ≥99.0%) (80 g, 137 mmol), potassium chloride (KCl) (Sigma-Aldrich, ≥99.0%) (2 g, 27 mmol), potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, ≥99.0%) (2.4 g, 17 mmol), and sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, ≥98.5%) (14.4 g, 10 mmol). The 0.1 M PBS stock solution was then diluted to be 10 mM before use. The 2-(diethylamino)ethyl methacrylate (DEAMEMA) (Sigma-Aldrich, 99%) and 2-(diisopropylamino)ethyl methacrylate (DPAEMA) (Sigma-Aldrich, 97%) were passed over aluminum oxide (activated, basic) (Sigma-Aldrich) to remove inhibitors prior to use. The 3.5 kDa MWCO Snakeskin dialysis tubing (Thermo Fisher Scientific), 12.4 kDa MWCO high retention seamless cellulose dialysis tubing (SigmaAldrich), 100 kDa MWCO Spectra-Por Float-ALyzer dialysis (SigmaAldrich), and Nonsterile Millex syringe filters with poly(ether sulfone) (PES) membrane (0.22, 0.45 μm pore diameter) were used as per instructions. Sodium bicarbonate, bovine serum albumin (BSA), calcein, and Triton X-100 solution were purchased from SigmaAldrich. DMEM with high glucose, with 4.0 mM L-glutamine, with sodium pyruvate and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Hyclone, GE Healthcare. Fetal bovine serum, penicillin/streptomycin (10 000 U/mL), Hochest stock solution (10 mg/mL), and alamarBlue reagent were obtained from Thermo Fisher Scientific. CellCarrier-96 Black, optically clear-bottom, tissue culture plates were obtained from PerkinElmer. Heparinized rat blood was ordered from Monash Animal Research Platform. 2.2. Characterization. 1H nuclear magnetic resonance (1H NMR) spectra were reported on an Agilent MR400 NMR spectrometer at room temperature. Chemical shifts (δH) were reported in parts per million (ppm). Deuterated chloroform (CDCl3: δH 7.25 ppm) and deuterium oxide (D2O) was used for 1 H NMR characterization. Gel permeation chromatographic (GPC) analysis was conducted with a Shimadzu system equipped with a CMB-20A controller system, an SIL-20A HT autosampler, and LC20AT tandem pump system, a DGU-20A degasser unit, a CTO-20AC column oven, an RDI-10A refractive index detector, and four Waters Styragel columns (HT2, HT3, HT4, and HT5). Each column was 300 mm × 7.8 mm2, providing an effective molar mass range from 100 to 4 × 106 Da. N,N-Dimethylacetamide (DMAc; with 4.34 g L−1 of lithium bromide (LiBr)) was used as an eluent with a flow rate of 1 mL/min at 80 °C. Number-average molecular weight (Mn) and polydispersity index (Đ) were calculated using Shimadzu LC Solution software. The GPC columns were calibrated with low dispersity poly(methyl methacrylate) (PMMA) standards (Polymer Laboratories). Transmission electron microscope (TEM) images were carried out on a Tecnai F30 TEM with cryotomography (FEI) operating at an acceleration voltage of 300 kV. To prepare cryo-TEM sample, small drops of particle solution were deposited onto copper TEM grids in liquid nitrogen by automated plunge freezer (FEI Vitrobot Mark III). Particle concentration was calculated with 5 measurements by using NanoSight (NS300, Malvern Instruments Ltd.) at 25 °C and, the amount of particles per frame were approximately 20−100 particles. The Nanoparticle Tracking Analysis (NTA) software C

