Article pubs.acs.org/Langmuir
Poly(N‑isopropylacrylamide)/Poly(dopamine) Capsules Yan Zhang,† Boon M. Teo,† Kenneth N. Goldie,‡ and Brigitte Stad̈ ler*,† †
Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark Center for Cellular Imaging & Nano Analytics, Biozentrum, University of Basel, Basel, Switzerland
‡
S Supporting Information *
ABSTRACT: Polymer capsules are an interesting concept considered in nanobiotechnology. Approaches that facilitate their assembly remain sought after. Poly(dopamine) (PDA) has been considered and successfully applied in this context. We recently demonstrated that PDA could be copolymerized with different types of poly(N-isopropylacrylamide) (pNiPAAm) to assemble mixed films on planar substrates. Herein, we transferred this approach onto colloidal substrates and characterized the film thickness depending on the film composition and template particles size. While the membrane of capsules assembled using 5 μm template particles exhibited strong dependency on the film composition, smaller templates led to capsules with similar membrane thickness. We then compared the permeability of different capsules using fluorescently labeled dextran and fluorescein. We found that the permeability of capsules was heavily dependent on the polymer composition and the template particle size. These fundamental findings contribute to the potential of these capsules, assembled in one-step, for biomedical applications.
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been used as a carrier for a multistep enzymatic reaction17 and the first in vitro drug delivery successes with mammalian cells.18,19 Further, Cu-containing PDA capsules have shown bactericidal activity.20 PDA capsules also exhibit an interesting environment-dependent21 and/or cargo-dependent permeability.22 PDA, deposited via a self-oxidative process at slightly basic pH, has, since its identification in 2007,23 attracted considerable interest for manifold (biomedical) applications.24,25 Currently, there are several hypotheses regarding the structure of this material.23,26−28 The possibility to modify PDA with amines and thiols has earlier been recognized. Further, mixed PDAbased film could be deposited using a variety of compounds by either simply premixing the molecule of interest with dopamine29−33 or by chemically modifying the reactant with dopamine34−37 prior to the film deposition. We recently demonstrated that the hydrophilic polymers poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) could be codeposited with DA to form mixed PDA/PEG(PVA) films on planar silica substrates without the need for amine or thiol groups.33 However, only PDA/PEG films allowed for the assembly of structurally intact polymer capsules. Further, we compared the formation of mixed PDA-based films using aminated and carboxylated low molecular weight poly(Nisopropylacrylamide) (pNiPAAm) on planar silica surfaces.31 Surprisingly, not only the aminated but also the carboxylated polymer was incorporated. In our most recent report, we also demonstrated that high molecular weight, highly branched pNiPAAm could be codeposited with dopamine to form mixed
INTRODUCTION Polymer capsules have recently attracted considerable interest for biomedical applications as drug carriers1 or for encapsulated catalysis toward therapeutic cell mimicry.2 Using the sequential deposition of interacting polymers (the layer-by-layer (LbL) technique) onto sacrificial silica colloids is the dominant approach to assemble these capsules. Although a variety of polymers and interactions including electrostatics, hydrogen bonding,3 or covalent linkages4 have been used to demonstrate the assembly of capsules,5 only a few systems including disulfide-stabilized poly(methacrylic acid) capsules6 or dextran sulfate/poly(L-arginine) capsules7 have reported drug delivery ability in vitro and in vivo, and vaccination applications have demonstrated particular promising potential.8−10 Apart from being considered as drug carriers, polymer capsules were also used in encapsulated