Microgel Surface Modification with Self-Assembling Peptides

Jul 27, 2016 - Mout , R.; Tonga , G. Y.; Ray , M.; Moyano , D. F.; Xing , Y.; Rotello , V. M. Environmentally Responsive Histidine-Carboxylate Zipper ...
0 downloads 0 Views 6MB Size
Download PDF
Article pubs.acs.org/Macromolecules

Microgel Surface Modification with Self-Assembling Peptides Kimberly C. Clarke*,† and L. Andrew Lyon*,‡ †

School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, United States Schmid College of Science and Technology, Chapman University, Orange, California 92866, United States

‡

S Supporting Information *

ABSTRACT: We describe the fabrication of peptide-coated microgels, where a fibrillizing peptide (RADA)4 self-assembles on the surface of hydrogel microparticles. The incorporation of an anionic comonomer into the microgel network is required for a stable colloidal dispersion to be obtained when particles are incubated with (RADA)4, suggesting that the assembly is dependent on Coulombic interactions. We further demonstrate the modification of the (RADA)4 shell by preparing coassemblies of (RADA)4 and a fluorescently labeled (RADA)4 peptide. Additionally, the (RADA)4 shell can be modified through postassembly conjugation of a cysteine residue or a non-natural amino acid bearing an alkyne moiety. Fluorescence and atomic force microscopy and circular dichroism spectroscopy were employed to characterize the assembly and modification of the peptide shell. Finally, our attempt to utilize a different fibrillizing peptide (Q11) in the formation of peptide-coated microgels was unsuccessful, demonstrating that the identity of the building blocks is important in the fabrication of these composite assemblies.

■

INTRODUCTION Colloidal particles have found versatile utility in biotechnological applications, including therapeutic delivery, disease targeting, and diagnostics.1−5 An important factor in their application is the ability to modify the particle surface, so that the particle interacts in a controlled and favorable manner within its intended environment.6,7 For example, the direct conjugation of polymers and biomolecules has been routinely used to enhance biocompatibility and site-specific targeting, respectively.8,9 Additional tactics include layer-by-layer (LbL) assembly onto the particle surface, where linear polymers or biomacromolecules, often of opposite charge, are deposited in alternating fashion onto the colloidal substrate.10 This allows for the controlled buildup of particle coatings that can be further modified if necessary. In summary, the ability to revise the surface properties of micro- and nanomaterials has extended and enhanced their applicability in biotechnology by facilitating a means to integrate them within their surroundings. Our group, among others, has investigated microgelssoft, polymeric colloidal particles, highly solvated with waterfor numerous biotechnological applications.11−14 Their high water content, facile synthesis, and favorable mechanical properties make microgels attractive in a range of biomaterials applications. Furthermore, microgel networks can be rendered environmentally responsive (e.g. temperature, pH, light) by synthesizing them from stimuli-responsive polymers.15 Our © XXXX American Chemical Society

group has worked extensively with temperature-responsive microgels synthesized from N-isopropylacrylamide (NIPAm).16 Comonomers such as acrylic acid (AAc) are commonly incorporated to add pH responsivity and functional groups for postsynthetic modifications. Further, microgel shells can be added directly to core microgel particles, and in so doing responsive elements and functional groups can be compartmentalized.17 Traditionally target molecules are directly conjugated to microgels using functional groups polymerized into the network.18−20 However, the ease and efficiency of comonomer incorporation can vary, potentially reducing the number and type of chemical handles that can be contained within a single particle.21,22 In addition, selectively modifying the microgel interface without altering the microgel network remains a challenge. Recently, an alternative approach to modify microgel interfaces was demonstrated in which microgels were employed as a substrate for LbL deposition.23 However, the potential for LbL-modified microgels in biomedical applications has yet to be fully investigated. Peptides and proteins serve a wide array of functions in biological systems, making them an important building block in biomaterials.24−26 Their diversity allows them to serve both biochemical and biophysical roles, helping to create functional Received: July 12, 2016

A

DOI: 10.1021/acs.macromol.6b01497 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules materials that more readily recapitulate the chemistry and mechanics of native tissues. Peptides have the ability to span multiple length scales, from the molecular to the macroscopic, because of their propensity to self-assemble into secondary structures (i.e., α-helices and β-sheets).27−31 This long-range assembly has been of particular interest in the development of cell and tissue scaffolding. Furthermore, the capacity to incorporate non-natural amino acids for selective bioconjugation reactions enhances the versatility of the peptide platform.24,32 In this work we demonstrate the modification of microgel surfaces using a self-assembling peptide, (RADA)4. (RADA)4 is a 16 amino acid peptide where the sequence arginine−alanine− aspartic acid−alanine is repeated four times. This peptide selfassembles into β-sheet fibrils and finally into a macroscopic hydrogel when exposed to physiological ionic strengths.33,34 Much of the attention (RADA)4 has received has focused on the use of (RADA)4 gels as cell culture substrates for tissue engineering.35−37 In the present study, (RADA)4 fibrils are observed to assemble on the surface of anionic, thermoresponsive microgels. Appending a cysteine residue to the peptide permits postassembly conjugation reactions through the thiol chemoligation site. In addition, we demonstrate that a nonnatural amino acid containing an alkyne can be added to (RADA)4 and subsequently used in a copper-catalyzed azide− alkyne cycloaddition (CuAAC) reaction. The modular nature of this biosynthetic construct permits independent tailoring of the interior particle and the exterior peptide shell. This new core/shell structure could have potential as a building block within extracellular matrix mimetic scaffolds, where its inclusion in the scaffolds would enable the properties of the bulk matrix to be controlled at a smaller length scale.15,38 Therefore, the properties of the bulk material would be independently addressable at the building block level, permitting a high degree of tailoring from easily accessible components.