DOI: 10.1021/acsanm.8b00338 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials C8H16−CH2−) to PDEAMA polymer peak at δ of 4.1−3.9 ppm (−COO−CH2−CH2−) and the PDPAEMA polymer peak at δ of 3.9−3.7 ppm (−COOH−CH2−CH2) and is given in Table S1 for all the polymers. 1H NMR (400 MHz, CDCl3): δ (ppm) 4.1−3.9 (PDEAEMA −COO−CH2−CH2−), 3.9−3.7 (PDPAEMA −COO− CH2−CH2−), 3.1−2.8 (PDPAEMA −N−CH−(CH3)2), 2.75−2.7 (PDPAEMA and PDEAEMA −CH2−CH2−N−), 2.7−2.4 (PDEAEMA −N−CH2−CH3−), 2.1−2.6 (backbone −C−CH2−C−), 1.25− 1.2 (CH3−C8H16−CH2), 1.2−0.9 (−N−CH2−CH3, −N−CH− (CH 3 )2 and backbone −CH 2 −C−CH 3 ), 0.9−0.7 (backbone −CH2−C−CH3) (Table S1 and Figures S3−S5). 2.6. Synthesis of Poly(2-diethylaminoethyl methacrylate)b-poly(ethylene glycol) (PDEAEMA-b-PEG). PDEAEMA-b-PEG was polymerized via RAFT polymerization with DEAEMA (0.5 g, 2.7 mmol), AIBN (0.49 mg, 3.0 μmol), poly(ethylene glycol)-4-cyano-4(phenyl carbonothioylthio)pentanoate (53 mg, 26.5 μmol), and 1,4dioxane (1.0 g). The mixture was subjected to freeze−thaw−pump cycles spanning 5, 10, 20, and 40 min then backfilled with nitrogen. The degassed reaction mixture was stirred in an oil bath at 60 °C for 15 h. The reaction was terminated by exposure of the reaction mixture to air. The mixture was purified by dialysis against 0.1 M PBS at pH 6 and then water. 1H NMR (400 MHz, D2O): δ (ppm) 7.9−7.3 (C2H5−C−), 4.4−4.0 (−COO−CH2−CH2), 3.75−3.45 (−COO− CH2−CH2−O−), 3.3−3.15 (−CH2−CH2−N−), 3.2 (−CH2−O− CH3− (under polymer peak)), 3.15−2.8 (−N−CH2−CH3), 2.5−2.4 (−C−CH2−CH2−COO−), 2.0−1.6 (backbone −CH2−C−), 1.25− 1.1 (−N−CH2−CH3) and (backbone−CH2−C−CH3), 1.11−0.7 (backbone−CH2−CH2−CH3). The molecular weight (Mn) of PDEAEMA-b-PEG was determined by NMR analysis to be approximately 7 kDa. 2.7. Purification of PDEAEMA, P(DEAEMA-r-DPAEMA), and PDPAEMA. To purify the crude polymers PDEAEMA, P(DEAEMAr-DPAEMA), and PDPAEMA polymerization mixtures were dialyzed in a 12.4 kDa MWCO dialyses tubing against 10 mM PBS pH 5.0 until they became homogeneous solutions and then for 24 h with at least 6 water exchanges for removing PBS. The solution obtained was lyophilized to yield white soft solid prior to use. The number-average molecular weight (Mn) (kDa) and polydispersity index (PDI) were determined for the water-soluble polymer by using DMac GPC (Table S1). 2.8. Polymer-Buffering Capability. To investigate polymerbuffering capability as a function of pH range and volume of 0.1 M NaOH, core polymer (0.5 mg/mL) in 150 mM NaCl at pH 3 was titrated against 0.1 M NaOH, and the pH was measured using a Mettler Teledo pH meter. 2.9. Conjugation of Cyanine-5 Amine to DMTMM Terminated Polymer. DMTMM coupling agent (20 mg, 72 μmol molar excess) was dissolved in 1 mL of water. Hydrophilic PDEAEMA (50 mg, 1.25 μmol), 3:1 (50 mg, 1.02 μmol), 1:1 (50 mg, 1.06 μmol), 1:3 (50 mg, 1.04 μmol), and PDPAEMA (50 mg, 1.04 μmol) were added in separated vials into DMTMM solution, and the mixtures were allowed to stir at room temperature for 24 h under a high stir rate. Cyanine-5 amine (Cy5) (1 mg, 1.53 μmol) was dissolved in 10 μL of DMSO and was then transferred into the mixture. The mixture was stirred for over 24 h in an oil bath at 50 °C. The polymer was dialyzed against water with multiple water exchanges until the exchange water was clear; then the polymers were lyophilized to yield blue soft solids. 2.10. Determination of Molar Ratio of Cy5 per Polymer. The absorbance (310 and 646 nm) of the resulting Cy5-polymer solution (1 mg/mL) was measured by UV−vis spectroscopy. Polymer concentration was measured from a standard curve of polymer concentration (PEG-b-PDEAEMA and core polymer at 1:5 w/w ratio) (Figure S6) versus absorbance at 310 nm (Figure S7). Cy5 concentration was calculated by Beer−Lambert Law equation with absorption coefficient, ε = 250 000 L/(mol cm). 2.11. Formation of pH-Responsive Nanoparticles and Determination of Particle Concentration. PDEAEMA (40 kDa), P(DEAEMA-r-DPAEMA) 3:1 (49 kDa), P(DEAEMA-rDPAEMA) 1:1 (47 kDa), P(DEAEMA-r-DPAEMA) 1:3 (48 kDa),