catalysis. The direct encapsulated the enzymes into the capsules has been considered but bears the risk of cargo lost or degradation.5 However, the permeability of the capsule’s polymer membrane can also be beneficial as demonstrated by the encapsulated synthesis of RNA.11 More recent developments in the field considered the assembly of subcompartmentalized systems including the use of liposomes,2 polymersomes,12 cubosomes,13 nanosized capsules,14 or the combination thereof15 as subunits within the LbL carrier capsules. All these examples indicate the potential of these polymer capsules. However, despite its popularity, the LbL technique is labor-intensive, time-consuming, and often struggles with aggregation issues and/or uncontrolled leakage of cargo, among others. As an alternative, poly(dopamine) (PDA) capsules have been first demonstrated in 2009,16 an approach which in particular reduced the labor related to their assembly. PDA capsules have © 2014 American Chemical Society
Received: February 11, 2014 Revised: March 26, 2014 Published: April 24, 2014 5592
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films on planar silica surfaces.32 For all the above-mentioned mixed films, we could show that the protein and liposome adsorption were affected and in the case of pNiPAAm-x mixed films, also the cell response. Herein, we studied the assembly of capsules consisting of PDA and aminated, carboxylated, or highly branched poly(Nisopropylacrylamide) (pNiPAAm-NH2, pNiPAAm-COOH, or pNiPAAm-HB) (Scheme 1). Specifically, we (i) assembled,
1X81 motorized inverted Olympus microscope and a 40× LD objective. The diameter and polydispersity index (PDI) of the 800 nm capsules were assessed by a dynamic light scattering (DLS) instrument (Zetasizer nano, Malvern Instruments) using a material refractive index of 1.590 and a dispersant (water at 25 °C) refractive index of 1.330. The polymer membrane thickness of the capsules was assessed using atomic force microscopy (AFM, Nanowizard 2, JPK Instruments AG, Germany). A small amount of the capsule solution was dried onto a glass slide and imaged by AFM using tapping mode (Olympus OMCL-TR400PSA-1 cantilevers). At least five capsules were measured, or at least two independently assembled batches were analyzed. The capsules were also visualized using transmission electron microscopy (TEM) by adsorbing 4 μL of the capsule suspension onto a carbon film mounted on 300 mesh copper grids (Quantifoil Micro Tools GmbH, Jena, Germany) for 5 min. The grid surface was rendered hydrophilic by glow discharge in a reduced atmosphere of air for 10 s prior to adsorption. The grid was blotted and left to air-dry. TEM images of the capsules were acquired digitally using a Philips CM10 microscope (FEI, Eindhoven, The Netherlands) fitted with a 2k × 2k side-mounted CCD Camera (Olympus SIS, Münster, Germany). The ζ-potential of 5 μm capsules was measured using a Zetasizer nano, Malvern Instruments, with a material refractive index of 1.590 and a dispersant (water at 25 °C) refractive index of 1.330. The permeability of the capsules was assessed by incubating 108 capsules mL−1, assembled from PDA, PDA/pNiPAAm-NH2 (10/1 mol %), or PDA/pNiPAAm-HB (2/3 wt %) using 5 μm, 2.5 μm, or 800 nm silica templates as described above, in a solution of fluorescently labeled dextran (DexFITC, 1 mg mL−1 in TRIS1 buffer) or fluorescein (FC, 0.25 mg mL−1 in TRIS1 buffer) for 24 h. 105 μL of these mixture was then added to 1 mL of TRIS2 buffer and instantly measured by flow cytometry (Accuri Cytometers Inc.) using an excitation wavelength of λ = 488 nm.
Scheme 1. Schematic Illustration of the Coating of Silica Particles with PDA or PDA/pNiPAAm-NH2(HB)a
a
Upon removal of the core, a polymer capsule is yielded.
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visualized and assessed the membrane thickness of capsules made of PDA, PDA/pNiPAAm-NH2, PDA/pNiPAAm-COOH, or PDA/pNiPAAm-HB, (ii) downscaled the capsule’s size, and (iii) assess the permeability of the different types and sizes of capsules.
