Self-Assembly of Telechelic Tyrosine End-Capped PEO Star Polymers

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Self-assembly of Telechelic Tyrosine EndCapped PEO Star polymers in Aqueous Solution Charlotte J. C. Edwards-Gayle, Francesca Greco, Ian W. Hamley, Robert P. Rambo, Mehedi Reza, Janne Ruokolainen, Dimitrios Skoulas, and Hermis Iatrou Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01420 • Publication Date (Web): 01 Dec 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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Self-assembly of Telechelic Tyrosine End-Capped PEO Star polymers in Aqueous Solution Charlotte J C Edwards-Gayle, Francesca Greco and Ian W Hamley*,a School of Chemistry, Food Biosciences and Pharmacy, University of Reading, Whiteknights, Reading RG6 6AD, U.K. Robert P Rambo Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, U.K. Mehedi Rezab and Janne Ruokolainen Department of Applied Physics, Aalto School of Science, P.O. Box 15100 FI00076 Aalto, Finland. Dimitrios Skoulasc and Hermis Iatroud Department of Chemistry, University of Athens, Panepistimiopolis Zografou, 157 71, Athens, Greece

* Author for correspondence. [email protected] a) orcid ID 0000-00024549-0926 b) orcid ID 0000-0003-0256-0629 c) orcid ID 0000-0002-2549-8867 d) orcid ID 0000-0001-9358-0769

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Abstract We investigate the self-assembly of two telechelic star polymer-peptide conjugates based on poly(ethylene oxide) (PEO) four-arm star polymers capped with oligotyrosine. The conjugates were prepared via N-carboxy anhydride (NCA)-mediated ring-opening polymerization from PEO star polymer macroinitiators. Self-assembly occurs above a critical aggregation concentration determined via fluorescence probe assays. Peptide conformation was examined using circular dichroism spectroscopy. The structure of self-assembled aggregates was probed using small-angle Xray Scattering (SAXS) and cryogenic transmission electron microscopy (Cryo-TEM). In contrast to previous studies on linear telechelic PEO-oligotyrosine conjugates which show self-assembly into βsheet fibrils, the star architecture suppresses fibril formation and instead micelles are generally observed, a small population of fibrils only being observed upon pH adjustment. Hydrogelation is also suppressed by the polymer star architecture. e. These peptide-functionalized star polymer solutions are cytocompatible at sufficiently low concentration. These systems present tyrosine at high density and may be useful in the development of future enzyme or pH-responsive biomaterials.

Introduction Conjugation of polymers and peptides can lead to interesting biomaterials with diverse applications, due to the combination of unique and novel properties of both components.1–8 A wide range of polymers can be synthesised inexpensively, and conjugated with peptides to produce materials with enhanced biocompatibility or bio-functionality. This can also lead to modified behaviour of the polymers, as these will be influenced by the nature and size of the incorporated peptide, and whether the peptide is attached laterally, forms part of the repeating unit, or whether it is attached to either one or both ends of the linear polymer. Therefore, a large range of novel materials can be generated from combining polymers and peptides, including peptide-functionalized polymer hydrogels,9–14 which have important applications for example as supports or matrices for cells with applications in tissue engineering,15–25 or in slow release drug delivery systems.26–31 2 ACS Paragon Plus Environment