and PDPAEMA (48 kDa) were referred to as the core component of the nanoparticles while PEG-b-PDEAEMA was termed the shell component. The core and shell component were mixed into 3 mL of 0.1 M PBS at pH 6 at a 1:5 shell/core mass ratio to achieve a total concentration of 1 mg/mL. The five mixtures were placed separately in 3.5 kDa MWCO Snaking dialysis tubing for 6 h and dialyzed against 0.1 M PBS pH 8. These mixtures were then transferred to 100 kDa MWCO dialysis tube for 24 h in 0.1 M PBS pH 8 with 5 buffer changes to remove excess polymer. Then the nanoparticles were removed from the dialysis tube and allowed to rest for 40 h. The five sets of nanoparticles were filtered though a 0.45 μm PES syringe filter prior to use. The Cy5 nanoparticle preparation was conducted as per standard pH-responsive particles formation however using Cy5polymer as core component of the nanoparticles. The particle concentration was measured using NanoSight. 2.12. Disassembly Studies and Particle Size. The disassembly and particle size of the nanoparticles were assessed using dynamic light scattering (Horiba Scientific, SZ-100) at a fixed scattering angle of 90° and at 37 °C. The detailed protocol can be found in the following publication.28 2.13. Critical Micelle Concentration (CMC) of pH-Responsive Nanoparticles. To investigate CMC of the particles the particle solution was diluted in PBS pH 8 ranging from 125 to 1250 times dilution. Mean particle diameter (nm) and particles concentration (particles/mL) were measured by NanoSight (NS300, Malvern Instruments Ltd.) at 25 °C. 2.14. Hemolysis Assay. A 5 mL portion of heparinized rat blood was obtained from salvaged animals 3 h before the assay. Each mL of blood was washed and spun with 50 mL of prechilled PBS at 3000 rpm at 4 °C until supernatant was clean. The supernatant was discarded carefully without disturbing the pellet. The blood was diluted to 0.05% w/w in PBS solution at different pH (6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4). Standard nanoparticles with different core polymer (2 × 109 particle/mL) were added to the blood solution (in triplicates) at different concentration in a V-shape-bottom 96-well plate (Corning, Sigma-Aldrich). The 100% hemolysis was reached by adding 2% Triton X-100 into the blood suspension, and the negative control was the blood with PBS. The plate was incubated at 37 °C for 1 h. The plate was spun at 3000 rpm at room temperature, and 100 μL of the supernatant was carefully transferred to a flat, clear-bottom 96-well plate (Corning). The UV absorbance was read at 540 nm. The percentage of hemolysis was calculated using the following equation:

ji Ab 540 sample − Ab 540 negative zyz z × 100% hemolysis (%) = jjj j Ab 540 100% − Ab 540 negative zz k {

2.15. Cell Viability Assay. NIH/3T3 mouse embryonic fibroblast cells were seeded at 10 000 cells per well in an optically clear-bottom 96-well plate and incubated overnight at 37 °C with 5% CO2. Standard nanoparticle solutions were added to the cells (in triplicates) to a final concentration of 0.19, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, and 50 × 109 particles/mL. Cells were incubated with particles for 4 h at 37 °C with 5% CO2. The media was replaced with fresh prewarmed media for overnight culture. alamarBlue reagent (Thermo Fisher) was incubated with cells as in the manufacture’s protocol for 6 h at 37 °C with 5% CO2. The fluorescence at 590 nm was read using a fluorescence plate reader (Clariostar, BMG).28 2.16. Investigation of Endosomal Escape Capability of pHResponsive Nanoparticles. The calcein escape assay was employed to investigate endosomal escape capability. NIH/3T3 cells were seeded at 10 000 cells per well (100 μL volume) in an optically clearbottom 96-well plate overnight. Standard nanoparticle solutions were added to cells to a final concentration of 0.5, 1.5, 5, and 15 × 109 particles/mL in culturing medium and incubated for 2 h at 37 °C with 5% CO2. Calcein solution was added to a final concentration of 100 μg/mL, and cells were incubated for another 2 h. The cells were carefully washed five times with FluoroBrite with 10% FBS to remove excess calcein. Live cell imaging was performed using an Olympus IX83 microscope with a 40 × 0.9 NA air objective with a standard D

DOI: 10.1021/acsanm.8b00338 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials “Pinkel” FITC filter set from Semrock. The detailed experimental protocol for assessment of endosomal escape is given in the following publication.28 2.17. Investigation of Colocalization of pH-Responsive Nanoparticles. Stably transfected NIH/3T3 cells with Rab5a-7 or Rab7a-7 or LAMP1 fused with mApple were seeded in an 8-well chamber (Lab-Tak II) as 40 000 cells per well, and cells were cultured overnight. Cy5 nanoparticles were added to cells to a final concentration of 5 and 15 × 109 particles/mL in culturing medium and incubated for 4 h at 37 °C with 5% CO2. Cells were carefully washed three times with prewarmed FluoroBrite with 10% FBS to remove excess particles. Cells were stained with 5 μg/mL HochestFluoroBrite solution for 15 min. Staining solution was replaced with fresh FluoroBrite with 10% FBS for imaging. Live cell imaging was performed using an Olympus IX83 microscope with a 60 × 1.3 NA silicone oil objective. The cells were imaged in a humidified incubation chamber with 5% CO2 and regulated at a temperature of 37 °C. All images were processed using Slidebook 6.0 and Fiji software. 2.18. Particle−Cell Association by Flow Cytometry. NIH/ 3T3 mouse embryonic fibroblast cells were maintained in DMEM supplemented with high glucose (GlutaMAX) and 20% fetal bovine serum. Cells were cultured at 37 °C in a humidified incubator with 5% atmospheric CO2. For flow cytometry, cells were seeded at 100 000 cells per well in 24-well plates with 400 μL of media. Cy5 nanoparticles were added to cells to a final concentration of 0.5, 1.5, 5, and 15 × 109 particles/mL in culturing medium and incubated for 4 h at 37 °C with 5% CO2. After incubation, the media was removed, and cells were washed three times on plate with 400 μL of PBS. The cells were detached and transferred to a 96-well V-bottom plate (Corning, Sigma-Aldrich) and spun at 400g for 5 min. The supernatant was discarded, and cells were resuspended in 150 μL of 1% BSA−PBS. Samples were run on a S1000EXi flow cytometer (Stratedigm, Inc.). Particle concentrations of 0.5, 1.5, 5, and 15 × 109 particles/mL were analyzed for each sample, and the data were analyzed using FlowJo 8.7 software (FlowJo, LLC).28 The association was normalized to the amount of Cy5 according to molar ratio of cy5 per polymer (Figure S7).

