Influence of Surface Chemistry on Particle Internalization into Giant

Jun 5, 2013 - (22, 25-27) One of the most advantages of GUV is the possibility to modulate the lipid components and environmental circumstances so as ...
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Influence of Surface Chemistry on Particle Internalization into Giant Unilamellar Vesicles Jiaojiao Liu,† Naiyan Lu,† Jingliang Li,§ Yuyan Weng,† Bing Yuan,*,† Kai Yang,*,† and Yuqiang Ma†,‡ †

Center for Soft Condensed Matter Physics and Interdisciplinary Research, Soochow University, Suzhou, 215006, P. R. China National Laboratory of Solid State Microstructures and Department of Physics, Nanjing University, Nanjing, 210093, P. R. China § Institute for Frontier Materials, Deakin University, Waurn Ponds, Vic 3216, Australia ‡

S Supporting Information *

ABSTRACT: Cellular uptake of materials plays a key role in their biomedical applications. In this work, based on the cell-mimic giant unilamellar vesicles (GUVs) and a novel type of microscale materials consisting of stimuli-responsive poly(N-isopropylacrylamide) microgel particles and the incorporated lipids, the influence of particle surface chemistry, including hydrophobic/hydrophilic property and lipid decorations, on the adsorption and consequent internalization of particles into GUVs was investigated. It is found that the decoration of particle surface with lipids facilitates the adsorption of particles on GUV membrane. After that, the hydrophobic property of particle surface further triggers the internalization of particles into GUVs. These results demonstrate the importance of surface properties of particles on their interactions with lipid membranes and are helpful to the understanding of cellular uptake mechanism.

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INTRODUCTION Engineered nano- and micromaterials have shown wide and increasing applications in different fields such as biomedicine and tissue engineering.1,2 However, to execute properly their biomedical functions, the interactions of such materials with the biological systems need to be understood. These include the adsorption and agglomeration of proteins on the surface of materials,3,4 the disturbance of materials to cell membranes,5−7 the transmembrane delivery and the potential cytotoxicity of the internalized particles.8,9 Among these processes, the cellular uptake and intracellular trafficking of the materials have attracted intensive interest, due to their biological significance as well as the importance in guiding the design and fabrication of new materials with optimal biomedical or bioengineering functions for disease diagnosis and therapy.2,10,11 The cellular uptake and intracellular distribution of materials depend critically on their characteristic properties such as size and shape, composition, charge, and especially the surface chemistry.1,2,12−16 However, how these properties influence their uptake pathway, intracellular location and translocation, biomedical functions and potential cytotoxicity, has not been fully understood.2 Both active and passive uptakes have been proposed for the cellular uptake of materials.17−19 To improve the particle recognition and uptake by cells, efforts has been devoted to the surface modification of materials with ligands that bind specifically to membrane receptors.12 However, the interactions between cells and particles without any targeting ligand are often poorly understood. Generally, a two-step model is considered for a passive uptake process. That is, the particle © XXXX American Chemical Society

uptake is triggered by the nonspecific adsorption of particles onto the plasma membrane, which is followed by internalization.20,21 Electrostatic interactions between particles and lipids have been considered to be one of the key factors governing both the adsorption and internalization processes.20,22 In addition, changes in the surface chemical properties of particles due to surface modification and/or functionalization have also been reported to be somewhat crucial.20,23,24 Giant unilamellar vesicles (GUVs) have been used widely as artificial systems to mimic cells.22,25−27 One of the most advantages of GUV is the possibility to modulate the lipid components and environmental circumstances so as to ensure that only particle−lipid interactions are probed.21 As one of the conventional stimuli-sensitive, namely “smart”, polymers, poly(N-isopropylacrylamide) (pNIPAM) microgel has been extensively studied in drug loading and release for advanced biomedicine applications owing to its good biocompatibility.28 However, it is still not fully understood how the surface chemistry of pNIPAM microgel particles influence their interactions with cell membranes. Some studies suggest that polymer dendrimers (such as polyamidoamine) are internalized into cells through the caveloae or clathrin pathway although they may also be passively translocated across the cell membrane without the aid of protein pathways.29 Received: April 25, 2013 Revised: June 5, 2013

