Article pubs.acs.org/Biomac

pH-Responsive PDMS‑b‑PDMAEMA Micelles for Intracellular Anticancer Drug Delivery Anja Car,*,† Patric Baumann,† Jason T. Duskey,† Mohamed Chami,§ Nico Bruns,†,‡ and Wolfgang Meier*,† †

Department of Chemistry, University of Basel, Klingelbergstrasse 80, 4056 Basel, Switzerland Adolphe Merkle Institute, University of Fribourg, Rte de l’Ancienne Papeterie, P.O. Box 209, 1723 Marly, Switzerland § Center for Cellular imaging and NanoAnalytics (C−CINA), Biozentrum, University of Basel, Mattenstrasse 26, 4058 Basel, Switzerland ‡

S Supporting Information *

ABSTRACT: A series of poly(dimethysiloxane)-b-poly(2(dimethylamino)ethyl methacrylate) (PDMS-b-PDMAEMA) block copolymers were synthesized with atom transfer radical polymerization (ATRP). In aqueous solution the polymers self-assembled into micelles with diameters between 80 and 300 nm, with the ability to encapsulate DOX. The polymer with the shortest PDMAEMA block (5 units) displayed excellent cell viability, while micelles containing longer PDMAEMA block lengths (13 and 22 units) led to increased cytotoxicity. The carriers released DOX in response to a decrease in pH from 7.4 to 5.5. Confocal laser scanning microscopy (CLSM) revealed that nanoparticles were taken up by endocytosis into acidic cell compartments. Furthermore, DOX-loaded nanocarriers exhibited intracellular pH-response as changes in cell morphology and drug release were observed within 24 h.



INTRODUCTION Amphiphilic block copolymers have rapidly become a hot topic in the drug delivery field due to their ability to form a variety of nanostructures such as micelles, rods, vesicles, etc. in solution.1−7 The self-assembly process is influenced by various parameters such as polymer concentration, molecular weight, volume ratio of the segments, and chemical nature of the hydrophilic and hydrophobic blocks.5 These factors allow the formation of nanoparticles that exhibit high level of complexity and consequently lead to multiple morphologies. The most explored delivery platforms are based on micelles and vesicles. The unique architecture of vesicles (polymersomes) enables loading of hydrophilic drugs into their cavity as well as hydrophobic drugs into the hydrophobic leaflet of their membrane.6,7 Furthermore, micelles possess high stability and can solubilize a broad spectrum of less water-soluble pharmaceuticals. In general, the prosperity of novel synthetic routes is reflected in the ability to obtain more sophisticated self-assemblies, such as Janus particles, bicontinuous rods, hexagonally packed hollow hoops, and others, studied in different applications.5,8 Investigation of nanocarriers for biomedical purposes is dominating the field and growing rapidly due to the discovery of the enhanced permeation retention effect (EPR) in tumors.9 Pathological vasculature is more permeable in comparison to healthy tissue; thus, an © XXXX American Chemical Society

accumulation of high molecular weight molecules and nanoparticles is possible in the interstitial space of tumors.10 Since the molecular weight of conventional drugs is only a few hundred Daltons, they easily escape from tumors. However, when encapsulated in a nanocarrier, the increased size allows entrance into the tumor vasculature but inhibits escape, leading to accumulation in the tumor tissue. Both the size of nanoparticles and the additional ability of drug delivery systems to release the active compound in a controlled manner play key roles in successful medical treatment. Hence, the development of different stimuli-responsive platforms to control the release of drugs upon applying external stimuli has attracted a lot of interest.6,11 In particular, a response to pH change is desirable, since defined pH gradients exist in biological systems, for example, among the extracellular medium at pH 7.4, endosomes at pH 6.0−6.5, and lysosomes at pH 4.5−5.0.12,13 Also, the more acidic environment in cancer cells compared to healthy cells make pH responsive systems a good supplement to the EPR effect when targeting cancerous tissues. Recently, amphiphilic block copolymers comprising of a protonable poly(2-(dimethylamino)ethyl methacrylate) Received: March 10, 2014 Revised: July 18, 2014

A

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Figure 1. Schematic representation of DOX-release from PDMS-b-PDMAEMA micelles in response to a drop in pH.

to 300 nm. The micelles did not disassemble at low pH. Additionally, only in the case of block copolymer with the shortest PDMAEMA, a minor fraction of bigger agglomerates was detected at low pH. At the same time, the platform was able to release the therapeutic cargo in response to a drop in pH (Figure 1). The high stability of this particulate system favors a slow release of an anticancer drug.

(PDMAEMA) block and PDMAEMA homopolymers have been extensively studied in drug and gene delivery.14 Different self-assemblies were produced varying from micelles to polyplex systems.15 For example, Adams et al. showed that spherical micelles, wormlike micelles, and vesicles can be formed by adjusting the relative length of PDEAMA blocks.16 In a later publication, the same group demonstrated the impact of pH on the formation of poly(ethylene oxide)-b-poly(N,N-diethylaminoethyl methacrylate) nanostructures and subsequent loading of several dyes into vesicles.17 The self-assembly of poly(2(dimethylamino)ethyl methacrylate)-b-poly(glutamic acid) (PDMAEMA-b-PGA) double hydrophilic block copolymers was investigate by Lecommandoux and co-workers.18 The authors demonstrated that the self-assembly process can be selectively triggered by variation of the pH or temperature. Apart from vesicles/micelles, other aggregated structures were also reported. For example Meier’s group synthesized polyethylenegylcol-b-polymethylcaprolactone-b-poly(2(dimethylamino)ethyl methacrylate) triblock copolymer (PEGb-PMCL-b-PDMAEMA) where elongated structures were observed in solution.19 Furthermore, polystyrene-b-poly(2(dimethylamino)ethyl methacrylate) (PS-b-PDMAEMA) diblocks with PDMAEMA blocks ranging from 3 to 47 units exhibited a self-assembly behavior that depended on the solvent used.20 In this publication, authors showed that polymer with extremely short PDMAEMA block assembly into spherical micelles in the mixture of dioxane/water. By tuning the solvent and pH, hexagonal and pearl necklace morphologies were also observed. A more comprehensive overview of different stimuliresponsive methacrylate based delivery systems behavior in solution, and their functional applications, is reported in a recent review paper.21 In general, the drug release mechanism in pH-responsive nanocarriers is based on the protonation of pH sensitive groups causing swelling of the system, a pH-triggered hydrolysis, or dissolution of the polymers.22−26 In this work, we present pH-responsive nanoparticles based on amphiphilic block copolymers that are comprised of polydimethysiloxane (PDMS) in combination with stimuliresponsive PDMAEMA blocks of various lengths. The polymers assembled into micelles with sizes ranging from 80



