Biomimetic Polymersomes as Carriers for Hydrophilic Quantum Dots

ARTICLE pubs.acs.org/Langmuir

Biomimetic Polymersomes as Carriers for Hydrophilic Quantum Dots Gong-Yan Liu, Xiang-Sheng Liu, Shan-Shan Wang, Chao-Jian Chen, and Jian Ji* MOE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China

bS Supporting Information ABSTRACT: For polymersomes to achieve their potential as effective delivery vehicles, they must efficiently encapsulate therapeutic agents into either the aqueous interior or the hydrophobic membrane. In this study, cell membrane-mimetic polymersomes were prepared from amphiphilic poly(D,Llactide)-b-poly(2-methacryloyloxyethylphosphorylcholine) (PLA-b-PMPC) diblock copolymers and were used as encapsulation devices for water-soluble molecules. Thioalkylated zwitterionic phosphorylcholine protected quantum dots (PC@QDs) were chosen as hydrophilic model substrates and successfully encapsulated into the aqueous polymersome interior, as evidenced by transmission electron microscopy (TEM) and flow cytometry. In addition, we also found a fraction of the PC@QDs were bound to both the external and internal surfaces of the polymersome. This interesting immobilization might be due to the ion-pair interactions between the phosphorylcholine groups on the PC@QDs and polymersomes. The experimental encapsulation results support a mechanism of PLAb-PMPC polymersome formation in which PLA-b-PMPC copolymer chains first form spherical micelles, then worm-like micelles, and finally disk-like micelles which close up to form polymersomes.

’ INTRODUCTION Polymersomes, self-assembled vesicles of amphiphilic block copolymers, have attracted considerable attention due to their potential applications in biomedicine as drug and gene delivery carriers, artificial cells, and bioreactors.1 17 Of all applications, perhaps the utilization of the polymersome as delivery vehicle for various therapeutic agents is the most promising.5 Compared to polymeric micelles, polymersomes possess an aqueous interior separated from the outside by the hydrophobic membrane, which gives them advantages in carrying both hydrophobic and hydrophilic molecules.18 Nevertheless, polymersomes must efficiently encapsulate substrates into either the aqueous interior or the hydrophobic membrane to achieve their potential use as effective delivery vehicles. For this reason, there are many reports of incorporation of gold or quantum dot nanoparticles (NPs) into the polymersome membrane wall to investigate the encapsulation capacity.19 23 NPs have bigger size than fluorescent dyes or drugs allowing easier visualization of their localization under transmission electron microscopy, and they are suitable models to characterize the polymersome loading properties.19 However, the utilization of polymersome interior cavities for encapsulation of water-soluble naoparticles such as quantum dots (QDs) is limited. In consideration of potential pharmaceutical applications, the ability of encapsulating hydrophilic substrates is also an important issue for polymersomes. Recently, a biomimetic polymersome was developed by our group based on poly(D,L-lactide)-b-poly(2-methacryloyloxyethylphosphorylcholine) (PLA-b-PMPC) diblock copolymers.24 Phosphorylcholine (PC) is an interesting kind of zwitterionic molecular segment, which is present at the end of the lipid and on the external r 2011 American Chemical Society

surface of cell membranes. Surface modification with zwitterionic phosphorylcholine (PC) can prevent protein adsorption and blood clotting due to PC’s excellent biocompatibility and high hydrophilicity.25 30 Herein, thioalkylated zwitterionic phosphorylcholine protected quantum dots (PC@QDs) were chosen as hydrophilic model substrates to explore the encapsulation behavior of the biomimetic polymersome. Through visualizing their location in polymersomes by TEM, we proposed a possible ionpair interaction between the PC groups on the quantum dot nanoparticle and polymersome corona. Furthermore, the polymersome formation mechanism was confirmed based on the encapsulation results.

