Whole Protein Imprinting over Magnetic Nanoparticles Using

Letter pubs.acs.org/acsapm

Cite This: ACS Appl. Polym. Mater. XXXX, XXX, XXX−XXX

Whole Protein Imprinting over Magnetic Nanoparticles Using Photopolymerization ́ ia Griffete*,† Charlotte Boitard,† Aazdine Lamouri,‡ Christine Meń ager,† and Neb́ ew †

Physico-chimie des Electrolytes et Nanosystèmes Interfaciaux, PHENIX, Sorbonne Université, CNRS, F-75005 Paris, France ITODYS, UMR 7086, University Paris Diderot, F-75013 Paris, France

‡

ACS Appl. Polym. Mater. Downloaded from pubs.acs.org by UNIV OF SOUTHERN INDIANA on 04/13/19. For personal use only.

S Supporting Information *

ABSTRACT: Photopolymerization using UV-light at 365 nm was used for the first time to imprint whole proteins. Combined with a grafting approach onto functionalized magnetic nanoparticles, a new and faster way to synthesize magnetic protein imprinted polymers with outstanding adsorption capacities was developed.

KEYWORDS: magnetic nanoparticles, molecular recognition, protein imprinted polymer, photopolymerization, diazonium salt chemistry

S

widely employed are polymerization initiators able to create free radicals when submitted to specific conditions, e.g., ultraviolet (UV) light17 or the presence of some metallic catalysts.18 While photopolymerization (Pp) using a thiocarbamate function was already employed in molecular imprinting,19 to the best of our knowledge, it was never applied to the imprinting of fragile biomacromolecules such as whole proteins in their native configurations. Proteins being easily denatured when submitted to harsh conditions, Pp was never thought suitable to synthesize PIP despite Creed mentioning in the early 1980s that amino acid ionization due to UV irradiation was never observed for wavelengths higher than 300 nm,20,21 and Panikkanvalappil22 showing more recently that irradiating cells using UV light at 365 nm induced no damage to intracellular proteins. Considering these papers, there was a strong interest in using Pp to imprint whole proteins. We here present a novel synthetic pathway using Pp to obtain magnetic PIP nano-objects (M-PIP). Four different proteins of various molecular weights (Mw) and isoelectric points (pI), namely the green fluorescent protein (GFP), bovine serum albumin (BSA), ovalbumin (OVA), and lysozyme (Lyz), were successfully imprinted. The spatial arrangement of methylene-bis-acrylamide (MBAM) and acrylamide (AM) monomers around proteins, combined with MION functionalization using the diazonium salt chemistry, allow the use of Pp to obtain M-PIP. The effectiveness of

ynthetic nano-objects able to specifically bind macromolecules are of great interest nowadays for nanomedicine. Over the past years, molecularly imprinted polymers became of utmost importance. Indeed, synthetic antibodies can be created by imprinting a target molecule in a polymer matrix.1,2 Because of unique interactions existing between functional monomers and template, molecular imprinting creates specific recognition sites. Despite some challenges caused by the fragile nature of proteins, synthetic pathways were developed to obtain protein imprinted polymers (PIP).3−5 Mostly used for analytical6,7 or diagnostic8 purposes at the beginning, they are now increasingly employed in nanomedicine. Some PIP have recently been successfully used to target growth factors,9 either to inhibit them10,11 or as the recognition part of a targeted drug delivery system.12 Combining PIP to magnetic iron oxide nanoparticles (MION) could open doors to a novel class of synthetic materials for nanomedicine applications. Indeed, MION could be used to facilitate the manipulation of drug imprinted polymers once injected in vivo and successfully accumulate them in the area where the drug they contained is needed13 or even actively release it using their hyperthermia properties.14 Indeed, when submitted to an alternative magnetic field, MION will dissipate energy as heat and thus locally increase the temperature.15 Moreover, once coupled to MION, PIP will be easier to collect by the application of an external magnet, and time-consuming synthetic stages, such as washing steps using centrifugation, will be reduced. To ensure coupling between MION and PIP, the particle surface is functionalized, mostly to allow a grafting-from polymerization approach.16 When imprinting small molecules, functionalization agents © XXXX American Chemical Society

Received: February 4, 2019 Accepted: April 10, 2019 Published: April 10, 2019 A

DOI: 10.1021/acsapm.9b00109 ACS Appl. Polym. Mater. XXXX, XXX, XXX−XXX

Letter

ACS Applied Polymer Materials

Figure 1. Preparation of M-PIP for various proteins using photopolymerization. Template proteins were either green fluorescent proteins, bovine serum albumin, ovalbumin or lysozyme, and monomers were acrylamide and methylene-bis-acrylamide.

