Pentafluorophenyl-based Single-Chain Polymer Nanoparticles as a Versatile Platform towards Protein Mimicry

Pentafluorophenyl-single chain polymer nanoparticles are readily conjugated with functional amines enabling facile SCNP modification, adjustment of physicochemical properties, and even protein mimicry.


Introduction
Protein-like synthetic polymers aim at combining the modularity and scalability of polymer synthesis 1 with the specific functions of proteins, such as catalytic activity, signalling, transport and storage. [2][3][4][5] Since proteins are able to perform a remarkable number of intricate functions under physiological conditions based on their structural conformation, replication of protein function and shape has become a primary goal for researchers worldwide. Mimicking natural proteins by synthetic polymer chemistry may be approached by several different routes. A promising strategy is the bioconjugation of peptides and proteins with polymers. 6,7 By combining the structural properties of the synthetic backbone with highly specific functions of a protein domain, hybrid materials are obtained with a range of versatile properties. 8 One of the first striking examples of a protein-polymer conjugate is the attachment of ( poly)ethylene glycol chains onto bovine serine albumin, reducing toxicity and improving pharmacokinetic profile. 9 Since then, a wide variety of chemistries has been used to create polymer-protein hybrids, 10 introducing functionalities such as catalysis 4 and biosensing 11 into synthetic polymeric materials. Although protein-polymer conjugates can mimic specific characteristics, there is little resemblance to the structural features and shape of a protein. Therefore, the folding of synthetic polymers, like the spatial arrangement of a protein, has become a primary goal towards protein mimicry.
Single-chain polymer nanoparticles (SCNPs) are prepared through exclusive intramolecular crosslinking of individual polymer chains. [12][13][14][15][16][17][18][19][20][21][22][23] Size and dispersity of SCNPs are directly related to the size and size distribution of the polymers from which they are prepared, and can therefore be controlled very accurately, yielding nanoparticles in the 5-20 nm size range, even resembling size and structure of proteins. [24][25][26] In order to approximate specific protein characteristics, control over the self-recognizing motifs determining secondary and tertiary structure, [27][28][29][30][31] as well as catalytic activity have been studied. [32][33][34] Current applications of protein-like SCNPs have focused on the development of enzyme-like materials, [33][34][35][36] and applications in the field of drug delivery, for example aiming at biocompatibility and targeted therapeutics delivery. [37][38][39][40][41] In order to achieve protein mimicry, besides controlling monomer sequence and chain folding, equipping SCNPs with different amino acids or peptide moieties further approaches the composition of natural proteins. SCNPs with protein-like characteristics can either be prepared through functional precursor polymers, as is the case with poly(γ-glutamic acid) precursors for example, [42][43][44] or by post-formation functionalization of SCNPs. [45][46][47] Post-formation functionalization enables modification of polymers, 48 nanoparticles 49,50 and nanogels 51,52 without markedly altering their size and structure. This strategy has been utilized to conjugate peptides onto SCNPs for targeting purposes, by covalently attaching the peptide via amidation to carboxylic acid-functional SCNPs, 38 as well as for peptide delivery by reversibly binding the peptide via disulfide linkages. 38,45 Even proteins have been attached onto SCNPs via disulfide linkages or NHS (N-hydroxysuccinimide) ester chemistry. 46 Exploiting post-formation functionalization of SCNPs to mimic proteins would allow careful tuning of the surface properties, while maintaining the SCNP structure intact.
Pentafluorophenyl (PFP) esters have found widespread application in bioconjugation chemistry. 53 These activated esters are soluble in a range of organic solvents and less susceptible to hydrolysis than NHS esters. 54,55 PFP-functional polymers have been successfully used in post-polymerization functionalization reactions with both primary and secondary amines. 55 Conjugation of antibodies to PFP-acrylate polymer brushes was achieved by Son et al., enabling capture and purification of target proteins. 56 In the stepwise compaction of N-substituted maleimide copolymers, the PFP-moiety was used as intramolecular crosslinking reaction to prepare SCNPs. 30,57 Furthermore, Palmans and co-workers used poly(PFP-acrylate) to prepare polymer precursors for intramolecular self-assembly of SCNPs. 58 Recently, PFP-polymers were also used to synthesize SCNPs with increased catalytic activity, even working inside HeLa cells. 59,60 In this study, we report the preparation and characterization of PFP-functional SCNPs via thiol-Michael addition crosslinking, and their use as a flexible platform to easily modify and functionalize SCNPs, while maintaining the SCNP backbone structure unaltered. Various functional amines, including amino acids and peptides, are conjugated onto the SCNPs to yield protein-like materials, while at the same time allowing for facile conjugation of fluorescent labels, click-functionality, and affording control over polarity, hydrophilicity and biocompatibility.

