Efficient Way to Generate Protein-Based ... - ACS Publications

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Efficient Way to Generate Protein-Based Nanoparticles by in-Situ Photoinitiated Polymerization-Induced Self-Assembly Chao Ma, Xiaoman Liu,* Guangyu Wu, Pei Zhou, Yuting Zhou, Lei Wang, and Xin Huang* MIIT Key Laboratory of Critical Materials Technology for New Energy Conversion and Storage, State Key Laboratory of Urban Water Resource and Environment, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China S Supporting Information *

ABSTRACT: Protein-based nanoparticles with tailored properties by using different functional proteins as building blocks have many actual and potential applications in biomedicine, biotechnology, and nanotechnology. In this study, we demonstrated a facile and efficient way to synthesize protein-based nanoparticles by taking advantage of photoinitiated reversible addition−fragmentation chain transfer (RAFT) polymerization-induced self-assembly by using multi-RAFT modified bovine serum albumin (BSA) as a macro-RAFT agent. The growth of the PHPMA chains results in the increase of the hydrophobicity of the star BSA−PHPMA conjugates, and when reaching the critical aggregation concentration in aqueous solution, they will aggregate into nanoparticles via the hydrophobic interaction of PHPMA. The generated nanoparticles also showed excellent encapsulation ability toward both hydrophobic and hydrophilic components, and as a proof of concept, after loading cancer drug DOX or biomacromolecule DNA, the protease-mediated release of the encapsulants was demonstrated. It is anticipated that the described method may open up new opportunities for designing a variety of protein−polymer self-assembled nanostructures tailored to specific applications.

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rotein-based self-assembled nano-objects have attracted much attention due to their great biocompatibility and degradability as well as highly sophisticated functions which have showed broad applications in drug/gene delivery, protein therapy, nanoreactors, or artificial cells, etc.1−6 In the last few decades, various strategies to engineer self-assembled protein nanostructures were well demonstrated by using different driving forces including electrostatic interactions, metal−ligand interactions, molecular recognition, and protein−protein interactions etc.1,3,7 Among these studies, one typical way developed by polymer chemist was focusing on the direct selfassembly from the giant amphiphilic protein−polymer conjugates.8,9 Various hydrophobic polymers like polystyrene or poly(N-isopropylacrylamide) were conjugated to hydrophilic proteins, by covalent bonding between amino acid residues of the proteins and the functionalized end group of polymers or noncovalent interactions of enzyme−cofactor recognition like hemin−horseradish peroxidase and bioaffinity coupling like biotin−streptavidin,10 to form amphiphilic protein−polymer conjugates, which were capable of generating spheres, worms, and vesicle morphologies in aqueous solutions or emulsions.11−19 For example, by taking advantage of the BSA− poly(N-isopropylacrylamide) conjugate as a building block, a hollow microcapsule could be well generated based on the Pickering emulsion strategy which showed a promising application toward synthetic ensembles capable of genedirected protein synthesis, membrane-mediated tandem catalysis, enzyme-mediated membrane fusion, and predatory behavior etc.20−23 © XXXX American Chemical Society

In this regard, very recently, a new technique by utilizing the living polymerization of a solvophobic polymer to drive selfassembly in situ called polymerization-induced self-assembly (PISA) was widely established. A wide range of polymer nanoobjects can be produced efficiently without the need for the synthesis and purification of preformed amphiphilic block copolymers. Extensive pioneered studies have been investigated by Armes, Pan, and others24−47 based on reversible addition− fragmentation chain transfer (RAFT) dispersion polymerization by using a variety of monomer and solvent systems. Although research on aqueous PISA is growing rapidly, reports on the synthesis of biorelated or environmentally responsive nanomaterials by using biomacromolecules as starting building blocks via aqueous PISA are still very limited. Especially considering the biodegradability, immunogenicity, stability, and toxicity, protein should be a more attractive material for the formation of nano-objects compared with that of synthetic polymers toward the potential biomedicine and biological materials application.48 Of particular relevance, rare reports state that using site-specific modification of human serum albumin (HSA) as an atom transfer radical polymerization (ATRP) macroinitiator, followed by in situ ATRP of a water-soluble monomer, yields HSA conjugates of which a hydrophobic polymer resulted in self-assembling into HSA-based nanostrucReceived: June 7, 2017 Accepted: June 15, 2017

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DOI: 10.1021/acsmacrolett.7b00422 ACS Macro Lett. 2017, 6, 689−694