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RESULTS AND DISCUSSION Capsule Assembly. With the aim to demonstrate the feasibility to assemble polymer capsules with a membrane of PDA and pNiPAAm-x (x = NH2 or COOH), we coated 5 μm silica colloids with DA, DA/pNiPAAm-NH 2 , or DA/ pNiPAAm-COOH using three different concentrations of DA but keeping the coating time as well as the molar ratio between DA and pNiPAAm-x (10:1) constant. Upon removal of the silica template, polymer capsules were obtained. Figure 1 shows representative DIC microscopy images in water as well as TEM and AFM images in air of the different types of capsules (Figure 1a: DA; Figure 1b1: mixtures of DA/pNiPAAm-NH2; and Figure 1b2: mixtures of DA/pNiPAAm-COOH). Using 1 mg mL−1 DA only, did not yield stable capsules in water, while the addition of pNiPAAm-NH2 improved the quality of the capsules and to a lesser extent when pNiPAAm-COOH was added. This suggested that both PDA and pNiPAAm-x were present in the capsule’s membranes. Further, capsules assembled from 1 mg mL−1 DA (with or without pNiPAAmx) entirely disintegrated upon drying and were not further considered. The use of 2 or 3 mg mL−1 DA (with or without pNiPAAm-x) yielded stable nonaggregated capsules in water, with similarly looking polymer membranes in TEM and AFM. Only capsules assembled from 2 mg mL−1 DA only and 2 mg mL−1 DA with pNiPAAm-COOH remained largely intact upon drying, while in all the other cases, the polymer membrane was damaged. This result points towards different membrane properties depending on the components used for their assembly. Unexpectedly, the polymer membrane of capsules assembled from 3 mg mL−1 DA was also damaged upon drying,
EXPERIMENTAL SECTION
Materials. Aminated poly(N-isopropylacrylamide) (pNiPAAmNH2, MW = 2500 Da), carboxylated poly(N-isopropylacrylamide) (pNiPAAm-COOH, MW = 2000 Da), dopamine hydrochloride (DA), hydrofluoric acid (HF), tris(hydroxymethyl)aminomethane (TRIS), sodium chloride (NaCl), fluorescein (FC), fluorescein isothiocyanate dextran (DexFITC, 3000−5000 Da), ethanol, and chloroform (purity ≥99.5%) were purchased from Sigma-Aldrich. Silica particles (5 μm, 2.5 μm, and 800 nm) were obtained from Microparticles GmbH, Germany. Highly branched poly(N-isopropylacrylamide) (pNiPAAmHB, MW = 44 100 Da) has been synthesized as previously described.32 Two types of TRIS buffer were used throughout all the experiments: TRIS1 consisted of 10 mM TRIS (pH 8.5), and TRIS2 consisted of 10 mM TRIS and 150 mM NaCl (pH 7.4). The buffer solutions were made with ultrapure water (Milli-Q gradient A 10 system, resistance 18 MΩ cm, TOC < 4 ppb, Millipore Corporation). Capsule Assembly. 100 μL of silica particles (5 wt %) was washed 2× in TRIS1 buffer (1060 g, 30 s). The particles were suspended in either 300 μL of DA solution (1, 2, or 3 mg mL−1) or solutions containing a mixture of DA and pNiPAAm-NH2(COOH) in a molar ratio of 10/1 with a final DA concentration of 1, 2, or 3 mg mL−1, or a mixture of DA and pNiPAAm-HB in different wt %, all in TRIS1. The polymerization was allowed to proceed for 8 h (2.5 μm silica), 19 h (800 nm silica), or 24 h (5 μm silica) with constant shaking. Following this, the mixture was centrifuged (1060g, 30 s) and washed 3× in TRIS1 buffer. Then, the silica core was dissolved using a 2 M HF solution for 2 min, followed by several washing cycles in ultrapure water (4500g, 3 min). The resulting capsules were visualized using a 5593
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measurement by two. Figure 2 presents the membrane thickness as assessed from AFM images. The membrane
Figure 2. Polymer membrane thickness of the different capsules as assessed by AFM is plotted. The molar ratio DA/pNiPAAm-x is 10/1 using 5 μm silica templates.