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components from Zeiss. All the experiments were carried out at room temperature of 22 °C unless stated otherwise. Interactions between PNIPAM/Lipogel Particles and GUVs. A volume of 500 μL of GUV solution was pretransferred to a homemade chamber cell, with a PDMS-coated glass coverslip as substrate, and stabilized for about 5 min for GUV immobilization (to a density of ∼4 GUVs/mm2).34 After that, 100 μL of pNIPAM (or lipogel) particle dispersion was injected slowly. The following interactions between particles and GUVs were monitored in situ with the confocal microscope. Rh-B Delivery Test. A volume of 100 μL of lipogel dispersion was mixed with 50 μL of 4 mM Rh-B solution for Rh-B loading, and then added to the confocal chamber containing preimmobilized GUVs (∼4 GUVs/mm2) or cells (∼3 cells/mm2), which was incubated at 37 °C for confocal observations.

In this study, we investigate the interactions between materials and membranes based on pNIPAM microgel particles and GUVs, and focus on the correlation between surface chemistry, including hydrophobic/hydrophilic property and lipid decorations, of the particles with the adsorption and consequent internalization of particles into GUVs. The pNIPAM particles demonstrate unique structural transition behaviors due to lipid incorporation (namely, “lipogels”) and/ or temperature-triggered volume phase transition, which would significantly help us gain some novel insights into the cellular uptake mechanism.

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EXPERIMENTAL SECTION

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Materials. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamineN-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) were purchased from Avanti Polar Lipids and used as received. Rhodamine B (Rh-B), chloroform (99.7%), ethanol (99.0%), and sucrose (analytical reagent) were purchased from Shanghai Chemical Reagents Company and used without further purification. PNIPAM particles were synthesized as reported (φ ∼ 0.5).30 HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C and equilibrated in 4% CO2 and air. Preparation of GUVs. Cell-sized GUVs were prepared following the conventional electroformation method.31 A solution of lipids (DOPC/DOTAP = 95:5 with 0.5 mol % (relative to lipid mixture, to visualize the membrane in red fluorescence) Rh-PE) in chloroform was deposited onto two ITO-coated glass slides and dried under vacuum overnight for GUV preparation. The GUVs were then performed in 0.1 M sucrose buffer in a homemade electroformation chamber, with the two glass slides as electrodes, under alternating voltages. The obtained vesicles (∼0.02 mg lipid mL−1) with a unilamellar structure and a size distribution of 10−30 μm were well dispersed from each other due to the positively charged DOTAP lipids. Preparation of Lipogels (i.e., Lipid Incorporation of pNIPAM Particles). Lipogels were fabricated by a solvent-exchange method.32,33 An amount of 0.2 mg lipid (DOPC labeled by 1 mol % NBDPE, green fluorescence) was dissolved in chloroform (2.0 mg mL−1) and then dried under a stream of N2 gas to create a lipid film. After being completely dried overnight under vacuum, the lipid film was rehydrated with a 0.1 mL mixture of 40 vol % ethanol and 60 vol % pNIPAM aqueous suspensions (containing about 1010 pNIPAM particles). A volume of 1 mL of distilled water was then added to the mixture. A micelle-to-bilayer transition and liposome deposition behavior occurred during this solvent-exchange process. The bulk solution was centrifuged at 6000 rpm for 10 min. The wash and centrifugation were repeated three times to remove the excessive lipids. The precipitates were resuspended in 500 μL of 0.1 M sucrose buffer for use. Characterizations. The zeta potential of pNIPAM particles was determined using a zeta potential analyzer (Zetasizer Nano ZS90, Malvern Instrument Ltd., U.K.). Morphology of the pNIPAM particles without or with lipid incorporation was characterized on a scanning electron microscope (Raith Pioneer) after being frozen-dried. Optical observation was performed on an inverted confocal laser scanning microscope (Zeiss, LSM 710) equipped with a 100× oil objective. NBD conjugated lipids (for lipogel labeling) were excited by an argon ion laser (EX 488 nm), and their fluorescence was observed through filter set 44 (EM BP 530/50 nm). Cationic GUVs labeled with rhodamine-conjugated phospholipids were excited by a He−Ne laser (EX 543 nm), and their fluorescence was observed through filter set 20 (EM BP 575−640 nm). The transmission channel illuminated with a halogen lamp was acquired in the meantime. The temperature of the system was set and stabilized with the temperature control