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ACS Macro Letters tures with tunable morphologies from spheres to worms and then to vesicles.49 Despite this study, the further investigation on biomacromolecule-based PISA behaviors still needs our great effort to answer questions, such as, rather than the sitespecific protein initiator, how about the PISA behaviors by using a star multisites protein initiator. Inspired by recent advancements in photoinduced electron transfer−reversible addition−fragmentation chain-transfer polymerization (PET-RAFT),44−46,50−54 the mild and friendly reaction condition with high polymerization efficiency provided a suitable technique to conduct the biomacromolecule-based polymerization process;55,56 herein, we demonstrated a PISA process by taking advantage of the photoinduced RAFT technique and bovine serum albumin (BSA) as a model protein (Scheme 1). Differing from the reported PISA behavior Scheme 1. Schematic Illustration of the Synthesis of BSA Macro-CTAs and in-Situ Photoinitiated PolymerizationInduced Self-Assembly of BSA-Based Nanoparticles

Figure 1. Characterization of BSA−CTAs by (a) MALDI-TOF mass spectra of native BSA (black line) and BSA−CTA8 (red line), (b) DLS of native BSA and BSA−CTA8, and (c) SDS-PAGE of BSA−CTA8: lane 1, native BSA: lane 2, and protein marker: lane 3.

by using site-specific modified protein, a multisite modified BSA with trithiol RAFT agent was synthesized as a star macroCTA (BSA−CTA), and 2-hydroxypropyl methacrylate (HPMA) was used as a polymerized monomer. Upon polymerizing, the spherical nanoparticles with size ranging from 164 to 255 nm were generated throughout the whole process by using the star macro-CTA. Significantly, the generated protein-based nanoparticles showed excellent stability against diluting and centrifuging, as well as strong encapsulation ability toward both hydrophobic and hydrophilic components. Therefore, as a potential drug delivery carrier, as a proof of concept, a protease-triggered release of the loaded components was demonstrated. The synthesis of BSA−CTAs was conducted in the solvent of water/DMSO mixture (9/1, v/v) at room temperature. BSA which is similar to human serum albumin (HSA) by 76% in space structure and chemistry composition is chosen as a model protein since serum albumin is not only ubiquitous in living organisms but also relatively inexpensive and easily available, as well as widely applied for surgery, burns, hemodialysis, and drug delivery.57−59 We employed the primary amino group (−NH2) on the external surface of BSA for multisite modification with a mercaptothiazoline-activated trithiolRAFT agent (Figures S1 and 2) which was synthesized according to our previously reported procedure.21 The conjugation of the trithiol RAFT agents onto BSA was confirmed by MALDI-TOF MS (Figure 1a). The molecular weights of native BSA and BSA−CTAs were determined to be 65 500 and 67 700 Da, respectively, which indicated that the trithiol-RAFT agent was successfully attached to BSA, and the molar ratio of the BSA:trithiol-RAFT agent was calculated to be 1:8 in one BSA−CTAs conjugate (BSA−CTA8). Also, from the DLS study, there was an obvious hydrodynamic diameter increase for the BSA−CTA8 compared with that of native BSA

(Figure 1b). After freeze-drying, the native BSA and BSA− CTA8 were analyzed by SDS-PAGE (Figure 1c), from which a band with higher molecular weight than that of native BSA was observed, indicating the successful conjugating RAFT agents onto BSA as well. In addition, by monitoring the chromophore corresponding to the RAFT at 320 nm, the number of conjugated RAFT agents per BSA could also be calculated, which gave a consistent conclusion with that of MALDI-TOF MS (Figures S3 and 4 and Table S1). Similarly, by varying the molar ratio of the used BSA and RAFT agent, BSA−CTA5 (5 conjugated RAFT agents per BSA in average) was also synthesized (Figures S5 and 6 and Table S1). The BSA-based PISA process was studied by using 2hydroxypropyl methacrylate (HPMA) as a polymerized monomer and the water-soluble ruthenium complex Ru(bpy)3Cl2 as a photoredox catalyst. The ability and mechanism of the employed photoinitiated RAFT polymerization to mediate polymerizations with good control over the molecular weight and molecular weight distributions have been widely investigated by Xu and Boyer et al.50 We performed the photoinitiated RAFT polymerization reaction with the molar ratio of [HPMA]:[BSA−CTA 8 ]:[Ru(bpy) 3 Cl 2 ·6H 2 O] = 2350:8:0.27, under 5% (w/w) content of HPMA in the system at room temperature using a blue LED light (460 nm, 3 mW/ cm2). The change of the turbility of the solutions from transparent to opaque (Figure S7) indicated the growth of hydrophobic PHPMA from BSA. This was further confirmed by directly analyzing the solution with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure S8), and new bands with higher molecular weights than that of BSA 690