thickness of capsules assembled from 2 mg mL−1 DA showed differences. The addition of pNiPAAm-NH2 to the coating yielded capsules with a ∼10 nm thicker membrane as compared to PDA capsules. The addition of pNiPAAm-COOH led to capsules with similar membrane thickness as PDA capsules. This implies the presence of the pNiPAAm-x polymer in the film and also that using pNiPAAm-NH2 yielded thicker capsules, likely due to the interaction of the PDA via the amines. On the other hand, for capsules assembled from 3 mg mL−1 DA, the membrane thickness was ∼15 nm larger as compared to capsules assembled from 2 mg mL−1 DA, showing that higher monomer concentration led to thicker polymer capsules. This finding is in agreement with prior results found for PDA deposition on planar silica surfaces using a DA concentration of 2 or 3 mg mL−1 and 24 h deposition times.38 However, the presence of pNiPAAm-x did not affect the membrane thickness when 3 mg mL−1 DA was used in the mixture. This is probably due to the fact that the high DA concentration caused the PDA polymerization to proceed too fast for the enhanced incorporation of the pNiPAAm-x polymer. Finally, the membranes of capsules assembled via the deposition of PDA followed by the adsorption of pNiPAAm-NH2 at 24 and 39 °C were analyzed. In the former case, capsules with a membrane thickness similar to the PDA deposition only were yielded. In the latter case, the polymer membrane was found to be thinner than in all the other assessed cases. This was surprising since we previously showed that at 39 °C, large amounts of pNiPAAm-NH2 were deposited as monitored by quartz crystal microbalance with dissipation monitoring.31 We hypothesized that precipitation onto planar substrates followed by gentle washing required weaker interactions between PDA and pNiPAAm-NH2 for the latter component to remain adsorbed. On the other hand, when deposition onto particles was performed, several centrifugal washing steps were required. During the washing steps, considerable amount of the pNiPAAm was probably removed. The thinner membrane might even point towards removal or compression of PDA upon exposure to elevated temperature. In a next step, we characterized the capsule assembly using the highly branched pNiPAAm (pNiPAAm-HB) mixed with DA as contrast to the low molecular weight pNiPAAm considered so far. We compared capsules assembled from 1,
Figure 1. Capsules: DIC microscopy, TEM and AFM images of capsules assembled using (a) DA (DA concentration (i) 1, (ii) 2, and (iii) 3 mg mL−1), (b) a mixture of (1) DA/pNiPAAm-NH2 and (2) DA/pNiPAAm-COOH (DA concentration (i) 1, (ii) 2, and (iii) 3 mg mL−1), and (c) subsequent deposition of (1) PDA-pNiPAAm-NH2 at 24 °C and (2) PDA-pNiPAAm-NH2 at 39 °C. The molar ratio DA:pNiPAAm-x was 10:1 using 5 μm silica templates. The scale bar is 20 μm for the DIC images, 1 μm for the TEM images, and 2 μm for the AFM images.
indicating that the membrane is not very flexible, hinting towards noncovalent interactions in the films. However, since the applications in mind are for capsules in liquid, this aspect is not affecting their usefulness. Since we have previously demonstrated that higher amounts of pNiPAAm-x were deposited above the lower critical solution temperature (LCST) of pNiPAAm, we attempted to coat the silica particles with bare DA or subject the mixtures to elevated temperature. However, these did not yield stable capsules, demonstrating only few-intact capsules but many aggregated PDA “flakes” (Supporting Information Figure S1). Alternatively, we explored the option to first coat the silica particles with PDA and then deposited pNiPAAm-NH2 at 24 and 39 °C (Figure 1c). In both cases, nonaggregated capsules were yielded. With the goal to assess the dry thickness of the polymer membrane, the capsules were dried and imaged using AFM. The membrane thickness was then calculated using a line scan over the edge of the capsules and dividing the height 5594