RESULTS AND DISCUSSION 1. Structural Characterization of PNIPAM Particles, GUVs, and Lipogel Spheres. The optical micrograph, scanning electron microscopy (SEM) image, and zeta potential profile of the as-fabricated pNIPAM microgel particles are presented in the Supporting Information (Figure S1). The pNIPAM particles, with a size of 2.7 ± 0.1 μm, have a core− shell structure with a hydrophilic surface. The particles are positively charged, with a zeta potential of ∼14.6 mV. Fluorescence image of GUVs, which are also positively charged due to the existence of DOTAP components, is visualized by Rh-PE in red. In one of our previous reports, we demonstrated the conformation of a lipogel, consisting of a pNIPAM particle and incorporated lipids, as a three-layer structure (Figure 1A).35 The outermost is a layer of separated lipid assemblies (i.e., vesicles and micelles) that are decorated on the surface of the

Figure 1. Confocal micrographs, including fluorescence, transmission and merged channels, of a model lipogel consisting of pNIPAM particle and incorporated lipids, at 22 (A) and 37 °C (B). The green fluorescence originates from lipids (labeled with NBD-PE). White lines were added for eye direction. (C, D) Transmission images of a native pNIPAM particle at 22 and 37 °C, respectively. The scale bar represents 2 μm. B

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pNIPAM particle. This layer flares brightly in fluorescence (due to the NBD-PE labeled lipids) and is hardly distinguishable in the transmission image. The middle layer consists of densely distributed polymer aggregates (which is obviously visible in the transmission image), with little lipid incorporation. A portion of loosely distributed polymer chains, rich in lipid molecules, is located in the core region of the lipogel. The existence of lipid decorations at the particle surface was also confirmed by SEM images (Figure 2). New spots referring to lipid assemblies adsorbed on the sphere substrate were obviously visualized in the lipogel image.

Scheme 1. Schematic Representation of the Volume Phase Transition and Morphological Transformation of pNIPAM and Lipogel Particles upon Temperature Modulation (between 22 and 37 °C, crossing the LCST of pNIPAM)a

Figure 2. SEM images showing the surface morphology of pNIPAM particles without (A−C) or with (D−F) lipid incorporation. The increased spots in (E) and (F), marked with red arrows, refer to the freeze-dried lipid assemblies decorated on the surface of the pNIPAM spheres. (B, E) and (C, F) are enlarged images of part or fragment of spheres, respectively.

The lipogel is colored green due to the fluorescence of the NBDlabeled lipids. a

of the GUVs surviving was disturbed by lipogel spheres, by way of adsorption and/or embedding. As shown in Figure 3A, the transmission and merged confocal images obviously demonstrate the adsorption of a lipogel sphere on the GUV surface. Furthermore, the intensity profiles of the lipid fluorescence

One of the most characteristic properties of the lipogel is the thermally induced transformation behavior, due to the volume phase transition of pNIPAM when crossing its lower critical solution temperature (LCST) of ca. 32 °C.35 Below the LCST, pNIPAM polymer is fully water-soluble as a result of hydrogen bonding between the amide groups of polymer molecules and the surrounding water molecules (cf. Figure S1-D). The corresponding lipogel presents as a hybrid spherical scaffold surrounded with a layer of adsorbed lipid assemblies, as described in the paragraph above (Figure 1A). Above the LCST, the hydrogen bonds break down and water is expelled from the vicinity of the polymer chains, leading to a significant volume reduction of the pNIPAM particle (Figure 1C, D). As a result, the lipogel sphere consisting of the collapsed polymer scaffold and the incorporated lipid molecules has a much smaller size (Figure 1B). In contrast to the structure of the lipogel at room temperature (below the LCST) which has an outmost layer of separated lipid assemblies adsorbed on the pNIPAM scaffold, at a higher temperature above the LCST (e.g., body temperature of 37 °C), a layer of bulk lipids are decorated on the hydrophobic surface of the pNIPAM particle.35 The temperature-triggered phase transition of the lipogel between the two states can be reversibly modulated and repeated for more than 10 times. Such temperature-triggered transition is illustrated in Scheme 1. 2. Interactions between Lipogels and GUVs. 2.1. Adsorption of Lipogels on GUVs at 22 °C. Lipogel dispersions were injected into the chamber cell of a confocal microscope, in which GUVs had been immobilized onto the cover-glass substrate at a density of ∼4 GUVs/mm2. After 2 h incubation at a constant temperature of 22 °C, the interactions between lipogels and GUVs were observed. Many of the GUVs were not broken upon lipogel injection, and a percentage of about 20%

