pH-Responsive Nanoemulsions for Controlled Drug Release

Feb 15, 2014 - TEM was performed using a JEM-100CX II microscope operated at 80 kV. ...... Mason , T. G.; Wilking , J. N.; Meleson , K.; Chang , C. B...
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pH-Responsive Nanoemulsions for Controlled Drug Release Feng Liu,†,‡,⊥ Shudong Lin,†,‡,⊥ Zuoquan Zhang,∥ Jiwen Hu,*,†,‡ Guojun Liu,*,†,§ Yuanyuan Tu,†,‡ Yang Yang,†,‡ Hailiang Zou,†,‡ Yangmiao Mo,†,‡ and Lei Miao†,‡ †

Guangzhou Institute of Chemistry, Chinese Academy of Sciences, Guangzhou 510650, P. R .China Key Laboratory of Cellulose Lignocellulosics Chemistry, Chinese Academy of Sciences, Guangzhou 510650, P. R .China § Department of Chemistry, Queen’s University, 90 Bader Lane, Kingston, Ontario, Canada K7L 3N6 ∥ Department of Radiology, the Fifth Affiliated Hospital, Sun Yat-Sen University, Zhuhai 519000, China ‡

ABSTRACT: Three ternary graft copolymers bearing polystyrene (PS), poly(ethylene glycol) methyl ether (MPEG), and poly(acrylic acid) (PAA) side chains were synthesized and characterized. At pH = 7.4, these copolymers stabilized doxorubicin (DOX)-containing benzyl benzoate (BBZ) nanoemulsion droplets in water and formed a compact polymer layer to inhibit DOX release. Upon lowering the solution pH to 5.0, the AA groups dissociated less and became less soluble. Moreover, the neutralized AA groups formed presumably Hbonded complexes with the EG units, reducing the solubility of the EG units. This dual action drastically shifted the hydrophilic and hydrophobic balance of the copolymer and caused the original stabilizing polymer layer to rupture and the nanoemulsion droplets to aggregate, releasing DOX. The rate and extent of DOX release could be increased by matching the numbers of PAA and MPEG chains per graft copolymer. In addition, these nanoemulsions were not toxic and entered human carcinoma cells, releasing DOX there. Thus, these nanoemulsions have potential as drug delivery vehicles.

I. INTRODUCTION Breaking an oil into fine droplets in water with a dispersant produces an oil-in-water emulsion. An emulsion is stable because the interfacial tension is reduced by an amphiphilic dispersant layer that forms at the oil/water interface. Commonly used dispersants include surfactants,1 diblock copolymers,2 particles,3 or graft copolymers.4−6 If the radii of the dispersed oil droplets are less than 100 nm, a nanoemulsion is produced.7 Compared to traditional microscaled emulsions that have droplet radii typically larger than 100 nm, nanoemulsions possess reduced opacity, enhanced stability, and increased ability to penetrate biological membranes including skins. Compared to solid particles, they are more deformable and softer and may exhibit new and different pathways for cellular uptake and dispersal.7 Thus, nanoemulsions have great potential in the controlled release of drugs,8,9 fragrances,10 and cosmetic agents.11 Despite this potential, nanoemulsions are not widely used in consumer products because they are energetically costly to produce and scalable techniques for their production have become available only recently.12 Emulsions that break or change their dispersion state when externally perturbed are stimuli-responsive. The stimuli used so far have included changes in a system’s temperature,13 the ionic strength,14 sample irradiation,15 or the introduction (or removal) of CO2.16,17 To achieve the aforementioned responses, surfactants,16 diblock copolymers,13 or particles17 that are stimuli-responsive have normally been used as dispersants. However, we are not aware of reports on the use © 2014 American Chemical Society

of graft copolymers to produce nanoemulsions in general or stimuli-responsive nanoemulsions in particular, despite the existence of vast literature on stimuli-responsive block and graft copolymer micelles or vesicles.18,19 We report in this paper the use of stimuli-responsive ternary graft copolymers as well as two binary graft copolymers that serve as reference compounds for the former group to prepare nanoemulsions. Evidently, a binary graft copolymer that bears both oil- and water-soluble side chains can stabilize an emulsion because the oil-soluble chains can stretch into the droplets and the water-soluble chains can extend into the aqueous phase to reduce the oil/water interfacial tension. Alternatively, oil droplets can be stabilized in at least three ways by a ternary graft copolymer that consists of three types of side-chains. First, such a copolymer can bear water-soluble and oil-soluble sidechains as well as chains that are soluble in neither oil nor water. In this case, the insoluble side-chains can form a membrane separating the oil droplets from their surrounding water phase, yielding an encapsulated emulsion droplet or a capsule.6 In the second case, two of the three types of side-chains may extend into the oil droplets while the third type protrudes into the aqueous phase. In the third case, the second situation is reversed: two types of side-chains stretch into the aqueous phase while the third type extends into the oil droplet.20 Received: December 16, 2013 Revised: February 12, 2014 Published: February 15, 2014 968

