Letter pubs.acs.org/macroletters
Photo-PISA: Shedding Light on Polymerization-Induced SelfAssembly Jianbo Tan,*,† Hao Sun,‡ Mingguang Yu,§ Brent S. Sumerlin,*,‡ and Li Zhang*,† †
Department of Polymeric Materials and Engineering, School of Materials and Energy, Guangdong University of Technology, Guangzhou 510006, China ‡ George and Josephine Butler Polymer Research Laboratory, Center for Macromolecular Science and Engineering, Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, Florida, United States § Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, and Key Laboratory of Designed Synthesis and Application of Polymer Material, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China S Supporting Information *
ABSTRACT: Herein we report an aqueous photoinitiated polymerizationinduced self-assembly (photo-PISA) for the preparation of a remarkably diverse set of complex polymer nanoparticle morphologies (e.g., spheres, worms, and vesicles) at room temperature. Ultrafast polymerization rates were achieved, with near quantitative monomer conversion within 15 min of visible light irradiation. An important feature of the photo-PISA is that diblock copolymer vesicles can be prepared under mild conditions (room temperature, aqueous medium, visible light), which will be important for the preparation of functional vesicles loaded with biorelated species (e.g., proteins). As a proof of concept, silica nanoparticles and bovine serum albumin (BSA) were encapsulated in situ within vesicles via the photo-PISA process.
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a more facile approach to reproducibly synthesize pure phase nano-objects.13−15 Although research on aqueous PISA is growing rapidly, reports of the synthesis of biorelated or environmentally responsive nanomaterials by aqueous PISA is still limited. This may be attributed to the high reaction temperature (70 °C) required for most aqueous PISA formulations, since a thermal initiator is usually employed as the radical source. High reaction temperatures could lead to denaturation of globular proteins, antibodies, or enzymes and, also, the precipitation of most environmentally responsive polymers (e.g., CO2-responsive, thermoresponsive) in water. Efforts have been devoted to carrying out aqueous PISA at low temperatures. Jiang et al.20 demonstrated a photoinduced dispersion polymerization of diacetone acrylamide (DAAM) at room temperature in water, with no higher order morphologies (e.g., worms or vesicles) other than spheres being reported. Liu et al.21 reported an aqueous dispersion polymerization by using a redox initiator, and only spherical particles were obtained. Despite these successes, preparation of higher order morphologies via aqueous PISA at room temperature remains challenging. Very recently, Yeow et al.22 reported a visible light-mediated
lock copolymer nanomaterials have attracted much attention due to their broad applications in drug/gene delivery,1 imaging,2 biomineralization,3 nanoreactors,4 and catalysis.5 Self-assembly of block copolymers in a selective solvent is an attractive method to prepare polymer nanomaterials with different morphologies, including spheres, worms, vesicles, lamellae, sunflowers, and so on.6−12 However, traditional self-assembly methods are usually conducted at low polymer concentrations (15 wt % in this case). In addition to various morphologies (spheres, worms, and vesicles), some intermedi-
alcoholic PISA, and worm-like morphology was obtained, but in all cases, the rate of polymerization was low (only 40−70% conversion was achieved within 39 h irradiation). Furthermore, the use of organic solvents and heavy metal catalysts may limit application of this method in biological areas. Herein we report an example of aqueous photo-PISA of 2-hydroxypropyl methacrylate (HPMA) for the preparation of a remarkably diverse set of complex polymer nanoparticle morphologies (e.g., spheres, worms, and vesicles) at room temperature, as shown in Scheme 1. We believe that the photo-PISA offers considerable scope for the preparation of various biorelated or stimuli-responsive nanomaterials. Scheme 1. Scheme for Preparation of Various Nano-Objects via Aqueous Photo-PISA