■

Table 1. Polymer Feed Ratios of Microgel Syntheses microgel

mol % NIPAm

mol % NIPMAm

mol % AAc

mol % BIS

[monomer]T (mM)

1 2 3 4 5 6

22 93 78 90 98 49

66 0 0 0 0 49

10 5 20 10 0 0

2 2 2 0 2 2

140 100 100 100 100 100

to the monomer solution. The reaction was initiated by adding 1.0 mL of filtered APS solution (1 mM) to the flask and continued for 6 h under a N2 blanket. After cooling to room temperature, the microgel solution was filtered (0.8 μm Acrodisk filter) and purified by ultracentrifugation. Microgel aliquots were centrifuged at 136000g to form a pellet, the supernatant was removed, and the microgels were redispersed in DI water. This process was repeated four times. Ultralow cross-linked (ULC) microgels were synthesized in an analogous fashion, except the reaction was performed without the addition of BIS to the monomer solution (Microgel 4).39 Purified microgels were lyophilized and redispersed in DI water before use. Microgel Characterization. Dynamic light scattering (DLS, Wyatt) was used to determine the hydrodynamic radii (RH) of the microgels. Microgels before and after coating with (RADA)4 peptides were diluted in pH 3 formate buffer (10 mM, 5 mM ionic strength) and pH 7 HEPES buffer (10 mM, 5 mM ionic strength). Each run consisted of 20 acquisitions, and five runs were collected for each sample. The data presented are the average and standard deviation of the five runs. Preparation of Peptide-Coated Microgels. Microgels were redispersed in DI water and diluted to 0.75 mg/mL. (RADA)4, (RADA)4C, (RADA)4Pr, or Q11 was dissolved in DI water and diluted to 0.5 mg/mL. TMR in DMSO was added to (RADA)4 or (RADA)4C solutions to make up 0.5 wt % of the total peptide. Microgel and peptide solutions were cooled to 4 °C. (RADA)4, (RADA)4C, or (RADA)4Pr was added to the microgel solution and mixed at 4 °C for 1 h. The microgel solution volume was always double the peptide solution volume. The resulting mixture was dialyzed against DI water at 4 °C in cellulose ester membranes (MWCO 1000 kDa). Flocculation resulted upon addition of Q11 to the microgel solution, so this mixture was not mixed for the full hour or dialyzed. Fluorophore Conjugation to (RADA)4C- and (RADA)4PrCoated Microgels. For postassembly conjugation of 5iodoacetamidofluorescein (5-IAF) to (RADA)4C on the microgel surface, 5-IAF (1.0 mM stock solution in DMF) was added to (RADA)4C-coated microgel 1 at room temperature for 2 h. 5-IAF was conjugated at 10-fold molar excess to 1.0 wt % of the theoretical quantity of (RADA)4C added during the coating process. As a control, (RADA)4-coated microgel 1 and uncoated microgel 1 were also mixed with 5-IAF for 2 h to confirm labeling via reaction with the cysteine. A coumarin azide (7-hydroxy-3-azidocoumarin, CAz) was conjugated to the alkyne of the non-native amino acid on (RADA)4Pr via CuAAC. CAz40 and the ligand, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA),41 were synthesized according to established methods and generously provided by the Finn group at Georgia Tech. CuSO4 (100 μM), ascorbic acid (11 mM), THPTA (500 μM), and CAz (78 μM) were mixed with 1.0 mL of (RADA)4Pr-coated microgel 1 for 2 h on a shaker table at room temperature in the dark. Concentrations listed are the final concentration in the mixture. Following each coupling reaction the mixtures were dialyzed against DI water in the dark at 4 °C in cellulose ester membranes (MWCO 1000 kDa). Circular Dichroism. A JASCO-J810 spectropolarimeter was used to determine the conformation of the (RADA)4 peptides and (RADA)4-coated microgels. (RADA)4 and (RADA)4C stock solutions were diluted with DI water to a final concentration of 20 μM; (RADA)4Pr was diluted to 40 μM with DI water. TMR was added to a solution of plain (RADA)4 to have a final concentration of