PFP-polymer
Prior to polymerization, PFPMA was filtered over neutral alumina to remove inhibitor. PFPMA and XMA were copolymerized in molar ratios of 10 : 1. In a typical polymerization experiment, PFPMA (3.38 g, 13.4 mmol), XMA (0.35 g, 1.52 mmol), RAFT agent CPADB (12.5 mg, 0.045 mmol) and AIBN (1.47 mg, 0.009 mmol) were combined with 1,4-dioxane (6 mL) in a polymerization flask fitted with a rubber septum. After purging for 15 minutes with nitrogen, the flask was placed in a preheated oil bath at 80°C. After reaching 60-70% conversion, dichloromethane (2 mL) was added to the cooled flask and the polymer was precipitated twice in cold n-hexane. 1

PFP-SCNP functionalization
In all functionalization experiments, PFP-SCNPs were dissolved in a solvent compatible with the reagents and products under nitrogen atmosphere, followed by addition of TEA and a primary amine (see Table 1). The solution was heated to 45°C and stirred until full conversion was observed by 19 F NMR measurements. For example, for SCNP-F2, 175 mg of PFP-SCNP (0.63 mmol PFPMA units) was dissolved in 4 mL THF. Under nitrogen atmosphere, TEA (265 mg, 2.52 mmol, 4 eq.) in 3 mL DMSO was added to the solution, followed by 1-aminoglycerol (575 mg, 6.30 mmol, 10 eq.) in 6 mL DMSO. The reaction was stirred overnight at 45°C, followed by dialysis against H 2 O. Functionalized SCNPs were isolated by lyophilization.
For SCNP-F5-F7, PFP-SCNPs were first partially reacted with 1-aminoglycerol. For example, for SCNP-F5, a solution of 75 mg PFP-SCNP (0.25 mmol PFP) and 140 μL TEA in 7 mL THF was nitrogen purged and heated to 45°C. 1-Aminoglycerol (20 mg, 0.85 eq.) in 6 mL DMSO was added and the heated solution was stirred overnight. Full amine conversion of the reaction mixture was determined by 19 F NMR measurements. Subsequently, L-alanine (H NMR and 19 F NMR measurements and 221 mg, 2.5 mmol, 10 eq.) in 3 mL PBS were added and the mixture was stirred overnight. Subsequently, 1-aminoglycerol (107 mg, 4.5 eq., 1.12 mmol) in 2 mL DMSO were added. After full conversion of the PFP moieties, the product was dialysed against water and lyophilized.

Nickel affinity chromatography
The nickel affinity column was loaded with 10 mg of SCNP-F7 or SCNP-F8 in buffer solution and incubated on a roller bank overnight. The fractions were subsequently eluted with washing buffer (15 mL) and elution buffer (2 × 15 mL). Fractions were desalted using centrifugal desalting devices and lyophilized.

Fluorescent labeling via click conjugation
To a solution of SCNP-F3 (10 mg, 6.4 µmol alkyne units) in 3 mL THF nitrogen purged, azide Alexa Fluor 488 (0.5 mg in 2 mL DMF), 2 µL N,N-diisopropylethylamine and 1.2 mg CuI in 1.2 mL DMF were added. The solution was stirred overnight at 60°C. After isolation by dialysis and lyophilisation, SCNP-F3a was obtained as an orange solid.