Figure 1. Investigation of pHlexi nanoparticle properties and morphology. (a) Intensity size distribution of pHlexi nanoparticles in PBS pH 8. (b) pH disassembly profile of pHlexi nanoparticles as a function of PBS pH range and mean particle size. DLS measurement errors were averaged from three readings. Note: color code for part b is the same as for part a, with symbols corresponding to PDEAEMA (▼), 3:1 (●), 1:1 (■), 1:3 (▲), and PDPAEMA (⧫). Cryo-electron microscope (Cryo-EM) images of (c) 3:1, (d) 1:1, (e) 1:3, and (f) PDPAEMA pHlexi nanoparticles. Note: dark nonspherical structures are due to the TEM grid.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of pHlexi Nanoparticles. PDEAEMA, PDEAEMA-r-PDPAEMA (3:1, 1:1 and 1:3), and PDPAEMA, which form the core of the pHlexi particles, were all synthesized by RAFT polymerization. The molecular weights of the polymers were between 40 and 49 kDa, and their 1H NMR and GPC characterization are given in the Supporting Information (Figures S1−S5, Table S1). These polymers all undergo a change from a hydrophobic (deprotonated) state above their pKa to a hydrophilic (protonated) state below their pKa, and have a similar buffering capacity (Figure S8, Table S2). A diblock copolymer, PDEAEMA-b-PEG (Mn 7 kDa), which stabilizes the pHlexi particles was synthesized by polymerizing DEAEMA monomer using a PEG-containing macro-chain-transfer-agent (macroCTA) (average Mn = 2 kDa). The nanoparticles were formed by dialyzing a solution of both polymers at a mass ratio of 1:5 (PEG-b-PDEAEMA/PDEAEMAx-r-PDPAEMAy) against PBS at pH 8. The core polymers and the PDEAEMA component of the diblock copolymer become hydrophobic under these conditions. It is proposed the nanoparticle structure comprises a hydrophilic shell formed by the PEG-b-PDEAEMA while the random copolymer PDEAEMAx-r-PDPAEMAy forms a hydrophobic core. DLS (Figure 1a and Table S4) data indicate the majority of the pHlexi nanoparticles, PDEAEMA, 3:1, 1:1, and 1:3, have particle diameters in the range 130−160 nm. However, PDPAEMA particles had a slightly larger size with an average diameter size of 210 nm. We believe it is possible that

the slightly larger size of the PDPAEMA nanoparticle could be due to more polymer being incorporated into each nanoparticle, as the nanoparticles take significantly longer to disassemble. This observation was confirmed by cryo-electron microscopy (cryo-EM) (Figure 1c−f, Figure S13). The cryoEM images show well-defined spherical structures for all pHlexi nanoparticles; interestingly, the contrast between core and corona of nanoparticles was enhanced with an increase of PDPAEMA in the core polymer.27,28 Nanoparticle Tracking Analysis (NTA) was employed to characterize the stability of the nanoparticles at different particle concentrations. The critical micelle concentration (CMC) of pHlexi particles was measured through a plot of mean particle diameter size versus the particle concentration (particles/mL) as shown in Figure S10. It was found the CMC was between 2 and 4 × 108 particles/mL depending on the particle formulation. This is well below the particle concentrations used in cell experiments throughout this study. DLS measurements were also used to ensure that the particles remained stable at physiological pH for the duration of subsequent cell association experiments (Figure S22). The disassembly profile of pHlexi nanoparticles was investigated to probe where in the endosomal/lysosomal trafficking pathway the particles are likely to disassemble. Particle size was measured by incubating an aliquot of particles E