EXPERIMENTAL SECTION

Materials. Chemicals were purchased from Sigma-Aldrich, unless noted otherwise. NIPAm and NIPMAm were recrystallized from hexanes (BDH Chemical). N,N′-Methylenebis(acrylamide) (BIS) and acrylic acid (AAc, Fluka) were used as received. The surfactant, sodium dodecyl sulfate (SDS), and initiator, ammonium persulfate (APS), were used as received. (RADA)4 peptides (the N- and Ctermini were acetylated and amidated, respectively) were purchased from Anaspec (Ac-(RADA)4-Am, denoted as (RADA)4) and Genscript (TMR-(RADA)4-Am, Ac-(RADA)4C-Am, and Ac(RADA)4Pr-Am, denoted as TMR, (RADA)4C, and (RADA)4Pr, respectively). Q11 was generously supplied by Tom Barker’s group at Georgia Tech. (RADA)4, (RADA)4C, (RADA)4Pr, and Q11 were dissolved in DI water and sonicated for 30 min before use; (RADA)4TMR was dissolved in DMSO (2.5 mg/mL). The following materials were used in the preparation of buffers: 4-(2-hydroxyethyl)piperazine1-ethanesulfonic acid (HEPES), formic acid, and sodium chloride. All water was distilled and deionized to a resistance of 18 MΩ (Barnstead E-pure). Microgel Synthesis. Microgels were synthesized via aqueous freeradical precipitation polymerization. The feed ratios for each of the microgels used in this study are presented in Table 1. As an example, the synthesis of microgel 1 is described here. NIPAm (22 mol %), NIPMAm (66 mol %), and SDS (1 mM) were dissolved in 99 mL of DI water and filtered (0.2 μm Acrodisk filter) into a three-neck reaction flask. The monomer solution was heated to 70 °C in an oil bath with a stir bar spinning at 450 rpm. A thermometer and condenser were attached, and N2 was bubbled through the solution. The solution was refluxed for 1 h under the above conditions. Approximately 10 min prior to initiation, AAc (10 mol %) was added B

DOI: 10.1021/acs.macromol.6b01497 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

Figure 1. AFM amplitude images: (a) microgel 1 and (b) microgel 1 + (RADA)4. (c) Bright-field and (d) fluorescence microscopy images of microgel 1 + (RADA)4 + TMR (scale bar = 2 μm). 0.5 wt % of the total mass of peptide. Microgel 1 before and after coating was diluted to 0.17 mg/mL, except microgel 1 + (RADA)4Pr, which was characterized as prepared. Microgels 2 and 3 (0.5 mg/mL in DI water) before and after peptide coating were used as prepared. Aliquots of microgel samples were centrifuged to form a pellet, and the supernatant was collected and analyzed by CD. All samples were placed in a quartz cuvette (1 mm path length) and scanned from 250 to 190 nm. Data presented are the average of four scans. Atomic Force Microscopy. Microgel aliquots (1 μL diluted with 25 μL of DI) before and after peptide coating were dried on a glass coverslip and imaged using an Asylum MFP-3D atomic force microscope (AFM). Imaging was performed in air operating in intermittent contact mode using silicon nitride cantilevers (k = 42 N/ m, Nanoworld). Data were processed through software written in the IgorPro environment (Wavemetrics). Optical Microscopy. The presence of fluorescently labeled TMR(RADA)4, (RADA)4C, or (RADA)4Pr localized on microgels was visualized using fluorescence microscopy (Olympus IX-71). Microgel aliquots (1 μL) were diluted with 25 μL of DI, dried on glass coverslips, and imaged with a 100× oil immersion objective. The exposure was set to 250 ms for all fluorescence images. For fluorescence intensity analysis of microgel 1 coated with (RADA)4 + TMR, (RADA)4C + 5-IAF, and (RADA)4Pr + CAz, aliquots of asprepared coated microgels (5 μL) were diluted with DI water (10 μL) and dried on glass coverslips. For plain microgel 1, a stock solution of 0.5 mg/mL in DI water was used. Five images were collected for each sample, and ImageJ was used to calculate the average fluorescence intensities of each image. The final average and standard deviation of the five images are provided in Table S1. Bright-field microscopy was also used to confirm the presence of microgels in each sample. Images were acquired using a 100× oil immersion objective.

residues. A (RADA)4 peptide shell assembles onto the surface of microgel 1 by simply mixing the two solutions. AFM amplitude images reveal fibrils on the surface of the particles after incubation with (RADA)4 (Figure 1b). For reference, an image of unmodified microgel 1 is presented in Figure 1a. Numerous studies with (RADA)4 gels have utilized coassemblies where plain (RADA)4 is doped with modified (RADA)4 peptides before gelation.34,36,42 In essence this would permit the inclusion of a variety of functional ligands to be coassembled into a single construct. As a proof of concept, we coassembled (RADA)4 with a rhodamine-labeled (RADA)4 peptide (TMR) onto the surface of microgel 1. Particles present in the bright-field image (Figure 1c) also appear red in the fluorescence image (Figure 1d), indicating successful coassembly onto the particles. DLS data show good agreement in hydrodynamic radii before and after coating, suggesting that the peptide shell is quite thin (Table 2). Table 2. Hydrodynamic Radii (nm) of Microgel 1 (M1) Coated with (RADA)4/(RADA)4C RH pH 3 M1 M1 M1 M1 M1