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Capping hydrophilic polymers with hydrophobic end groups creates telechelic polymers. Telechelic polymers possess useful rheological behaviours due to their noncovalent end group associations. This is exploited in associative polymers,32,33 which are widely used as thickeners and viscosity modifiers, and in polyethylene glycol (PEG)-based polymers, such as Pluronics, which are widely used polymeric surfactants (which can also form hydrogels at sufficiently high concentration in aqueous solution).34 Pluronics have a linear triblock polymer architecture. Tetronics are analogous polymers but with a four-arm star architecture.34 PEG- or PEO-(poly ethylene oxide)- based polymers have low toxicity and high biocompatibility. They have been proposed to have the ability to be inert spacers within peptide sequences to improve stability and circulation time of actives, for applications such as gene delivery,35 cell signalling36 or drug delivery.31 Polymer-peptide conjugates with a 4-arm star architecture have been prepared previously for several applications. Iatrou et al. synthesized 4-arm PEO star molecules functionalised with poly(γ-benzyl-L-glutamate).37 Reineke and coworkers described the preparation of 4-arm PEG peptide star molecules modified with heparin binding peptides and examined the cytocompatibility and affinity of these peptides for heparin which could have applications as DNA delivery vehicles.38 There have been few studies on the self-assembly properties of telechelic peptide-polymer conjugates. Heise and co-workers have recently investigated PEG-oligotyrosine diblock copolymers. PEG2000-Tyr6 was found to undergo thermo-responsive gelation at relatively low concentration.39 The hydrogel was found to be biodegradable and not cytotoxic. Interestingly, conjugates containing higher molecular weight PEG, PEG5000 with varying lengths of poly(tyrosine) werefound to not form hydrogels. Hydrogelation was thought to be driven by the amphiphilic balance of PEO and tyrosine, and the hydrogen bonding in the β-sheet secondary structure and phenolic groups.39 The Hamley group reported on the gel-sol transition of PEO conjugates with hydrophobic dipeptides, including dityrosine and diphenylalanine.11 These conjugates were synthesised by coupling dipeptides to bisaminopropyl PEO with a molar mass of 1500 g mol-1. The Fmoc [Fluorenylmethyloxycarbonyl] protecting group, which was used in the synthesis, was retained in some of the prepared conjugates, 3 ACS Paragon Plus Environment


at one or both termini, influencing the hydrophile-lipophile balance thus resulting in a significant impact on the self-assembly behaviour. One conjugate with a di-tyrosine cap and a C-terminal Fmoc protecting group was found to undergo a gel-sol transition period near body temperature. This was driven by loss of β-sheet structure associated with extended fibril formation.11 Recently the selfassembly of three tyrosine-capped PEO and poly(L-alanine), (PAla), polymers has been examined.40 These polymers were synthesised by living NCA (N-carboxy anhydride) polymerization techniques which allows control of polymer chain length, and conjugates containing PEO with a mass of 2 kg mol-1 or 6 kg mol-1 , or poly(alanine) were prepared. All three polymer conjugates were shown to assemble into β-sheet fibrillar structures imaged by cryo-TEM. Interestingly, hydrogel formation was not observed for any samplesup to moderately high concentration.37 The Lendlein group also worked with similar telechelic conjugates of DAT (desaminotyrosine) or DATT (desaminotyrosyl-tyrosine), with a linear PEG (3 kg mol-1) midblock and also four arm-PEG conjugates, which were shown to self-assemble at high concentration.41 The same group investigated star-shaped and linear oligo-ethylene glycol/ DATT conjugates.41 Moreover, the structural, conformation and rheological properties of gelatin (which has collagen-based helical structure) functionalised with either DAT or DATT at lysine residues was examined.42,43 Fibrillar hydrogels have also been reported for PAla-PGlu-PAla, [PGlu: poly(L-glutamic acid)] in aqueous buffer.44 Lui et al described the synthesis of 4-arm poly(tert-butyl acrylate) functionalised with peptide sequences modified from the protein-derived antigen E2 of the human papillomavirus (HPV) proteinderived antigen E2. These star polymers were shown to self-assemble into large particles between 500 nm-1 µm. in size. They were able to function as a self-adjudicating vaccine delivery system, demonstrated in mice.45 Here, we investigate the self-assembly in aqueous solution of conjugates comprising telechelic PEO star-shaped polymers capped with short tyrosine sequences at native pH and pH 12 This pH was selected as it is above the pKa of tyrosine (10.1) and it has previously been