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ACS Applied Nano Materials in PBS ranging from pH 4.2 to pH 8.0 at 37 °C (Figure 1b). Particle disassembly was assessed by measuring the size of the particles, the count rate in the DLS (low counts indicating less particles), and the quality of the correlation function. The disassembly pH of PDEAEMA nanoparticles was similar to the pH reported in our previous work, disassembling at pH 7.27,28 This disassembly point correlates to the pKa of the DEAEMA monomer, and suggests disassembly is driven by protonation of the DEAEMA components. The pKa of the polymers are shown the Supporting Information, Table S2. It is important to note the pKa in the disassembly experiment will be lower as pKa of these monomers is temperature dependent. The three sets of hybrid pHlexi nanoparticles (3:1, 1:1, and 1:3) showed progressively lower disassembly pH as more DPAEMA was contained in the particle. This was as expected as DPAEMA has a lower pKa; however the disassembly pHs were lower than expected. The disassembly pH values of 3:1, 1:1, and 1:3 particles were at 6.6, 6.2, and 5.8, respectively. PDPAEMA particles disassembled at pH 4.9, which is significantly lower than the pKa of DPAEMA at approximately 6.4 (Table S2). This suggests additional interactions stabilized the particles, e.g., hydrophobic interactions of the isopropyl groups with the polymer backbone. All pHlexi nanoparticles showed a region of instability, where partial swelling and/or aggregation of the particles occurred before its disassembly point. This rearrangement was over a wider pH range for the particles with more DPAEMA, suggesting enhanced stability to polymer rearrangement when more DPAEMA is present. The disassembly of the nanoparticles is governed by the core polymer, with the shell polymer likely remaining physically entangled within the particle structure. It does cause some rearrangement at pH 7.0−7.2 as indicated by higher polydispersity for some nanoparticles at those pHs (Table S3). 3.2. Assessing Cytotoxicity of pHlexi Nanoparticles in Cultured NIH/3T3 Cells. To investigate cell behavior of the pHlexi nanoparticles it is important to first assess cell toxicity. To evaluate the biocompatibility of pHlexi nanoparticles, cell viability tests on NIH/3T3 cells were conducted using the alamarBlue assay (Figure 2). The cells showed >90% viability when exposed to 3:1 and 1:1 particles, up to a concentration of 50 × 109 particles/mL. The 1:3 nanoparticles showed some toxicity >15 × 109 particles/mL, and PDPAEMA particles showed some toxicity at concentrations >6.5 × 109 particles/

mL. However, PDEAEMA had significantly higher toxicity with viability starting to decrease at 6.5 × 109 particles/mL, resulting in a decrease of viability to 70% at 15 × 109 particles/ mL. Based on these results, further cell experiments were conducted at 0.5 × 109, 1.5 × 109, 5 × 109, and 15 × 109 particles/mL. 3.3. Investigation of Membrane Destabilization via Hemolysis Assay. To understand the cell interactions of the nanoparticles, their membrane destabilization capacity was examined by a red blood cell hemolysis assay. Rat red blood cells and pHlexi nanoparticles were coincubated in buffers at defined pHs from 6.0 to 7. This range was chosen as it mimics the pH transition from the bloodstream to the endosomal compartments. The amount of hemoglobin released during the incubation period was quantified as a measure of red blood cell lysis (Figure 3). As expected, the pH at which hemolysis

Figure 3. Hemolysis assay of pHlexi nanoparticles at various pHs ranging between 6.0 and 7.4 that mimic biologically relevant pHs.

occurred decreased with increasing proportion of DPAEMA. An increase in DPAEMA correlates with a decrease in DEAEMA so the pH at which maximum hemolysis occurs increases with increasing DEAEMA proportion. The block copolymer proportion was the same in all particles. However, interestingly, the maximum membrane destabilization was observed about 0.5 pH units above particle disassembly pH, with lower hemolysis observed at the disassembly pH. This suggests that some rearrangement occurs in the particles prior to disassembly, and this rearrangement plays an important role in endosomal escape. The higher toxicity observed for the PDEAEMA particles in Figure 2 may be partly explained by the hemolytic potential of the PDEAEMA particles at pH 7.4. If the particles are able to disrupt the plasma membrane at physiological pH, this is likely to affect the viability of the cells. 3.4. Endosome Escape Capability of pHlexi Nanoparticles. To test the ability of pHlexi nanoparticles to disrupt endosomes, calcein, a membrane-impermeable fluorophore, was used as a tracer to monitor the integrity of endosomes following incubation with particles at various concentrations (0.5, 1.5, 5, and 15 × 109 particles/mL). As shown in Figure 4f, NIH/3T3 cells incubated with calcein alone (control) showed a punctate distribution of fluorescence, indicating calcein remained internalized within endosome compartments. This is consistent with previous work by our group and others.28−30 In contrast, cell coincubated with calcein and pHlexi nanoparticles at 15 × 109 particles/mL showed calcein fluorescence throughout the cytosol indicating particle-induced escape of the calcein from the endosomes (Figure 4). We have previously shown that while these particles are effective at