DOI: 10.1021/acsmacrolett.7b00422 ACS Macro Lett. 2017, 6, 689−694

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ACS Macro Letters

In contrast to the widely reported micelle-like nanoparticles generated based on the self-assembly of regular amphiphilic blocks, the hydrophobic intertwining of the PHPMA made a random aggregation of the star BSA−PHPMA into nanoparticles, as shown in the SEM nitrogen elemental mapping image (Figure S10). By treating the BSA−PHPMA nanoparticles with protease (1 mg/mL) for 60 min to remove the BSA located on the external surface of the nanoparticles, the wide distribution of nitrogen element inside the nanoparticles was still observed, suggesting that some of the BSA−PHPMA conjugates should be buried inside the nanoparticles which could not be removed by protease after 1 h incubation. Accordingly, rather than forming other higher-order morphologies, the further polymerization inside the formed nanoparticles induced the growth of the size mainly. As shown in Figure 2c−h upon different reaction time from 1 to 6 h, the hydrodynamic diameters of the formed nanoparticles could increase from 164 to 220 nm determined by DLS (Figure S11). On the other hand, after forming the nanoparticles, the further polymerization will induce the free BSA−PHPMA conjugates in the aqueous solution to aggregate into new nanoparticles. This was confirmed by the performed control experiment (Figure 3a): after initiating polymerization for 30 min, the reaction was stopped, and the formed nanoparticles were removed by centrifuging; then the left supernatant solution was reinitiated to continue polymerizing for another 30 min, and the BSA−PHPMA nanoparticles could be collected again, well suggesting the continuous formation of the nanoparticles from the free BSA−PHPMA in the aqueous solution. The two times collected particles were analyzed by TEM as shown in Figure 3b,c, and they both had good spherical morphologies. During the whole procedure, the decrease of the content of BSA in the aqueous solution was monitored by fluorescence spectra (Figure S12). Also, the turbidimetry study by monitoring the UV absorbance at the wavelength of 560 nm (Figure 3d) showed that there was a continuous formation of the nanoparticles within the first 3 h, reaching a plateau when the polymerization finished. The time to reach the same turbidity was dependent on the concentration of used BSA−CTA8 in the system. The low concentration of the BSA−CTA8 needs longer polymerization time to generate a higher hydrophobic volume ratio in the conjugate to reach the CAC and then shows a longer time to reach the same turbility compared with that of a high concentration of the BSA−CTA8 system. For example, different concentrations of BSA−CTA8 from 2 to 10 mg/mL were employed (Figure 3e), and as expected, 2 mg/mL of the BSA− CTA8 needs the longest polymerization time to reach the certain turbidity. To further investigate the BSA−PHPMA nanoparticle formation process, after 1 h polymerization, the MW of PHPMA obtained from both the aggregated BSA−PHPMA nanoparticles and the free BSA−PHPMA conjugates in the aqueous solution was conducted. The obvious increase in the MW of the PHPMA from the free BSA−PHPMA conjugates in the aqueous solution (Figure 3g, red curve, 22.5 K) compared with that of the PHPMA from the BSA−PHPMA nanoparticles (Figure 3f, blue curve, 17.8 K) suggests that the polymerization inside the formed nanoparticles was slower than that in the aqueous solution. This is acceptable given that the nanoparticles formed in this studied system are based on the random aggregation of the star BSA−PHPMA conjugates which is different from the micelle-like nanoparticles formed by the self-

appeared after polymerization. Moreover, after removing BSA from the free BSA−PHPMA conjugates in the aqueous solution by protease, the successful chain extensions were clearly confirmed by proton nuclear magnetic resonance (1H NMR, Figure S9) and gel permeation chromatography (GPC) (Figure 2a) from which the molecular weight of the grafted PHPMA

Figure 2. (a) GPC traces of the PHPMA chains obtained from the free BSA−PHPMA conjugates in the aqueous solution under different polymerization time, which corresponded to 0.5, 1, 1.5, 2 and 3 h. (b) Monomer conversion vs time curve (black squares) and corresponding Mw/Mn vs time curve (red circles) for the PHPMA (in a) with different polymerization time. Representative TEM images of the BSA−PHPMA nanoparticle morphologies observed for a series of polymerization times, which corresponded to (c) 1 h, (e) 2 h, and (g) 6 h. (d), (f), and (h) are the corresponding SEM images. Scale bars = 500 nm.