■

+ + + +

(RADA)4 + TMR (RADA)4C (RADA)4C + TMR (RADA)4C + 5IAF

261 282 259 267 258

± ± ± ± ±

36 56 37 46 37

RH pH 7 352 365 344 355 345

± ± ± ± ±

63 92 66 63 64

Microgel 1 was initially selected because it has a response temperature of 38 °C, close to physiological temperature.43 After the initial observation of the peptide coating, we explored the use of alternative microgel polymer compositions in the assembly. Microgels 2 and 3 (synthesized with 5 and 20% AAc, respectively) demonstrated successful coatings with (RADA)4 + TMR (Figure 2a−d). Surface localized fibrils can be observed in AFM images of microgels 2 and 3 following incubation with (RADA)4 + TMR (Figures S1 and S2). Ultralow, self-crosslinked microgels (microgel 4) copolymerized with AAc also show evidence of a (RADA)4 peptide shell (Figure 2e,f and

RESULTS AND DISCUSSION Microgels are soft, hydrated, polymeric particles. Typical modifications to microgels are achieved synthetically through the incorporation of comonomers or the addition of a polymer shell. Functional groups incorporated into the network can subsequently be employed for postsynthetic conjugation reactions. In the present study we explore the utility of selfassembling peptides for the modification of microgel surfaces. One such peptide is (RADA)4, which assembles into β-sheet fibrils due to the alternating hydrophobic and hydrophilic C

DOI: 10.1021/acs.macromol.6b01497 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

Figure 2. Microscopy images of microgels 2−4 after coating with (RADA)4 + TMR: bright-field (a, c, e) and fluorescence (b, d, f) (scale bar = 2 μm).

thiol for the conjugation of 5-IAF. (RADA)4C and microgel 1 were mixed, dialyzed, and then reacted with 5-IAF. Following a second round of dialysis, the particles were analyzed by fluorescence microscopy. As expected, the particles fluoresce green, indicating successful conjugation of the fluorophore (Figure 4b). Mixtures of microgel 1 and plain (RADA)4-coated microgel 1 with 5-IAF did not result in significant labeling (Table S1). In addition, the (RADA)4C coating can be observed in AFM amplitude images (Figure 4a), similar to the plain (RADA)4 coating. Next, (RADA)4C and TMR were mixed and coassembled on the surface of microgel 1. Subsequent labeling with 5-IAF demonstrated the potential to incorporate functionality both during and after surface assembly (Figure 4d−f and Table S1). Aliquots of each solution were centrifuged to form a pellet. Each pellet was colored in accord with the fluorophores either coassembled or conjugated to the (RADA)4C shell: unlabeled is colorless, TMR labeled is pink, 5-IAF labeled is yellow, and TMR/5-IAF is orange (Figure S6). Following the successful conjugation using (RADA)4C, we sought to utilize a noncanonical amino acid for a bioconjugation reaction. Noncanonical amino acids can be employed to add new functional groups not commonly found within peptides and proteins, eliminating side reactions.24,32,44 A variety of nontraditional ligation strategies have been explored recently, including the CuAAC reaction.44 This conjugation reaction is efficient and fast and forms a stable product. An amino acid with a propargyl group was added to (RADA)4. Following assembly onto microgel 1 (AFM Figure

AFM Figure S3). However, microgels lacking AAc (microgels 5 and 6) resulted in flocculation when the peptide and microgel solutions were mixed. (RADA)4 fibrils are observed to extend between particles following incubation of (RADA)4 + TMR with microgel 5 (Figure 3a and Figure S4). A second uncharged

Figure 3. AFM amplitude images of microgels 5 (a) and 6 (b) after incubation with (RADA)4.

particle, microgel 6, was also tested for assembly of (RADA)4 on the surface. Particle aggregates are present when an aliquot is dried on a glass substrate (Figure 3b and Figure S5). A stable colloidal dispersion of peptide-coated microgels is observed only when AAc is polymerized into the microgel network, suggesting that the anionic microgel network is important in stabilizing the colloidal dispersion. We next explored postassembly conjugation to (RADA)4 shells assembled on a microgel surface. A cysteine residue was added to the C-terminus of a (RADA)4 peptide to provide a D

DOI: 10.1021/acs.macromol.6b01497 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

Figure 4. (a) AFM amplitude image of microgel 1 + (RADA)4C; (b) bright-field and (c) fluorescence microscopy images of microgel 1 + (RADA)4C after labeling with 5-IAF; (d) bright-field and (e, f) fluorescence microscopy of microgel 1 + (RADA)4C + TMR after labeling with 5IAF (scale bar = 2 μm).

