Fabrication of Polyion Complex Vesicles with Enhanced Salt and

Jul 1, 2014 - Integrating catalytic functions into polymeric vesicles through enzyme entrapment is appealing for bioreactor fabrication, yet there are...
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Fabrication of Polyion Complex Vesicles with Enhanced Salt and Temperature Resistance and Their Potential Applications as Enzymatic Nanoreactors Sayan Chuanoi,† Yasutaka Anraku,† Mao Hori,† Akihiro Kishimura,*,‡,§ and Kazunori Kataoka*,† †

Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Faculty of Engineering and §Center for Molecular System, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan

‡

S Supporting Information *

ABSTRACT: Integrating catalytic functions into polymeric vesicles through enzyme entrapment is appealing for bioreactor fabrication, yet there are critical issues regarding the regulation of solute transport through membranes and enzyme loading without denaturation. Polyion complex vesicles (PICsomes) with semipermeable membranes and the propensity to form in water can overcome these issues; however, cross-linking is required for sufficient physiological stability. Herein, we report the first successful fabrication of non-cross-linked PICsomes with sufficient stability at physiological salinity and temperature by tuning the hydrophobicity of the aliphatic side chains in the pendant group of the constituent polyelectrolytes. Dynamic light scattering and transmission electron microscopy revealed that the intervesicular fusion and disintegration of the PICsomes was prevented and a narrow distribution was maintained at physiological salinity and temperatures. Furthermore, their application as enzymatic nanoreactors was verified even in the presence of proteases. As such, the potential utility of the PICsomes in biomedical fields was established.

1. INTRODUCTION Submicrometer-sized vesicles that can be dispersed in aqueous milieu have attracted much interest due to their ability to load guest molecules with various properties (size, solubility, surface charge, etc.), making them potentially useful for versatile biological applications.1−5 One promising application is as water-dispersible bioreactors in which enzymes and other catalytic materials are encapsulated for the production of biologically relevant substances under physiological conditions for diagnostic and therapeutic purposes.2,6−10 Enzyme-loaded vesicles for such purposes should be able to tolerate physiological stimuli (ionic strength, temperature change, protease attack, etc.), allow substrates to permeate their membranes, and to retain the activity of the loaded enzymes. Conventional synthetic vesicles, primarily fabricated from polymers and lipids, do not adequately meet all of the aforementioned requirements.11 For example, conventional polymersomes, vesicles made of amphiphilic block copolymers, such as poly(acrylic acid)-block-polystyrene (PS), poly(ethylene oxide)-block-polybutadiene, and so on, exhibit physiological and thermal stabilities, and can encapsulate bioactive macromolecules;1,12 however, they lack membrane permeability.1 Furthermore, although polymersomes formulated from PSblock-poly(L-isocyanoalanine(2-thiophen-3-yl-ethyl)amide)13,14 and other stimuli-sensitive polymers,15 and polymersomes containing transmembrane proteins2 have been verified to possess enhanced water permeability, their preparation is © 2014 American Chemical Society

complicated and includes the use of organic solvents, heating, and sonication,16 which may cause denaturation or deactivation of enzymes upon loading. Thus, these drawbacks limit the potential bioreactor applications. Recently, we prepared polyion complex vesicles (PICsomes) in aqueous media via electrostatic self-assembly of oppositely charged block- and homoionomers. PICsomes have a unilamellar vesicle wall, tunable size, and are semipermeable to solutes.17,18 They also have potential to act as bioreactors due to the facile encapsulation of proteins by simple mixing.19 Nevertheless, a critical issue in the application of PICsomes as bionanoreactors is their susceptibility to elevated temperatures and salt concentrations.17 Indeed, cross-linking of the PIC layer imparts excellent physiological stability to PICsomes providing long blood circulation time and selective tumor accumulation.17,20,21 However, cross-linking is not always favorable, since it complicates the preparation and results in less biodegradability and long-term retention in the body. Furthermore, crosslinking may hamper the activity of encapsulated enzymes. Therefore, a novel approach is needed for the development of physiologically stable PICsomes that do not require crosslinking in order to establish the broad utility of PICsomes in versatile practical applications. Received: January 25, 2014 Revised: May 27, 2014 Published: July 1, 2014 2389

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Herein, we report the first example of non-cross-linked PICsomes less than 100 nm in size that are stable at elevated temperatures and high salt concentrations. The PICsomes were prepared through elongation of the aliphatic spacer in the pendant group of the homocatiomer paired with a block aniomer, poly(ethylene glycol)-b-poly(aspartic acid). Finally, we demonstrated that the novel, robust PICsomes with octyl side chains could encapsulate enzymes without disrupting the original enzymatic activity. Furthermore, the PICsomes acted as nanoreactors even in the presence of protease, demonstrating their utility under physiologically relevant conditions.