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shown that this influences the peptide conformation and interactions between peptide chains.46 The creation of tyrosine-functionalised conjugates is of interest in the development of biomaterials, responsive to tyrosine-specific enzymes, for example. We aim to investigate what effect the star architecture has on the self-assembled nanostructure, peptide conformation and gelation behavior compared to analogous linear PEO-oligotyrosine conjugates. In our conjugates, the central core PEO star molecular mass is either 10 kg mol-1 or 20 kg mol-1 in total i.e. 2.5 kg mol-1 or 5 kg mol-1 per arm respectivelyTyrosine is considered to be hydrophobic according to the majority of hydrophobicity scales47,48 (excluding the Kyte-Doolittle scale49), and is capable of undergoing π-π stacking (aromatic) interactions. Tyrosine is involved in biological interactions including cell signalling, where it is responsive to a large number of tyrosine kinase and phosphatase enzymes, which have the ability to phosphorylate or dephosphorylate tyrosine residues. These interactions are vital for a large number of signalling cascades, and are of clinical interest because dysregulation is highly linked to a number of cancers.50,51 As well as reporting on self-assembly of the conjugates in aqueous solution, the peptide conformation is also examined using a combination of spectroscopic and X-ray diffraction methods. Finally the cytocompatibility of the conjugates is examined.

Experimental Section Materials Boc-Tyr(tBu)-OH, (99%) was purchased from Bachem. Thionyl chloride (99.7%, Acros Organics) was distilled prior to use. 4-arm poly(ethylene oxide) star amine terminated with number-average molar mass Mn = 1.0 x 104 g/mol, and 4-arm poly(ethylene oxide) amine terminated hydrochloric salt with total molar mass, Mn = 2.0 x 104 g/mol were purchased from Aldrich. The PEO star with Mn = 1.0 x 104 g/mol was used as received, while the other one was neutralized with NaOH before being used 5 ACS Paragon Plus Environment


as macroinitiator. The solvent purification was performed using standard high vacuum techniques reported elsewhere.52

Synthesis of tert-Butyl protected N-carboxy Anhydride of L-Tyrosine (Tyr(tBu)-NCA) The synthesis of tert-butyl protected N-carboxy anhydride of L-Tyrosine (Tyr(tBu)NCA) has been reported elsewhere.40

a) Synthesis of [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 4-arm star All manipulations and polymer synthetic procedures were performed under high vacuum in custommade glass reactors, equipped with break-seals, high vacuum stopcock, glass-covered magnets and constrictions for the addition of reagents under the guidelines of the high vacuum techniques. The reactions used for the synthesis of the polymers are shown in Scheme 1.


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Scheme 1: Reactions used for the synthesis of the polymers. x is either 4-5 in the case of the PEO star with Mn= 9.95 x 103 g/mol or 4 for PEO with Mn= 19.9 x 103 g/mol.

The custom-made glass reactor was initially attached to the vacuum line through a ground joint and was evacuated and flame dried several times. After that, it was transferred to the glove box and 1.5 g of 4-arm poly(ethylene oxide) star amine terminated with average Mn= 9.95 x 103 g/mol (0.15 mmol) was added to the apparatus. Then, it was attached again to the vacuum line, evacuated and the apparatus was left overnight. The next day, 20 mL of highly dry benzene was distilled and the