Figure 5. (a) Bright-field and (b) fluorescence images of microgel 1 + (RADA)4Pr after the conjugation of CAz (scale bar = 2 μm).

S7), the propargyl was reacted with a coumarin azide (CAz) in the presence of copper, ascorbic acid, and the ligand THPTA. Only the triazole product is fluorescent,40,45 so a fluorescence signal is indicative of successful conjugation. Bright-field and fluorescence microscopy images of microgel 1 coated with (RADA)4Pr following the CuAAC reaction are presented in Figure 5. When excited with UV light, the presence of blue fluorescence is observed, indicating the formation of the triazole product (Table S1). (RADA)4 is known to assemble into β-sheets.33,34 However, it was unknown whether the secondary structure would be maintained on the microgel surface. CD spectroscopy was used to provide insight into the possible presence of β-sheet secondary structure on (RADA)4-coated microgels. CD spectra of (RADA)4, (RADA)4C, and (RADA)4Pr in DI water present typical bands for β-sheets (Figure 6a). The bands present in the

(RADA)4Pr spectrum are less intense, suggesting that the alkyne functionality may interfere to some degree in the assembly of the peptide. Microgel 1 coated with (RADA)4, (RADA)4C, and (RADA)4Pr display similar bands, suggesting that the peptide fibrils retain the β-sheet structure on the surface of the particles (Figure 6b). CD spectra of microgel 1 alone and supernatants collected after centrifuging the particles do not show any bands indicative of secondary structure. Furthermore, the spectra of (RADA)4-coated microgels 2 and 3 also have bands indicative of β-sheets (Figures 6c and 6d, respectively). The supernatants from microgels 2 and 3 show the presence of β-sheets, likely due to some (RADA)4 disassembling from the particle surface. A decrease in the amount of peptide in the supernatants, as indicated by a decrease in the magnitude of the supernatant CD spectra, E

DOI: 10.1021/acs.macromol.6b01497 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

Figure 6. CD spectra in DI water of (a) (RADA)4 peptides and (b−d) microgels 1, 2, and 3, respectively, uncoated and after incubation with (RADA)4 peptides.

Figure 7. Microscopy images of microgel 1 after mixing with Q11: (a) AFM amplitude image; (b) bright-field image (scale bar = 5 μm).

complexation or for the preparation of emulsions.31,51−53 However, to our knowledge, no examples exist where the surfaces of hydrogel-based particles have been modified by the self-assembly of peptide architectures as described here. The peptide shell provides a route to control the surface properties of microgel particles as well as lending new topographical features to the interface. Moreover, enzymatic peptide substrates can be introduced onto the self-assembling peptide, permitting specific bioconjugation reactions to occur at the particle interface.7 We hypothesize that this particular type of construct would be useful within extracellular matrix scaffolding as a modular building block to enhance existing properties or impart new properties to the bulk material, including, but not limited to, sequestration or release of biomolecules, control over concentration gradients, crosslinking to alter the mechanical environment, and control over matrix architecture. Numerous properties could therefore be defined by the building blocks incorporated within the scaffold, rather than attempting to individually address each property during the synthesis of the bulk material alone.

suggests that (RADA)4 is specifically associated with the particles. In addition to (RADA)4, we were curious whether additional self-assembling peptides were capable of coating microgels. We focused on Q11, a β-sheet forming peptide with the sequence Ac-QQKFQFQFEQQ-Am.42,46 The residues alternate between hydrophobic (phenylalanine, F) and hydrophilic (glutamine, Q), but unlike (RADA)4, Q11 has fewer charged residues. When Q11 was mixed with microgel 1, flocculation was immediately observed. Microscopy of a dried aliquot revealed large aggregates and entanglements (Figure 7). Additionally, all microgels added to a Q11 solution resulted in aggregation (data not shown). These results suggest that the identity of the peptide is also crucial to the formation of stably coated microgels. In this work we presented the coating of microgel particles with (RADA)4, a β-sheet self-assembling peptide, and subsequently performed surface localized reactions with functional groups displayed on the peptide shell. The selfassembly of peptides has been achieved on hard colloidal particles, including gold nanoparticles and quantum dots via covalent bond formation, amino acid−metal coordination, or electrostatic interactions.6,47−50 The assembly of peptides has also been demonstrated at liquid−liquid interfaces as a result of

■

CONCLUSIONS In this work we demonstrated the facile assembly of a (RADA)4-based shell onto the surface of microgels. IncorpoF

DOI: 10.1021/acs.macromol.6b01497 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