EXPERIMENTAL SECTION

Materials. Monocarbinol terminated poly(dimethylsiloxane) (AB146681; molecular weight 5000 g mol−1) was purchased from ABCR (Germany). 2-(Dimethylamino)ethyl methacrylate, α-bromoisobutyryl bromide, triethylamine (TEA), tetrahydrofuran (anhydrous) (THF), toluene (anhydrous), dichloromethane, diethyl ether, anhydrous magnesium sulfate, NaHCO3, basic alumina, and copper(I) bromide (99.99%) were obtained from Sigma-Aldrich (Germany). Pyridine-2-carboxaldehyde (99%) and n-propylamine were purchased from Alfa Aesar (Switzerland), and doxorubicin hydrochloride (DOX) from Beijing Zhongshuo Pharmaceutical (China). Sulforhodamine B (SRB) was from Sigma-Aldrich, and Biotech CE dialysis tubes (MW cutoff 100 kDa) were provided by Spectrum Laboratories (Germany). Track etched polycarbonate membrane Nuclepore was obtained from Whatman (U.K.) and Dulbecco’s phosphate buffered saline (DPBS) buffer from Sigma-Aldrich Chemie (Germany). Dulbecco’s Modified Eagle’s Medium (DMEM) and HeLa cells were provided from SigmaAldrich (U.S.A.), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and Hoechst 3342 from Promega (U.S.A.), and Lysotracker Deep red and CellMask Deep from LifeTechnologies (U.S.A.). All reagents were of the highest grade commercially available and used without any further purification unless otherwise stated. Synthesis of PDMS-b-PDMAEMA Block Copolymers and Formation of Self-Assemblies. Block copolymers were synthesized by atom transfer radical polymerization (ATRP), according to a previously described procedure with slight changes as described below.27 Briefly, monofunctional PDMS-Br macroinitiator (Mw 5400 g mol−1 and PDI 1.12) was prepared from hydroxyl-terminated PDMS by using α-bromoisobutyryl bromide and TEA (ratio 1:3:6) in solution of anhydrous THF with the final polymer concentration of 10 wt % in solution. First, TEA was added to the stirred solution, followed by dropwise addition of α-bromoisobutyryl bromide. The reaction was stirred at room temperature for 18 h. Then, the polymer solution was filtered to remove salts. THF was removed on a rotary evaporator, and afterward, the macroinitiator was dissolved in dichloromethane and B

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−3 μm. Micrographs were recorded at 4K × 4K CMOS camera (TVIPS, Germany). Light Scattering. Dynamic light scattering (DLS) and static light scattering (SLS) studies were carried out using a commercial goniometer (ALV-Langen) equipped with He−Ne laser (λ = 633 nm) at scattering angles between 30° and 150°. An ALV-5000/E correlator calculated the photon intensity autocorrelation function, g(t), at T = 293 K. The refractive index increment, dn/dc, was obtained at the corresponding temperature and wavelength of the light scattering experiments, using a commercial ALV-DR-1 differential refractometer. Radius of gyration (Rg), second viral coefficient (A2) and weight-average molecular weight (Mw) were obtained by analyzing the results in Guinier plot (Supporting Information Figure S3). Effect of pH on Surface (ζ) Potential and Particle Size (Titration). The influence of pH on hydrodynamic radius (Rh) and ζpotential of particles was investigated in the range pH 3−11. The pH values were controlled by MPT-2 autotitrator connected to a fully automated Nano ZSP zetasizer (Malvern Instruments), which enables simultaneous measurement of ζ-potential and Rh. In Vitro Doxorubicin Release. In vitro DOX release was investigated at different pH (5.5 to 7.4) in a PBS buffer solution. One to three milliliters of solution containing DOX-loaded particles were introduced into CE dialysis tubes (MW cutoff 100 kDa; Spectrapor). Dialysis tubes were closed and exposed to 800 mL (or 80 mL for cumulative release studies) of solutions, for a defined period of time and pH, at 37 °C in the dark. The amount of released drug was calculated from the residual drug content that remained in the dialysis tube or in the case of cumulate release studies in permeate solution, by applying spectrophotometric measurements at λmax = 485 nm, using an extinction coefficient of 9.6 × 103 M−1cm−1. The latter was experimentally determined from a dilution series of DOX (Supporting Information Figure S4). Cell Culture and Viability MTS Assay. HeLa cells (2000 cells per well) were seeded in a 96-well plate, and incubated at 37 °C, 5% CO2 for 24 h in DMEM containing 10% fetal calf serum and 1% penicillin/ streptomycin. At 24 h, neat or DOX loaded colloidal solutions were added to triplicate wells at concentrations ranging from 50 to 300 μg/ mL (DOX loaded particles contained the same amount of polymer as the pristine platform with DOX concentrations ranging from 5 to 30 μg determined by absorbance) and compared to DOX controls (0−50 μg/mL). Cells were cultured in the presence of polymeric micelles or DOX for an additional 24, 48, or 72 h. Cell growth inhibition was measured at each time point using the MTS assay: briefly 20 μL MTS assay solution was added to each well and incubated for 3 h at 37 °C as per the supplier’s instructions. Cell viability was determined by absorbance at 490 nm of each well measured with a microplate reader (SpectraMax M5e, Molecular Devices, U.S.A.) and calculated as a percent of live cells compared to a PBS control (0% growth inhibition). All samples were corrected against controls containing only media or media containing the dosed amount of doxorubicin at each concentration (due to doxorubicin absorbance at 490 nm leading to increased background signal at higher concentration). Nanoparticles Uptake into Cells As Followed by Confocal Laser Scanning Microscopy (CLSM). HeLa cells were grown at a density of 5 × 104 cells per well in 8-well Lab-Tek plates (Nalgene Nunc International, U.S.A.) for 24 h in DMEM growth medium to allow attachment to the surface. The medium was subsequently removed, and cells were incubated with 300 μL of a 100 μg/mL polymer solution and grown for 24, 48, and 72 h. Cells were stained with Lysotracker Deep red (0.1 μM, 30 min; stains lysosomes), CellMask Deep red (5 mg/mL, 5 min; stains the cell membrane), or Hoechst 3342 (5 mg/mL, 10 min, stains the nucleus) and washed twice with PBS buffer. Cells were reconstituted in PBS followed by imaging with a confocal laser scanning microscope (Carl Zeiss LSM510, Germany) equipped with a 63× water emulsion lens (Olympus, Japan). The measurements were performed in multitrack mode with the gain and contrast adjusted individually for each fluorescent dye (Hoechst 3342 was excited at 405 nm, Deep Red at 633 nm and Doxorubicin HCl at 543 nm). Images were recorded using Carl Zeiss LSM software (version 4.2 SP1). Control images were

