有机化学英文文献6

CHEN ET AL. VOL. 5 ’ NO. 4 ’ 2671–2680 ’ 2011 www.acsnano.org 2671 March 04, 2011 C 2011 American Chemical Society Highly Compressed Assembly of Deformable Nanogels into Nanoscale Suprastructures and Their Application in Nanomedicine Huabing Chen,†,‡, ) Hongda Zhu,†,‡, ) Jingdong Hu,†,‡ Yanbing Zhao,†,‡ Qin Wang,§ Jiangling Wan,†,‡ Yajiang Yang,§ Huibi Xu,†,‡ and Xiangliang Yang†,‡,^,* †College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China, ‡National Engineering Research Center for Nanomedicine, Wuhan 430074, China, and §School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China ) These authors contributed equally to this work. ^ Present address: College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. INTRODUCTION Pickering emulsion is an emulsion that is stabilized by solid particles residing be- tween an oily phase and aqueous phase. The assembly of the nanoparticles such as silica, microgel, Au, and polystyrene na- noparticles into microscopic suprastruc- tures at oil-in-water (O/W) interfaces for stabilizing Pickering emulsions has been studied as an intriguing focus in field of chemical industry and material sciences.1-4 Because of the preferential residing of nanoparticles at O/W interfaces, the micro- scopic suprastructures consisting of nano- particles provide significant advantages over amphiphilic molecules or copolymers with high drug loading, strong kinetic hin- drance to droplet-droplet coalescence, tunable interfacial permeability, enhanced controlled release of therapeutic molecules, and so on.4-8 It is highly valuable to explore the applicable potential of the supra- structures with these advantages in nano- medicine. However, themicroscopic supras- tructures consisting of these nanoparticles can only induce the formation and stabiliza- tion of microscopic emulsion droplets. It is difficult to fabricate nanoscale suprastruc- tures for stabilizing nanoscale emulsion dro- plets using these nanoparticles, because the stronger interfacial hindrance is required to resist the droplet-droplet coalescence and the relatively higher interfacial curva- ture of nanodroplets can induce the spa- tially confined interface upon the formation of nanoscale emulsions.9 Especially, the con- fined nanoscopic space at the O/W interface of nanodroplets does not allow nanoparti- cles to perfectly arrange at the O/W inter- face. Consequently, the intact assembly of nanoparticles into nanoscale suprastruc- tures at the spatially confined space of O/W interfaces becomes the major chal- lenge in the development of nanodroplets for nanomedicine. Recently, ideas about the assembly of flex- ible DNA chains and porous nanoparticles into nanoarchitectures imply an intriguing strategy to fabricate the nanoarchitectures by usingdeformable or soft buildingblocks.10-14 * Address correspondence to yangxl@mail.hust.edu.cn. Received for review October 26, 2010 and accepted March 4, 2011. Published online 10.1021/nn102888c ABSTRACT Assembly of nanoparticles as interfacial stabilizers at oil-in-water (O/W) interfaces into microscopic suprastructures for stabilizing Pickering emulsions is an intriguing focus in the fields of chemical industry and material sciences. However, it is still a major challenge to assemble nanoscale suprastructures using nanoparticles as building blocks at O/W interfaces for fabricating nanoscale emulsion droplets with applicable potential in nanomedicine. Here, we show that it is possible to fabricate the nanodroplets by assembling highly deformable nanogels into the nanoscale suprastructures at spatially confined O/W interfaces. The compressed assembly of the nanogels induced the formation of the nanoscale suprastructures upon energy input at the nanoscale O/W interface. The hydrogen bonding interaction between the nanogels at the O/W interface are possibly responsible for the stabilization of the nanoscale suprastructures. The nanoscale suprastructures are further employed to stabilize the paclitaxel-loaded nanodroplets, which are found to provide sustained release of the drug, enhanced in vitro cytotoxicity, and prolonged in vivo blood circulation. Furthermore, the tissue distribution and antitumor efficacy studies show that the nanodroplets could induce a higher drug accumulation at the tumor site and enhance tumor growth inhibition when compared with the commercial product. This approach provides a novel universal strategy to fabricate nanoscale suprastructures for stabilizing nanodroplets with built-in payloads using deformable nanoparticles and displays a promising potential in nanomedicine. KEYWORDS: nanogels . nanoscale suprastructures . nanodroplets . paclitaxel . nanomedicine A RTIC LE CHEN ET AL. VOL. 5 ’ NO. 4 ’ 2671–2680 ’ 2011 www.acsnano.org 2672 The spherical poly(N-isopropylacrylamide) (PNIPAM)- based nanogels were found to have significant de- formability of swelling-shrinkage in response to an external stimulus (e.g., temperature) in our previous studies,15,16 and displayed different physicochemical properties from those of the solid nanoparticles.7,17 The shrinkage capacity of the nanogels is expected to provide a possibility of achieving the compressed assembly of nanogels into the nanoscale suprastruc- tures at O/W interfaces. Here, we synthesized poly(N- isopropylacrylamide-co-allylamine) (PNIPAM-co-AA) na- nogels, where N-isopropylacrylamide was used as the scaffold of network-like nanogels with deformability.15,16 Allylamine was copolymerized to improve the hydro- philicity of PNIPAM-based nanogels at 37 �C, because PNIPAM can display rapid dehydration above 32 �C.18 The nanogels could first form microscopic suprastruc- tures for stabilizing Pickering emulsions at O/W inter- faces and further be compressed into nanoscale suprastructures upon energy input (e.g., ultrasonication) (Figure 1). The nanoscale suprastructures were further used to stabilize the nanodroplets with active mol- ecules in the oily phase, which were further evaluated as the nanocarrier for cancer therapy. RESULTS AND DISCUSSION The PNIPAM-co-AA nanogels were synthesized using N-isopropylacrylamide and allylamine at a ratio of 7:1 (Supporting Information). The PNIPAM-co-AA nanogels with positive zeta potentials in aqueous solution displayed a significant size change from 188.4 nm at 28 �C to 67.5 nm at 42 �C (Figure S1a-c). The volume of the nanogels at 42 �C is only about 4.6% of that at 28 �C, which implies that the nanogels have significant shrinkage capability. The lyophilized PNIPAM-co-AA nanogels at the shrunken state were further found to have an average diameter of 48.4 nm using a field small-angle X-ray scattering system. TEM imaging also validated that the nanogels had a spherical morphology and were highly shrunken at 37 �C (Figure S1d and S1e). It reveals that the nanogels have a highly deformable ability via hydration or dehydration, which allows the nanogels to significantly swell or shrink in aqueous solution.19 The shrinkage ability is expected to provide the nanogels with an ability to perfectly arrange at O/W interfaces in a spatially confined space. To demonstrate the preferential distribution of the PNIPAM-co-AA nanogels at O/W interfaces, FITC as a fluorescent dye was conjugated to PNIPAM-co-AA nanogels via amide, which were first used to stabilize the microscopic suprastructures for stabilizing the Pickering emulsions droplets.20,21 The hydrophobic organic solvents such as isopropyl myristate (IPM) and hexane could act as the oily phase of Pickering emulsions. FITC-labeled PNIPAM-co-AA nanogels were used to stabilize pharmaceutically acceptable IPM droplets. Fluorescent imaging showed that a yellow- green color existed around the droplets and was possibly ascribed to the FITC-labeled PNIPAM-co-AA nanogels around microscopic emulsion droplets (Figure 2a). Nile red (1.0 μg/mL) as a red hydrophobic fluorescent dye was encapsulated into oily droplets of the Pickering emulsions stabilized by the microscopic suprastructures of PNIPAM-co-AA nanogels. The red fluorescence from nile red validated the presence of spherical oily droplets inside the Pickering emulsions (Figure 2b). Furthermore, FITC-labeled PNIPAM-co-AA na- nogels were used to stabilize the oily droplets containing Figure 1. Schematic illustration of the highly compressed assembly of nanogels into the nanoscale suprastructures at O/W interfaces which are used to stabilize the nanodroplets with active molecules for nanomedicine. A RTIC LE CHEN ET AL. VOL. 5 ’ NO. 4 ’ 2671–2680 ’ 2011 www.acsnano.org 2673 nile red for differentiating the interfacial microscopic suprastructures and interior oily droplets (Figure 2c). The red fluorescence from nile red was observed in the interior of droplets, and simultaneously a yellow-green color from FITC-labeled PNIPAM-co-AA nanogels coex- isted surrounding the droplets. It indicated that the microscopic suprastructures consisting of the nanogels located at O/W interfaces of the Pickering emulsions and could be differentiated from inner oily droplets. TEM imaging was further used to validate the droplet morphology of the microscopic Pickering emulsions.22,23 Figure 2e and Figure S2a showed that the Pickering emulsions displayed a capsule-like structure, which implied that the microscopic suprastructures might surround the oily droplets and matched well with their fluorescent imaging and optical imaging (Figure 2d). But the microscopic Pickering emulsions had an aver- age droplet size of 1.5 μm and a broad size distribution Figure 2. Fluorescent and TEM imaging of the Pickering emulsions and nanodroplets. (a) Fluorescent images of the Pickering emulsions stabilized by the microscopic suprastructures of FITC-labeled PNIPAM-co-AA nanogels. (b) Fluorescent image of the Pickering emulsions containing nile red, stabilized by the microscopic suprastructures of PNIPAM-co-AA nanogels. (c) Fluorescent