After cross-linking, the TEM samples were prepared using two different methods, namely the conventional method (conventional TEM) and the cross-sectional method (cross-sectional TEM). For conventional TEM, cross-linked particles were centrifuged using ultrafiltration against pure water (a poly(ether sulfone) ultrafiltration membrane with a molecular weight cutoff (MWCO) of 300000 for 100 nm scale particles and a MWCO of 100000 for 70 nm scale particles was utilized) to remove phosphate salts. Next the particles were dropped onto the copper grids, stained with a 50 v/v % ethanol solution containing 2 wt % uranyl acetate, and dried at RT. For the cross-sectional TEM, cross-linked particles were concentrated by centrifugation, fixed with a 2 wt % osmic acid solution, embedded in an epoxy resin (EPON 812), cut with an ultramicrotome to a thickness of 50 nm, stained with a 50 v/v % ethanol solution containing 2 wt % of uranyl acetate, and dried at RT. Number-based diameter distributions were determined from conventional TEM images with a scale bar using ImageJ software (available online at http://rsb.info.nih.gov/ij/download.html). A line on the scale bar was manually drawn in the image opened with ImageJ, and the program’s built-in Analyze → Set Scale command was used to generate a pixel to nanometer conversion value. Next, the diameters of single particles were measured by drawing lines by hand, and were expressed as nanometer values calculated using the Analyze → Measure command. For each sample, the values obtained from 100 particles were utilized to provide the histograms using KaleidaGraph. 2.2.5. Cryo-TEM Measurement. Enzyme-loaded PICsomes were cross-linked and purified by the method shown in 2.2.4. TEM samples were prepared on microgrids (coated with a thin film of Formvar (polyvinyl formal) and carbon; purchased from JEOL, Tokyo, Japan). The particles were dropped onto the microgrids, and vitrified by rapid immersion into liquid ethane near its freezing point. Vitrification was performed on an EM GP (Leica Microsystems, Vienna, Austria). The vitrified specimen was transferred to a JEM-2100F transmission electron microscope (JEOL, Tokyo, Japan) for imaging using a cryotransfer holder (G-914, Gatan, Pleasanton, CA 94588, U.S.A.). The microscope was equipped with a Field emission gun (ZrO/W(100)) and a 4K × 4K CCD camera (UltraScan, Gatan, Pleasanton, CA 94588, U.S.A.). The temperature of the sample was maintained below −170 °C throughout the analysis. Number-based diameter distributions were determined by the same method described in section 2.2.4. 2.2.6. Fluorospectrometry. The fluorescence measurements were performed using a NanoDrop ND-3300 fluorospectrometer (NanoDrop Technologies; U.S.A.). 2.3. Syntheses and Characterization. 2.3.1. Synthesis of Poly([8-aminooctyl]-α,β-aspartamide) (Homo-P(Asp-C8)). As shown in Scheme S1, the synthesis of Homo-P(Asp-C8) was carried out using a modified version of a previously reported method.17 First, n-butylamine was used to initiate the polymerization of BLA-NCA, yielding poly(β-benzyl-L-aspartate) (Homo-PBLA). Next, HomoPBLA lyophilized from CH2Cl2 and benzene was dissolved in a mixture of DMAc and NMP (DMAc/NMP = 1.6:1 (v/v)). The Homo-PBLA solution was added dropwise to a DMAc solution of C8 (50 mol equiv per BLA units) and stirred at 50 °C for 25 min under an argon atmosphere. The experimental conditions, namely the choice of solvent and temperature, were chosen to maintain the solubility of C8 throughout the reaction. The reaction solution was added dropwise to cold acetic acid (20% (v/v); 2 mol equiv per amine group) for neutralization and was subsequently dialyzed against 0.01 M HCl for 36 h and against deionized water for 12 h at 4 °C using a Spectra/Por dialysis membrane (MWCO of 6−8000). The dialyzed solutions were lyophilized to give the salt form of the homocatiomers. Detailed information on the synthesis and characterization (GPC and 1H NMR; Figures S1 and S2) of Homo-P(Asp-C8) is available in the Supporting Information. 2.3.2. Preparation of PICsomes. Homo-P(Asp-C8), Homo-P(AspC5), and PEG−PAsp were individually solubilized in 10 mM PB (0 mM NaCl, pH 7.4) at a concentration of 1.0 mg/mL. Next, the solutions were passed through a 0.22-μm membrane filter to remove dust. Homo-P(Asp-C8) and Homo-P(Asp-C5) solutions were added in one portion to a solution of PEG−PAsp at RT at an equal unit ratio