Figure 2. Nano-objects characterizations. (a) FTIR spectra and (b) thermograms of bare, initiator-functionalized, and M-PIP (after template protein extraction for FTIR spectrum).

weight loss of 7%, corresponding for 2% to the initiator. This slight value is due to the small size of the functionalizing agent. At the same time, weight losses corresponding to organic compounds for M-PIP were respectively 20% and 11% before and after template extraction, demonstrating the existence of the polymer layer and the effectiveness of the extraction steps. Synthesis of the polymer coating did not influence the crystal structure of MION (SI, Figure S2), which also maintained their magnetic properties after their functionalization (SI, Figure S3). Finally, PIP-coated MION were imaged using TEM. The polymer is clearly visible around nanoparticles as displayed in SI, Figure S4. A polymer shell formed around individualized MION, and these core−shell structures seemed to aggregate during either the synthesis or the magnetic decantation of washing steps, as the size of the aggregates observed by TEM is coherent with the size measured using dynamic light scattering (225 and 210 nm, respectively, SI, Figure S5). Adsorption properties of the as-synthesized nano-objects were then assessed to optimize the experimental protocol and produce M-PIP with the best possible adsorption performances. First, adsorption capacities (Qmax) and imprinting factor (IF) of nano-objects obtained from reaction mixtures containing functional monomer AM and cross-linking agent MBAM in different molar ratios were compared. The best ratio was found to be 5.54 as it led to the higher IF (SI, Figure S6). Second, we observed that it was necessary to let cross-linking agents organize themselves around proteins to be imprinted before starting polymerization. Indeed, M-PIP synthesized from prepolymerization mixtures containing MBAM present higher Qmax than when MBAM is added as the UV light is switched on (SI, Figure S7). In the first case, MBAM molecules may already have prearranged themselves around proteins to be printed. A rigid structure can appear from the first instant of UV irradiation, sufficient to imprint proteins in

MION surface functionalization and polymer synthesis was assessed and adsorption properties of M-PIP were demonstrated by rebinding experiments, the as-prepared nano-objects displaying outstanding adsorption capacity and selectivity. M-PIP were synthesized as displayed in Figure 1. First, MION synthesized using a coprecipitation method were covalently bond to a diethyldithiocarbamate function, using the specific chemistry of diazonium salts,23 to later initiate the Pp24,25 (Supporting Information (SI), Figure S1). Modified MION were added to a reaction medium containing prepolymerization complexes formed by proteins to be printed and monomers. Proteins were either GFP, BSA, OVA, or Lyz. Pp was then allowed to proceed at room temperature under UV light irradiation (365 nm) for different time. Magnetic nonimprinted polymer nano-objects (M-NIP) were synthesized using the same protocol without template proteins. To assess the effectiveness of the different synthesis steps, Fourier-transform infrared (FTIR) spectra of bare, initiatorfunctionalized and PIP-coated MION were recorded (Figure 2a). MION present peaks at 628 and 580 cm−1, corresponding to the Fe−O vibration. FTIR spectrum of initiator-functionalized MION depicts new peaks around 2900 cm−1 and 1630 cm−1, corresponding respectively to the stretching of C−H and CO bonds from the carboxylic acid function. One can also denote in Figure 2a the absence of a peak corresponding to NN bond of diazonium salts, confirming the modification of the diazonium salt structure. Moreover, a peak appears around 1400 cm−1 on the FTIR spectrum of M-PIP corresponding to C−N vibrations of acrylamide, along with another one around 1100 cm−1 characteristic of C−C elongations in polymer chains. All these different IR peaks suggest a successful synthesis of polymer coatings. To further characterize nano-objects, thermogravimetric analysis (TGA) was performed and thermograms are shown in Figure 2b. Initiator-functionalized nanoparticles displayed a B


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Figure 3. Adsorption isotherms of different proteins on the corresponding M-PIP and M-NIP. (a) GFP, m = 0.2 mg. (b) BSA, m = 0.8 mg. (c) OVA, m = 0.8 mg. (d) Lyz, m = 0.9 mg. All experiments: V = 2 mL, 24 h.

Figure 4. (a) Adsorption kinetics for GFP on M-PIP, Ci = 0.75 μg/mL. (b) Adsorption selectivity of magnetic GFP-imprinted polymers (PIP), Ci = 11 μmol/L. All: V = 2 mL, m = 0.2 mg, room temperature.

presented in Figure 3. As one can see, M-PIP (black curves) adsorbed more proteins than M-NIP (orange curves) for the four proteins used as template. This confirms the imprint’s presence and their ability to recognize proteins in their native configuration. Photopolymerization seems to be a robust way to synthesize PIP, as it can be applied to a wide range of proteins having different Mw, configurations, and pI. The adsorption equilibrium of proteins on corresponding M-PIP and M-NIP were found to correspond to a linearized Langmuir isotherm (SI eq S-2). By fitting experimental curves presented in Figure 3 with this model, association constants (K) and Qmax values were found for the adsorption of proteins on corresponding M-PIP and M-NIP (SI, Table S1). From the isotherms in Figure 3, one can suppose that acrylamide interacts more strongly with GFP (Figure 3a) than any other protein, maybe because this protein is the less charged one in the solvent used for synthesis and therefore the most likely to be bound by a noncharged monomer while forming

their native configuration. While in the second case, protein denaturation may occur before the formation of a polymer sufficiently rigid to maintain the protein configuration. These results highlight the importance of the reaction mixture composition, as already mentioned in the literature.26 Finally, as irradiation time directly influences the polymer thickness and therefore Qmax, we synthesized both M-NIP and magnetic GFP-imprinted polymers with irradiation time ranging from 2 to 5 h. As one can see in SI, Figure S8, the amount of protein adsorbed (Q) by M-NIP do not vary significantly from a 2 h to a 5 h irradiation, while it dramatically increases for M-PIP as displayed in Figure 3a. After only 2 h of irradiation, polymer may not be synthesized homogeneously on all magnetic nanoparticles, explaining the wider range covered by Q values. Optimal irradiation time was found to be 5 h. To assess how robust the experimental protocol was, adsorption capacities of M-PIP and M-NIP toward different proteins were determined using rebinding experiments, C