image of the Pickering emulsions containing nile red, stabilized by the microscopic suprastructures of FITC- labeled PNIPAM-co-AA nanogels. (d) Optical image of the Pickering emulsion stabilized by themicroscopic suprastructures of FITC-labeled PNIPAM-co-AA nanogels. (e) TEM images of the Pickering emulsions stabilized by the microscopic supras- tructures of PNIPAM-co-AA nanogels (dashed circle indicates the capsule-like morphology). (f) TEM images of the nanodroplets stabilizedby the nanoscale suprastructures of PNIPAM-co-AA nanogels (dashed circle indicates the capsule-like morphology). A RTIC LE CHEN ET AL. VOL. 5 ’ NO. 4 ’ 2671–2680 ’ 2011 www.acsnano.org 2674 (polydispersive index is 0.396) (Figure S2b). Figure 2e also showed that the microscopic suprastructures around Pickering emulsion droplets had a thickness of 150-200 nm, which is comparable to the particle size of the nanogels at the swollen status in aqueous solution as shown in Figure S1a. In addition, the Pick- ering emulsions with a microscopic droplet size dis- played a significant temperature-responsive size change (Figure S2c), which should be attributed to the tem- perature-responsive nanogels at O/W interfaces. Further- more, the Pickering emulsions also avoided the phase separation of the emulsions at 37 �C due to the presence of the amino group in the nanogels.7 Generally, the PNIPAM-based polymers can be dehydrated and dis- play phase separation when the temperature is above 32 �C. The amine group from allylamine could prevent the nanogels from precipitation because of the en- hancement of their hydrophilicity. It implies that the nanogels could display a state of dehydration at 37 �C and maintained amphiphilic properties at O/W inter- faces as well, which was very important for their stability. Then, the nanogels can form the microscopic suprastructures as shown in Figure 1 and maintain their original size at O/W interfaces of the Pickering emulsions at room temperature and also afford the steric hindrance to droplet-droplet coalescence for the stabilization of Pickering emulsions.4 Interestingly, we found the presence of the nano- scale suprastructures at O/W interfaces when we further disintegrated the above emulsions into nanoscale emul- sion droplets using ultrasonication (Figure 1). Figure 2f showed the TEM images of the nanodroplets stabilized by nanoscale suprastructures of PNIPAM-co-AA nano- gels. The nanodroplets showed a capsule-likemorphol- ogy that was similar to that of the above emulsions. But their droplet size was significantly decreased to about 137.0 nm and the thickness of nanoscale suprastruc- tures only ranged from 30 to 60 nm (Figure 2f). This thickness wasmuch lower than that of themicroscopic suprastructures of the Pickering emulsions, and it was similar to the particle size of the shrunk nanogels (Figure S1a and S1e). The FITC-labeled PNIPAM-co-AA nanogels were used to construct fluorescent nanoscale suprastructures to stabilize the nanodroplets, and the fluorescence imaging (Figure S3) showed that the fluorescent morphology of nanodroplets was signifi- cantly different with that of Pickering emulsions stabi- lized by the microscopic suprastructures in Figure 2a because of their small diameters. In addition, the nano- droplets had a narrow size distribution (Figure S2d) and only showed a slight change of droplet size when the temperature was increased. The PNIPAM-co-AA nano- gels at O/W interfaces might be significantly shrunken with the simultaneous expulsion of water from the interior gel network when the emulsion droplets were disintegrated by ultrasonication, even though no other stimulus (e.g., temperature) was applied. The ultraso- nicationmight trigger the dehydration of the nanogels and subsequently induce the transformation of the microscopic suprastructures into nanoscale suprastruc- tures when the PNIPAM-co-AA nanogels were forced to rearrange at O/W interfaces of the nanodroplets. To validate the presence of the nanoscale supra- structures, we employed the nanodroplets as a tem- plate to encapsulate inorganic superparamagnetic iron oxide nanoparticles (SPIO) clusters using evaporable hexane containing hydrophobic SPIO as an oily phase andthenanoscale suprastructuresas stabilizers (Supporting Information, Figure S4a and S4b).3,24,25 Here, SPIOwere used to differentiate the hydrophobic oily phase and nanoscale suprastructures because of their strong TEM imaging contrast (Figure S4a). The hydrophobic SPIO are expected to reveal the oily microstructure within the nanodroplets. TEM imaging in Figure 3a showed that tens of hydrophobic SPIO aggregated into the clusters with the diameters ranging from 50 to 100 nm, which were surrounded by the nanoscale suprastruc- tures with a thickness of about 50 nm. Themorphology and size (average diameter of 149.2 nm from DLS) of these nanoscale suprastructures were similar to those of the above nanodroplets. The