2. EXPERIMENTAL SECTION 2. 1. Materials. β-Benzyl-L-aspartate N-carboxy anhydride (BLANCA) was obtained from Chuo Kaseihin Co. Inc. (Tokyo, Japan), and used as received. α-Methoxy-ω-amino poly(ethylene glycol) (MeOPEG-NH2) (Mn = 2000; Mw/Mn = 1.05) was purchased from NOF Co. Ltd. (Tokyo, Japan), and purified by ion-exchange chromatography using CM Sephadex C-50 (GE Healthcare; U.S.A.).17 1,5Diaminopentane (C5) and 1,8-diaminooctane (C8) were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). For the removal of water, C5 was distilled over CaH2 under reduced pressure and C8 was dehydrated using a Dean−Stark apparatus. N-Methyl-2pyrrolidone (NMP) and N,N-dimethylacetamide (DMAc) were purchased from Wako Pure Chemical Industries (Osaka, Japan). N,N-Dimethylformamide (DMF) and dichloromethane (CH2Cl2) were purchased from Kanto Chemical Co. Inc. (Tokyo, Japan). NMP, DMAc, DMF, and CH2Cl2 were distilled prior to use. Cy3 and Cy5 monoreactive dye packs were purchased from GE Healthcare (Tokyo, Japan) and used as received. The homocatiomer poly([5aminopentyl]-α,β-aspartamide) (Homo-P(Asp-C5), degree of polymerization (DP) of P(Asp-C5) = 82 (Scheme S1), block-aniomer PEG-b-poly(α,β-aspartic acid) (PEG-P(Asp), Mn of PEG = 2000, DP of P(Asp) = 75, and Cy3-labeled PEG-P(Asp) were prepared as previously reported.17 1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC) was purchased from Wako Pure Chemical Industries (Osaka, Japan). TokyoGreen-β gal (TG-β-gal) was purchased from Sekisui Medical Co. Ltd. (Tokyo, Japan). βgalactosidase (β-gal) and bovine pancreas trypsin were obtained from Sigma (St. Louis, MO, U.S.A.). EDC, TG-β-gal, and β-gal were used as received. 2.2. Methods. 2.2.1. 1H NMR. The 1H NMR spectra were recorded using a JNM-AL 400 (JEOL; Japan) spectrometer operating at a frequency of 400 MHz. The analyses were carried out at 25 °C using D2O as a solvent. 2.2.2. Size Exclusion Chromatography (SEC). SEC was carried out using a high-performance liquid chromatography (HPLC) system (JASCO; Japan) equipped with a column (Superdex 200−10/300GL; GE Healthcare; U.S.A.) and fluorescent detector. Phosphate buffer (PB) solution (10 mM, pH 7.4) containing 500 mM NaCl was used as the eluent at a flow rate of 0.75 mL/min at room temperature (RT). 2.2.3. Dynamic Light Scattering (DLS). The DLS measurements were conducted at 25 °C using a Zetasizer Nano-ZS system (Malvern Instruments; Malvern, U.K.) equipped with an He−Ne laser operating at 633 nm. Analyses were performed at an angle of 173°. In this study, the intensity-averaged hydrodynamic diameter (z-averaged size) and polydispersity index (PDI) were calculated using the Cumulant method.17 2.2.4. Transmission Electron Microscopy (TEM). TEM measurements were conducted using a HITACHI H-7000 electron microscope or a JEOL JEM-1400 electron microscope, operating at 75 kV or 120 kV, respectively. Copper grids (400 mesh size) were coated with a thin film of collodion and carbon. Prior to TEM measurements, all samples were cross-linked by adding 10 mM PB of EDC (10 mg/mL) to a 10 mM PB solution of the sample (molar ratio of EDC to carboxylate groups contained in PEG−PAsp = 10), followed by 24 h incubation, for ease of purification. 2390