Figure 3. (A) Confocal micrographs, including fluorescence, transmission, and merged images, of lipogels interacting with GUVs after incubation for 2 h. For clarity, only one representative vesicle is shown. The green and red images come from the fluorescence of lipids originated from the lipogel (initially labeled with NBD-PE) and GUV (initially labeled with Rh-PE), respectively. The spherical spot, marked with white arrows, refers to the localization of lipogel. (B) Intensity profiles of fluorescence signals from the NBD-PE (green histogram) and Rh-PE (red histogram), marked by a red arrow in the left graph. The distance starts from the beginning point of the arrow. The portions marked with dashed squares refer to the presence of lipids (originated from GUV) that transferred to and covered the surface of lipogel. The temperature of the system is kept at 22 °C. C

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signals originated from both the lipogel (green) and the GUV (red), as shown in Figure 3B, indicate the lipogel is surrounded by GUV lipids. Furthermore, the fluorescence images in green and red channels in Figure 3A indicate that lipid exchange occurred between lipogel and GUV as a result of their interactions. Other phenomena induced by the introduction of lipogel, such as compartment formation in the GUVs, were also observed frequently. However, the GUVs with anchored lipogels stayed stable over hours after formation. It is noted that both GUVs and lipogels are positively charged. The adsorption of lipogel on GUV hence indicates that the electrostatic force is not a vital or predominant factor for the interaction between the lipogel particles and GUVs. 2.2. Internalization of Lipogels into GUVs at 37 °C. When the temperature of the system was elevated to the body temperature of 37 °C, something different happens. Figure 4

Figure 5. Confocal micrographs of a pea-shaped GUV containing a lipogel sphere within it at 37 °C. The red image originates from the fluorescence of lipids from the lipogel and GUV (both labeled with Rh-PE). White arrows point out the localization of the lipogel.

competition between the adsorption of lipogel on GUV and the deformation of the lipid bilayer, during the particle transmembrane process.36 2.3. Interactions between Native PNIPAM Particles and GUVs. As control experiments, plain pNIPAM microgel particles without lipid incorporation were also employed to test their interactions with GUVs under identical conditions, at both 22 and 37 °C. However, neither adsorption nor internalization of the particles was observed (Supporting Information, Figure S3), even after a long time of 5 h. 2.4. Drug Delivery by Lipogels into GUVs or Cells. The lipogel uptake process was also employed for drug delivery applications in which Rh-B was preloaded into lipogels as a model drug.37 Figure 6 shows the Rh-B delivery into GUVs and

Figure 4. Confocal micrographs of a model GUV interacting with lipogels after incubation for 2 h at 37 °C. The green and red images refer to the same meaning as in Figure 3. White arrows point out the localization of lipogel.

shows the interaction between GUVs and lipogels after incubation at 37 °C for 2 h. It can be obviously found that the lipogel is no longer adsorbed on the GUV surface but internalized into the GUV and a similar lipid exchange between the lipogel and GUV occurs as well. Based on our observations, the internalization of lipogel into GUV takes place in two steps. First, after 1 h incubation at 37 °C, lipogel adsorption on the GUV surface happened (not shown). Then after another one hour, many of the adsorbed lipogels further entered the GUVs and were finally internalized within them. In a control experiment we increased the temperature of system in Figure 3 from 22 to 37 °C, and found that after 1 h incubation, lipogel internalization happened as well. To further confirm the localization of lipogels in GUVs, zstack confocal images of the GUV−lipogel composite at different directions were performed (Figure S2, cf. Supporting Information). From these images, we can obtain the locations of lipogel to GUV at various cross sections. It can be obviously distinguished that the internalized lipogel was still linked with the GUV membrane, but not dissociated from it. Furthermore, lipid exchange was also found in this lipogel internalized system. Many of the NBD-labeled lipids (green) were transferred from lipogels to GUVs. At the same time, the lipogels taken up by GUVs were surrounded by Rh-PE (red) that was derived from the membranes of GUVs. Due to the existence of surrounding lipids, the lipogel was possibly internalized by GUV in an endocytosis-like pathway.24 However, no fission occurred during this internalization process. Besides the formation of compartments within GUVs induced by the adsorption of lipogels at 22 °C as mentioned above, the morphology of GUVs at 37 °C was frequently transformed to, for example, pealike shapes, followed by the disintegration of them within tens of minutes (Figure 5), upon the disturbance by the lipogel. This phenomenon reflects the

Figure 6. Rh-B delivery of lipogels into GUVs (A) or HeLa cells (B), after incubation for two hours at 37 °C. The lipogel was labeled with NBD-PE (green) and loaded with Rh-B fluorophores (red) as a model drug in advance. GUV was labeled with Rh-PE. Lipogel was pointed out with white arrows.