Cytotoxicity assay
hCMEC/D3 cells ( passage 30) were seeded on collagen I coated 96-well plates at 10 × 10 3 cells per well in 100 µL EndoGro medium and incubated for 24 h at 37°C in a humidified 5% CO 2 -containing atmosphere. SCNP-F2 was dissolved in EndoGro medium and filtered with a 0.2 µm filter. The stock solution was diluted to the desired concentrations (500, 200, 100, 50, 10 and 1 µg mL −1 ) and 100 µL was added to the cells. The measurements were performed in triplicates. For the reference and the positive control, 100 µL of EndoGro medium was added. The medium of the positive control was aspirated 30 min prior to the assay and replaced by 70% methanol. After 24 and 48 h of incubation, the cells were washed with HEPES buffer and 100 µL of resazurin sodium salt (440 mM) in PBS was added to the cells. The fluorescence intensity was analyzed after 4 h for hCMEC/D3 cells incubation at 37°C in a humidified 5% CO 2 -containing atmosphere with a Tecan infinite m200 plate reader at an excitation and emission of 560/ 590 nm (gain of 70).

Results and discussion
Protected thiol monomer (XMA) 63 and pentafluorophenyl methacrylate monomer (PFPMA) 55 were synthesized according to literature procedures and copolymerized via RAFT polymerization. The polymerization followed first order kinetics and both monomers were consumed at comparable rates as observed by 1 H NMR spectroscopy, yielding copolymers (PFPpolymer) with PDI ∼ 1.4, as determined by size exclusion chromatography (SEC) measurements ( Fig. 1 and Fig. S1 †).  PFP-SCNP formation was carried out via thiol-Michael addition crosslinking as reported previously. 62 Full deprotection of the xanthate moieties in precursor polymer with ethanolamine was confirmed by 1 H NMR and 19 F NMR spectroscopy ( Fig. 1b and Fig. S2 †). The reaction proceeded more slowly than in earlier investigated XMA copolymers, but nonetheless remained much favored over amine substitution of PFP groups under the selected reaction conditions. 39,62 Within 2.5 h, the xanthate moieties are fully deprotected as observed by the disappearance of signals corresponding to the xanthate moiety at 4.6 ppm and the shift of the CH 2 -S signal in 1 H NMR spectroscopy (Fig. S2 †). At the same time, the PFP moieties are functionalized only to a limited extend (∼7%), as observed by 19 F NMR spectroscopy (data not shown).
Intramolecular crosslinking of the deprotected PFP-Polymer with 1,4-butanediol diacrylate, followed by endcapping of residual thiol moieties with methyl acrylate, yielded PFP-SCNPs, displaying 30-65% size reductions in comparison to the polymer precursor as determined by SEC analysis (Scheme 1 and Fig. 1a). The extent of size reduction increased with increasing precursor chain length (Fig. S3 †). DLS measurements revealed particles of approximately 14 nm in diameter. Potential residual thiols were reacted with methyl acrylate. Additional signals of the crosslinker (signals d and f in Fig. 2) and of the methyl acrylate endcapper (signal e in Fig. 2) are identified in the 1 H NMR spectra of PFP-SCNPs. The 19 F NMR signal corresponding to the ortho-fluoro atoms displays a broadening upon crosslinking, which is not observed for the meta-and para-fluoro peaks (see Fig. S4 †). This peak broadening might be assigned to increased crowding around the pentafluorophenyl rings upon chain compaction. The restricted mobility may lead to additional line-broadening of the ortho-fluorine atoms. Furthermore, increased dipoledipole relaxation between the fluorine and hydrogen nuclei due to their increased vicinity upon compaction further contributes to the increased signal width. 64 PFP-SCNPs were functionalized with n-butyl amine (SCNP-F1) to achieve acrylamides, which are known for their thermoresponsiveness. 65,66 Complete functionalization was confirmed by disappearance of the signals in 19 F NMR measurements (Fig. S5 †), whereas in the 1 H NMR spectrum n-butyl amide signals appeared (Fig. S6 †). In addition, FT-IR measurements further confirmed PFP substitution by the disappearance of signals at 1000 cm −1 and 1515 cm −1 , assigned to C-F bonds and the ring vibration of the PFP moiety, as well as by the emergence of signals in the amide region at 1658 cm −1 .