’ EXPERIMENTAL SECTION Materials. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was synthesized according to the literature.31 The PLA73-b-PMPC21 diblock copolymers used in this study with Mn = 11 800 and PDI = 1.18 were synthesized by atom transfer radical polymerization (ATRP) of the MPC monomer using PLA-Br as a macroinitiator. A detailed description of the synthesis can be found in a previous paper24 and in the Supporting Information for this paper. TOPO-coated CdSe/ZnS core/shell quantum dots were kindly donated by Dr. Zhu Huiguang from Rice University. Thioalkylated zwitterionic phosphorylcholine (HS-PC) was synthesized in our group as described before.25 FITC-labeled BSA (FITC-BSA) was prepared by reacting fluorescein isothiocyanate with bovine serum albumin at room temperature, and free FITC was removed Received: June 2, 2011 Revised: November 7, 2011 Published: November 10, 2011 557

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Figure 1. Amphiphilic PLA-b-PMPC diblock copolymer (a); schematic, fluorescent, and TEM images of the biomimetic polymersome (b, c, and d). Scale bar represents 10 μm in (c).

Fluorescence Emission Measurements. Fluorescence measurements were recorded from Perkin-Elmer LS 55 fluorescence spectrometer. The excitation wavelength was 488 nm, and the emission was monitored from 500 to 700 nm. Excitation and emission slit widths were both maintained at 5.0 nm, and spectra were accumulated with a scan speed of 200 nm/min. Fluorescence Microscopy. An Olympus IX71 inverted fluorescence microscope with a 100 oil objective was used to visualize blank or PC@QD-loaded PLA-b-PMPC polymersomes at room temperature. Fluorescent labeling was performed by simply adding a drop of Rhodamine 6G/water solution (0.2 mg mL 1) into the PLA-b-PMPC polymersome aqueous solution (1 mL) and vortexed for 30 s. Then the sample was directly observed by fluorescence microscopy equipped with a color video recorder. UV vis. UV vis spectra were carried out with a UV vis Shimadzu UV-2505 spectrometer using 1 cm path length quartz cuvette. Spectra were collected within a range of 400 800 nm. Flow Cytometry. Polymersomes with or without PC@QDs encapsulated were analyzed in a Coulter Epics Elite flow cytometer equipped with a 488 nm excitation laser.

by dialysis. All other reagents and solvents were purchased from domestic suppliers and used as received.

Synthesis of Water-Soluble HS-PC-Protected QDs (PC@QDs). 100 μL of 11-mercaptoundecylphosphorylcholine (HS-PC, 0.4M) in PBS solution (pH 7.4) was added to 1 mL of TOPO-coated QDs (2 μM) in chloroform, and the mixed solution was stirred for 2 h. Then another 900 μL of PBS was added, and a milk-like suspension was formed. The suspension was centrifuged at 12 000 rpm for 5 min. The color of the supernatant became bright orange, suggesting the water-soluble PC@QDs formed. Excess HS-PC was removed by centrifugation at 16 000 rpm for 15 min with added acetone. The PC@QDs have good stability and can be stored in PBS for more than 4 months.

Preparation of PLA-b-PMPC Polymersomes in Aqueous Solution. The polymersome was prepared by a solvent-injection method.32,33 Briefly, 50 mg of PLA-b-PMPC copolymers was first dissolved in a 1 mL mixture of methanol and chloroform (1:1, v/v). Then 50 μL of the obtained solution was injected into 10 mL of deionized water and stirred vigorously at room temperature for 2 h. Dialysis was performed against deionized water to remove methanol. Chloroform was completely removed by evaporation at 4 °C overnight. PLA-b-PMPC polymersome solutions were stored at 4 °C to minimize degradation.

Encapsulation of Hydrophilic PC@QDs or FITC-BSA into PLA-b-PMPC Polymersome Aqueous Interior. Hydrophilic

’ RESULTS AND DISCUSSION

PC@QDs were encapsulated during the polymersome formation. Briefly, 50 μL of PLA-b-PMPC organic solution was injected into a 10 mL PBS solution (pH 7.4) with 0.1 μM PC@QDs. The following steps were the same as the polymersome preparation. Unencapsulated PC@QDs were filtered to remove using amicon Ultra Free-MC centrifugal filters (0.1 μm filter). Hydrophilic FITC-BSA were loaded by injecting a 50 μL PLA-b-PMPC organic solution into a 10 mL PBS solution (pH 7.4) with 1 mg of FITC-BSA. Unencapsulated protein was removed by dialysis for 3 days. Dynamic Light Scattering (DLS). The intensity-average diameter and size distribution of aggregates were measured using a laser particle-size analyzing system (Brookhaven 90 plus, Brookhaven Instruments Corp.) at the scattering angle of 90°, and the wavelength was set as 658 nm during the whole experiment. Transmission Electron Mcroscopy (TEM). For TEM measurement, a drop of the polymersome solution was placed onto a carboncoated grid, washed with a negative stain solution (2% uranyl acetate solution), and blotted with filter paper. The specimens were observed with a transmission electron microscope (JEM 1230, JEOL).