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emulsions by human carcinoma cells and their release of DOX in these cells, suggesting the potential of these nanoemulsions in drug delivery. Although the use of nanoemulsions stabilized by graft copolymers as vehicles for drug delivery is new, graft copolymers have been tested in other forms as carriers for hydrophobic drugs. For example, drugs have been loaded into the cores of micelles that were self-assembled from ternary5 and binary26,27 graft copolymers. Analogously, micelles have also been prepared from a diblock copolymer that consisted of a hydrophilic corona-forming graft copolymer block and a hydrophobic core-forming poly(methyl methacrylate) block and used for drug delivery.28,29 More recently, wormlike singlechain micelles of graft copolymers that bear dense and strongly repelling amphiphilic diblock copolymer side-chains have been used for drug delivery.30 Inside these individual polymer chains that are also known as core−shell cylindrical brushes, the insoluble block of the side-chains collapsed and became wrapped around the backbone. Meanwhile, the soluble outer block stretched into water to stabilize the cylindrical core that was loaded with a hydrophobic drug. Aside from physically entrapping drugs in the insoluble hydrophobic domains of graft copolymer multichain or single-chain micelles, drugs have also been attached, via readily cleavable bonds, onto copolymer backbones that also bore hydrophilic side-chains in addition to the drug.31,32 In one further case, tubular capsules have been prepared from cylindrical brush polymers bearing triblock copolymer side chains.33−35 After the cross-linking of the middle block, the block that was attached to the backbone was degraded to yield the tubular core. The tubular capsules were then used in drug delivery. To the best of our knowledge, however, this report describes the first case in which graft copolymer-stabilized nanoemulsions have been investigated as drug delivery agents.

In this study, the third mode mentioned above has been adopted to stabilize nanoemulsions using a series of pHresponsive ternary graft copolymers, which were poly(glycidyl methacrylate)-g-[(polystyrene-r-poly(acrylic acid)-r-poly(ethylene glycol) methyl ether)] (abbreviated as PGMA-g(PS-r-PAA-r-MPEG)) (Scheme 1). Evidently, the PS chains were introduced for their selective solubility in the oil phase and the PAA chains were used because of their pH responsiveness. Scheme 1. Chemical Structure for PGMA-g-(PS-r-PAA-rMPEG)

We used PGMA-g-(PS-r-PAA-r-MPEG) rather than PGMAg-(PS-r-PAA) to prepare pH-responsive nanoemulsions because nanoemulsions prepared from the former were anticipated to have better pH responsiveness and a better chance to rupture or undergo a structural change within the physiologically relevant pH range between 5.0 and 7.4.21,22 It is well-known that long PAA chains are water-soluble under basic conditions due to deprotonation, but become insoluble in water under acidic conditions. However, the apparent pKa value of PAA is rather small at 4.7.23 Thus, a PGMA-g-(PS-r-PAA)-stabilized nanoemulsion may not readily de-emulsify at pH = 5.0, for example. On the other hand, acrylic acid (AA) groups that have been protonated can undergo H-bonding with ethylene glycol (EG) units and preformed PAA/PEG complexes of sufficiently high molecular weights have poor solubility in water even at pH = 7.0.24 Thus, the solubility of not only the AA units but also the EG units is diminished as the pH decreases in a system containing both PAA and MPEG coronal chains. The diminished solubility of both types of coronal chains with pH could drastically shift the hydrophilic-to-hydrophobic balance and was anticipated to readily cause structural changes to the dispersion even at pH = 5.0, for example. Drug-loaded nanoemulsions were prepared in this study by dispersing doxorubicin-loaded benzyl benzoate into nanodroplets in water. Here benzyl benzoate (BBZ) was used as the oil phase because it is “generally recognized as safe” by the United States Food and Drug Administration (FDA).25 Doxorubicin (DOX) was chosen because it is not only an anticancer drug but also a good fluorophore. The prepared nanoemulsions were stable at pH = 7.4 but ruptured, coarsened, or aggregated at pH = 5.0, thus releasing DOX. We further discovered the internalization of these nano-

II. EXPERIMENTAL SECTION Materials. Doxorubicin hydrochloride (DOX·HCl) was purchased from Dalian Meilun Biotechnology Co., Ltd., China. Triethylamine (99%), benzyl benzoate (BBZ, 99%) and N,N-dimethylformamide (DMF, 99%) were purchased from Aladdin Reagents of China and used as received. The five graft copolymers denoted as GP1-GP5 used in this project were prepared according to a previously reported procedure and their properties will be discussed in the Results and Discussion section.20 Nanoemulsion Formation. To prepare nanoemulsions, 10 mg of a graft copolymer and 5 mg of DOX·HCl were dissolved into a DMF (0.80 mL) solution containing triethylamine (28 μL). BBZ (0.050 mL) was then added and the resultant mixture was stirred at 30 οC for 30 min. Under mechanical stirring at 1600 rpm, the DMF/BBZ mixture was added dropwise into 15 mL water with pH adjusted to 11.0 by adding a 20 mg/mL aqueous NaOH solution. The mixture was further stirred at 30 οC for 30 min before the stable nanoemulsion formed was dialyzed (cutoff molecular weight: 14 kDa) against PBS (1.0 L, 10 mM of phosphate, pH = 7.4). The buffer was changed several times over 2 days to remove the free DOX and DMF from the dialysis solution.36 DOX-Loading Content. To determine DOX-loading content, a nanoemulsion sample stabilized with a known amount of a graft copolymer was first freeze-dried to remove water and BBZ. After the mixture was redissolved in DMF, the absorbance of DOX at 480 nm was measured using a spectrophometer (UV-1750, Shimadzu, Japan). The DOX concentration was calculated from this absorbance value using a pre-established calibration curve and then used to evaluate the DOX-loading content, which was defined as the weight percentage of DOX in a sample consisting of DOX and the graft copolymer. 969

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Scheme 2. Synthetic Route toward PGMA-g-(PS-r-PAA-r-MPEG)