(4) Scheinberg, D. A.; Villa, C. H.; Escorcia, F. E.; McDevitt, M. R. Conscripts of the Infinite Armada: Systemic Cancer Therapy using Nanomaterials. Nat. Rev. Clin. Oncol. 2010, 7, 266−276. (5) Ghosh, P.; Han, G.; De, M.; Kim, C. K.; Rotello, V. M. Gold Nanoparticles in Delivery Applications. Adv. Drug Delivery Rev. 2008, 60, 1307−1315. (6) Algar, W. R.; Prasuhn, D. E.; Stewart, M. H.; Jennings, T. L.; Blanco-Canosa, J. B.; Dawson, P. E.; Medintz, I. L. The Controlled Display of Biomolecules on Nanoparticles: A Challenge Suited to Bioorthogonal Chemistry. Bioconjugate Chem. 2011, 22, 825−858. (7) Walper, S. A.; Turner, K. B.; Medintz, I. L. Enzymatic Bioconjugation of Nanoparticles: Developing Specificity and Control. Curr. Opin. Biotechnol. 2015, 34, 232−241. (8) Otsuka, H.; Nagasaki, Y.; Kataoka, K. PEGylated Nanoparticles for Biological and Pharmaceutical Applications. Adv. Drug Delivery Rev. 2003, 55, 403−419. (9) Ruoslahti, E. Peptides as Targeting Elements and Tissue Penetration Devices for Nanoparticles. Adv. Mater. 2012, 24, 3747− 3756. (10) Becker, A. L.; Johnston, A. P. R.; Caruso, F. Layer-by-Layer Assembled Capsules and Films for Therapeutic Delivery. Small 2010, 6, 1836−1852. (11) Smith, M. H.; Lyon, L. A. Multifunctional Nanogels for siRNA Delivery. Acc. Chem. Res. 2012, 45, 985−993. (12) Brown, A. C.; Stabenfeldt, S. E.; Ahn, B.; Hannan, R. T.; Dhada, K. S.; Herman, E. S.; Stefanelli, V.; Guzzetta, N.; Alexeev, A.; Lam, W. A.; Lyon, L. A.; Barker, T. H. Ultrasoft Microgels Display Emergent Platelet-Like Behaviours. Nat. Mater. 2014, 13, 1108−1114. (13) Saxena, S.; Hansen, C. E.; Lyon, L. A. Microgel Mechanics in Biomaterial Design. Acc. Chem. Res. 2014, 47, 2426−2434. (14) Milani, A. H.; Freemont, A. J.; Hoyland, J. A.; Adlam, D. J.; Saunders, B. R. Injectable Doubly Cross-Linked Microgels for Improving the Mechanical Properties of Degenerated Intervertebral Discs. Biomacromolecules 2012, 13, 2793−2801. (15) Bradley, M. Chemistry at the Polymer-Particle Interface for the Design of Innovative Materials. Soft Matter 2012, 8, 1268−1274. (16) Hendrickson, G. R.; Smith, M. H.; South, A. B.; Lyon, L. A. Design of Multiresponsive Hydrogel Particles and Assemblies. Adv. Funct. Mater. 2010, 20, 1697−1712. (17) Jones, C. D.; Lyon, L. A. Synthesis and Characterization of Multiresponsive Core-Shell Microgels. Macromolecules 2000, 33, 8301−8306. (18) Blackburn, W. H.; Dickerson, E. B.; Smith, M. H.; McDonald, J. F.; Lyon, L. A. Peptide-Functionalized Nanogels for Targeted siRNA Delivery. Bioconjugate Chem. 2009, 20, 960−968. (19) Nayak, S.; Lee, H.; Chmielewski, J.; Lyon, L. A. Folate-Mediated Cell Targeting and Cytotoxicity Using Thermoresponsive Microgels. J. Am. Chem. Soc. 2004, 126, 10258−10259. (20) Meng, Z. Y.; Hendrickson, G. R.; Lyon, L. A. Simultaneous Orthogonal Chemoligations on Multiresponsive Microgels. Macromolecules 2009, 42, 7664−7669. (21) Farley, R.; Saunders, B. R. A General Method for Functionalisation of Microgel Particles with Primary Amines using Click Chemistry. Polymer 2014, 55, 471−480. (22) Singh, N.; Lyon, L. A. Synthesis of Multifunctional Nanogels using a Protected Macromonomer Approach. Colloid Polym. Sci. 2008, 286, 1061−1069. (23) Wong, E. W.; Richtering, W. Layer-by-Layer Assembly on Stimuli-Responsive Microgels. Curr. Opin. Colloid Interface Sci. 2008, 13, 403−412. (24) Tang, W.; Becker, M. L. Click” Reactions: A Versatile Toolbox for the Synthesis of Peptide-Conjugates. Chem. Soc. Rev. 2014, 43, 7013−7039. (25) Woolfson, D. N.; Mahmoud, Z. N. More than Just Bare Scaffolds: Towards Multi-Component and Decorated Fibrous Biomaterials. Chem. Soc. Rev. 2010, 39, 3464−3479. (26) Boekhoven, J.; Stupp, S. I. 25th Anniversary Article: Supramolecular Materials for Regenerative Medicine. Adv. Mater. 2014, 26, 1642−1659.