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of COO− and NH3+ groups. The resulting solutions were subsequently mixed using a vortex mixer for 2 min to generate the PIC solutions. For ease of discussion, the abbreviations C8-PICsomes and C5PICsomes are hereafter used to describe the PICsomes prepared from complexation of PEG−PAsp with Homo-P(Asp-C8) and HomoP(Asp-C5), respectively. 2.3.3. Determination of PICsome Yield. PEG−PAsp-Cy3 was used as the anionic component for the PICsome formation. Resulting solutions of Cy3-labeled C8- and C5-PICsomes were cross-linked (abbreviated as Cy3-labeled X-C8- and X-C5-PICsomes, respectively) using EDC (see detailed procedure in section 2.2.4). Then, the solutions were analyzed using SEC (Ex/Em = 520/550 nm) without purification. 2.3.4. Evaluation of Salt Resistance of PICsomes. A buffer solution of NaCl (10 mM PB) was added to the solutions of C8- and C5PICsomes (final NaCl concentration = 150 mM) followed by gentle pipetting. After 24 h incubation at 25 or 37 °C, NaCl was removed by dialysis using a Mini Dialysis Kit (GE Healthcare; U.S.A.; MWCO of 8000) against PB buffer at 25 °C for 24 h. The size and PDI of resulting PICsomes before and after NaCl removal were analyzed by DLS at the corresponding incubation temperature. 2.3.5. Evaluation of Thermal Stability of PICsomes. All PICsomes were prepared at RT, as described in section 2.3.2. The temperature of the PICsome solutions was rapidly elevated to 25, 37, 50, or 70 °C over a few minutes and was maintained using a heat block (DryThermo Unit DTU-1B; Taitec Corporation; Japan). After 24 h of incubation, the solutions were cooled to 25 °C over 30 min and maintained at 25 °C for 24 h using the heat block. The size and PDI of the resulting PICsomes were analyzed by DLS before and after cooling at the incubation temperature. 2.3.6. Preparation of Cy5-Labeled β-gal (Cy5-β-gal). Cy5-β-gal was prepared by mixing Cy5 monoreactive dye (Cy5-NHS) with β-gal, followed by stirring at RT for 2 h. Finally, unreacted Cy5-NHS was removed using a Sephadex G-25 M PD-10 column (GE Healthcare; USA). The resulting labeled β-gal had approximately three Cy5 groups per enzyme. 2.3.7. Preparation of Cy5-β-gal-loaded PICsomes. A solution of Cy5-β-gal (1 mg/mL) was added to the solutions of C8- or C5PICsomes, followed by vigorous vortex mixing for 2 min to obtain Cy5-β-gal-loaded C8- and C5-PICsomes,22 respectively (final concentration of Cy5-β-gal = 0.1 mg/mL). 2.3.8. Determination of Cy5-β-gal Loading in PICsomes. Encapsulation of Cy5-β-gal was verified by SEC after the cross-linking of Cy5-β-gal-loaded C8- and C5-PICsomes (Cy5-β-gal-X-C8- and Cy5-β-gal-X-C5-PICsomes, respectively) using EDC. Next, nonencapsulated Cy5-β-gal was removed by ultrafiltration to facilitate the SEC measurement. Empty Cy3-labeled X-C8- and X-C5PICsomes were used as controls after the cross-linking and purified under the same conditions. The SEC traces of the samples were recorded using a fluorescent detector (Cy5: Ex/Em = 650/670 nm and Cy3: Ex/Em = 520/550 nm). 2.3.9. Evaluation of Enzymatic Activity. The enzymatic activities of non-cross-linked Cy5-β-gal-C8- and Cy5-β-gal-C5-PICsomes were evaluated. The Cy5-β-gal-C8- and Cy5-β-gal-C5-PICsomes were subjected to 150 mM NaCl at 37 °C, followed by the addition of trypsin (final concentrations of Cy5-β-gal and trypsin were 50.0 μg/ mL and 500.0 μg/mL, respectively). After incubation at 37 °C for 24 h, TG-β-gal, a nonfluorescent substrate, was added to the incubated solutions (final concentrations of TG-β-gal and Cy5-β-gal were 4.75 μg/mL and 0.025 μg/mL, respectively). Finally, the hydrolysis of TGβ-gal into fluorescent TG, catalyzed by Cy5-β-gal loaded in PICsomes, was evaluated by monitoring the increase of the fluorescence intensity at 515 nm, following incubation at 37 °C for 0, 1, 2, and 3 h. The enzymatic activities were calculated from the linear slope of the fluorescent intensity versus time and were expressed in enzymatic units per milliliter of reaction medium (U/mL). One unit of activity was defined as the amount of enzyme that catalyzed the conversion of 1 μmol of substrate per minute.