DOX Release. Two methods were used to follow DOX release from the nanoemulsions. In method 1, the pH of DOX-loaded GP3stabilized nanoemulsion (DOX concentration: 118 μg/mL) at pH 7.4 was adjusted to approximately 5.0 with 0.10 M dilute hydrochloric acid. The solution was stirred for 6 h before the fluorescence intensity of the DOX emission was measured using a F4600 FL spectrophotometer (Hitachi) with an excitation wavelength of 480 nm. This fluorescence intensity was compared with that of its precursory solution at pH = 7.4 to judge if DOX had been released. In method 2, 4.0 mL of a DOX-loaded nanoemulsion (DOX concentration: 118 μg/ mL) was transferred into a dialysis bag (cutoff molecular weight: 14 kDa) and dialyzed against 100.0 mL of a phosphate-citric acid buffer (200 mM Na2HPO4 and 100 mM citric acid mixture, pH = 5.0). This dialysis procedure was performed at 37 °C in an incubator shaker (ZHWY-200B, Shanghai Zhicheng, China). At selected time intervals, 3.0 mL of solution surrounding the dialysis bag was collected for UV− vis analysis to determine the DOX concentration, while the removed solution was replenished with a similar volume of fresh buffer solution. The cumulative amount of drug released was calculated from the increase in the DOX absorbance value. Cell Counting Kit-8 (CCK-8) Assay. A CCK-8 assay was used to test the cytotoxicity of the nanoemulsions toward human hepatocyte LO2 cells. This experiment involved dispensing 100 μL of a RPMI (Roswell Park Memorial Institute) 1640 medium containing 4 × 103 LO2 cells that were supplemented with 10 vol % fetal bovine serum into each well of a 96-well plate. The cells were incubated for 24 h at 37 °C in a humidified atmosphere containing 5 vol % CO2. Subsequently, the media were replaced with 100 μL of a fresh medium containing 1−500 μg/mL of the BBZ-loaded GP3 nanoemulsions. After 48 h of incubation, 10 μL of a CCK-8 solution containing [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] or WST-8 was added to each well and the mixture was incubated for 1 h at 37 °C. WST-8 is reduced by dehydrogenases in living cells to give a water-soluble product (formazan), which absorbs at 450 nm. The amount of the formazan dye present was determined from the absorbance at 450 nm of each well and was directly proportional to the number of living cells. The average absorbance of three duplicated wells was calculated for each concentration. The relative cell viability R (%) was calculated according to eq 1: R (%) = (A treat /Acontrol ) × 100%

was determined via confocal laser scanning microscopy (CLSM). For these experiments, Bel-7402 cells were placed in cell culture dishes (dish diameter = 35 mm, glass bottom diameter = 15 mm, Nest Biotechnology, Shanghai, China) at a density of 5 × 103 cells per dish. The cells were subsequently incubated at 37 °C in a humidified atmosphere containing 5 vol % CO2 for 24 h to allow them to adhere to the cell bottom. Then the cells were incubated for 9 h with 1.0 mL of a culture medium containing either 85 μL of DOX-loaded nanoemulsions of DOX concentration at 118 μg/mL or 85 μL of free DOX at 118 μg/mL. The culture medium was then removed and the adhered cells were washed thrice with a phosphate buffered saline (PBS) solution. Subsequently, the cellular nuclei in each dish were stained for 20 min with 200 uL of a 0.5 μg/mL Hoechst 33342 solution (Beyotime Biotech, China). The cells were then washed with PBS thrice again before they were observed with a LSM-710 microscope (Carl Zeiss, Germany). DOX and Hoechst 33342 were excited at 485 and 352 nm, respectively. Meanwhile, the DOX and Hoechst 33342 samples exhibited emission peaks centered ∼595 and ∼455 nm, respectively. Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS). TEM was performed using a JEM-100CX II microscope operated at 80 kV. The hydrodynamic diameters (Dh) of the capsules and their polydispersity (PDI) were determined by DLS using a Malven Zetasizer Nano System. The measurements were conducted in a 3.0 mL quartz cuvette, using an 800 nm diode laser at 25 °C and a 90° scattering angle. Each set of Dh and PDI values represented the average of five measurements. Nanoemulsion Destabilization at pH = 5.0. We followed the destabilization of the nanoemulsions using TEM and DLS. For TEM analysis, 1.0 mL of a nanoemulsion at pH = 7.4 (10 mM of phosphate, PBS) was rapidly mixed with 6.0 mL of a phosphate-citric acid buffer (200 mM Na2HPO4 and 100 mM citric acid mixture, pH = 5.0). At selected times, 0.5 mL of the diluted nanoemulsion at pH 5.0 was collected and quickly aero-sprayed onto nitrocellulose-coated 200mesh copper grids. Residual volatile residues such as water were removed by vacuum drying. To selectively stain the PAA chains for TEM observation, a droplet of an aqueous 2 wt % uranyl acetate solution was added onto the grid for 30 min. This droplet was subsequently gently wicked away with filter paper, and the stained droplet samples were rinsed ten times with water droplets. For DLS analysis at pH = 7.4, 1.0 mL of a nanoemulsion was clarified by passing through a 1.0 μm filter. To follow nanoemulsion size change with time at pH 5.0, 1.0 mL of a nanoemulsion at pH = 7.4 was pushed through 1.0 μm filter before 6.0 mL of the phosphate-citric acid buffer was quickly added. At selected times, 1.0 mL of the nanoemulsion mixture at pH 5.0 was taken and analyzed by DLS. Cryogenic Transmission Electron Microscopy (Cryo-TEM). A drop of a nanoemulsion was dispensed onto a copper TEM grid that was covered by a porous Formvar film. The excess solution was