Preparation and Characterization of Biomimetic PLA-bPMPC Polymersome. Recently, we reported on cell membrane-

mimetic polymersomes self-assembled from PLA-b-PMPC diblock copolymer (Figure 1a,b).24 As shown in Figure 1b, the blue color represents hydrophobic PLA membrane and the yellow color indicates hydrophilic PMPC shell, which comprises the external and internal surfaces of the polymersome. Polymersomes were prepared via an easy solvent-injection method which leads to polymersomes with large size and broad size distribution. The morphology and size of the polymersomes were characterized by scattering and microscopy techniques. Fluorescent and TEM images confirmed the hollow structure of the polymersomes with micrometer-size (Figure 1c,d). The average diameter of the polymersomes estimated from fluorescent images was 3.5 ( 1.6 μm. However, DLS results showed that there were two size fractions, suggesting micrometer-sized and nano-sized polymersomes both existed (Figure S2a). The nano-sized polymersome was further proved by TEM shown in Figure S2b. 558

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Figure 2. DLS result (a) and TEM images (b, c, and d) of PC@QD-encapsulated polymersomes.

Figure 3. Emission spectra of PC@QD, PC@QD-encapsulated polymersomes, and blank polymersome (a). Fluorescence image of PC@QDencapsulated polymersomes (b). Scale bar represents 10 μm.

To estimate the potential application of PLA-b-PMPC polymersomes as effective delivery vehicles, water-soluble quantum dots (PC@QDs) were selected as hydrophilic model substrates to be encapsulated into the polymersome. Since the size of the PC@QD was larger than most drugs and dyes, such nanoparticles might affect the size and morphology of the polymersome or even prevent their formation. DLS results shown in Figure 2a reveal that the size distribution of PC@QD-encapsulated polymersomes was similar to that of empty ones (Figure S1a), indicating the encapsulation did not disturb the vesicular structure. Figure 2b shows the TEM photographs of micrometer- and

Encapsulation of Water-Soluble PC@QDs into PLA-bPMPC Polymersomes. Novel water-soluble PC@QDs were

synthesized by protecting hydrophobic CdSe/ZnS quantum dots with thioalkylated phosphorylcholine (11-mercaptoundecylphosphorylcholine, HS-PC). The HS-PC ligand combines the stability of a strong QD-S linkage with the excellent water solubility of zwitterionic phosphorylcholine, as schematically illustrated in Figure S3. Figure S4 shows that the PC@QDs with sizes ∼5 nm were stabilized well by small molecules of thioalkylated zwitterions. The characteristic emission and absorbance peaks of PC@QD are shown in Figure S5. 559

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nano-sized polymersomes, which agreed with the DLS results. Figures 2c and 2d were magnified TEM images of the micrometer-sized and nano-sized polymersomes, respectively. A large number of PC@QDs can be seen as dark spots in these polymersomes, which demonstrated the successful encapsulation. In addition, the TEM image represented a different transmission in the interior compared to the polymersome wall, enabling the wall thickness to be clearly seen in Figure 2c. The successful encapsulation of water-soluble PC@QDs was further evidenced by fluorescence spectroscopy and microscopy. As shown in Figure 3a, blank polymersomes showed no characteristic emission signal at λmax = 600 nm originating from QD. However, by comparison with polymersome-free PC@QD, the emission peak at 600 nm of the polymersomePC@QD sample indicates successful incorporation of the quantum dots into the polymersome. The fluorescence image of the polymersome shows red fluorescence in the inner cavity, which demonstrated PC@QDs were loaded into the polymersome interior (Figure 3b). Flow cytometry is a powerful technique for fluorescenceactivated screening and sorting of cells. Recently, Nallani et al. have sorted catalytically active polymersome nanoreactors with ∼300 500 nm diameter by flow cytometry.34 Since the polymersome that we prepared has a micrometer size which is close to cells in diameter, the fluorescent PC@QD-loaded polymersomes can also be detected by flow cytometry. As shown in Figure 4a, blank polymersomes only displayed low background fluorescence intensity. However, we observed two populations of polymersomes with different fluorescence intensities after the