Letter


ones than what was previously obtained. As this synthesis method proved to be robust toward proteins of various Mw and pI, we strongly believe in its applicability to a whole new range of other proteins, which could be helpful for applications in nanomedicine.

prepolymerization complexes (see SI for pI). Interestingly, the high similarity of BSA and OVA amino acid sequences is reflected in the similar adsorption capacities observed for corresponding M-PIP (Figure 3b,c). The higher nonspecific adsorption observed for OVA may be due to the proteins different Mw, as smaller proteins can more easily diffuse inside the polymer and nonspecifically adsorb there. Curiously, the synthetic pathway proposed here led to higher K values than what was obtained in our previous work using a different method to initiate polymerization,27 while IF are not significantly different (SI, Table S2). This highlightsthe fact that while 1 g of photopolymerized smaller particles have higher surface to volume ratio and consequently present more nonspecific adsorption sites, more imprints can be accessible because of a thinner polymer coating. Adsorption specificity is therefore an intrinsic property of the polymer and do not depend on its synthesis pathway, as other properties may. To further investigate protein−PIP interactions, we carried out a kinetic study with magnetic GFP-PIP. As one can see in Figure 4a, equilibrium is reached in less than 2 h. Considering there is specific and nonspecific adsorption sites, an adsorption model describing the possible simultaneous binding on both kind of sites may be better suited than the widely used ones considering only one site for adsorption while fitting kinetics data.28 Because of the size of nano-objects, adsorption sites can be considered as independent ligands, and global adsorption kinetics can be described by the sum of kinetics for both kind of adsorption sites, each one following either a pseudo-first- or a pseudo-second-order kinetics model (see SI for more details). Experimental data were fitted with the four possible combinations of kinetics models, as well as with models considering only one type of adsorption sites. Kinetics parameters and correlation coefficient values are displayed in SI, Table S3. They suggest that a pseudo-first-order model is the most suitable to describe interactions between template protein and M-PIP, and that a two-ligand kinetics model may be more accurate to describe this kind of adsorption data. Interestingly, specific adsorption is slower than the nonspecific one, as the rate constant is lower. It may be due to the need for proteins to have a specific orientation to adsorb inside the imprints. Then, we chose to assess the selectivity of GFP-PIP while using OVA and Lyz as competitive proteins. As displayed in Figure 4b, GFP-PIP adsorbed more GFP (17.1 μmol/g) than the other proteins (OVA, 8.6 μmol/g, and Lyz, 9.3 μmol/g), even if one can denote the presence of some nonspecific adsorption. The absence of significant differences between adsorption capacities of GFP-PIP and NIP toward either OVA or Lyz indicates a purely nonspecific adsorption due to the nature of the polymer and not to the imprints. M-PIP obtained through Pp are able to specifically adsorb the template protein. Finally, to assess the regenerative properties of M-PIP, adsorption−desorption cycles were carried out 5 times as described in SI. No significant changes in the Q values of MPIP were recorded (SI, Figure S9). This points out a great regeneration capability of the particles presented here. In conclusion, we developed a novel synthetic pathway for PIP coatings on MION using Pp. M-PIP are synthesized combining a grafting approach from the MION surface, functionalized using the diazonium salt chemistry and for the first time the use of UV-light to initiate polymerization. The obtained material display high adsorption capacities toward template proteins, and affinity constants nearer to biological

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsapm.9b00109. Experimental section, nano-objects characterization, and adsorption performances data for magnetic PIP nanoparticles (PDF)

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AUTHOR INFORMATION

Corresponding Author

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

Charlotte Boitard: 0000-0002-8381-8144 Nébéwia Griffete: 0000-0003-4888-6261 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Aude Michel and Delphine Talbot for their help while characterizing the different nano-objects. The authors also would like to thank the Dahan Group from the Curie Research Institute who provided the GFP. There is no funding to acknowledge.

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ABBREVIATIONS MION, magnetic iron oxide nanoparticles; (M)-PIP, (magnetic)-protein imprinted polymer nano-objects; (M)-NIP, (magnetic)-non imprinted polymer nano-objects; GFP, green fluorescent protein; BSA, bovine serum albumin; OVA, ovalbumin; Lyz, lysozyme; pI, isoelectric point; Pp, photopolymerization; FTIR, Fourier-transformed infrared spectroscopy; TGA, thermogravimetric analysis; TEM, transmission electron microscopy; AM, acrylamide; MBAM, methylene-bisacrylamide.

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REFERENCES

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Whole Protein Imprinting over Magnetic Nanoparticles Using

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