HeLa cells by lipogel (labeled with NBD-PE) after incubation for 2 h at 37 °C. It is observed that under both conditions the lipogel internalization happened, and at the same time red fluorophores (referring to the preloaded Rh-B) were found within the targeted GUV or cell. These results indicate the feasibility to use such lipogels as vehicle to transfer molecules into cells for drug delivery or gene therapies. 3. Discussion. In summary, no adsorption of the native pNIPAM particles onto GUVs happened at either 22 or 37 °C; however, with lipid incorporation (i.e., for the lipogel spheres), adsorption of the lipogels occurred to ∼20% of the surviving GUVs at 22 °C, while at an elevated temperature of 37 °C; that is, after the phase transition of pNIPAM polymers, not only adsorption but also internalization happened to ∼10% of the GUVs for the lipogels. It is inferred that the interaction D

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Then the interaction process between the particle and membrane was simulated by dissipative particle dynamics (DPD, cf. Supporting Information).38 For a native pNIPAM particle at 22 °C, a repulsive effect occurs between the hydrophilic particle surface and the identically charged lipid molecules within the lipid membrane. In this case, it is difficult for the particle to adhere to the membrane, although it has been reported that a hydrophobic particle always tends to embed itself into a lipid membrane (Figure 7A). However, the lipids decorated on the particle

manners between particle and membrane are significantly influenced by the chemical properties and molecular modification with lipids of the particle surface. At 22 °C, the difference of a lipogel system from that of the native pNIPAM particles mainly lies on the layer of lipids decorated on the pNIPAM particle’s surface. The impacts of such an electric neutral lipid layer might be in two sides: on one hand, it might shield the electrostatic repulsions between the identically charged pNIPAM particles and GUVs to some extent; on the other hand, it is possible to astrict the primarily free polymer chains on the surface of the pNIPAM particles, as it has been reported that surface modification with hydrophilic polymers (such as PEG) interrupts the adsorption between particles and membrane.22,23 These might be the reasons leading to the adsorption of lipogels onto GUVs at 22 °C. However, at an elevated temperature of 37 °C, adsorption was not observed still for the native pNIPAM particle system, although under such conditions the pNIPAM particles had contracted to much condensed spheres with no more hydrophilic polymer brushes around due to the volume phase transition of pNIPAM. This means that the shielding effect on the electrostatic repulsions between pNIPAM particle and GUV membranes due to the lipid covering might be a predominant factor facilitating the adsorption of lipogel spheres onto GUVs. When the temperature was changed from 22 to 37 °C, many of the preadsorbed lipogels were further internalized by GUVs. It is noted that the significant structural transitions in lipogel surface during such temperature elevation process mainly lies on the exposure of the hydrophobic groups of pNIPAM chains on the lipogel surfaces. Therefore, the hydrophilic/hydrophobic property of the pNIPAM scaffold surface can be regarded as a key factor determining whether or not the particle internalization happens. To verify this assumption of the role of surface chemistry of particles in the particle internalization process and further understand the underlining mechanism, computer simulations were employed. For the sake of simplicity, a hydrophobic sphere (of about 10 nm in size, cf. Supporting Information) was used to mimic a part of the pNIPAM particle (in dark blue as shown in Scheme 2), and its surface was randomly decorated with points with varying chemical properties (in light blue) that correspond to different conditions of the experiments. An uncharged lipid bilayer (containing 5 mol % lipids as shown in green and orange to represent the positively charged DOTAP molecules) was employed to mimic the membrane of a GUV.