3. RESULTS AND DISCUSSION 3.1. Preparation and Physicochemical Characterization of PICsomes Prepared from Homo-P(Asp-C8). PICsome formation is quite sensitive to the length of alkyl spacers in the homocatiomers that are paired with PEG−PAsp. Previously, we studied a series of PICsomes prepared from a combination of PEG−PAsp and poly(aspartamide) with varying lengths of alkyl spacers (from ethyl to hexyl; Homo(PAsp-Cx: x ≈ 2−6).18 Notably, the only micelle structure was found in PICsome systems generated from catiomers with shorter alkyl spacers (x ≈ 2−4), while the catiomers with longer alkyl spacers (x = 5−6) yielded a clear PICsome structure, indicating that there was a minimum spacer length required to stabilize the PIC lamella in the formation of vesicular structures. Nevertheless, the PICsomes generated from Homo(PAsp-C5) and Homo(PAsp-C6) could not tolerate physiological salt concentrations (150 mM NaCl). Accordingly, we hypothesized that the longer aliphatic alkyl spacer in the homocatiomer may stabilize the PICsome lamella structure through more favorable side chain packing to form tight ion pairs surrounded by the microenvironment with a lowered dielectric constant in the PICsome membrane. Homocatiomer Homo-P(Asp-C8) (DP of P(Asp-C8) = 95), which had an octyl spacer in the side chain, was expected to yield effective chain packing, and thus the hydrophobic microenvironment capable of stabilizing polyion pairs in the PIC membrane with block aniomer PEG−PAsp (DP of PAsp = 75; Scheme 1). As a control, the homocatiomer bearing a Scheme 1. Formation of C8-PICsomes with Improved Physiological Stability for Bionanoreactor Applications

pentyl side chain, Homo-P(Asp-C5) (DP of P(Asp-C5) = 82), was chosen to mix with the same block aniomer to form PICsomes (C5-PICsomes), which were reported to be sensitive to physiological levels of salt (150 mM NaCl).15 The formation of PICsomes and their size distributions were directly studied using TEM (Figure 1). According to the number-averaged size histograms obtained from TEM images (Figure 1a), narrow unimodal distributions of PICsomes were confirmed. Notably, PICsomes generated from Homo-P(AspC8) (C8-PICsome) had a smaller diameter (∼59 nm) than C5PICsomes (∼88 nm), indicating that the size decreased as the length of the aliphatic spacer increased. These trends were confirmed by the DLS of the PICsome solution (see Supporting Information for details; Figure S3). Negatively stained TEM images (Figures 1b-i and ii (high magnification) and S4 (low magnification)) revealed the formation of hollow vesicles in C8-PICsomes (dark arrows in Figure 1b-i) and C5PICsomes (Figure 1b-ii).17 However, there were relatively smaller particles (white arrows in Figure 1b-i) that did not 2391

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suggested that the C8-PICsomes were unilamellar vesicles, in which the thickness of the membranes was comparable to that of the C5-PICsomes (10−15 nm, as shown in Figure 1d-ii and the previous report17). C8-PICsomes were composed of relatively smaller vesicles than the C5-PICsomes (Figure 1a). Further, it might have been difficult for the smaller vesicles to deform into concaved shapes in order for the staining materials to pool in the center during the drying process, resulting in the negligible dark region in the center of the negatively stained TEM images (Figure 1b-i). To the best of our knowledge, C8PICsomes are the smallest unilamellar vesicles prepared on the basis of PIC formation (detailed mechanism for small PICsome formation is discussed in section 3.6). 3.2. Stability of C8-PICsomes under Physiological Salt Concentrations. The effect of 150 mM NaCl on C8-and C5PICsomes was evaluated, as this is the key ionic strength for evaluating the physiological stability of PICs that are often destabilized by Na+ and Cl−.28 After addition of NaCl at 25 °C, the solution of C5-PICsomes promptly became turbid, whereas the solution of C8-PICsomes remained clear. As evaluated by DLS (Figure 2a), C8-PICsomes increased slightly in z-averaged

Figure 1. Formation of PICsomes: (a) Number-averaged size histograms obtained by measuring the diameter of C8- (i) and C5(ii) PICsomes from TEM images shown in (b). (b) TEM images of negatively stained C8- (i) and C5- (ii) PICsomes. The dark and white arrows indicate C8-PICsomes with and without clear dark concave centers. (c) TEM images of positively stained C8-PICsomes obtained using high (left) and low (right) magnification. (d) Cross-sectional TEM images of C8- (i) and C5- (ii) PICsomes.