ration of acrylic acid into the microgel network was found to be an important factor in the formation of a stable colloidal solution of coated particles. Two routes were explored for functionalizing the peptide coating: coassembly of modified peptides and postassembly functionalization via a cysteine residue and an alkyne-modified amino acid. Both methods permit tailoring of the peptide shell. The surface localized fibrils have a β-sheet secondary structure as confirmed by CD, indicating that the microgels do not interfere with the peptide self-assembly. An alternative β-sheet assembling peptide, Q11, was utilized to investigate the generality of the assembly, but a stable dispersion of Q11-coated particles was not obtained. The microgel core/peptide shell is a modular composite, where each component can be independently modified. In this way a variety of functional constructs can be tailor-made for applications. We are currently interested in the pursuit of new building blocks to modify and enhance extracellular matrix scaffolds for tissue engineering. The colloidal particles presented in this work would allow us to leverage the unique properties of both biological (i.e., biochemical and biophysical roles) and synthetic materials (i.e., chemical complexity), ideally providing access to a larger repertoire of tools through which we can build better matrices.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b01497. Atomic force microscopy images, bright-field and fluorescence images, fluorescence intensity measurements, image of microgel pellets (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (K.C.C.). *E-mail: [email protected] (L.A.L.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors acknowledge Dr. Mike Smith and Dr. Grant Hendrickson for assistance in microgel synthesis. We also acknowledge the Georgia Tech School of Chemistry and Biochemistry for funding.

■

ABBREVIATIONS NIPAm, N-isopropylacrylamide; AAc, acrylic acid; 5-IAF, 5iodoacetamidofluorescein; CAz, coumarinazide; CuAAC, copper-catalyzed azide−alkyne cycloaddition; CD, circular dichroism; AFM, atomic force microscopy.

■

REFERENCES

(1) Loomis, K.; McNeeley, K.; Bellamkonda, R. V. Nanoparticles with Targeting, Triggered Release, and Imaging Functionality for Cancer Applications. Soft Matter 2011, 7, 839−856. (2) Louie, A. Multimodality Imaging Probes: Design and Challenges. Chem. Rev. 2010, 110, 3146−3195. (3) von Maltzahn, G.; Park, J.-H.; Lin, K. Y.; Singh, N.; Schwoppe, C.; Mesters, R.; Berdel, W. E.; Ruoslahti, E.; Sailor, M. J.; Bhatia, S. N. Nanoparticles that Communicate in vivo to Amplify Tumour Targeting. Nat. Mater. 2011, 10, 545−552. G

DOI: 10.1021/acs.macromol.6b01497 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

(48) Mout, R.; Tonga, G. Y.; Ray, M.; Moyano, D. F.; Xing, Y.; Rotello, V. M. Environmentally Responsive Histidine-Carboxylate Zipper Formation between Proteins and Nanoparticles. Nanoscale 2014, 6, 8873−8877. (49) Prasuhn, D. E.; Blanco-Canosa, J. B.; Vora, G. J.; Delehanty, J. B.; Susumu, K.; Mei, B. C.; Dawson, P. E.; Medintz, I. L. Combining Chemoselective Ligation with Polyhistidine-Driven Self-Assembly for the Modular Display of Biomolecules on Quantum Dots. ACS Nano 2010, 4, 267−278. (50) Pinaud, F.; King, D.; Moore, H.-P.; Weiss, S. Bioactivation and Cell Targeting of Semiconductor CdSe/ZnS Nanocrystals with Phytochelatin-Related Peptides. J. Am. Chem. Soc. 2004, 126, 6115− 6123. (51) Bitton, R.; Chow, L. W.; Zha, R. H.; Velichko, Y. S.; Pashuck, E. T.; Stupp, S. I. Electrostatic Control of Structure in Self-Assembled Membranes. Small 2014, 10, 500−505. (52) Bai, S.; Pappas, C.; Debnath, S.; Frederix, P. W. J. M.; Leckie, J.; Fleming, S.; Ulijn, R. V. Stable Emulsions Formed by Self-Assembly of Interfacial Networks of Dipeptide Derivatives. ACS Nano 2014, 8, 7005−7013. (53) Shimanovich, U.; Efimov, I.; Mason, T. O.; Flagmeier, P.; Buell, A. K.; Gedanken, A.; Linse, S.; Akerfeldt, K. S.; Dobson, C. M.; Weitz, D. A.; Knowles, T. P. J. Protein Microgels from Amyloid Fibril Networks. ACS Nano 2015, 9, 43−51.