Figure 7. DPD simulation results after the particle−membrane interactions with various chemical properties of the particle surface: (A) a repulsion effect occurs between the decorated points on particle surface and the positively charged lipids within the membrane; (B) an attraction effect lies between the decorated points and the lipid beads; (C) a hydrophilic-to-hydrophobic transition occurs to the decorated points on the particle surface. The bottom image is a cross-sectional image of the above one. All results were obtained by simulations in about 3 × 105 steps (27 μs).

Scheme 2. Coarse-Grained Model of a Part of pNIPAM Particle (A) and Molecular Structure of a pNIPAM Polymer Drawn in the Same Colors (B)a

surface may decrease the repulsive interaction and even render an attractive effect between the decorated points on the particle surface and the membrane. Under this condition, the particle can be adsorbed on the membrane immediately (Figure 7B). Moreover, similar to the situations of the phase transition of pNIPAM from 22 to 37 °C, such a hydrophilic-to-hydrophobic transition of the decorated points would significantly facilitate the embedding of the particle within the lipid bilayer followed by the adsorption of it (Figure 7C). These results confirm the experimental observations and further emphasize the importance of particle surface chemistry in determining the particle− membrane interactions.

a

Dark blue, the hydrophobic scaffold of the particle; light blue, randomly decorated points on particle surface with various chemical properties during the particle−membrane interactions; red, hydrated water molecules at room temperature below the LCST of pNIPAM. E

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Understanding biophysicochemical interactions at the nano-bio interface. Nat. Mater. 2009, 8, 543−557. (2) Zhao, F.; Zhao, Y.; Liu, Y.; Chang, X.; Chen, C.; Zhao, Y. Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials. Small 2011, 7, 1322−1337. (3) Statz, A. R.; Meagher, R. J.; Barron, A. E.; Messermith, P. B. New peptidomimetic polymers for antifouling surfaces. J. Am. Chem. Soc. 2005, 127, 7972−7973. (4) Mei, Y.; Gerecht, S.; Taylor, M.; Urquhart, A. J.; Bogatyrev, S. R.; Cho, S.-W.; Davies, M. C.; Alexander, M. R.; Langer, R. S.; Anderson, D. G. Mapping the interactions among biomaterials, adsorbed proteins, and human embryonic stem cells. Adv. Mater. 2009, 21, 2781−2786. (5) Demina, T.; Grozdova, I.; Krylova, O.; Zhirnov, A.; Istratov, V.; Frey, H.; Kautz, H.; Melik-Nubarov, N. Relationship between the structure of amphiphilic copolymers and their ability to disturb lipid bilayers. Biochemistry 2005, 44, 4042−4054. (6) Amri, C. E.; Lacombe, C.; Zimmerman, K.; Ladram, A.; Amiche, M.; Nicolas, P.; Bruston, F. The plastcins: membrane adsorption, lipid disorders, and biological activity. Biochemistry 2006, 45, 14285−14297. (7) Yue, T.; Zhang, X. Molecular understanding of receptor-mediated membrane responses to ligand-coated nanoparticles. Soft Matter 2011, 7, 9104−9112. (8) Kim, T.-W.; Chung, P.-W.; Slowing, I.-I.; Tsunoda, M.; Yeung, E. S.; Lin, V. S.-Y. Structurally ordered mesoporous carbon nanoparticles as transmembrane delivery vehicle in human cancer cells. Nano Lett. 2008, 8, 3724−3727. (9) Kirchner, C.; Liedl, T.; Kudera, S.; Pellegrino, T.; Munoz-Javier, A.; Gaub, H. E.; Stolzle, S.; Fertig, N.; Parak, W. J. Cytotoxicty of colloidal CdSe and CdSe/ZnS nanoparticles. Nano Lett. 2005, 5, 2331−2338. (10) Ding, H. M.; Tian, W. D.; Ma, Y. Q. Designing nanoparticle translocation through membranes by computer simulations. Acs Nano 2012, 6, 1230−1238. (11) Mu, Q.; Hondow, N. S.; Krzeminski, L.; Brown, A. P.; Jeuken, L. J. C.; Routledge, M. N. Mechanism of cellular uptake of genotoxic silica nanoparticles. Part. Fibre Toxicol. 2012, 9, 29. (12) Ding, H. M.; Ma, Y. Q. Role of physicochemical properties of coating ligands in receptor-mediated endocytosis of nanoparticles. Biomaterials 2012, 33, 5798−5802. (13) Yang, K.; Ma, Y. Q. Computer simulation of the translocation of nanoparticles with different shapes across a lipid bilayer. Nat. Nanotechnol. 2010, 5, 579−583. (14) Ding, H. M.; Ma, Y. Q. Interactions between Janus particles and membranes. Nanoscale 2012, 4, 1116−1122. (15) Huang, K.; Jacobs, A.; Rzayev, J. De novo synthesis and cellular uptake of organic nanocapsules with tunable surface chemistry. Biomacromolecules 2011, 12, 2327−2334. (16) Chen, T.; Guo, X.; Liu, X.; Shi, S.; Wang, J.; Shi, C.; Qian, Z.; Zhou, S. A Strategy in The Design of Micellar Shape for Cancer Therapy. Adv. Healthcare Mater. 2012, 1, 214−224. (17) Iversen, T.-G.; Skotland, T.; Sandvig, K. Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nano Today 2011, 6, 176−185. (18) Janson, J.; Ashley, R. H.; Harrison, D.; Mclntyre, S.; Butler, P. C. The mechanism of islet amyloid polypeptide toxicity is membrane disruption by intermediate-sized toxic amyloid particle. Diabetes 1999, 48, 491−498. (19) Geiser, M.; Rothen-Rutishauser, B.; Kapp, N.; Schurch, S.; Kreyling, W.; Schulz, H.; Semmler, M.; Hof, V. I.; Heyder, J.; Gehr, P. Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells. Environ. Health Perspect. 2005, 113, 1555−1560. (20) Graf, C.; Gao, Q.; Schutz, I.; Noufele, C. N.; Ruan, W.; Posselt, U.; Korotianskiy, E.; Nordmeyer, D.; Rancan, F.; Hadam, S.; Vogt, A.; Lademann, J.; Haucke, V.; Ruhl, E. Surface functionalization of silica nanoparticles supports colloidal stability in physiological media and facilitates internalization in cells. Langmuir 2012, 28, 7598−7613.