blocks increasing accordingly with longer polymerization time was calculated based on PMMA standards. The molecular weight of grafted PHPMA increased from 10.8 kg/mol after 0.5 h reaction, to 28.6 kg/mol after 2 h reaction, and finally to 37.5 kg/mol after 3 h reaction (monomer conversion ca. 90%), which together showed good control of the polydispersity (Mw/Mn) ranging from 1.25 to 1.44 (Figure 2b). As a consequence, with the polymerization proceeding, the increase of the hydrophobic volume of PHPMA in the star BSA−PHPMA conjugates will damage the balance of the conjugates in the aqueous solution, and when reaching the critical aggregation concentration of the star BSA−PHPMA conjugates, they will aggregate into nanoparticles (Figure 2c,d). 691

DOI: 10.1021/acsmacrolett.7b00422 ACS Macro Lett. 2017, 6, 689−694

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ACS Macro Letters

Figure 3. (a) Schematic illustration showing that after removing the generated nanoparticles by centrifuging the nanoparticle could again form in the left supernatant upon reinitiating polymerization, and the corresponding TEM image of the collected nanoparticles after 0.5 h polymerization (b), and the collected nanoparticles generated upon reinitiating polymerization for 0.5 h (c) (scale bars = 500 nm). (d) Turbidimetry study of the solution at different polymerization time by UV−vis spectra monitoring the absorbance at wavelength of 560 nm. (e) Plotting of polymerization time when reaching the same turbidity (A560 nm = 0.25) against concentration of BSA−CTA8 in the solution (2, 4, 6, and 10 mg/mL, respectively). (f) GPC traces of the grafted PHPMA chains obtained from the BSA−PHPMA nanoparticles at different polymerization time, corresponding to 1, 2, and 6 h, respectively, under the condition of [HPMA]:[BSA−CTA8]:[[Ru(bpy)3Cl2·6H2O]] = 2350:8:0.27 with 5% (w/w) content of HPMA. (g) GPC traces of the grafted PHPMA chains obtained from the free BSA−PHPMA conjugates in the aqueous solution at different polymerization time, corresponding to 0.5 h (black line) and 1 h (red line), respectively, under the same reaction condition with (f).

assembly of amphiphilic block copolymer, and the core of the BSA−PHPMA nanoparticle could not take a role effectively in the monomer partition. Therefore, the increase in the polydispersity of the PHPMA in the formed nanoparticles from 1.61 (1 h) to 1.98 (6 h) (Figure 3f) should be contributed by the later formed BSA−PHPMA nanoparticles, despite the that two PHPMA chains coupling termination could not be ruled out. Moreover, to extend the synthesis of the BSA− PHPMA nanoparticles, different reaction conditions were employed either using 15% (w/w) solids content or using another RAFT agent anchored BSA (BSA−CTA5) at 5% (w/ w) solid content. The SEM images in Figures S13 and 14 showed that the spherical nanoparticles could still be generated widely with the mean hydrodynamic diameter ranging from 190 to 255 nm (Figure S15). Esterase-like activity toward aryl esters showed by native BSA that the maintaining of the catalytic activity of BSA after polymerization reaction was studied by monitoring the hydrolysis process of 4-nitrophenyl acetate with a UV−vis spectrophotometer at the wavelength of 405 nm (Figure 4a). The activity of native BSA, BSA−CTA8, and BSA−PHPMA nanoparticles at different reaction times (1, 2, and 6 h) was tested. Despite that there was a 10−20% decrease on the catalytic activity of the BSA−PHPMA after forming nanoparticles compared with that of native BSA, it was acceptable considering the steric hindrance especially for the BSA− PHPMA conjugates which were buried inside nanoparticles. In general, the well-maintained catalytic activity of BSA suggested that the described strategies should be a mild technique and contributed another way for studying the various protein-based self-assemblies. Moreover, it should be mentioned that the widely generated BSA-based nanoparticles showed a very good stability against diluting and could withstand the centrifugation−redispersion procedure without any obvious aggregation (Figure S16).

Figure 4. (a) Esterase activity of BSA after treatment under various conditions, (b) schematic illustration of loading and release behavior of DOX/DNA-SYBR Green I, (c) plot showing release of DOX from nanoparticles triggered by using different amounts of protease, and (d) plot showing release of DNA from nanoparticles triggered by using different amounts of protease.