(1)

where Atreat and Acontrol denoted the absorbance of the cells treated with the nanoemulsion-containing medium and that of a blank culture medium as a negative control, respectively. DOX Distribution within Bel-7402 Cells. After DOX or DOXloaded nanoemulsions were incubated with human hepatoma (liver cancer) Bel-7402 cells, the distribution of the drug within these cells 970

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Table 1. Recipes for Preparing and Molecular Characteristics of the Graft Copolymers sample

mass feed ratioa

molar feed ratiob

Mn,theor, (kg/mol)c

Mn,SEC, (×106g/mol)d

Mw/Mnd

Mn,SEC, (×106g/mol)e

Mw/Mne

GP1 GP2 GP3 GP4 GP5

1.2:4.0:0:4.0 1.2:4.0:3.0:4.0 1.2:4.0:3.0:2.5 1.2:4.0:5.0:2.0 1.2:4.0:17.2:0

41:6.3:0:5.1 41:6.3:1.1:5.1 41:6.3:1.1:3.1 41:6.3:1.7:2.5 41:6.3:6.0:0

56 75 67 76 139

1.5 2.1 1.8 2.3 2.5

1.20 1.23 1.25 1.21 1.20

2.0 1.7 2.1 2.1

1.22 1.24 1.20 1.19

a

Mass feed ratios as defined by P(GMA-N3)/PS−CCH/PtBA−CCH/MPEG−CCH during the click chemistry synthesis of the graft copolymers. bMolar feed ratios [P(GMA-N3)]/[PS−CCH]/[PtBA−CCH]/[MPEG−CCH] as calculated from the mass feed ratios. c Number average molecular weights calculated by assuming 100% grafting of the precursors. Mn,theor = Mn (P(GMA-N3)) + x × 41 × 4000 + y × 41 × 18000 + z × 41 × 5000. The terms x, y, and z denote the molar fractions of PS, PAA, and MPEG, respectively. dSize-exclusion chromatography (SEC) molecular weight and polydispersity index values determined in DMF for the graft copolymers before they were hydrolyzed (bearing PtBA chains). The SEC system was calibrated with narrowly dispersed polystyrene standards. eMolecular weight and polydispersity index values determined in DMF for the graft copolymers after they were hydrolyzed (bearing PAA chains).

Scheme 3. Schematic Depiction of Nanoemulsion Formation and Destabilizationa

a

The nanoemulsion was prepared by dispersing DMF/BBZ/DOX in water at pH 11.0. After DMF escape into the aqueous phase, the DOX solubility drastically decreased and DOX existed in the BBZ droplets (A→B). Upon dialysis against a pH = 7.4 buffer, the free DOX molecules in the aqueous phase would have been removed (B→C). After pH reduction to 5.0, the droplets ruptured to different extents depending on the MPEG/ PAA molar ratios for the chains that stabilized the droplets (C→D). Polymers with MPEG/PAA molar ratio approaching 1 might become insoluble in water and fall off the emulsion droplets to yield ill-defined fragments. The polymer fragments and emulsion droplets then fused together to eventually yield large aggregates (D→E). DOX escaped more readily from the ruptured or partially ruptured emulsion droplets than from the intact droplets. quickly blotted away with a piece of filter paper before the grid was immersed into liquid propane that was cooled to 90 K to vitrify the nanoemulsion film. The grid was subsequently transferred into a liquid nitrogen reservoir and mounted onto a cryogenic sample holder. TEM observations were performed at −173 °C using a JEM-2010 cryo-TEM instrument operating at 200 kV. This instrument was equipped with a Gatan 655 cryoholder and a high-resolution cooled Gatan 832 CCD digital camera. Atomic Force Microscopy (AFM). Nanoemulsions were aerosprayed onto freshly cleaved mica surfaces before they were dried under high vacuum to remove any volatile residues. The dried polymer layer was observed using a MultiMode 8 SPM AFM system (Bruker) using the ScanAsyst mode.

III. RESULTS AND DISCUSSION Graft Copolymers. Three PGMA-g-(PS-r-PAA-r-MPEG) samples (GP2-GP4), one PGMA-g-(PS-r-MPEG) or GP1 sample, and one PGMA-g-(PS-r-PAA) or GP5 sample were synthesized following reported methods20 and used to disperse nanoemulsions at pH = 7.4. Three PGMA-g-(PS-r-PAA-rMPEG) samples were used mainly to investigate the effect of varying the molar ratio between the PAA and MPEG chains on the stability of the nanoemulsions at pH = 5.0. The binary graft copolymers were used mainly as reference compounds or controls for the ternary graft copolymers. To synthesize the precursory copolymers, alkyne end-capped MPEG, PS, and poly(tert-butyl acrylate) (denoted as MPEG971

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dynamic radii Rh were analyzed by DLS at the scattering angle of 90°. The Rh and polydispersity values of nanoemulsions stabilized by different graft copolymers are summarized in Table 2. Their Rh values were ∼100 nm, and they were therefore nanoemulsions.