(27) Fichman, G.; Gazit, E. Self-Assembly of Short Peptides to Form Hydrogels: Design of Building Blocks, Physical Properties and Technological Applications. Acta Biomater. 2014, 10, 1671−1682. (28) Maude, S.; Ingham, E.; Aggeli, A. Biomimetic Self-Assembling Peptides as Scaffolds for Soft Tissue Engineering. Nanomedicine 2013, 8, 823−847. (29) Frederix, P. W. J. M.; Scott, G. C.; Abul-Haija, Y. M.; Kalafatovic, D.; Pappas, C. G.; Javid, N.; Hunt, N. T.; Ulijn, R. V.; Tuttle, T. Exploring the Sequence Space for (Tri-)Peptide SelfAssembly to Design and Discover New Hydrogels. Nat. Chem. 2014, 7, 30−37. (30) Hudalla, G. A.; Sun, T.; Gasiorowski, J. Z.; Han, H.; Tian, Y. F.; Chong, A. S.; Collier, J. H. Gradated Assembly of Multiple Proteins into Supramolecular Nanomaterials. Nat. Mater. 2014, 13, 829−836. (31) Rozkiewicz, D. I.; Myers, B. D.; Stupp, S. I. Interfacial SelfAssembly of Cell-Like Filamentous Microcapsules. Angew. Chem., Int. Ed. 2011, 50, 6324−6327. (32) Ngo, J. T.; Tirrell, D. A. Non-Canonical Amino Acids in the Interrogation of Cellular Protein Synthesis. Acc. Chem. Res. 2011, 44, 677−685. (33) Zhang, S.; Holmes, T. C.; DiPersio, C. M.; Hynes, R. O.; Su, X.; Rich, A. Self-Complementary Oligopeptide Matrices Support Mammalian Cell Attachment. Biomaterials 1995, 16, 1385−1393. (34) Yokoi, H.; Kinoshita, T.; Zhang, S. Dynamic Reassembly of Peptide RADA16 Nanofiber Scaffold. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 8414−8419. (35) Wang, X.; Horii, A.; Zhang, S. Designer Functionalized SelfAssembling Peptide Nanofiber Scaffolds for Growth, Migration, and Tubulogenesis of human Umbilical Vein Endothelial Cells. Soft Matter 2008, 4, 2388−2395. (36) Koutsopoulos, S.; Unsworth, L. D.; Nagai, Y.; Zhang, S. Controlled Release of Functional Proteins through Designer SelfAssembling Peptide Nanofiber Hydrogel Scaffold. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 4623−4628. (37) Chau, Y.; Luo, Y.; Cheung, A. C. Y.; Nagai, Y.; Zhang, S.; Kobler, J. B.; Zeitels, S. M.; Langer, R. Incorporation of a Matrix Metalloproteinase-Sensitive Substrate into Self-Assembling Peptides-A Model for Biofunctional Scaffolds. Biomaterials 2008, 29, 1713−1719. (38) Clarke, K. C.; Douglas, A. M.; Brown, A. C.; Barker, T. H.; Lyon, L. A. Colloid-Matrix Assemblies in Regenerative Medicine. Curr. Opin. Colloid Interface Sci. 2013, 18, 393−405. (39) Bachman, H.; Brown, A. C.; Clarke, K. C.; Dhada, K. S.; Douglas, A. M.; Hansen, C. E.; Herman, E. S.; Hyatt, J.; Kodlekere, P.; Saxena, S.; Spears, M. W., Jr.; Welsch, N.; Lyon, L. A. Ultrasoft, Highly Deformable Microgels. Soft Matter 2015, 11, 2018−2028. (40) Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.; Wang, Q. A Fluorogenic 1,3-Dipolar Cycloaddition Reaction of 3Azidocoumarins and Acetylenes. Org. Lett. 2004, 6, 4603−4606. (41) Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation. Angew. Chem., Int. Ed. 2009, 48, 9879−9883. (42) Jung, J. P.; Gasiorowski, J. Z.; Collier, J. H. Fibrillar Peptide Gels in Biotechnology and Biomedicine. Biopolymers 2010, 94, 49−59. (43) Clarke, K. C.; Lyon, L. A. Modulation of the Deswelling Temperature of Thermoresponsive Microgel Films. Langmuir 2013, 29, 12852−12857. (44) Sletten, E. M.; Bertozzi, C. R. Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality. Angew. Chem., Int. Ed. 2009, 48, 6974−6998. (45) Kislukhin, A. A.; Hong, V. P.; Breitenkamp, E.; Finn, M. G. Relative Performace of Alkynes in Copper-Catalyzed Azide-Alkyne Cycloaddition. Bioconjugate Chem. 2013, 24, 684−689. (46) Tian, Y. F.; Devgun, J. M.; Collier, J. H. Fibrillized Peptide Microgels for Cell Encapsulation and 3D Cell Culture. Soft Matter 2011, 7, 6005−6011. (47) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Self-Assembled Monolayers of Thiolates on Metals as a Form of Nanotechnology. Chem. Rev. 2005, 105, 1103−1169. H

DOI: 10.1021/acs.macromol.6b01497 Macromolecules XXXX, XXX, XXX−XXX