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2, or 3 mg mL−1 DA mixed with different wt % pNiPAAm-HB. Figure 3a shows representative DIC and AFM images of these
similar thickness, confirming the deposition of mixed films. The similar thicknesses also point towards a limit for the incorporation of pNiPAAm-HB. These findings indicated the importance of the initial building block composition on the properties of the deposited film, which will in turn provide a basis to fine-tune the mixed coatings. With the aim to support the fact that the assembled capsules had a different membrane composition, ζ-potential measurements were performed at pH 4, 7, and 9 and compared (Table 1). Capsules assembled using 2 and 3 mg mL−1 DA mixed with Table 1. ζ-Potential of Different Capsules Measured at Different pH (Average (STD) Is Shown) ζ-potential (mV) 10 mM NaOAc, pH 4
10 mM Tris, pH 7
10 mM Tris, pH 9
30.4(1.7) 31.0(0.5)
−7.9(0.9) −14.0(1.5)
−37.4(0.4) −39.9(0.4)
23.3(0.1) 12.5(2.1)
−6.2(1.7) −8.3(2.5)
−25.0(0.5) −23.8(1.4)
27.3(3.6) 31.5(2.8)
−11.0(1.8) −11.0(1.5)
−31.4(4.5) −35.7(4.4)
3.8(1.5) 4.4(0.9)
−5.4(2.8) −3.5(4.9)
−16.8(1.3) −11.0(5.3)
28.8(2.9) 10.5(0.4)
−15.5(3.9) −11.7(0.7)
−39.3(1.5) −21.9(0.4)
DA 2 mg mL−1 3 mg mL−1 DA/pNiPAAm-NH2 2 mg mL−1 3 mg mL−1 DA/pNiPAAmCOOH 2 mg mL−1 3 mg mL−1 DA/pNiPAAm-HB 2 mg mL−1 3 mg mL−1 DA-pNiPAAm-NH2 24 °C 37 °C
pNiPAAm-NH2, pNiPAAm-COOH, or pNiPAAm-HB as well as pristine PDA capsules and PDA capsules subsequently coated with pNiPAAm-NH2 at 24 or 37 °C were used. When considering pristine PDA capsules, the measured ζ-potential was decreasing from ∼30 to ∼−38 mV with increasing pH, independent of the used DA concentration. While the trend of the ζ-potential has been observed before, our positive ζpotential was considerably higher than previously reported values.22 A similar trend for the change in ζ-potential was observed when pNiPAAm-COOH was codeposited with PDA, indicating that only small amounts of pNiPAAm-COOH were incorporated into the films, in agreement with our previous findings.31 On the other hand, when pNiPAAm-NH2 was used, the ζ-potential at pH 4 and 9 was found to be overall lower and higher, respectively, compared to pristine PDA. Further, using 3 mg mL−1 DA in the mixture led to lower ζ-potential at pH 4 than using 2 mg mL−1 DA. These observations were supporting the deposition of a mixed film, probably due to the fact that the quinones reacted with the amine groups, which reduced the amount of functional groups that could contribute to the ζpotential. When considering pNiPAAm-HB which does not have any charged end groups, the monitored ζ-potential started at ∼4 mV at pH 4, decreased to ∼−3−4 and ∼−11−17 mV at pH 7 and pH 9, respectively, independent of the DA concentration used. This finding points towards the presence of a mixed PDA/pNiPAAm-HB film. Finally, when considering capsules assembled by first depositing PDA followed by pNiPAAm-NH2 adsorption at 24 or 37 °C, the change in ζpotential was similar to pristine PDA capsules for 24 °C, likely
Figure 3. Capsules: (a) DIC microscopy and AFM images of capsules assembled from DA/pNiPAAm-HB mixtures with different wt % using 5 μm silica templates. The scale bar for the DIC images is 20 μm and for the AFM images 2 μm. (b) Polymer membrane thickness of these capsules as assessed by AFM is plotted. The dashed line represents the polymer membrane thickness for capsules assembled using 2 mg mL−1 DA.