Figure 2. alamarBlue cell viability assay results performed on NIH/ 3T3 cells in the presence of pHlexi nanoparticles after 24 h of incubation at various concentrations. F

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Figure 5. Quantification of pHlexi particle-induced endosomal escape of calcein from NIH/3T3 fibroblast cells with particle concentration at (a) 0.5, (b) 1.5, (c) 5, and (d) 15 × 109 particles/mL.

the association of PDPAEMA particles, while the escape induced by 1:1 particles was significantly less than that induced by PDPAEMA particles. One factor that could govern the endosomal escape behavior of the different particles is the degree of association with the cells. To determine cellular association, Cy5 labeled particles at 0.5, 1.5, 5, and 15 × 109 particles/mL were incubated with NIH/3T3 fibroblast cells, and cell binding was detected using flow cytometry. Particles prepared with different core composition contained varying amounts of Cy5 amine, and hence had different fluorescence intensities. The particle association was normalized to the relative amount of Cy5 in the particles (degree of labeling, DOL). The DOL for each particle was calculated by ratioing the absorbance at 646 nm (Cy5) with the absorbance of the particles at 310 nm (polymer) (Figure S7). The normalized fluorescence signal allows a direct comparison of association across all particles, as shown in Figure 6. As expected, the relative fluorescence intensity of the cells increased with an increase in particle concentration, showing higher association with higher particle concentration. PDEAEMA particles had the highest association; however it should be noted at high concentrations there was some toxicity, and thus cell numbers were reduced.

Figure 4. Fluorescence microscopy images showing the percentage of diffuse calcein fluorescence (green) throughout the NIH/3T3 cells with incubating (a) PDEAEMA, (b) 3:1, (c) 1:1, (d) 1:3, and (e) PDPAEMA at concentration of 15 × 109 particle/mL. (f) Calcein control image. Scale bar = 10 μm.

inducing the endosomal escape of small molecules like calcein, we did not detect fluorescence from the polymer in the cytosol. The percent of cells showing endosomal escape was assessed by counting the number of cells with diffuse fluorescence in a 5 × 5 mosaic of cell images (total area 2.5 mm2) (Figure 4, Figures S24−S28) at the center of the well. Cells treated with PDEAEMA and PDPAEMA particles showed ∼90% of the cells had endosomal escape at 15 × 109 particles/mL, while cells treated with 3:1 and 1:3 particles exhibited ∼70% endosomal escape, and cells treated with 1:1 particles had only 15%. It should be noted that at 15 × 109 particles/mL PDEAEMA particles show a degree of cytotoxicity, and this could affect the endosomal escape behavior. Calcein experiments that were conducted at 0.5, 1.5, and 5 × 109 particles/ mL demonstrated a threshold particle concentration was required for endosomal escape (Figure 5). The most promising candidate for inducing effective endosomal escape was particles with the 3:1 core, as they displayed high levels of endosomal escape behavior down to 5 × 109 particles/mL, but with significantly reduced toxicity compared to PDEAEMA particles. While the PDEAEMA, 3:1, 1:1, and 1:3 particles had similar sizes, suggesting particle size was not a defining property of endosomal escape, PDPAEMA particles were slightly larger, and thus size may play a role in enhancing the escape properties. However, it should be noted that the association of 1:1 particles to cells was significantly higher than

Figure 6. Association of the pHlexi nanoparticles with NIH/3T3 cells. Normalized cell fluorescence intensity as a result of Cy-5 nanoparticle association at increasing particle concentration: (a) 0.5, (b) 1.5, (c) 5, and (d) 15 × 109 particles/mL. Error bars are the standard deviation of the experiment conducted in triplicate. G

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Figure 7. Multichannel cellular images showing (i) 3:1 and (ii) 1:3 Cy-5 labeled pHlexi nanoparticles colocalization in NIH/3T3 cells transfected (a) Rab5a, (b) Rab7a, and (c) LAMP1 biomarkers. Scale bar = 10 μm.