SPIO clusters had a high relaxivity of 121.3 mM-1 s-1 (Figure S4c) and also Figure 3. (a) TEM images of SPIO clusters stabilized by the nanoscale suprastructures (insert, same sample at various magnifications). (b) Schematic illustration of SPIO clusters stabilized by the nanoscale suprastructures of the PNIPAM-co-AA nanogels. A RTIC LE CHEN ET AL. VOL. 5 ’ NO. 4 ’ 2671–2680 ’ 2011 www.acsnano.org 2675 showed no significant change of particle size during 30 days (data is not shown), which implied that the nano- scale suprastructures exhibited good encapsulation and stabilization for hydrophibic SPIO clusters.24,25 The formation and stabilization of SPIO clusters vali- dated the presence of the nanoscale suprastructures at the O/W interface of nanodroplets as proposed in Figure 3b. The compressed assembly of the nanogels into the nanoscale suprastructures during ultrasonication of the Pickering emulsions possibly attributed to the shrinkage of nanogels at O/W interfaces. According to the Young-Laplace equation for the spherical oil droplets in an O/W emulsion,26 ΔP ¼ 2γ r (where ΔP is the pressure difference across the O/W interface, γ is the interfacial tension of the oil droplet, and r is the radius of the oil droplet). The proposed formation mechanism of the nanoscale suprastructures of PNIPAM-co-AA nanogels can be described as fol- lows: Firstly, for the microscopic Pickering emulsions without a strong energy input (ΔP is kept constant), the thickness of the microscopic suprastructures of PNI- PAM-co-AA nanogels at O/W interfaces was equal to the particle size of the nanogels at the swelling state. Then, the nanogels could form the interfacial layers at O/W interfaces for decreasing the interfacial tension at O/W interfaces. When the Pickering emulsions were disintegrated into the nanodroplets by ultrasonica- tion, ΔP was increased by the input of ultrasonication energy, and the value of γ could not be decreased since the nanogels had located at O/W interfaces in the Pickering emulsions. So, the radius of the emulsion droplets have to be further decreased according to the equation. Consequently, the packing density or vo- lume of nanogels adsorbed per unit area at O/W interfaces was significantly increased with the de- crease in droplet size. The increase of their density or volume in the spatially confined space can trigger the squeeze and dehydation of the nanogels around dro- plets, because the input of power could overcome the osmotic pressure of the nanogels in the aqueous side of O/W interfaces and then lead to the release of water from the nanogels. Finally, the squeeze of the nanogels induced the formation of the nanoscale suprastruc- tures and also allowed the nanogels to perfectly arrange around nanodroplets with relatively high in- terfacial curvature. In order to explore the stabilization mechanism of the nanoscale suprastructures, we probed the pre- sence of hydrogen bonding interaction between the nanogels at O/W interfaces using urea, which was used to break hydrogen bonds existing between the nano- gels (Figure 4a and 4b).18,27,28 Urea was expected to penetrate into the nanoscale suprastructures from the aqueous phase, effectively interact with the nanogels via hydrogen bonds, and break the existing hydrogen bonds between the nanogels.27 The nanodroplets with- out urea only had a slight increase of droplet size during 30 days, but the addition of urea resulted in the quick coarsening of droplets after storage for 4 days and phase separation after 7 days at 25 or 37 �C (Figure 4a Figure 4. Validation of hydrogen bonds between the nanogels in the nanoscale suprastructures at O/W interfaces. (a) Influence of urea (4.0mol/L) on the stability of the nanodroplets stabilizedby the nanoscale suprastructures of PNIPAM-co-AA nanogels at 25 �C. (b) Proposed interaction mechanism of urea with side chains of the nanogels in the nanoscale suprastructures at O/W interfaces. A RTIC LE CHEN ET AL. VOL. 5 ’ NO. 4 ’ 2671–2680 ’ 2011 www.acsnano.org 2676 and Figure S5). It shows that the hydrogen bond interaction is possibly one of the most important mechanisms for the stability of the nanoscale supra- structures. Additionally, we also found that the micro- scopic suprastructures formation of the Pickering emulsions hardly relied on the interaction of hydrogen bonds between the nanogels, but the nanoscale su- prastructures for stabilizing the nanodroplets ob- viously depended on this interaction (Supporting Information). This interaction possibly induced the interlock of the nanogel network, which is advanta- geous to the perfect surface coverage of droplets with high interfacial curvature for avoiding droplet-droplet coalescence and the dissociation of the nanogels from the nanoscale suprastructures.29,30 The nanodroplets contain hydrophobic oily droplets stabilized by the nanoscale suprastructures, which possi- bly act as a reservoir for drug delivery in nanomedic