capsules. As previously mentioned, 1 mg mL−1 DA did not yield intact capsules. However, when mixed with pNiPAAmHB, the capsules’ appearance considerably improved, even more than for capsules incorporated with pNiPAAm-NH2 in their mixed membrane. In general, all the tested mixed coatings yielded intact nonaggregated capsules in water. Upon drying, only low pNiPAAm-HB (DA/pNiPAAm-HB 2/1) or high DA concentrations (3 mg mL−1 DA) led to broken capsules. In the other cases, the capsules remained intact, even when only 1 mg mL−1 DA was used. Figure 3b compares the polymer membrane thickness of these capsules as assessed by AFM. First, keeping the wt % between DA/pNiPAAm-HB constant but increasing the amount of DA led to thicker capsules as expected. On the other hand, keeping the DA concentration constant (2 mg mL−1) but varying the wt % between DA/ pNiPAAm-HB yielded the thickest capsules for the lowest amount of pNiPAAm-HB. They exhibited similar thickness as pristine PDA capsules. Capsules assembled from DA/ pNiPAAm-HB 2/2 or 2/3 wt % had thinner membranes of 5595
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measured by AFM. It was found that they were in general considerably thinner than when 5 μm template particles were used (Figure 4b), likely due to the shorter PDA coating times. Further, the differences in membrane thickness depending on the film composition were negligible, with the only exception of capsules assembled from DA/pNiPAAm-HB using 800 nm template particles. In this case, the polymer membrane was found to be thicker, probably due to the effect of the relatively high amount of pNiPAAm-HB incorporated in the film. We hypothesize that the curvature of the particles started to have an effect on the codeposition of DA with large macromolecules like pNiPAAm-HB. Capsule’s Permeability. The semipermeable property of the capsule’s polymer membrane is among the features that makes them interesting in drug delivery. Passive or triggered release of the encapsulated cargo from polymer capsules is expected to be a way to control/steer the therapeutic response. Typically, the cargo diffusion is controlled via the number or type of deposited polymer layers39 or the used cross-linker.40 There are fundamental approaches reported to (quantitatively) assess the cargo diffusion constants.41−43 Here, we aimed to qualitatively compare the permeability of capsules assembled from PDA, PDA/pNiPAAm-NH2, or PDA/ pNiPAAm-HB using 5 μm, 2.5 μm, or 800 nm silica templates. The capsules were incubated in a solution of fluorescently labeled dextran (DexFITC) or fluorescein (FC), and the fluorescent intensity of the capsules was monitored by flow cytometry right after mixing (Supporting Information Figure S3) and after 24 h (Figure 5). The values were normalized to
due to the overall rather small adsorption of pNiPAAm-NH2 in this case. On the other hand, when adsorbing pNiPAAm-NH2 at 37 °C, the change in ζ-potential was similar to capsules made of PDA/pNiPAAm-NH2, pointing towards a higher interaction of PDA with pNiPAAm-NH2 in this case. Downscaling of the Capsules. While 5 μm template particles provided fundamental insight into the possibility to assemble capsules with mixed PDA/pNiPAAm membranes, these capsules are typically too large for many envisioned drug delivery applications. Therefore, we assembled capsules using 2.5 μm and 800 nm diameter silica template particles. The 5 μm template particles did not exhibit any aggregation issues, but when applying the same protocol to the smaller particles, severe aggregation after the coating for 24 h was observed. Therefore, the coating times were shortened and different optimal times were identified depending on the silica template size: 8 h for 2.5 μm and 19 h for 800 nm. Figure 4a shows AFM
Figure 4. Downscaling of the capsules. (a) AFM images of PDA (i), PDA/pNiPAAm/NH2 (ii), or PDA/pNiPAAm/HB (iii) capsules assembled using 2.5 μm (top row) or 800 nm (bottom row) silica particles as templates. The scale bar is 2 μm. (b) Membrane thickness of the different capsules as measured by AFM (DA/pNiPAAm-NH2 10/1 mol %, DA/pNiPAAm-HB 2/3 wt %).