CCH, PS-CCH, and PtBA-CCH, respectively) were randomly grafted, as shown in Scheme 2, onto an azide-bearing poly(2-hydroxy-3-azidopropyl methacrylate) (PGMA-N3) backbone via “click” chemistry catalyzed by copper sulfate and scorbic acid (SA). The residual azide groups were then deactivated using excess propargyl alcohol. PAA was obtained via poly(tert-butyl acrylate) (PtBA) hydrolysis catalyzed by ZnBr2, which was shown not to affect the integrity of other ester groups.20 These polymers have been previously carefully characterized by 1H NMR and size exclusion chromatography (SEC),20 and these analyses indicated that the precursory alkyne-endfunctionalized chains were quantitatively grafted to the P(GMA-N3) chains under our reaction conditions. Thus, the feed molar ratios equaled the grafted side chain molar ratios and were listed in Table 1. While the PGMA backbone had 41 repeat units, the repeat unit numbers for the PS, PtBA, and MPEG side chains were 40, 142, and 114, respectively. The total molar fraction x + y + z of the grafted PS, PAA, and MPEG chains among the GMA units was lower than 32%. The binary graft copolymers were prepared analogously except for the omitted use of one precursory side-chain polymer.20 All of the five graft copolymers possessed an average of 6.3 PS side chains along the PGMA backbone that was 41 units long. The respective numbers of MPEG and PAA chains per graft copolymer were 5.1 and 0 for GP1; 5.1 and 1.1 for GP2; 3.1 and 1.1 for GP3; 2.5 and 1.7 for GP4; as well as 0 and 6.1 for GP5. All of the samples had moderately low size-exclusion chromatography (SEC) polydispersity indices of ∼1.20 based on polystyrene standards (Table 1). Nanoemulsions. To prepare the DOX-loaded nanoemulsions, 5.0 mg of DOX·HCl and 10.0 mg of a given graft copolymer were first dissolved in a N,N-dimethylformamide (DMF, 0.80 mL)/benzyl benzoate (BBZ, 0.050 mL) solvent mixture containing 28 μL of triethylamine. Triethylamine was used to neutralize the HCl existing in the DOX·HCl salt and to turn DOX·HCl into DOX, which bears a neutralized amino group and is far more soluble in the organic phase than DOX· HCl. DMF was used because even the neutralized DOX had a low solubility of ∼1 mg/mL in BBZ. The mixture was then added under vigorous stirring into 15 mL of water with its pH adjusted to 11.0. A pH value higher than the pKa value of 8.3 for the DOX amino group was used mainly to ensure the preferential solubilization of DOX in organic phase.36 Our speculation was that the added organic phase was broken down into droplets due to mechanical shearing (A→B, Scheme 3). The droplet/water front shrank as a result of a DMF flux leaving the droplet. This flux brought the internal graft copolymer chains to the droplet/water interface to form an interfacial layer (B, Scheme 3). The exit of DMF also drastically reduced the solubility of DOX in the organic phase and caused the DOX to aggregate. The final droplet would have contained mostly BBZ and DOX aggregates. The escaped DMF and DOX were subsequently removed by dialyzing the nanoemulsions against pH = 7.4 PBS, in which DOX has a solubility of ∼0.1 mg/mL.37 Emulsions thus prepared remained stable when left unstirred for one year, our maximum observation period, without undergoing phase separation (droplets coalescing into a bulk oil phase) or creaming (settling to the bottom as dispersed emulsion droplets due to higher density of BBZ than water), suggesting the small size of the oil droplets. Their hydro-

Table 2. DLS Analysis Results for Nanoemulsion Droplets That Were Stabilized by Different Graft Copolymers sample

polydispersity

Rh (nm)

GP1 GP2 GP3 GP4 GP5

0.11 0.12 0.12 0.13 0.13

95 97 100 104 110

The nanoemulsions were readily obtained here without resorting to extreme shearing because we prepared the emulsion using the solvent displacement method.12 In this method, a much excess of the water-miscible sacrificial solvent DMF was initially mixed with BBZ. Thus, the initial oil droplets containing DMF were substantially larger than ∼100 nm and not much shearing stress was required to create these larger droplets.7 The nanoemulsion samples were also analyzed by AFM. For AFM analysis, nanoemulsion droplets were atomized into micrometer-sized droplets by aero-spraying38 to facilitate the quick evaporation of water and the residual volatile components were then removed under vacuum. We imagine that the polymer layer on the BBZ/DOX nanodroplet surface shrank during this process and eventually froze into a rigid nanocapsular structure after the PAA, MPEG, or PS concentration became sufficiently high. In addition, the GP3stabilized nanoemulsion was analyzed via cryo-TEM. For cryoTEM, a thin film of the GP3 nanoemulsion was immersed into liquid propane to quickly vitrify the water phase and the polymer layer. A contrast between this polymer layer and the background was obtained by lightly defocusing the image. Figure 1 shows a cryo-TEM and an AFM image of the GP3stabilized nanoemulsion. A clear contrast between the polymer layer and the core in the cryo-TEM image (Figure 1a) suggested the structure of vitrified nanoemulsion droplets for the particles. Furthermore, the particles were mostly round as revealed by AFM. The presence of some bowl-shaped particles in the AFM image resulted probably from the collapse of the graft copolymer layer during AFM specimen preparation involving BBZ evaporation. Our quantitative analysis yielded the cryo-TEM diameter of 50 ± 17 nm and the AFM diameter of 60 ± 10 nm. The AFM diameter was larger probably for a contribution from the finite size of the AFM probe tip. These values were substantially smaller than the hydrodynamic diameter Dh of 200 nm probably for several reasons. First, DLS yielded the z-average diameters, which emphasized contributions from the larger emulsion droplets and were supposed to be larger than the number-average diameters determined from TEM and AFM. The presence of large particles and the relatively wide distribution of the shrunken capsules were pretty evident in Figure 1a. Second, some shrinkage of the droplets and thus the stabilizing polymer layer during microscopic specimen preparation probably occurred. All the side chains of the graft copolymer were initially solubilized and no dense insoluble polymer membrane existed on the surface of a nanoemulsion 972

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Figure 1. (a) Cryo-TEM and (b) AFM images of specimens of GP3-stabilized nanoemulsions.