neutralized with a saturated NaHCO3 solution. After filtration and removal of dichloromethane on a rotary evaporator the macroinitiator was washed with methanol in a separatory funnel until the yellowish color vanished. In the final step, the residual methanol was removed under vacuum on a Schlenk line. The ligand was synthesized according to previous procedures described in the literature.28 Inhibitor present in DMAEMA was removed by passing the monomer through a basic alumina column prior to use. The polymerization of DMAEMA was mediated by copper(I) bromide/N-(N-propyl)-2-pyridilmethanimine in a toluene solution at 90 °C. Ratios between macroinitiator, copper(I) bromide, and ligand were kept constant during polymerization (1:1:2), and the amount of DMAEMA monomer was varied according to the degree of polymerization. The reaction time was between 3.5 and 5 h. The copper present in the polymerization solution was removed by passing the polymer solution through a basic alumina column. The scheme of the reaction can be found in the Supporting Information (Scheme/ Figure S1). Self-assemblies were prepared by a film rehydration method with an initial concentration of 3 mg mL−1 of polymer in chloroform. After solvent evaporation, PBS buffer (pH = 7.4) was added to the polymer film. The solution was exposed for 1 min to an ultrasound bath to facilitate the transfer of polymer chains into the liquid phase. Afterward, colloidal solutions were magnetically stirred at 300 rpm overnight and extruded through a 0.4 μm track etch polycarbonate membrane followed by adjustment to the desired pH. DOX-loaded and SRB-loaded particles were prepared in a similar way as described previously. Instead of neat PBS buffer, a solution of DOX with a concentration of 1 mg mL−1 in PBS or a 50 μg mL−1 solution of SRB in PBS was added to the polymer film and rehydration was performed as previously described. Because DOX is difficult to dissolve in PBS buffer, an ultrasound bath at 40 °C was used to facilitate drug solubilization. Loaded nanocarriers were extruded through a 0.4 μm pore size membrane. Nuclear Magnetic Resonance (NMR) and Gel Permeation Chromatography (GPC) Characterization of Block Copolymers. 1 H-nuclear magnetic resonance (1H NMR) spectra were recorded in CDCl3 using a Bruker 250 MHz instrument. The compositions and the number-average molecular weight Mn of PDMS-b-PDMAEMA copolymers were determined from the characteristic proton signals of each block and of the end-groups (Supporting Information Figure S2). The degree of polymerization was calculated from the integral of the PDMS protons (peak “d” in Supporting Information Figure S2a) and the integral of the PDMAEMA protons (peak “a” in Supporting Information Figure S2b). GPC experiments were performed on a Viscotek GPC max system equipped with four Agilent PLgel columns (10 Mm guard; Mixed-C; 10 μm, 100 Å; 5 μm, 103 Å; Agilent Technologies Chromatography Division, Germany). THF was used as an eluent at 45 °C, with a flow rate of 0.7 mL min−1. Molecular weight characteristics of the copolymers were calculated using a calibration curve constructed with monodisperse polystyrene standards. Toluene was used as an internal standard. Transmission Electron Microscopy (TEM). Transmission electron microscopy (TEM) of was performed on a Philips EM400 electron microscope operated at 100 kV. A drop of the copolymer solution was deposited on a carbon-coated copper grid negatively stained with 2% uranyl acetate solution. Excess solution was blotted away using a strip of filter paper and the sample was allowed to dry at room temperature and atmosphere before observation. Cryo-Transmission Electron Microscopy (Cryo-TEM). A 4 μL aliquot of sample was adsorbed onto glow-discharged holey carboncoated grid (quantifoil, Germany), blotted with Whatman filter paper and vitrified into liquid ethane at −178 °C using a vitrobot (FEI company, Netherlands). Frozen grids were transferred onto a Philips CM200-FEG electron microscope using a Gatan 626 cryo-holder. Electron micrographs were recorded at an accelerating voltage of 200 kV and a nominal magnification of 50000×, using a low-dose system (10 e−/Å2) and keeping the sample at −175 °C. Defocus values were C

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Table 1. GPC and NMR Data of PDMS-b-PDMAEMA Block Copolymers name

sample

predicted Mn (g mol−1)

predicted (wt %) PDMAEMA

exptl. (wt %) PDMAEMA

NMR Mn (g mol−1)

GPC Mn (g mol−1)

PDI

PDMS74-b-PDMAEMA5 PDMS74-b-PDMAEMA13 PDMS74-b-PDMAEMA22

AB5 AB13 AB22

7000 8000 10000

20 30 45

13 27 39

6300 7600 9000

7400 7800 10800

1.09 1.16 1.15

recorded with the same setting as cells containing polymeric micelles. Transmission images were recorded simultaneously with the fluorescence images, using the 543 nm laser.