Figure 5. Fluorescent intensity of different sized capsules exposed to DexFITC or FC for 24 h (DA/pNiPAAm-NH2 10/1 mol %, DA/ pNiPAAm-HB 2/3 wt %). Higher normalized intensity corresponds to lower permeability.
the 5 μm capsules for each of the tested cargo. There are two possibilities what low normalized fluorescent intensities could correspond to (1) low normalized fluorescent intensity values correspond to more permeable capsules, due to the fast penetration of the cargo molecules across the polymer membranes, i.e., fast cargo release upon dilution of the samples for the flow cytometry measurements, or (2) low normalized fluorescent intensity values are indicative of low permeability due to the limited access of the cargo to the capsule’s interior.
images (top row: 2.5 μm template; bottom row: 800 nm template) of the intact nonaggregated capsules obtained. The nonaggregated nature of the 800 nm capsules was confirmed by DLS (Supporting Information Table S1). Because of their size, the capsules assembled using 2.5 μm templates could not be analyzed by DLS, but by optical microscopy (Supporting Information Figure S2), and intact nonaggregated capsules were observed. The membrane thickness of these capsules was 5596
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showed a decreasing permeability for PDA < PDA/ pNiPAAm-NH2 < PDA/pNiPAAm-HB capsules. Taken together, these fundamental findings are important aspects to be considered when employing these types of films as coatings toward biomedical applications.
When comparing the normalized intensities for the PDA capsules at time 0 and 24 h, it is observed that decreasing size led to higher and lower normalized intensity, respectively. We hypothesized that this is due to the fact that the permeability increases with decreasing capsule size, allowing initial fast diffusion of the fluorescent cargo to the capsule’s interior, hence, higher initial normalized intensities measured in that case. On the other hand, after 24 h, the cargo diffused faster out of the capsules with decreasing size upon contacting the sheath fluid of the flow cytometer. Similar observations were made of the other types of capsules. Because of this, we believe that the more likely case is that lower normalized intensity after 24 h was indicative for more permeable capsules. Further, using flow cytometry as a read-out method allows for the monitoring of the initial change in fluorescence due to change in the amount of trapped fluorescent cargo. Although it is not possible to distinguish between cargo trapped in the void and the polymer membrane with this approach, it allows assessing the permeability in a more reliable way than microscopy techniques. The need to focus and to remove the fluorescence from the background for the latter technique is likely affecting the outcome. Also, flow cytometry is analyzing several thousands of capsules in a very short time, something that is not achievable with microscopy techniques. When considering the 5 μm capsules, the normalized fluorescent intensity of the capsules decreased from PDA > PDA/pNiPAAm-NH2 > PDA/pNiPAAm-HB for both tested cargo. The previously assessed polymer membrane thickness revealed that PDA/pNiPAAm-HB capsules were the thinnest ones (∼22 nm) and therefore probably also the most permeable. Although PDA capsules (∼27 nm) had a thinner membrane than PDA/pNiPAAm-NH2 capsules (∼36 nm), the permeability seemed higher in the latter case. The measured normalized fluorescent intensity of the 2.5 μm capsules was similar, independent of the type of polymer membrane, indicating similar permeability of the capsules. This is in good agreement with the previously assessed membrane thickness which was found to be similar (∼11 nm). When considering the 800 nm capsules, the normalized fluorescent intensity of the capsules increased from PDA < PDA/ pNiPAAm-NH2 < PDA/pNiPAAm-HB for both cargo tested, but more pronounced for DexFITC than FC. The previously measured polymer membrane thicknesses are in good agreement with this finding, with PDA and PDA/pNiPAAm-NH2 capsules having similar thickness (∼11 nm) and PDA/ pNiPAAm-HB being slightly thicker (∼17 nm) and due to that, probably less permeable, especially toward larger cargo, DexFITC in this case.
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ASSOCIATED CONTENT
* Supporting Information S
Microscopy image of capsules assembled at 39 °C, table of the diameter and PDI of 800 nm capsules, microcopy images of 2.5 μm capsules, and the initial fluorescence intensity of different capsules. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail
[email protected] (B.S.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a Sapere Aude Starting Grant from the Danish Council for Independent Research, Technology and Production Sciences, Denmark.