Figure 2. (a) Fluorescence spectra of DOX-containing GP3 capsules at pH = 7.4 and 6 h after the pH was reduced to 5.0. (b) Plots of the amount of DOX that had been released from the capsules versus their dialysis time against a pH = 5.0 buffer. The error bars denote the standard deviations among data obtained from three parallel trials for each sample. The solid lines were drawn to guide the eye.

% that have been reported previously for graft copolymer micelles.27,39 In addition, the loading efficiency of for this sample (defined as the mass ratio between the loaded and the initially added DOX) was also high at 62 wt %. We have also determined the DOX loading contents for GP1 and GP5 nanoemulsions, and they were 21.8 and 25.1 wt %, respectively. The DOX loading content increased from the GP1 to GP3 and then to the GP5 nanoemulsions, probably because of the Rh increase. Further, the PAA content increased in this order as well. PAA could complex with the amino group of DOX and carboxyl/amine complexation had been previously used to increase DOX loading capacities.40 In Vitro Release of DOX. The in vitro pH-triggered release of DOX from the nanoemulsions was demonstrated by two experiments. In the first experiment, the DOX fluorescence was measured for a DOX/GP3 nanoemulsion sample at pH = 7.4. Hydrochloric acid was then added into the system to lower the solution pH to 5.0. The DOX fluorescence was subsequently measured 6 h after the hydrochloric acid had been added. The fluorescence spectra that were recorded under these two conditions are compared in Figure 2a. The DOX fluorescence intensity increased by ∼5 folds at pH = 5.0. To elucidate the cause for this fluorescence intensity increase, we performed a control experiment. We compared the fluorescence intensities of an aqueous DOX solution at 36 μg

droplet. This relatively dilute polymer layer should shrink during AFM specimen preparation to form a solid film. Sample vitrification was supposed to be fast during cryo-TEM specimen preparation. Still, the cooling probably started from outer rim of a droplet (heat conductivity coefficient of water is larger than that of BBZ) and the cooling front might still push the initially not dense polymer layer inward, shrinking the initial stabilizing polymer layer. This substantially larger DLS diameter than the microscopic diameters might be a direct consequence of the nanoemulsion structure. The difference between these two sets of parameters was much smaller for nanocapsules when the inner liquid was separated from water by an insoluble polymer membrane.6 DOX Loading Content. To determine the amount of DOX loaded into the nanoemulsion, a given volume of a GP3stabilized nanoemulsion containing a known amount of GP3 was purified via dialysis against PBS and subsequently freezedried to remove water and BBZ. The solid was then redispersed into DMF and analyzed spectrophotometrically to obtain the DOX amount from the DOX absorbance value at 480 nm. The DOX amount and the graft copolymer amount were then used to calculate the DOX loading content, which was defined as the ratio between the weight of DOX and the total weight of DOX and the graft copolymer. For GP3-stabilized nanocapsules, the loading content was 23.7 wt %. This value was higher than the typical DOX loading contents ranging between 2.7 and 7.9 wt 973

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mL−1 in pH = 5.0 and 7.4 buffers. The fluorescence intensities in these two buffers were equal. We suggest that the DOX fluorescence intensity was higher at pH = 5.0 because DOX was released from the BBZ phase after the pH reduction. As mentioned before, even neutral DOX had a limited solubility of ∼1 mg/mL in BBZ and should have formed a nanoprecipitate inside the BBZ droplets and thus its fluorescence intensity was low due to self-quenching.41,42 After its release into the aqueous phase, self-quenching was reduced and thus the fluorescence intensity increased. While the fluorescence result of Figure 2a suggested release of DOX, the released DOX amount was quantified in the second set of experiments. These experiments involved dialyzing 4.0 mL nanoemulsion that was initially at pH = 7.4 and placed in a dialysis bag against 100.0 mL of an acetate buffer at pH = 5.0. The DOX concentration in the solution surrounding the dialysis bag was determined at various intervals by DOX absorbance analysis at 480 nm and used to calculate the amount of DOX that had been released. Figure 2b compares the DOX release profiles for nanoemulsions stabilized by the different copolymers. While little DOX was released in a control experiment that involved dialyzing a GP3 nanoemulsion against PBS (pH = 7.4), the DOX release rate at pH = 5.0 increased upon changing the carrier from a GP1- to a GP4-based nanoemulsion. This trend demonstrated our ability to tune the rate and extent of DOX release by gradually reducing the MPEG/PAA molar ratio from 4.6 to 1.5. We further note that the GP1 nanoemulsions barely released DOX at pH = 5.0. This behavior was reasonable because GP1 contained no pH-responsive PAA chains and the emulsions were expected to withstand pH changes. The release of DOX in this case was probably caused by the increased solubility of DOX from ∼0.1 to ∼0.4 mg/mL as the solution pH was decreased from 7.4 to 5.037 and thus the increased driving force for DOX to exit the BBZ droplets at pH = 5.0. We finally note that the GP5 nanoemulsion had a modest DOX release rate that fell between those of the GP1 and GP2 nanoemulsions at pH = 5.0. This trend was reasonable, because the GP5 nanoemulsions bore pH-responsive PAA coronal chains but no MPEG chains. Consequently, hydrogen bonding between the PAA and MPEG chains was not an available route for inducing destabilization of the GP5 nanoemulsions. DOX Release Mechanism at pH = 5.0. As depicted in frame C of Scheme 3, DOX in BBZ should exist mostly as aggregats even at pH = 7.4 due to the low DOX solubility of ∼1 mg/mL in BBZ and small total BBZ volume. Little DOX escaped from the droplets at this pH because DOX solubility in water was low at ∼0.1 mg/mL. Reducing the pH to 5.0 should further increase the extent of the DOX amino group protonation and increase the DOX solubility in the aqueous phase to ∼0.4 mg/mL.37 While this solubility was still low, the total volume of aqueous phase was large. Thus, the driving force for DOX to release greatly increased. At sufficiently long times, the maximum released DOX amount at equilibrium should be governed by the coefficient of DOX partition between the BBZ and aqueous phases and their volume ratio. Traditionally, two mechanisms have been proposed to explain DOX release from pH-responsive block copolymer capsules36 when they were subjected to a sudden pH change. First, the capsule wall could rupture or degrade due to the pH change, leading to accelerated DOX release. Second, the individual neutral DOX molecules could diffuse across the hydrophobic walls. Since pH and ion concentration gradients