RESULTS AND DISCUSSION Synthesis of PDMS-b-PDMAEMA Block Copolymer. Three PDMS-b-PDMAEMA diblock copolymers were synthesized by ATRP from a PDMS-macroinitiator. The feed of hydrophilic monomer was varied to obtain PDMAEMA blocks of various lengths in order to influence further self-assembly process. The characteristics of the block copolymers, as measured by GPC and NMR, are summarized in Table 1 and Figure 2. The reaction yielded block copolymers with molecular

Figure 3. Cytotoxicity of AB5, AB13, and AB22 polymers. HeLa cell growth inhibition versus concentration of polymer solutions after 24 h of incubation.

delivery polycations such as poly(ethyleneamine) or PDMAEMA homopolymers due to30 a higher number of positive charges having a higher probability to complex with oppositely charged moieties on the cell surface. Since AB13 and AB22 block copolymers showed high level of cytotoxicity only AB5 carriers were suitable for drug delivery studies and therefore paper mainly focuses on AB5 self-assemblies. Self-Assembly Studies. Light-scattering method was used to analyze the self-assemblies in buffer. Results for AB5 particulates yielded a value of 0.86 for the ratio between Rg and Rh(ρ) (Table 2). This value was below 1, which was lower than expected for a vesicular morphology (ρ = 1.0) but slightly higher than the value determined for homogeneous spheres.31 This indicates AB5 particulates may form hollow kinds of structures but homogeneous spheres cannot be excluded. A possible explanation for this could also be slight agglomeration occurring at pH 7.4 as PDMAEMA slowly approaches its isoelectric point. As a consequence, results of Rh are higher than in well monodisperse samples. Hence, it was important to understand the behavior of the particles in PBS before incorporating additional compounds, such as DOX, into the system. Since PDMAEMA is charged, the ions in PBS form a diffuse layer of counterions around the cationic chains and may have an influence on the structures formed by self-assembly. Furthermore, the second virial coefficient (A2) reflects the energy of binary interaction between solvent molecules and polymer segments. The experimentally determined A2 is close to zero. This indicates a repulsive behavior between PDMS and PDMAEMA blocks and confirms that PBS buffer is a suitable solvent for the self-assembly of these amphiphilic block copolymers. Since the behavior of polyelectrolytes in solution is much more complex compare to uncharged systems we additional used TEM and Cryo-TEM (vide inf ra) to better understand the morphology of the particles.

Figure 2. GPC traces of PDMS macroinitiator and PDMS-bPDMAEMA block copolymers.

weights close to the targeted Mn all having a small polydispersity index (PDI). The GPC chromatograms of the copolymers reveal a clear shift toward higher molecular weight when compared to the trace of the PDMS-Br macroinitiator. The GPC traces of the diblock copolymer were unimodal and symmetrical. No tailing in the low molecular weight region was observed. Although no kinetic studies of the polymerization were performed, these results indicate that the polymerizations were well-controlled. Cytotoxicity of Pristine Polymers. PDMS is a nontoxic and biocompatible polymer, while cationic PDMAEMA exhibits cytotoxicity.14,29 Therefore, the effect of pristine PDMS-bPDMAEMA particulates on the viability of HeLa cells were evaluated using the MTS assay (Figure 3). Particles prepared from AB5 showed good cell viability up to 300 μg mL−1. AB13 and AB22 resulted in very poor cell survival rates even at low polymer concentrations. In brief, cytotoxicity of polymers increased with the molecular weight of the PDMAEMA hydrophilic charged block. This correlates to previous literature on nonviral gene D

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Table 2. Light-Scattering Data of AB5 at pH = 7.4 in PBS Rg (nm)

pH = 7.4 AB5

120

Rh (nm)

Mw(g mol−1)

Nagg

140

1.6 × 10

2.5 × 10

9

5

A2 (mol dm3g−2)

ρ

5.5 × 10−10

0.86

Table 3. Rh of AB5 and AB5-DOX Particulates upon Incubation at pH 7.4 and 5.5 at 37 °C 1 day

a

2 days

AB5

Rh at pH = 7.4 Rh at pH = 5.5

155 ± 16 nm (100%)a 167 ± 20 nm (100%)

160 ± 13 nm (100%) 145 + 18 nm (100%)

AB5-DOX

Rh at pH = 7.4

192 ± 22 nm (100%)

205 ± 24 nm (100%)

Rh at pH = 5.5

1. peak 165 ± 45 nm (91%) 2. peak 62 ± 7 nm (9%)

1. peak 215 ± 65 nm (94%) 2. peak 546 ± 38 nm (6%)

3 days 150 ± 18 nm (100%) 1. peak 140 ± 11 nm (98%) 2. peak 348 ± 31 nm (2%) 1. peak 178 ± 21 nm (96%) 2. peak 420 ± 21 nm (4%) 1. peak 240 ± 41 nm (61%) 2. peak 469 ± 36 nm (36%) 3. peak 5425 ± 338 nm (3%)

Relative peak intensity.