CONCLUSION In this work, influence of particle surface chemistry including the hydrophobic/hydrophilic property and lipid decorations on particle internalization into GUV was investigated based on the volume phase transitions of the temperature-sensitive pNIPAM particles. At room temperature below the LCST of pNIPAM, no adsorption happens between the plain pNIPAM particles and GUVs; however, a lipid decoration of the pNIPAM particle (i.e., for the lipogel) facilitates the adsorption of lipogel spheres to GUVs. Such an adsorption is supposed to be due to the presence of lipid coating layers, which somewhat shields the electrostatic repulsions between the pNIPAM particles and GUVs and increases the possibility of contact between them after which the adsorption happens with the fusion and exchange of lipid molecules. On the other hand, at an elevated temperature above the LCST, a further internalization into GUVs (although without dissociation) occurs for the lipogels, which is probably facilitated by the phase transition of pNIPAM polymers from hydrophilic to hydrophobic. Computer simulations were employed to confirm these assumptions. The present study adds a new depth to the classic researches on particle uptake mechanism, and provides a new pathway for the investigations on membrane processes such as endocytosis and drug delivery.

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ASSOCIATED CONTENT

S Supporting Information *

Additional data on the structural characterization of pNIPAM particles (e.g., optical micrograph, SEM image and zeta potential); molecular structure of pNIPAM with hydrated water at room temperature; fluorescence image of GUV; confocal and z-stack images of a model GUV with internalized lipogel after incubation for two hours at 37 °C; interaction between GUVs and native pNIPAM particles at 22 and 37 °C; selection of particle size in the simulations; detailed computer simulation method. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(B.Y.) Tel/Fax: 86 512 65220239. E-mail: yuanbing@suda. edu.cn. (K.Y.) Tel/Fax: 86 512 65220239. E-mail: yangkai@ suda.edu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Science Foundation of China (Nos. 91027040, 31061160496, 21106114, and 11104192, 21204058), the National Basic Research Program of China (No. 2012CB821500), and the Natural Science Foundation of Jiangsu Province of China (No. BK2012177). K. Yang thanks the support of the Key Project of Chinese Ministry of Education (No. 210208) and the Applied Basic Research Program (No. 2010CD091). The authors thank Prof. Zexin Zhang (Soochow University) for pNIPAM synthesis.

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REFERENCES

(1) Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. F

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dx.doi.org/10.1021/la4015652 | Langmuir XXXX, XXX, XXX−XXX