encapsulation of PC@QDs (Figure 4b). One population, with similar fluorescence intensity as blank polymersome, was believed to be empty polymersomes or polymersomes enclosing very few QDs with not enough fluorescence intensity to be detected by flow cytometry. The other population had very high fluorescence intensity and made up more than 80% of total polymersomes, which we assigned to polymersomes loaded to high capacity with QDs. Figure S6 displays the mean QD fluorescence intensity of the blank and PC@QD-loaded polymersomes. We can see that the fluorescence intensity of the PC@QD-loaded polymersomes was about 46 times that of empty polymersomes. These experiments indicated that PLA-b-PMPC polymersomes can encapsulate PC@QDs very effectively. Mechanism of PLA-b-PMPC Polymersome Formation. After encapsulation, it was interesting to find that a fraction of the PC@QDs was bound to both the external and internal surfaces of the polymersome, enabling the polymersome membrane wall to be clearly seen (Figure 5a). In general, phosphorylcholine (PC) groups on a flat surface prefer to have a balanced charge and an antiparallel orientation for electrostatic energy and dipole minimization.35 However, PC groups on different surfaces may have electrostatic interactions between them. In this study, the surfaces of the quantum dot and the polymersome both have PC groups. The interesting immobilization of PC@QDs on the curved polymersome membrane might due to the ion-pair interactions between the net positively charged quaternary ammonium and negatively charged phosphate groups on the QD nanoparticle and polymersome corona, as shown in Figure 5b. It should be stressed here that the surface-circling immobilization was also found on nano-sized polymersomes (Figure S7). To confirm the effect of the ion-pair interaction on surface immobilization, we chose another nonzwitterionic hydrophilic molecule (FITC-BSA) as an encapsulation substrate. After removal of unencapsulated protein by exhaustive dialysis, UV/vis spectroscopy revealed successful association of FITCBSA with the polymersomes (Figure S8A). Compared to blank polymersomes (Figure S2b), Figure S8B indicates the FITC-BSA has been loaded into the polymersome, which can be seen as dark spots in the interior. It is obvious that no protein was immobilized to either the outer or inner surfaces of the polymersome. It is well-known that proteins are polyampholytes as they carry both positive and negative charges on the surface. However,

Figure 4. Histogram of blank (a) and PC@QD-loaded (b) polymersomes.

Figure 5. Immobilization of PC@QDs. (a) TEM image showed a fraction of the PC@QDs were located on the external and internal interfaces of the polymersome around the membrane wall. (b) The proposed ion-pair interactions between PC groups on the QD and polymersome corona. 560

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Scheme 1. Formation Mechanism of PLA-b-PMPC Polymersome

FITC-BSA was evidently unable to form ion-pair interactions with PMPC segments on the polymersome surface. In fact, protein resistance is an important property of zwitterionic PC molecules. This result indirectly proved that such ion-pair interactions may only happen between PC groups on different surfaces. There are two different proposed mechanisms of polymersome formation.36 38 In mechanism 1, amphiphilic copolymers first self-assemble into spherical micelles, then worm-like micelles, and finally disk-like micelles which subsequently close up to form polymersomes.39 41 In mechanism 2, small spherical micelles are formed rapidly and then grow up to large micelles by a phase-separation process, which then reorganize into polymersomes.42,43 The formation mechanism is expected to affect the efficiency of encapsulation of water-soluble molecules within the polymersomes. Mechanism 1 involves a closing process and allows trapping water-soluble molecules within the interior during the final polymersome formation step. On the contrary, mechanism 2 precludes encapsulation during polymersome formation, leading to very low loading efficiency. Our experimental encapsulation results showed that PC@QDs can be loaded into PLA-b-PMPC polymersome effectively. Thus, the PLA-b-PMPC polymersomes are believed to form by mechanism 1 and the immobilization of PC@QDs on the curved polymersome surfaces might assist in this process, as shown in Scheme 1. When amphiphilic PLA-b-PMPC diblock copolymer was injected into aqueous PC@QDs solution, the copolymers will aggregate into spherical micelles, then worm-like micelles, and then disk-like micelles with some PC@QDs adsorbed on the micelle surface by the ion-pair interactions. Then the disk-like or bilayer-like micelles close up and encapsulate surrounding PC@QDs within. These results indicate that biomimetic PLAb-PMPC polymersomes can be used as carriers for hydrophilic molecules.