Figure 4. TEM images of particles prepared from (A) AB5, (B) AB13, and (C) AB22 at pH = 7.4 and (D) AB5, (E) AB13, and (F) AB22 at pH = 5.5 (after 3 days).

pH-Induced Morphology Changes of Pristine and DOX-Loaded Micelles. Pristine particles, as well as DOXloaded nanocarriers, were incubated for 3 days in pH 7.4 or 5.5 at 37 °C. These conditions were chosen to mimic the extracellular environment in the body, as well as the acidic conditions of cell compartments. Since an acidic pH causes protonation of the tertiary amine side groups of the PDMAEMA blocks, size and morphology changes of the particles were expected. Moreover, the presence of DOX could also influence morphological changes of self-assemblies. Therefore, the solutions were analyzed by dynamic light scattering (DLS) from 1 to 3 days (Table 3). During the first 2 days of incubation, pristine particles showed a non pH dependent single peak comprised of a narrow size distribution. After 3 days, a small amount of larger aggregates were detected at pH 5.5 but not at pH 7.4. DOX-loaded particles at pH 7.4 were larger than empty ones (300 and 400 nm in diameter, respectively). They remained stable for the first 2 days at pH 7.4 but showed minor aggregation on the third day. In contrast, acidic conditions caused the appearance of two particle fractions on day one, and after the third day of incubation, three size populations were found. These results suggest the main fraction of the nanoparticles, at pH = 5.5, kept the same size particles as those measured at pH 7.4. However, appearance of agglomerates also showed that some disassembly may occur or that some of their chains with lower degree of polymerization lead to aggregate formation. Another explan-

ation of detecting agglomerating fraction in the loaded system compared to neat particles also arises from the possibility of DOX aggregation. Some research groups have reported selfassociation behavior of DOX in aqueous solution that can be greatly influenced by counterions.32 This is in agreement with the results showing that DOX is released at lower pH and after few days show strong tendency to agglomerate (see Supporting Information Figure S5). It is worthy to mention that the size of neat AB5 nanoparticles was also monitored over the period of one month at pH 5.5 and 7.4. The results are comparable with the data obtained over the first 3 days (main fraction of the particles corresponds to the size measured at the day one). In addition, self-assemblies prepared from AB13 and AB22 block copolymers measured over longer period of the time showed stable size at both pH values and no agglomeration was observed. This indicates that longer PDMAEMA leads to more stable particles. Thus, the appearance of a very small fraction of agglomerates in the case of neat AB5 can be attributed to the chains with lower degree of polymerization, as previously mentioned. TEM was used to visualize the morphology of self-assembled structures at different pH values (Figure 4). At pH 7.4 spherical particles were observed. Their size was dependent on the length of the PDMAEMA block. AB5 formed particles with a diameter of approximately 300 nm compared to particles obtained from block copolymer AB13 (180 nm), and AB22 (60−80 nm). E

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Figure 5. TEM images of AB5-DOX loaded particles after (A) 1 day, (B) 2 days, and (C) 3 days incubation at pH= 7.4 and (D) 1 day, (E) 2 days, and (F) 3 days incubation at pH = 5.5.

After incubation at pH 5.5 for 3 days AB13 and AB22 particles appeared to be intact, while some AB5 particulates disintegrated into smaller pieces. This is in contrast to the DLS results, which showed the majority of AB5 nanocarriers to retain their size. Therefore, the broken pieces of AB5 at pH 5.5 could be due to nanoparticles in their protonated state (at pH = 5.5) being “softer” in comparison to particles prepared at pH 7.4 and breaking during TEM sample preparation. AB13 and AB22 self-assemblies retained their structure most likely because the longer PDMAEMA blocks have higher degree of mobility and formed more stable particles. This statement is well supported by cryo-TEM images (Figure 6) showing intact particles at both pH values. TEM micrographs of DOX-loaded AB5 self-assemblies at pH 7.4 and pH 5.5 (Figure 5) remained intact during incubation at 37 °C for 3 days. In acidic conditions, however, aggregates were observed in addition to particles after the second and third day. As discussed previously, these aggregates could be related to the released DOX. Cryo-TEM was used to visualize the shape and the architecture of the self-assembled structures in their native environment. The most representative images revealed homogeneous spheres (Figure 6). However, in the AB5 samples, assemblies with a hollow-like and multicompartment structure were found but this fraction was present in the minority (see Supporting Information Figure S6). The presence of such particles was not detected in the cases of AB13 and AB22 samples. The size of the particles decreased with increasing lengths of the hydrophilic PDMAEMA. Hence, we conclude that PDMS-b-PDMAEMA polymers assemble into micelles with size ranging from 60 to 300 nm in diameter. Since the results obtained with light-scattering and TEM measurements showed some discrepancy, cryo-TEM images proved that AB5 nanocarriers stay intact upon lowering pH down to 5.5. These results suggested that the PDMAEMA block length mainly influences the particle size and not the morphology of particles. pH Effect on ζ-Potential and Particle Size. Stimuliresponsive polymers change properties in response to environmental changes. Thus, for the comprehensive understanding of the pH-responsive PDMS-b-PDMAEMA nanoparticles, it is important to study the physicochemical properties of such nanostructures in a broad pH range. To this end, solutions containing micelles were titrated in the pH range from 3 to 11.

Figure 6. Cryo-TEM images of self-assembled polymers AB5, AB13, and AB22 at pH 7.4 (A, C, and D respectively) and AB5 at pH 5.5 (B) after 3 days.