Experiments with PFP substitution by ethanolamine proved insufficient to grant water-solubility to the nanoparticles. PFP-SCNPs were therefore reacted with 1-aminoglycerol to obtain water-soluble and biocompatible SCNPs (SCNP-F2), which was confirmed similarly by FT-IR and 1 H NMR spectroscopy ( Fig. 3a and Fig. S7 † respectively). Surprisingly, DLS measurements indicated structures of 100 nm in diameter for SCNP-F2, as depicted in Fig. 3b, whereas the size of SCNP-F1 remained unaltered after functionalization (∼10 nm). Upon sodium chloride addition, however, two populations of 100 nm and 10 nm became apparent. With increasing amounts of salt, the 10 nm species became more pronounced, suggesting the earlier observed larger aggregates formed as a result of electrostatic interactions between the amide diols. TEM imaging of SCNP-F2 confirmed particles of 10 nm in diameter (Fig. 3c). Furthermore, SEC elution displayed a single Scheme 1 SCNP formation by intramolecular chain collapse of PFP-polymer after aminolysis. size population for SCNP-F2 (Fig. 3d). The relative sizes of PFP-SCNP and SCNP-F2 cannot be compared by SEC as they are obtained by relative calibrations in different solvent systems with different standards.
As a first step towards future biomedical applications, cellular toxicity of SCNP-F2 was evaluated on the brain endothelial cell line hCMEC/D3. No significant decreases in metabolic activity were observed, even after 48 hours of incubation with increasing concentrations of SCNPs up to 500 µg mL −1 (Fig. S8 †). These preliminary results indicate the potential of aminoglycerol-functionalized SCNPs for biological use and point to further investigations into cellular compatibility and intracellular localization.
In order to utilize SCNPs in bioconjugations, propargylamine was conjugated onto the PFP-SCNPs as an orthogonal handle for efficient click-functionalization (SCNP-F3). Given that click chemistry can be performed on these SCNPs, the alkyne pendant may for example be utilized to add radioactive labels for tracking the particles in in vivo studies. 67 1 H NMR spectroscopy confirms signals of the alkyne bonds at 2.2 and 3.9 ppm, respectively (see Fig. S9 †), indicating replacement of approximately 10% of the PFP moieties by propargyl amine. The presence and accessibility of the alkyne moieties were verified by a copper catalyzed azide-alkyne Huisgen click reaction with azide-functional Alexa Fluor 488. The click reaction resulted in an orange, fluorescent solid and, in accordance with rhodamine conjugation, SEC chromatography revealed UV-vis and fluorescence signals at the elution time of the click product (SCNP-F3a; Fig. 4a and Fig. S10a †).
With the aim of approaching protein-mimicry, functionalization with amino acids was pursued next, as summarized in Scheme 2. PFP-SCNPs were reacted with 0.05 molar equivalents of tyrosine methyl ester, an amino acid with a protected carboxylic acid, and accordingly, 5% conversion of PFP-esters was observed by 19 F NMR spectroscopy (Fig. S11 †).
In order to modify PFP-SCNPs with natural amino acids such as alanine, PFP-SCNPs were first partially functionalized with 1-aminoglycerol to render the particles water-soluble and then reacted with alanine in a buffered solution at pH = 7 to minimize potential hydrolysis. The sequential addition of 1-aminoglycerol and alanine was followed by 1 H and 19 F NMR spectroscopy revealing 30% alanine substitution, which is in agreement with the prospected degree of functionalization. Excess 1-aminoglycerol was added after conjugation of the respective amino acids to ensure full conversion of PFP moieties, yielding SCNP-F5. Successful conjugation of alanine and 1-aminoglycerol onto the SCNPs was confirmed by 1 H NMR spectroscopy revealing among others new amide signals, as well as a carboxylic acid signal at 12.5 ppm (Fig. S13 †). In control experiments without alanine, no significant PFP hydrolysis was detected. Interestingly, the zeta potential of SCNP-F5 decreased to −23 mV as compared to −13 mV for the fully 1-aminoglycerol functionalized SCNP-F2 (Table 1), due to the presence of carboxylate groups.
Hence, the sequential addition of functional amines may be conducted to facilitate conjugation of non-water-soluble components or to economize the use of precious substituents. Analogously, the peptides glutathione (GSH) and hexa-histidine (His 6 ) were added onto partially modified aminoglycerol SCNPs, yielding SCNPs-F6 and SCNP-F7, respectively. His 6functionalized SCNP-F7 showed comparable size and zeta potential to the alanine-functional SCNP-F5. Carboxylic acid