The macro-CTA was synthesized by esterification of monomethoxy poly(ethylene glycol) (mPEG113) and 4-cyano-4(ethylthiocarbonothioylthio) pentanoic acid (CEPA). Figure S1 shows the 1H NMR spectra of mPEG113, CEPA, and mPEG113CEPA. The efficiency of the esterification can be calculated by the area ratio of the signal at 4.25 ppm and the signal at 2.66 ppm, indicating that about 96% of mPEG113 is converted into mPEG113-CEPA. Sodium phenyl-2,4,6-trimethylbenzoylphosphinate (SPTP) was chosen as the water-soluble photoinitiator, which rapidly decomposes under UV or visible light. Notably, visible light irradiation is often preferred to avoid potential decomposition of the macro-CTA (405 nm visible light used in the present case).23 The aqueous photo-PISA of HPMA was carried out at 25 °C with mPEG113-CEPA as a macro-CTA with a molar ratio between macro-CTA and photoinitiator being fixed at 3.0. High monomer conversions (>99%) were observed
Figure 1. (a) Kinetics of aqueous photo-PISA of HPMA at 25 °C using mPEG113-CEPA as the macro-CTA where the concentration of HPMA was 10 wt % and the target DP was 300; (b) Plots of Mn and Mw/Mn vs conversion according to the GPC data obtained for a subset of the kinetic data described above. 1250
DOI: 10.1021/acsmacrolett.5b00748 ACS Macro Lett. 2015, 4, 1249−1253
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ACS Macro Letters
Figure 2. Phase diagram for the aqueous photo-PISA of HPMA using mPEG113-CEPA as the macro-CTA. Phase regions consist of spheres (S), worms (W), lamellae (L), unilamellar vesicles (ULV), jellyfish (J), branched worms (BW), and multilamellar vesicles (MLV).
Figure 3. (a) TEM image of unpurified mPEG113-PHPMA360 vesicles prepared by aqueous photo-PISA of HPMA with the addition of 3.0 g silica sol (30% solids concentration) at the beginning; (b, c) TEM images of sample (a) purified by centrifugation at 15000 × g; (d−f) TEM images of sample (a) purified by centrifugation at 2000 × g.
ate “jellyfish” morphologies were also observed (e.g., target DP of 200 and 25 wt % HPMA, Figure S4f). GPC results (Figure S5) indicated well-defined diblock copolymers were obtained by aqueous photo-PISA using 10 or 20 wt % monomer concentrations. A significant increase in polydispersity was observed when targeting the DP of 400 due to the presence of dimethacrylate cross-linker in the monomer.15 It is noteworthy that small peaks at higher elution volume are observed in all cases, which can be attributed to the presence of a small amount of nonfunctional mPEG113. Diblock copolymer vesicles have broad applications in catalysis, drug delivery, and enzymatic reactions.1,5,30 It is desirable to develop a facile approach to encapsulate antibodies,
enzymes, or nanoparticles into vesicles with a high solids concentration. For conventional PISA, the high reaction temperature may restrict the encapsulation of proteins or thermally sensitive materials. The present photo-PISA is a facile and rapid approach to prepare diblock copolymer nano-objects with different morphologies under mild conditions (visible light, aqueous medium, and room temperature), and it serves as a promising strategy to prepare thermosensitive or biorelated vesicles via in situ encapsulation. As a proof of concept, we first employed silica nanoparticles as model encapsulated species to simplify characterization by TEM. The encapsulation process is straightforward and involves simply adding the commercial silica nanoparticles to the polymerization recipe to obtain silica1251
DOI: 10.1021/acsmacrolett.5b00748 ACS Macro Lett. 2015, 4, 1249−1253
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Figure 4. (a) Activity of (1) native BSA, (2) BSA in the supernatant of the product prepared by photo-PISA, (3) BSA of sample (2) with further incubation at 70 °C for 2 h (i.e., intentional denaturation). (b) TEM image of mPEG113-PHPMA360 vesicles prepared by photo-PISA in the presence of BSA. (c) Centrifuge tube containing sedimented mPEG113-PHPMA360 vesicles loaded with fluorescein-labeled BSA.