Simultaneous Rh and ζ-potential measurements were analyzed (Figure 7). All types of particles possessed positive surface ζpotential between 20 to 30 mV at pH ≤ 7.4. The charge was inversely proportional to the pH with ζ-potential reaching 0 mV at the isoelectric point (IEP). AB5, the block copolymer with the shortest pH-responsive block, had the lowest IEP (at pH = 8.6). Longer PDMAEMA segments led to higher IEP values. The IEP of AB13 was at pH = 9, and AB22 nanoparticles at pH = 9.3. Beyond the IEP solutions become unstable leading to precipitation of the polymers. The DLS data revealed that AB5 particulates have an Rh of approximately 150 nm at physiological pH. Micelles prepared F

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Figure 7. pH dependence of ζ-potential and size of nanocarriers. (T = 25 °C).

from AB13 and AB22 are smaller, with an Rh of 110 and 40 nm, respectively. This is in agreement with the TEM data and cryoTEM presented above. It also corresponds to previous empirical results that an increase in hydrophilic content of amphiphilic block copolymers results in a size decrease of the particles.33 The Rh indicates that all PDMS-b-PDMAEA particles are stable at physiological pH 7.4 and do not disintegrate at lower pH. In more basic conditions the size of the nanoparticles gradually increases due to a loss of charge and particles start to precipitate. A comparison of the pH-profiles of AB5 and AB5-DOXloaded platform is presented in Figure 8. Small deviation of ζpotential is observed between the neat and the loaded nanocarrier. DOX is a weak base and a certain amount could be released during the measurement leading to deviation of the surface potential.34 Interaction of nonencapsulated DOX with the particle surface is less probable, since both systems were positive charged at present conditions. In addition, these results

indicate a stable delivery system at lower pH. In comparison to the neat platform, the size of DOX-loaded particles was larger with a higher polydispersity, which can be attributed to the influence of DOX on the self-assembly process. pH Effect on In Vitro DOX Release. In order to understand the pH-triggered release of therapeutic cargo from the carriers we evaluated the release of DOX at varying pH values in vitro (Figure 9). First, AB5 micelles loaded with an

Figure 9. pH-dependent release of DOX from AB5 particulates.

initial concentration of 1 mg mL−1 DOX were intensively dialyzed while the concentration of DOX in the dialysis tube was monitored by UV/vis spectroscopy. The average encapsulation efficiency of DOX was 30% after 2 days of dialysis at pH 7.4. For cumulative release studies, samples were dialyzed and the concentration of DOX was measured over time (Figure 9). After 1 day, the buffer of the pH 7.4 experiments was exchanged against fresh buffer of either pH 7.4 or pH 5.5. At

Figure 8. pH dependence of ζ-potential and size of (A) AB5 and (B) AB5-DOX loaded particles (T = 37 °C). G

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pH 7.4 the micelles released 5% of the drug within the first 24 h, and 20% within 72 h. A gradual pH change from 7.4 to 5.5 after the first day resulted in a release of approximately 80% DOX by the third day. Immediate exposure of the particles to pH 5.5 led to an enhanced release within the first 24 h and the release of approximately 90% of the drug within 48 h. These data prove that drug release is much faster at lower pH in comparison to higher pH values. Since we could not identify disassembly of the delivery system at lower pH values, by previously mentioned characterization methods, it is assumed to be the passive release of drug attributed to the higher degree of protonation. At lower pH, the increased charge density on PDMAEMA causes intensive repulsion between hydrophilic chains (swelling), which induces tension on the hydrophobic core. However, PDMS has an extremely low glass transition temperature and, consequently, a high flexibility at room temperature. Thus, the effect on the DOX release is not instantaneous and rather slow when compared to a system where disintegration of the carriers takes place. Observed results of slow drug release are in accordance with the literature data.35 In this work, release of DOX from dually (redox and pH) responsive micelles was reported. Authors demonstrated that within 24 h 20% of the drug at pH 7.4 and 60% at pH 5.5 are liberated from the micelles. After adding glutathione, enhanced release of the DOX was observed (75%) already at pH 7.4, and at pH 5.5, most of the DOX was released within 10 h. This micellar system represents an excellent comparison between different release mechanisms of the drug from the particles since it used the same material. It is well demonstrated that drug release in a system, which does not disassemble is much slower in the presence of external stimuli. Furthermore, for better understanding of release mechanism also cumulative release on AB13 and AB22 carriers was performed and discussed (see Supporting Information, Figure S7). In addition, the dialysis of free DOX at pH 7.4 is reported in Figure 9 in order to validate the experimental setup. DOX diffuses out of the dialysis tube within the first hours of dialysis. Thus, the dialysis membrane does not pose a diffusion barrier that significantly influences the experimental results of DOXrelease from delivery platform. In summary, the release studies suggested that the DOXloaded delivery system was able to retain the drug at physiological conditions and release it upon lowering the pH. The release profile under a gradual pH change is complex, and it seems that the concentration profile follows a mixed zero and first order kinetic. Delivery of DOX to Cells. The possibility to use the AB5 platform as an intracellular drug-delivery system was investigated by monitoring the cytotoxicity of DOX-loaded micelles to HeLa cells. Ideally, the drug-loaded system would be nontoxic when outside of the cells and become highly cytotoxic when taken up into the acidic compartment of the cell. Figure 10a and b illustrates the time effect on the cytotoxicity of AB5 and AB5-DOX-loaded nanoparticles at pH 7.4 over 72 h and compares DOX concentration encapsulated in particles to free DOX. The cytotoxicity profile of nonencapsulated DOX was determined after 24 h of cell incubation and is highly toxic at concentration above 5 μg mL−1. This is not the case for encapsulated DOX, showing only 40% cell growth inhibition at this regime. Comparable toxicity results were only obtained at much higher encapsulated DOX concentrations (approximately 30 μg mL−1), suggesting that only part of the DOX is released within the first 24 h. These results agreed with in vitro release

Figure 10. Cytotoxicity of DOX-loaded PDMS-b-PDMAEMA particles. HeLa cell growth inhibition is shown versus the concentration of (a) pristine AB5 particles after 24, 48, and 72 h and (b) DOX-loaded particles and DOX.