have been sustained across these hydrophobic walls, proton or ion transport across these membranes has been generally considered to be slow relative to DOX diffusion.37 We believe that the two mechanisms suggested for DOX release from polymer vesicles would have operated for DOX release from our nanoemulsion droplets as well. A reduction in pH probably disintegrated or ruptured some emulsion droplets and thus accelerated DOX release from these emulsion droplets. This pH reduction might also affect the rate of DOX release from droplets that maintained an intact stabilizing polymer layer but possessed different PS or/and coronal chain conformations due to complexation between MPEG and PAA. Effect of pH Reduction on Nanoemulsion Structure. DLS was used to follow the apparent Dh change and to gain insight into the structural evolution of a nanoemulsion sample after pH reduction to 5.0. After the GP3 emulsion sample was diluted 6-fold with a pH = 5.0 buffer to bring the final solution pH to 5.0, the scattering-intensity-based Dh distribution functions and polydispersity could be obtained 0, 5, and 15 min after the nanoemulsion dilution (Figure 3). After 15 min,

Figure 3. Scattering-intensity-based Dh distribution functions for a GP3 nanoemulsion (black line) before, as well as (red line) 5 min and (blue line) 15 min after nanoemulsion dilution by a pH = 5.0 buffer.

the particles became too large for resolution by our DLS instrument and no Dh values were reported. The rapid increase in Dh from 200 nm for the nanoemulsion at pH = 7.4 to 226 nm at 5 min and then 1471 nm at 15 min suggested that the initial nanoemulsion droplets had fused and/or coarsened into larger particles. We also performed TEM analysis of GP3-based nanoemulsion samples that were collected 1, 5, and 30 min after the pH reduction and were then immediately aero-sprayed onto nitrocellulose-coated copper grids.20 The sprayed and probably somewhat shrunken polymer skeletons were subsequently stained with uranyl acetate for observation. Figure 4 shows the TEM images of the specimens thus prepared. Comparing the TEM images found in Figure 4a (which shows particles that were aero-sprayed from a pH = 7.4 buffer) and Figure 4b (which shows a specimen 1 min after the pH reduction), we note that the polymer skeleton on the seemingly intact nanoemulsion droplets in Figure 4b were significantly rougher than those in Figure 4a. This surface roughening might be a consequence of H-bonded complex formation between 974

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The latter parts of Scheme 3 depict our view of the destabilization of ternary graft copolymer nanoemulsions at pH = 5.0. Our hypothesis was that DOX would pass an intact stabilizing polymer layer very slowly and the rupturing of the polymer layer created pathways facilitating DOX release. Cytotoxicity of the GP3 Nanoemulsions. To further evaluate the viability of using the ternary graft nanoemulsions for drug delivery, we also tested the cytotoxicity of the GP3 nanoemulsions. For this test, human hepatocyte LO2 cells were incubated in 100 μL of a pH = 7.4 cultivating medium for 48 h at 37 °C with 5.0 μL of aqueous BBZ-loaded GP3 nanoemulsions at various concentrations. After this treatment, 10 μL of a dye precursor from a Cell Counting Kit-8 was added and the resultant mixture was incubated further. Subsequently, absorbance of the colored dye produced in each plate well was measured at 450 nm to quantify the live cells present. The cell survival rates after their incubation with GP3 nanoemulsions at different concentrations are plotted in Figure 5. The BBZloaded nanocapsules showed low cytotoxicity toward the LO2 cells, even at high concentrations reaching 500 μg/mL.

Figure 4. TEM images of GP3 nanoemulsion specimens aero-sprayed at (a) pH = 7.4 and at (b−d) pH = 5.0. The time delay after pH reduction was 1, 5, and 30 min for preparing the specimens with images shown in (b), (c), and (d), respectively. Also shown are TEM images of (e) GP5 and (f) GP1 nanoemulsions that had been aerosprayed 6 h after their pH reduction to 5.0. While the PAA domains of the other specimens were stained by uranyl acetate, the PS domains of the GP5 nanoemulsion specimen was stained by RuO4.