Figure 2. Stability of C8-PICsomes against physiological salinity. (a) Time-dependent, z-averaged size and PDI of C8-PICsomes after incubation at 25 °C, with (+) and without (−) 150 mM NaCl, as revealed by DLS. (b) TEM images of negatively stained C8-PICsomes after saline addition (25 °C) at 0, 6, and 24 h.

involve any dark contrast part in the center. In the negatively stained imaging, such higher contrast part is normally observed by the pooling of staining agents on the concaved part of the vesicles.23,24 Deformation involving concaved or irregular shapes is mainly caused in the drying process. In contrast, in the TEM images obtained with a positive staining method (Figure 1c), one can see the particles consisting of a rather bright part surrounded by a darker rim, regardless of the size. This was consistent with a vesicular structure because the brighter center was correlated with the inner hollow space in the positively stained images.25−27 To further confirm the vesicular structure of the C8-PICsomes of all sizes, a crosssectional TEM study was carried out (Figures 1d-i and ii). Indeed, the bright core surrounded by the dark ring was observed by cross-sectional TEM for all particles in the C8PICsome sample, clearly indicating the formation of vesicles. Furthermore, the thickness of the PIC membranes (dark ring) was estimated to be ∼12 nm (Figure 1d-i). These results

size from ∼70 to ∼95 nm upon salt addition before reaching a plateau at 6 h after addition (∼125 nm). On the other hand, C8-PICsomes incubated in the absence of NaCl changed in size only slightly. A narrow size distribution (PDI < 0.1) was maintained, regardless of NaCl concentration. TEM images (Figure 2b (high magnification) and S5a (low magnification)) showed the complete preservation of the vesicular structure of the C8-PICsomes after exposure to saline, although their size increased, as confirmed by DLS. Notably, just after treatment with 150 mM NaCl, the negatively stained TEM images showed that all C8-PICsomes had a clear concave region at the center, which was not observed for the relatively smaller C8PICsome fraction obtained in the absence of NaCl (see white arrows in Figure 1b-i). Since the size of the C8-PICsomes became larger upon addition of salt (Figure 2a,b), the vesicles might have collapsed more easily to reveal the concave area. By contrast, the morphology of the C5-PICsomes changed from 100 nm vesicles to μm-sized PIC aggregates, with sponge-like architectures, as seen in the cross-sectional TEM images 2392

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(Figure S5b). This result indicated that the C5-PICsomes could not tolerate the high salt concentration. Alternatively, C8PICsomes successfully acquired salt-resistance and increased slightly in size in response to NaCl. It is noteworthy that C8PICsomes were able to keep their submicrometer-scaled size and vesicle architecture even after addition of NaCl up to 300 mM, although a slight size increase to ∼130 nm was involved (Figure S6 and Table S2 in the Supporting Information). The detailed mechanism for morphological and size changes of PICsomes is discussed in section 3.7. 3.3. Thermal Stability of C8-PICsome. C8- and C5PICsomes were incubated at 37 °C for 24 h in 10 mM PB (0 mM NaCl) to evaluate their stability against physiological temperature. According to the DLS measurements, after incubation at 37 °C (Figure 3a), C8-PICsomes grew from 70 to 80 nm, similar to those incubated at 25 °C. By contrast, the z-averaged size and PDI of the C5-PICsomes increased over time as the temperature increased. TEM showed that the monodispersity of the C8-PICsomes was preserved and was accompanied by a subtle size increase, while C5-PICsomes became progressively larger and their polydispersity increased

after heating (Figures 3b and S7; high magnification and low magnification images, respectively). In order to obtain more information about the thermal stability of PICsomes, the temperature range was expanded. After incubation at 70 °C for 24 h, the size and polydispersity of C5-PICsomes continuously increased and finally gave a mixture of giant (μm scale) PICsomes and sub-μm scale PICsomes (Figures S8 and S9). C8-PICsomes incubated at 70 °C were ∼115 nm with a low PDI value (