which the PC@QDs adsorb to the PLA-b-PMPC at the micellar stage, prior to polymersome formation.

’ ASSOCIATED CONTENT

bS

Supporting Information. Detailed information on PLAb-PMPC polymersome and PC@QDs. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]; Ph: +86 571 8795 3729.

’ ACKNOWLEDGMENT Financial support from the NSFC-50830106, National Science Fund for Distinguished Young Scholars (51025312), The National Basic Research Program of China (2011CB606203), the Fundamental Research Funds for the Central Universities (2009QNA4039), and Open Project of State Key Laboratory of Supramolecular Structure and Materials (SKLSSM201103) is gratefully acknowledged. We also thank Dr. Zhang Yuying for valuable suggestions for flow cytometry measurements. ’ REFERENCES (1) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967–973. (2) Jain, S.; Bates, F. S. Science 2003, 300, 460–464. (3) Binder, W. H.; Barragan, V.; Menger, F. M. Angew. Chem., Int. Ed. 2003, 42, 5802–5827. (4) Du, J. Z.; Chen, Y. M.; Zhang, Y. H.; Han, C. C.; Fischer, K.; Schmidt, M. J. Am. Chem. Soc. 2003, 125, 14710–14711. (5) Christian, D. A.; Cai, S.; Bowen, D. M.; Kim, Y.; Pajerowski, Discher, D. E. Eur. J. Pharm. Biopharm. 2009, 71, 463–474. (6) Lomas, H.; Canton, I.; MacNeil, S.; Du, J. Z.; Armes, S. P.; Ryan, A. J.; Lewis, A. L.; Battaglia, G. Adv. Mater. 2007, 19, 4238–4243. (7) Blanazs, A.; Armes, S. P.; Ryan, A. J. Macromol. Rapid Commun. 2009, 30, 267–277. (8) Du, J. Z.; Tang, Y. Q.; Lewis, A. L.; Armes, S. P. J. Am. Chem. Soc. 2005, 127, 17982–17983. (9) Koide, A.; Kishimura, A.; Osada, K.; Jang, W.-D.; Yamasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2006, 128, 5988–5989. (10) Meng, F. H.; Zhong, Z. Y.; Feijen, J. Biomacromolecules 2009, 10, 197–209. (11) Zhou, Y.; Yan, D. Angew. Chem., Int. Ed. 2005, 44, 3223–3226. (12) Zhou, Y.; Yan, D. J. Am. Chem. Soc. 2005, 127, 10468–10469. (13) Menger, F. M.; Seredyuk, V. A.; Yaroslavov, A. Angew. Chem., Int. Ed. 2002, 41, 1350–1352. (14) Zhou, Y.; Yan, D. Angew. Chem., Int. Ed. 2004, 43, 4896–4899.

’ CONCLUSIONS Biomimetic PLA-b-PMPC polymersomes were developed to exploit their potential use as carriers for hydrophilic molecules. Hydrophilic PC@QDs were selected as water-soluble substrates and successfully encapsulated into the polymersome. TEM imaging revealed that most of the PC@QDs were located in the interior of the polymersome with some PC@QDs adsorbed around polymersome membrane. We hypothesized the surfacecircling immobilization was due to the ion-pair interactions between the phosphorylcholine groups on the quantum dot nanoparticle and polymersome corona. The encapsulation results are consistent with a polymersome formation mechanism in 561

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