Polymer Chemistry Paper
This journal is © The Royal Society of Chemistry 2020 signals became apparent after the functionalization with peptides, but no other distinct signals corresponding to the conjugated peptides could be identified by 1 H NMR spectroscopy, due to strongly overlapping amide, alkane and water signals. In order to confirm GSH conjugation to PFP-SCNPs, doubly 13 C-labeled GSH was incorporated (2%) in a similar manner. 13 C NMR spectroscopy revealed two additional signals at 41 and 172 ppm as compared to SCNP-F2, corresponding to the 13 C-enriched carbonyl and α-carbon respectively, thereby confirming successful conjugation (Fig. S14 †). The sequential substitution of PFP-ester moieties was further utilized to directly equip SCNPs with a fluorescent label. Rhodamine ethylenediamine was added to SCNPs with 70% 1-aminoglycerol substituted PFP moieties to yield SCNP-F8. Successful addition of the dye was confirmed by new UV-vis and fluorescence signals in SEC measurements at the elution volume corresponding to the SCNPs (Fig. 4a and S10b †).
Affinity chromatography with nickel-nitrilotriacetic acid (Ni 2+ -NTA) columns is a well-established method for purification of recombinant proteins from cell lysate. 68 Proteins modified with a polyhistidine-tag (usually His 6 -tag) display strong affinity to the nickel column and can thus be isolated from other proteins. With the aim of validating the integrity of peptides after coupling to the nanoparticles, SCNP-F7, which contains His 6 peptides in addition to 1-aminoglycerol moieties, was tested for affinity towards Ni 2+ -NTA affinity columns. Rhodamine-functionalized SCNPs (SCNP-F8) served as a negative control. SCNP-F8 fully eluted from the Ni 2+ -NTA column already upon flushing with the washing buffer (see Fig. 4b) and no further rhodamine elution was observed when flushing the column with imidazole-containing buffer (elution buffer). On the contrary, 1 H NMR analysis on the lyophilized fractions eluting from the SCNP-F7 loaded Ni 2+ -NTA column revealed that SCNP-F7 is only released upon eluting with an imidazolecontaining buffer. Consequently, the His 6 -conjugated SCNP-F7 interacts well with the affinity column, confirming successful peptide conjugation.

Conclusions
In this work, we demonstrate the development of PFP-SCNPs, based on covalent intramolecular chain collapse and the subsequent modification of these nanoparticles yielding highly modular, water-soluble SCNPs. Facile amine conjugation provides an easy way towards peptide-functionalization for more advanced biomedical applications. Furthermore, conjugation of a His 6 -tag peptide to the SCNPs, enabled to transfer a classical protein purification method to polymers. Besides direct addition of amino acids and peptides, also modification with simple, functional amines, such as ethanolamine as chemical analogue to serine, aids in mimicking protein appearance.
In principle, nearly any physicochemical function may be added to the SCNPs via amine post-formation in an easy and efficient manner. Additionally, parallel functionalization may be performed on a single type of nanoparticles, offering the possibility to alter particle functionality, without inherently changing particle size and dispersity, which would greatly facilitate comparative cell experiments, for example to study ligand density effects. Addition of alkyne groups onto the SCNPs enables click functionalization, giving way to radiolabeling of SCNPs for in vivo studies. Current work is focused on the effects of peptide conjugation on SCNP uptake and biodistribution behavior. As a protein mimic, amino acid decorated SCNPs may appropriate some of the numerous features proteins display, such as biocompatibility and receptor binding, greatly extending the potential and understanding of SCNPs in biomedical applications.

Conflicts of interest
The authors have no conflicts of interest to declare.