the protein. These results demonstrate that our photo-PISA strategy provides a very attractive platform for loading vesicles with antibodies or enzymes for biomedical applications under mild reaction conditions. Moreover, the present photo-PISA can also be a facile approach to prepare hybrid vesicles containing functional inorganic nanoparticles. In conclusion, we have expended the tremendous promise of RAFT aqueous dispersion polymerization by combining the traditional PISA process with photoinitiation. These polymerizations proceeded rapidly with exposure to visible light or sunlight, and complete monomer conversions could be achieved within 15 min of irradiation. Various mPEG113PHPMA nano-objects with complex morphologies were accessible by changing the reaction conditions, and a phase diagram was established. As a proof of concept, silica nanoparticles and a model protein were encapsulated into the mPEG−PHPMA vesicles via photo-PISA, with the latter demonstrating retention of activity during the polymerization process.
loaded vesicles. The presence of silica nanoparticles had no influence on the reaction, and complete monomer conversions were achieved within 30 min of irradiation. Figure 3a shows the TEM image of the obtained product, it can be clearly seen that a large number of silica nanoparticles are encapsulated in the vesicles, and some free silica nanoparticles are also bound to the surface of the vesicles. The free silica nanoparticles can be removed by careful centrifugation. When the as-synthesized product was purified by centrifugation at 15000 × g, some spherical microspheres with silica nanoparticles embedded inside were observed, as shown in Figure 3b,c. It seems that high centrifugation rates will collapse the structure of vesicles. When the as-synthesized product was purified by centrifugation at 2000 × g, the vesicles maintained their structures with almost no silica nanoparticles on the surface, as shown in Figure 3d−f. It is noteworthy that the membranes of vesicles were clearly observed, thus, it should not be the result of drying effect. To prove our ability to encapsulate biorelated species without losing their biological activity, we employed BSA as a model protein. The concentration of BSA in solution was measured by Bradford assay, while the activity of BSA was tested by hydrolysis of 4-nitrophenyl acetate. BSA in the supernatant demonstrated >90% activity as compared to native BSA (Figure 4a), suggesting that photo-PISA has no detrimental effect on the activity of BSA. As a comparison, the obtained product was also incubated at 70 °C for 2 h, a typical protocol for the synthesis of PHPMA-based vesicles via thermally initiated PISA. The activity of BSA decreased to around 35%, as compared to native BSA. Moreover, the product maintains its vesicular structure with the addition of BSA, as clearly shown in Figure 4b. To further prove the successful encapsulation of proteins by photo-PISA, we employed fluorescein-labeled BSA as a model protein, which fluoresces under 365 nm irradiation. It is noteworthy that the presence of fluorescein-labeled BSA disturbed the photo-PISA process under 405 nm irradiation and only a low monomer conversion was achieved. As an alternative approach, we exposed the reaction to sunlight, which resulted in complete monomer conversion within 30 min. After the third centrifugation−redispersion cycle, irradiation at 365 nm confirmed that the vesicle sediment was fluorescent, whereas the supernatant was nonfluorescent (see Figure 4c). A control experiment was also carried out by mixing the same amount of vesicles and fluorescein-labeled BSA for 30 min. After the third centrifugation−redispersion cycle, both the sediment and the supernatant were nonfluorescent (Figure S9), suggesting that the adsorption of proteins to the surface of vesicles is low and that the fluorescence observed after the photo-PISA process was the result of in situ encapsulation of
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.5b00748. Full experimental section, and further synthesis and characterization details for mPEG113-CEPA, mPEG113PHPMA using 1H NMR, GPC, and TEM; additional TEM images for various mPEG113-PHPMA nano-objects (PDF).
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]fl.edu. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are thankful for the support from the National Natural Science Foundation of China (Grants 21504017). J.T. thanks Prof. Zhaohua Zeng (Sun Yat-Sen University) for the great support of this work.
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