We were encouraged by the described facile and efficient way to generate the BSA−PHPMA nanoparticles, in particular, considering that the generated BSA-based nanoparticles were very similar in size and shape to biological nanostructures like exosomes or viruses. Also, the BSA−PHPMA nanoparticles showed very low cytotoxicity toward normal NIH 3T3 cells (Figure S17), and it should show a promising application toward the biomedicine or biology-related fields. Therefore, as a proof of concept, its potential as a drug delivery carrier was demonstrated. To encapsulate different drugs, the polarity of the nanoparticle interior was investigated first by using pyrene as a fluorescent probe of which the emission intensity ratio of the the first (I1) and third (I3) bands in the fluorescence spectrum was very sensitive to the change of the polarity of the 692

DOI: 10.1021/acsmacrolett.7b00422 ACS Macro Lett. 2017, 6, 689−694

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ACS Macro Letters surrounding.60 Prior to starting the polymerization, pyrene was added into the solution. Then, with the formation of the nanoparticles, the pyrene could be well encapsulated inside the nanoparticles, as seen from the change of I1/I3 value from 1.72 at the very beginning to 1.25 at 180 min (Figure S18), suggesting the decreased polarity of the interior of the nanoparticles with the increase of the molecular weight of the grafted PHPMA. Accordingly, a hydrophobic drug model of the fluorescence dye (Nile Red), as anticipated, could be well loaded inside the nanoparticles (Figure S19). Considering the generated nanoparticles were composed of hydrophilic BSA and hydrophobic PHPMA, we were wondering whether the hydrophilic substance could also be encapsulated during formation of the nanoparticles. Therefore, hydrophilic substances like small-molecule DOX or biomacromolecule (DNA stained by SYBR Green I) were added into the system (Figure 4b), both of which could be encapsulated into the particle as well, and the encapsulation efficiency could reach 13.5% and 11.6% for DOX and DNA, respectively (Figures S20 and 21). With these successful loadings of the different substances, as a consequence, the release of encapsulated DOX or SYBR green I-stained DNA was investigated in the presence of protease. It is well-known that protease widely exists inside lysosomes and could hydrolyze the BSA polypeptide chain, thus resulting in the disassembling of the nanoparticles. The release profiles were evaluated under different protease concentrations by fluorescence spectroscopy; as anticipated, higher concentrations of protease resulted in more efficient degradation of the nanoparticles and corresponding faster release rates of encapsulants (Figure 4c,d). In this study, we successfully describe a simple, mild, and efficient way to generate protein-based nanoparticles based on photoinitiated RAFT polymerization-induced self-assembly by using a multi-RAFT agent anchored BSA as a macro-RAFT agent and HPMA as a polymerized monomer. The well dispersed BSA−PHPMA nanoparticles could be generated widely under different solid content with the size ranging from 164 to 255 nm. The growing of the hydrophobic PHPMA chains could induce the free star BSA−PHPMA conjugates in the aqueous solution aggregating into nanoparticles continuously. Significantly, the generated nanoparticles showed excellent ability to carry various kinds of hydrophobic and hydrophilic substances. By loading cancer drug DOX or biomacromolecule DNA, the protease-mediated release of the encapsulants was demonstrated. Moreover, considering the various types of proteins in nature, by employing different proteins as building blocks which could easily bring different functions into the self-assembled structure, besides BSA, we also studied other functional proteins like lysozyme which could also generate the spherical morphology following the same procedure. This well suggested the potential universality of the demonstrated strategy applied to other proteins. Overall, it is anticipated that such a demonstrated mild and efficient strategy could open up other possibilities to investigate hierarchical hybrid protein-based self-assembly and to optimize the promising application in the biomedicine field.

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Full experimental section, 1H NMR and UV−vis spectra for trithiol RAFT agent, DLS data for native BSA and BSA−CTA, photoimages and additional SEM images for the formed protein nanoparticles, as well as the determination of loading efficiency etc. (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Xin Huang: 0000-0002-1858-2956 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank NSFC (21474025, 21504020), the Fundamental Research Funds for the Central Universities (HIT. NSRIF. 201632), Postdoctoral Science Foundation of China (2015M571401, 2016M600247), and Open Project of Key Laboratory of Microsystems and Microstructures Manufacturing (2016KM003) for financial support.

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