PAA and MPEG. We further note that the background was fairly clean in Figure 4a, but many fragments were seen in Figure 4b. These fragments probably corresponded to broken pieces of the nanoemulsion stabilizing polymer layers. Their appearance suggested the rupturing of some nanoemulsion droplets. Only some of the droplets ruptured, probably because of the chemical heterogeneity of the graft copolymers. Probably only those droplets that were stabilized by copolymer bearing comparable numbers of MPEG and PAA chains had ruptured. Five minutes after the pH reduction, we note that the background was still messy in Figure 4c. More importantly, some apparently “intact” droplets had fused together to form clusters, such as the one marked by a regular arrow. Moreover, some ill-defined fragments similar to that marked by the arrow with a circular end were also observed. The ill-defined structures must have formed from the fusion of the polymer fragments seen in Figure 4b. Going from Figure 4c to d (which shows a TEM image of a specimen that was aero-sprayed 30 min after pH reduction to 5.0), we noticed extensive aggregation of polymer fragments and emulsion droplets. We have also analyzed by TEM specimens of GP1 and GP5 nanoemulsions 6 h after they were brought down to pH 5.0 and the images are shown in Figure 4f and 4e, respectively. While intact nanoemulsion droplets existed in both samples, more polymer fragments existed in the background for the GP5 nanoemulsion specimen, suggesting rupturing of more GP5 nanoemulsions. These results were in agreement with the DOX release data that showed more DOX release from the GP5 than the GP1 nanoemulsions. Also, the TEM results explained why more DOX was released from the GP3 nanoemulsions.

Figure 5. LO2 cell survival rates after their equilibration at 37 °C for 48 h with 100 μL of culture media and 5.0 μL of BBZ-loaded GP3 nanocapsules in water at various concentrations. Error bars denote standard deviations from three parallel trials.

Release of DOX inside Cells. DOX as well as DOX-loaded GP1 and GP3 nanoemulsions containing an equal DOX amount were separately added into a pH = 7.4 culture medium and incubated with human hepatoma (liver cancer) Bel-7402 cells for 9 h at 37 °C.42 Subsequently, the cellular nuclei were stained with Hoechst 33342. The cells that adhered onto the glass culture dishes were observed by confocal laser scanning microscopy (CLSM). Figure 6 compares the images obtained for the three sets of samples. While the blue fluorescence from Hoechst 33342 allowed the localization of the cell nuclei, the pink fluorescence from DOX allowed us to view the DOX distributions within the cells. The free DOX entered the cellular nuclei after it was incubated with the cells, as observed by others.43 More interestingly, DOX molecules that were loaded into the GP3 nanoemulsions also reached the nuclei but the cellular nuclei were not reached by DOX samples that were loaded into the GP1 nanoemulsions. These in vitro experimental results can be reasonably explained as follows. DOX of the pH-responsive GP3 nanoemulsion could reach the nuclei because it was released into the cytoplasm of the cells after these emulsion droplets were taken up by the cells. The emulsion droplets ruptured there because the pH of the cytosol of cancerous cells could reach down between 4.5 and 6.5. On the other hand, the GP1 975

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Author Contributions ⊥

F.L. and S.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (No. 20474068, 51173204), the Leading Talents Program of Guangdong Province, the Guangzhou Invited-Talents Special Project for Addressing Challenging Problems (NO 11D34060038), the Program of the Comprehensive Strategic Cooperation between the Chinese Academy of Sciences and Guangdong Province (NO 2012B090400033), and the Intergration of Industry, Education and Research of Guangdong Province Project (2011A091000007) for financial support. We also thank Dr. Ian Wyman for proofreading this paper.

Figure 6. CLSM images of Bel-7402 cells after their incubation for 9 h at 37 °C with free DOX, a DOX-loaded GP1 emulsion, and a DOX loaded GP3 emulsion. The DOX dosage was the same for all samples, at 10 μg per dish.

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nanoemulsion droplets would not break down under these conditions. DOX was not detected in the nuclei in this case because the DOX-containing GP1 emulsion droplets could not enter the nuclei.

IV. CONCLUSIONS Three PGMA-g-(PS-r-PAA-r-MPEG) ternary graft copolymers have been synthesized and characterized. These copolymers were then used to stabilize BBZ droplets containing DOX in water at pH = 7.4 to yield oil-in-water nanoemulsions. These nanoemulsions were highly stable at this pH, and the stabilizing polymer layer effectively blocked the release of DOX into water. At pH = 5.0, some carboxylate groups of PAA became protonated and the protonated AA groups and the EG groups of MPEG chains should have formed insoluble H-bonded complexes. This complexation could have shifted the hydrophilic-to-hydrophobic balance of a graft copolymer and reduced its effectiveness as an emulsion stabilizer. Our TEM analysis suggested that some of the nanoemulsion droplets had ruptured at pH = 5.0, while other polymer layers surrounding the emulsion droplets roughened and probably became perforated. The partially ruptured emulsion droplets then aggregated into micrometer-sized network structures. The rupture of the droplets accelerated DOX release. The rate and extent of DOX release increased as the molar ratio between PAA and MPEG increased from 0.22 to 0.68 for GP2−GP4. More interestingly, the GP3 nanocapsules encapsulating BBZ possessed minimal toxicity to human LO2 cells. When incubated at pH = 7.4 with the cancerous Bel-7402 cells, DOX was able reach the nuclei. This result suggested that the GP3 nanoemulsions containing BBZ and DOX were able to enter Bel-7402 cells and to release DOX probably due to the lower pH of the cytosol of these cancerous cells. The released DOX subsequently entered the cellular nuclei. These results suggest the great potential for under-explored nanoemulsions as vehicles for drug delivery applications.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.H.). *E-mail: [email protected] (G.L.). 976

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