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REFERENCES
(1) De Koker, S.; Hoogenboom, R.; De Geest, B. G. Polymeric Multilayer Capsules for Drug Delivery. Chem. Soc. Rev. 2012, 41, 2867−2884. (2) Städler, B.; Price, A. D.; Chandrawati, R.; Hosta-Rigau, L.; Zelikin, A. N.; Caruso, F. Polymer Hydrogel Capsules: En Route toward Synthetic Cellular Systems. Nanoscale 2009, 1, 68−73. (3) Kharlampieva, E.; Kozlovskaya, V.; Sukhishvili, S. A. Layer-byLayer Hydrogen-Bonded Polymer Films: From Fundamentals to Applications. Adv. Mater. 2009, 21, 3053−3065. (4) Such, G. K.; Johnston, A. P. R.; Liang, K.; Caruso, F. Synthesis and Functionalization of Nanoengineered Materials Using Click Chemistry. Prog. Polym. Sci. 2012, 37, 985−1003. (5) Tong, W. J.; Song, X. X.; Gao, C. Y. Layer-by-Layer Assembly of Microcapsules and Their Biomedical Applications. Chem. Soc. Rev. 2012, 41, 6103−6124. (6) Zelikin, A. N.; Price, A. D.; Städler, B. Poly(Methacrylic Acid) Polymer Hydrogel Capsules: Drug Carriers, Sub-compartmentalized Microreactors, Artificial Organelles. Small 2010, 6, 2201−2207. (7) Dierendonck, M.; De Koker, S.; Vervaet, C.; Remon, J. P.; De Geest, B. G. Interaction between Polymeric Multilayer Capsules and Immune Cells. J. Controlled Release 2012, 161, 592−599. (8) Sexton, A.; Whitney, P. G.; Chong, S. F.; Zelikin, A. N.; Johnston, A. P. R.; De Rose, R.; Brooks, A. G.; Caruso, F.; Kent, S. J. A Protective Vaccine Delivery System for In Vivo T Cell Stimulation Using Nanoengineered Polymer Hydrogel Capsules. ACS Nano 2009, 3, 3391−3400. (9) De Koker, S.; Naessens, T.; De Geest, B. G.; Bogaert, P.; Demeester, J.; De Smedt, S.; Grooten, J. Biodegradable Polyelectrolyte Microcapsules: Antigen Delivery Tools with Th17 Skewing Activity after Pulmonary Delivery. J. Immunol. 2010, 184, 203−211. (10) De Geest, B. G.; Willart, M. A.; Lambrecht, B. N.; Pollard, C.; Vervaet, C.; Remon, J. P.; Grooten, J.; De Koker, S. SurfaceEngineered Polyelectrolyte Multilayer Capsules: Synthetic Vaccines Mimicking Microbial Structure and Function. Angew. Chem., Int. Ed. 2012, 51, 3862−3866. (11) Price, A. D.; Zelikin, A. N.; Wark, K. L.; Caruso, F. A Biomolecular “Ship-in-a-Bottle”: Continuous RNA Synthesis within Hollow Polymer Hydrogel Assemblies. Adv. Mater. 2010, 22, 720− 723.
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CONCLUSIONS We demonstrated the assembly of PDA/pNiPAAm capsules using different combinations of building blocks and qualitatively compared their permeability. We found that overall, larger template particles yielded capsules with thicker polymer membrane, and a more film composition dependent thickness was identified. Capsules assembled using smaller templates were thinner, and their film thickness was less dependent on the DA to pNiPAAm composition during the assembly. We further identified a template size dependent permeability of the capsules. Large capsules exhibited an increasing permeability for PDA > PDA/pNiPAAm-NH2 > PDA/pNiPAAm-HB capsules. The permeability of medium sizes capsules was largely independent of the film composition and small capsules 5597
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dx.doi.org/10.1021/la5005227 | Langmuir 2014, 30, 5592−5598