Interestingly, the amount of particle association with cells does not directly correlate with the degree of endosomal escape. The 1:1 particles showed significantly higher association with NIH/3T3 cells than PDPAEMA, 3:1, and 1:3 particles; however they showed significantly lower endosomal escape. While association and internalization are not always the same, we demonstrated in previous studies that they are strongly correlated for similar nanoparticles in this cell line.31 This suggests that the unique properties of each particle govern the endosomal escape properties. More efficient endosomal escape was observed with particles that disassemble at high (pH 7.2) or low (pH 4.9) pH, compared to less efficient escape for particles that disassemble between pH 5.8 and 6.6. This trend was confirmed with a repeat calcein experiment shown in Figures S14−S18. From the hemolysis results, it appears that membrane association plays a role in disrupting the membrane, but it is likely this interaction is more significant in the early endosome compared to the lysosome. This suggests other mechanisms are playing a role at low pH. Understanding these mechanisms will be the focus of further studies. Importantly, a control poly(ethyl glyoxylate) (PEtG) pHlexi particle that does not undergo disassembly but has the same surface chemistry was also tested for hemolysis and endosomal escape potential. The synthesis of the control particle is given in the Supporting Information along with particle characterization (Figure S19). These particles showed no hemolysis or endosomal escape, suggesting the disassembly of the nanoparticle is key to the particles inducing endosomal escape (Figures S20, S21, and S26). To investigate the subcellular localization of the particles, colocalization studies with Rab5a (early endosome), Rab7a (late endosome), and LAMP1 (lysosome) were conducted. Cy-5 labeled nanoparticles were incubated with NIH/3T3 cells that stably express a mCherry fusion of Rab5a, Rab7a, or LAMP1. As we have observed previously, the polymer signal

remains punctate, with no observable signal detected in the cytoplasm (Figure 7, Figure S9). This is in clear contrast to the diffuse fluorescence of the calcein that occurs when the particles induce endosomal escape. This indicates that minimal amounts of polymer can escape from the endosomes, and suggests that the destabilization of the endosomal membrane is only sufficient to allow small molecules (such a calcein) to escape, but the larger polymers are not able to do so efficiently. Minimal colocalization of the polymers was observed with the early or late endosomes, with almost complete colocalization with the lysosomes.

4. CONCLUSIONS In summary, a library of pHlexi particles was synthesized by a simple, one-pot assembly approach. The pH of disassembly was tuned by combining poly(ethylene glycol)-b-poly(2(diethylamino)ethyl methacrylate) (PEG-b-PDEAEMA) with random copolymers of 2-(diethylamino)ethyl methacrylate and 2-diisopropylamino)ethyl methacrylate with tunable pKa. All the particles were stable at pH > 7.2 but disassembled between pH 4.9 and 7.0, depending on the monomer composition. Hemolysis of these particles was also shown to be tunable based on the monomer composition; however it was observed to occur slightly higher than the pH at which the nanoparticles disassemble. The association of these particles with cells was found to be higher for PDEAEMA and 1:1 particles, and did not correlate to the endosomal escape behavior. Higher endosomal escape was found with particles that disassemble at high pH (PDEAEMA) or low pH (PDPAEMA), with the lowest escape observed in the 1:1 formulation. This suggests there are different pathways for inducing endosomal escape, as if PDPAEMA and PDPAEMA induced escape by the same mechanism; then the copolymer particles would exhibit the same levels of escape as the homopolymer. In contrast, we observe significantly less escape in this case. It was also H