studies, showing 30% (Figure 9, platform exposed to pH 5.5) drug release from particles within 24 h. This high concentration correlates to approximately 9 μg mL−1 of released DOX, and thus, the cytotoxicity correlates with the level of pristine DOX. In this way, the cytotoxicity of encapsulated DOX at 24 h was reduced and reached similar cytotoxicity after 48 h, where the majority of the DOX was released as suggested by cumulative studies. To ensure that the toxicity after 24 h was not an effect of the delivery system itself, we performed control experiments with nonloaded AB5 delivery system over a time span of up to 72 h (Figure 10a). Cell viability did not depend on incubation time. This proves that the cytotoxicity of the loaded AB5 particles is the result of the released DOX. This suggested that the platform was taken up by the cells into acidic compartments such as lysosomes (vide inf ra) with pH expected to be around 5.5. Thus, the carriers were able to release the drug upon environmental pH changes. Uptake of Particles into Cells. Cellular uptake of AB5 nanoparticles into HeLa cells was followed by confocal laser scanning microscopy (Figure 11). Cells were additionally stained with lysotracker to identify whether the nanocarieers were uptaken in acidic compartments of the cells (Figure 12). After 24 h of incubating the cells with particles, the acidic organelles (in green) overlay with the fluorescent DOX-loaded nanocarriers (in red), giving yellow areas in the merged image. Thus, the pH-resposive particulates did enter cells via lysosomes/endosomes. Micelles that encapsulated Sulforhodamine B (SRB) were administered to cells in order to visualize the location of the particles. In both cases, carriers accumulated in the perinuclear region. However, the drug-loaded delivery system was monitored directly by DOX fluorescence. The confocal microscopy images show that pristine and DOXloaded AB5 particles were internalized into the cells within 24 h. Previous literature precedence shows that nanoparticles of this type can internalize into cells within 2 h, rapidly escape the endosome and release the cargo.36 While no early time points were analyzed for this system it is assumed they internalized in the same time frame. Furthermore, the particle presence and lowered toxicity at 24 h supports the stability of these H

dx.doi.org/10.1021/bm500919z | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

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Figure 11. Confocal laser scanning microscopy images of HeLa cells that were exposed to nanoparticles. Uptake of pristine (SRB-stained) AB5particles (a−c) and DOX-loaded particles (d−f) after 24 h (a and d), 48 h (b and e), and 72 h (c and f). Blue channel = nucleus; green channel = cell membrane (a−c), acidic cell compartments (d−f) (endosomes and lysosomes); red channel = SRB-loaded particles and DOX-loaded particles, respectively; overlay image. Scale bars are 50 μm.

However, our results do correlate with the increase in toxicity of DOX loaded particles at a concentration of 5 μg mL−1 increasing from low toxicity (20%) at 24 h (Figure 10, -■-) to high toxicity (∼60%) at 72 h (Figure 10, -▲-). This could also be visualized by cell morphology changes indicating unhealthy or apoptotic cells. Therefore, the CLSM images support the hypothesis of enhanced DOX release over 72 h, which is in agreement with the viability studies presented above. This proved that drop in pH between the extracellular medium and the acidic cell compartments is beneficial in inducing DOX release from the pH-responsive AB5 platform.



CONCLUSIONS Stimuli-responsive drug delivery systems with controlled release of a drug are highly desirable, especially for delivery of anticancer drugs. Of particular interest are pH-responsive nanocarriers because they allow the exploitation of the various pH gradients within the body, for example, between healthy tissue and tumor tissue, or between the extracellular tissue and some cell compartments. In this context, nanocarriers capable of encapsulation, and pH-triggered release, of DOX were developed, and their potential as intracellular delivery system explored. In a first step, amphiphilic PDMS-b-PDMAEMA block copolymers were synthesized and their self-assembly in solution was studied. Micelles, with diameters between 80 and 300 nm, formed in PBS were stable over several days, and only minor changes were observed. The release of the DOX strongly depended on the pH of the surrounding medium. Nano-

Figure 12. Confocal laser scanning microscopy image of a HeLa cell after 24 h incubation with DOX-loaded AB5 particles, showing colocalization of acidic cell compartments and particles. Blue channel = nucleus; green channel = lysotracker; red channel = DOX; overlay image. Scale bars are 10 μm.

nanoparticles over time and their slow release mechanism of DOX. These results indicate that the drug was released from the nanocarrier and migrated into the nucleus. An explanation for why the time course in cells does not match the release assay is that the pH change in cells is not as abrupt as was tested in the assay leading to slower release kinetics. I