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(6) Ganta, S.; Devalapally, H.; Shahiwala, A.; Amiji, M. A review of stimuli-responsive nanocarriers for drug and gene delivery. J. Controlled Release 2008, 126, 187−204. (7) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-responsive nanocarriers for drug delivery. Nat. Mater. 2013, 12, 991−1003. (8) Such, G. K.; Yan, Y.; Johnston, A. P. R.; Gunawan, S. T.; Caruso, F. Interfacing Materials Science and Biology for Drug Carrier Design. Adv. Mater. 2015, 27, 2278−2297. (9) Shim, M. S.; Kwon, Y. J. Stimuli-responsive polymers and nanomaterials for gene delivery and imaging applications. Adv. Drug Delivery Rev. 2012, 64, 1046−1059. (10) Selby, L. I.; Cortez-Jugo, C. M.; Such, G. K.; Johnston, A. P. R. Nanoescapology: progress toward understanding endosomal escape of polymeric nanoparticles. WIREs Nanomed Nanobiotechnol 2017, 9, e1452. (11) Kelley, E. G.; Albert, J. N. L.; Sullivan, M. O.; Epps, T. H. Stimuli-responsive copolymer solution and surface assemblies of biomedical applications. Chem. Soc. Rev. 2013, 42, 7057−7071. (12) Bayles, A. R.; Chahal, H. S.; Chahal, D. S.; Goldbeck, C. P.; Cohen, B. E.; Helms, B. A. Rapid Cytosolic Delivery of Luminescent Nanocrystals in Lice cells with Endosome-Disrupting Polymer Colloids. Nano Lett. 2010, 10, 4086−4092. (13) Bareford, L. M.; Swaan, P. W. Endocytic mechanisms for targeted drug delivery. Adv. Drug Delivery Rev. 2007, 59, 748−758. (14) Canton, I.; Battaglia, G. Endocytosis at the nanoscale. Chem. Soc. Rev. 2012, 41, 2718−2739. (15) Iversen, T. G.; Skotland, T.; Sandvig, K. Endocytosis and intracellualr transport of nanoparticles: Present knowledge and need for future studies. Nano Today 2011, 6, 176−185. (16) Kang, Y.; Wang, C.; Liu, K.; Wang, Z.; Zhang, X. EnzymeResponsive Polymeric Supra-Amphiphiles Formed by the Complexation of Chitoscan and ATRP. Langmuir 2012, 28, 14562−14566. (17) Huang, D.; Wang, Y.; Yang, F.; Shen, H.; Weng, Z.; Wu, D. Charge-reversible and pH-responsive biodegradable micelles and vesicles from linear-dendritic sup[ramolecular amphiphiles for anticancer drug delivery. Polym. Chem. 2017, 8, 6675−6687. (18) Su, X.; Fricke, J.; Kavanagh, D. G.; Irvine, D. J. In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles. Mol. Pharmaceutics 2011, 8, 774−787. (19) Hu, Y.; Litwin, T.; Nagaraja, A. R.; Kwong, B.; Katz, J.; Watson, N.; Irvine, D. J. Cytosolic Delivery of Membrane-Impermeable Molecules in Dendritic Cells using pH-Responsive Core-Shell Nanoparticles. Nano Lett. 2007, 7, 3056−3064. (20) Chen, Q.; Lin, W.; Wang, H.; Wang, J.; Zhang, L. PDEAEMAbased pH-sensitive amphiphilic pentablock copolymers for controlled anticancer drug delivery. RSC Adv. 2016, 6, 68018−68027. (21) Yang, C.; Xiao, J.; Xiao, W.; Lin, W.; Chen, J.; Chen, Q.; Zhang, L.; Zhang, C.; Guo, J. Fabrication of PDEAEMA-based pH-responsive mixed micelles for application in controlled doxorubicin release. RSC Adv. 2017, 7, 27564−27573. (22) Lomas, H.; Canton, I.; MacNeil, S.; Du, J.; Armes, S. P.; Ryan, A. J.; Lewis, A. L.; Battaglia, G. Biomimetric pH Sensitive Polymersomes for Efficient DNA Encapsulation and Delivery. Adv. Mater. 2007, 19, 4238−4242. (23) Liang, K.; Such, G. K.; Zhu, Z.; Yan, Y.; Lomas, H.; Caruso, F. Charge-Shiftiing Click Capsules with Dual-Responsive Cargo Release Mechanisms. Adv. Mater. 2011, 23, H273−277. (24) Huang, X.; Huang, G.; Zhang, S.; Sagiyama, K.; Togao, O.; Ma, X.; Wan, Y.; Li, Y.; Soesbe, T. C.; Sumer, B. D.; Takahashi, M.; Sherry, A. D.; Gao, J. Multi-chromatic pH-activatable 19F-MRI nanoprobes with binary ON/OFF pH transitions and chemical-shift barcodes. Angew. Chem., Int. Ed. 2013, 52, 8074−8078. (25) Yu, H.; Zou, Y.; Wang, Y.; Huang, X.; Huang, G.; Sumer, B. D.; Boothman, D. A.; Gao, J. Overcoming Endosomal Escape by Amphotericin B-loaded Dual pH-Responsive PDMA-b-PDPA Micelleplexes for siRNA Delivery. ACS Nano 2011, 5, 9246−9255. (26) Wilson, J.T.; Keller, S.; Manganiello, M.J.; Cheng, C.; Lee, C. C.; Opara, C.; Convertine, A.; Stayton, P.S. pH-Responsive

demonstrated that while endosomal escape was dependent on particle concentration, even at the highest concentrations tested, the 1:1 particles were less efficient at inducing escape than PDEAEMA and 3:1 particles at 3 times lower particle concentrations. We have shown here that pHlexi particles can be easily engineered to disassemble across a physiologically relevant pH range, and thus have potential to provide important insights into the mechanism of endosomal escape and how it can be optimized in nanoparticle systems. In addition, pHlexi particles are simple to synthesize, exhibit pH dependent endosomal escape, and therefore have potential for application in therapeutic delivery.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b00338. Detailed experimental methodologies, along with characterization information and additional figures including 1 H NMR, UV−vis, absorbance spectra, polymerbuffering curve, multichannel cellular images, CMC curves, mean particle diameter, disassembly curves, cryoelectron microscope images, fluorescence microscopy images, pHlexi particle-induced endosomal escape quantification, particle size distribution, hemolysis assay, and cell viability vs endosomal escape (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Elizabeth R. Gillies: 0000-0002-3983-2248 Angus P. R. Johnston: 0000-0001-5611-4515 Georgina K. Such: 0000-0002-2868-5799 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Australian Research Council through the Future Fellowship Scheme (FT120100564, G.K.S.; and FT110100265, A.P.R.J.) and Centre of Excellence in Convergent Bio- Nano Science and Technology (A.P.R.J.). The authors thank Dr. E. Hanssen (Melbourne Advanced Microscopy Facility, Bio21 Molecular Science and Biotechnology Institute) for cryo-EM imaging.

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REFERENCES

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