dx.doi.org/10.1021/bm500919z | Biomacromolecules XXXX, XXX, XXX−XXX

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(4) Renggli, K.; Baumann, P.; Langowska, K.; Onaca, O.; Bruns, N.; Meier, W. Selective and responsive nanoreactors. Adv. Funct. Mater. 2011, 21 (7), 1241−1259. (5) Mai, Y.; Eisenberg, A. Self-assembly of block copolymers. Chem. Soc. Rev. 2012, 41 (18), 5969−5985. (6) Onaca, O.; Enea, R.; Hughes, D. W.; Meier, W. Stimuliresponsive polymersomes as nanocarriers for drug and gene delivery. Macromol. Biosci. 2009, 9 (2), 129−139. (7) Brinkhuis, R. P.; Rutjes, F. P. J. T.; van Hest, J. C. M. Polymeric vesicles in biomedical applications. Polym. Chem. 2011, 2 (7), 1449− 1462. (8) Walther, A.; Mueller, A. H. E. Janus Particles: Synthesis, SelfAssembly, Physical Properties, and Applications. Chem. Rev. (Washington, DC, U. S.) 2013, 113 (7), 5194−5261. (9) Matsumura, Y.; Maeda, H. A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res. 1986, 46 (12, Pt. 1), 6387−6392. (10) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an emerging platform for cancer therapy. Nat. Nanotechnol. 2007, 2 (12), 751−760. (11) Cabane, E.; Zhang, X.; Langowska, K.; Palivan, C. G.; Meier, W. Stimuli-responsive polymers and their applications in nanomedicine. Biointerphases 2012, 7 (1−4), 9. (12) Lee, E. S.; Gao, Z.; Bae, Y. H. Recent progress in tumor pH targeting nanotechnology. J. Controlled Release 2008, 132 (3), 164− 170. (13) Bawa, P.; Pillay, V.; Choonara, Y. E.; du Toit, L. C. Stimuliresponsive polymers and their applications in drug delivery. Biomed. Mater. (Bristol, U.K.) 2009, 4 (2), 022001/022001−022001/022015. (14) Agarwal, S.; Zhang, Y.; Maji, S.; Greiner, A. PDMAEMA based gene delivery materials. Mater. Today 2012, 15 (9), 388−393. (15) Qian, Y.; Zha, Y.; Feng, B.; Pang, Z.; Zhang, B.; Sun, X.; Ren, J.; Zhang, C.; Shao, X.; Zhang, Q.; Jiang, X. PEGylated poly(2(dimethylamino) ethyl methacrylate)/DNA polyplex micelles decorated with phage-displayed TGN peptide for brain-targeted gene delivery. Biomaterials 2013, 34 (8), 2117−2129. (16) Adams, D. J.; Butler, M. F.; Weaver, A. C. Effect of block length, polydispersity, and salt concentration on PEO-PDEAMA block copolymer structures in dilute solution. Langmuir 2006, 22 (10), 4534−4540. (17) Adams, D. J.; Adams, S.; Atkins, D.; Butler, M. F.; Furzeland, S. Impact of mechanism of formation on encapsulation in block copolymer vesicles. J. Controlled Release 2008, 128 (2), 165−170. (18) Agut, W.; Brulet, A.; Schatz, C.; Taton, D.; Lecommandoux, S. pH and temperature responsive polymeric micelles and polymersomes by self-assembly of poly[2-(dimethylamino)ethyl methacrylate]-bpoly(glutamic acid) double hydrophilic block copolymers. Langmuir 2010, 26 (13), 10546−10554. (19) Matter, Y.; Enea, R.; Casse, O.; Lee, C. C.; Baryza, J.; Meier, W. Amphiphilic PEG-b-PMCL-b-PDMAEMA triblock copolymers: From synthesis to physico-chemistry of self-assembled structures. Macromol. Chem. Phys. 2011, 212 (9), 937−949. (20) Zhu, Y. J.; Tan, Y. B.; Du, X. Preparation and self-assembly behavior of polystyrene-block-poly(dimethylaminoethyl methacrylate) amphiphilic block copolymer using atom transfer radical polymerization. eXPRESS Polym. Lett. 2008, 2 (3), 214−225. (21) Hu, J.; Zhang, G.; Ge, Z.; Liu, S. Stimuli-responsive tertiary amine methacrylate-based block copolymers: Synthesis, supramolecular self-assembly, and functional applications. Prog. Polym. Sci. 2014, 39 (6), 1096−1143. (22) Borchert, U.; Lipprandt, U.; Bilang, M.; Kimpfler, A.; Rank, A.; Peschka-Suess, R.; Schubert, R.; Lindner, P.; Foerster, S. pH-induced release from P2VP-PEO block copolymer vesicles. Langmuir 2006, 22 (13), 5843−5847. (23) Zhang, J.; Wu, L.; Meng, F.; Wang, Z.; Deng, C.; Liu, H.; Zhong, Z. pH and reduction dual-bioresponsive polymersomes for efficient intracellular protein delivery. Langmuir 2012, 28 (4), 2056− 2065.

particles prepared from AB5 block copolymer, loaded with DOX retained 80 wt % of the drug for 3 days at pH 7.4, while systems directly exposed to pH 5.5 released more than 80 wt % of the encapsulated drug within 48 h. Further cell studies showed that the nanocarriers were taken up by HeLa cells via endocytosis. The acidic pH in the endosomes/lysosomes caused intracellular release of DOX, which induced cell death. The results indicate that the therapeutic cargo can be released in a pH-dependent manner, showing that PDMS-b-PDMAEMA carrier can act as an efficient, stimuli-responsive drug delivery system. Several modifications can be envisioned to further improve and tune the system. Blending of PDMS-b-PDMAEMA block copolymers with other block copolymers or the same type of block copolymers could be designed to change the release kinetics of DOX, to influence the size of particles, or to further tailor the pH-triggered permeability of different drugs. Moreover, targeting ligands such as antibodies and peptides could be conjugated to the surface of the platform since the block copolymers possess reactive bromine-end groups: This would allow the development of targeted drug-delivery systems. As such modifications are well-known in the literature, the pHresponsive PDMS-b-PDMAEMA particles could be developed into a versatile pH responsive platform. Due to the fact that they retain their morphology at low pH and slowly release their therapeutic payload in the time course of several days, these particles could be effective in passive tumor targeting.



ASSOCIATED CONTENT

S Supporting Information *

Scheme of the synthesis, 1H NMR spectrum of PDMS macroinitiator, DMAEMA, and PDMS-b-PDMAEMA block copolymers. Guinier plot of static light scattering data of AB5 particles, determination of DOX extinction coefficient, additional Cryo-TEM images of AB5 nanoparticles and cumulative release of AB13-DOX and AB22-DOX loaded particles. This material is available free of charge via Internet at http://pubs. acs.org



AUTHOR INFORMATION

Corresponding Authors

*Tel.: +41 61 267 38 02. Fax: +41 61 267 38 55. Email: [email protected]. *Tel.: +41 61 267 38 28. Fax: +41 61 267 38 55. Email: anja. [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the Swiss National Science Foundation (SNSF) (P.B.), the Swiss Nanoscience Institute, an Marie Curie Actions Intra European fellowship (IEF) (p.n. 301398) (A.C.), and the Holcim Stiftung Wissen (N.B.). We thank Elisa Nogueira for editorial help. We thank Prof. Henning Stahlberg (Director of C-CINA) for his support.



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pH-responsive PDMS-b-PDMAEMA micelles for intracellular anticancer drug delivery.

A series of poly(dimethysiloxane)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMS-b-PDMAEMA) block copolymers were synthesized with atom transfer ra...
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