macroinitiator was dissolved. The solution was stirred for two hours and benzene was distilled off to dryness. This step is necessary in order to remove residual water in PEO, which can initiate NCA polymerization. Then the reactor was attached at the vacuum line and was left to pump overnight to dryness. 30 mL of highly pure DMF was distilled followed by dissolution of the macroinitiator. Subsequently, the apparatus was inserted again in the glove box, where 0.59 g (2.24 mmol) of Tyr(tBu)-NCA was added in the side ampoule of the apparatus, which was equipped with a ground joint and a constriction. The ground joint of the side ampoule of the apparatus containing the NCA was attached to the vacuum line and, after evacuation, 5 mL of highly pure DMF was distilled into the ampoule of the solid NCA in order to dissolve it and thus form an ampoule of the solubilized monomer. The reactor was removed from the vacuum line through heat sealing a constriction and the Tyr(tBu)-NCA was dissolved. The break-seal of the ampoule was ruptured, leading to the addition of the solution of the NCA to the solution of the macroinitiator. The polymerization lasted for 3 days and during this time the consumption of the monomer was monitored by FTIR through removal of an aliquot of the solution in the glove box. Periodically the solution was pumped to remove the CO2 produced from polymerization. After completion of the polymerization, the polymer was precipitated in diethylether and dried under high vacuum. The polymer was suspended in CH2Cl2 (10 mL) and an equal volume of trifluoroacetic acid (TFA) was added. The polymer was completely dissolved and was left to be deprotected for 1 hour at room temperature. Subsequently, an equimolar amount (with respect to the number of Tyr monomeric units) of triisopropyl silane was added. The solution was distilled in the vacuum line in order to remove all solvents. The solid that remained was dissolved in water and was dialyzed against 2 liters of MilliQ water with pH ~ 5 (adjusted with a dilute aqueous solution of HCl) twice, twice in Milli-Q water with pH ~ 9 (adjusted with a dilute aqueous solution of NaOH) and twice in pure Milli-Q water. Finally, the aqueous polymer solution was freeze-dried to produce the corresponding polymer. An amount 0.5 g of polymer was obtained.


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b) Synthesis of [poly(L-tyrosine4)-b-(PEO 5k)]4 4-arm star For the synthesis of the 4-arm star [poly(L-tyrosine3-4)-b-(PEO 5k)]4, the hydrochloric salt of 4-arm poly(ethylene oxide) star amine terminated with average Mn= 19.9 x 104 g/mol was used as macroinitiator. Initially, the polymer was neutralized by the removal of the hydrochloric acid. The macroinitiator was dissolved in water and was dialyzed against 2 liters of Milli-Q water with pH~10 (adjusted with a dilute aqueous solution of NaOH) twice and then two times in pure Milli-Q water. The solution was freeze-dried to give 4-arm amine terminated poly(ethylene oxide) with average Mn= 19.9 x 103 g/mol with the free amines. The synthetic method followed for the 4-arm star [poly(L-tyrosine4)-b-(PEO 5k)]4 was similar to that described above. Briefly, 1 g (0.05 mmol) of 4-arm poly(ethylene oxide) star amine terminated with average Mn= 19.9 x 103 g/mol and 0.197 g (0.748 mmol) of Tyr(tBu)-NCA were used.

Size Exclusion Chromatography Size-exclusion chromatography (SEC) was used to determine the Mn and Mw/ Mn values. The analysis was performed using a system that was composed of a Waters 600 high pressure liquid chromatographic pump, Waters Ultrastyragel columns (HR-2, HR-4, HR-5E and HR-6E), a Waters 410 differential refractometer detector and a Precision PD 2020 two angle (150, 900) light scattering detector at 60oC. A 0.1 N LiBr DMF solution was used as an eluent at a rate of 1 mL/min (SEC-TALLS). The dn/dc values of the samples were 0.080 for the sample [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and 0.078 for the sample [poly(L-tyrosine3-4)-b-(PEO 5k)]4. The concentrations measured were 6.0 x 10-3 g/ml.

NMR spectroscopy 1

H NMR spectroscopy (300 MHz) was measured using a Varian Unity Plus 300/54 spectrometer. The

spectra of the polymers were performed in deuterated DMSO.

Fourier Transform Infrared (FTIR) Spectroscopy – Synthesis Product Characterization 9 ACS Paragon Plus Environment


FTIR measurements were performed with a Perkin Elmer Spectrum One instrument, in KBr pellets at room temperature, in the range 450-4000 cm-1.

Sample Preparation The conjugate [poly(L-tyrosine3-4)-b-(PEO 5k)]4 was found to have greater solubility than [poly(Ltyrosine4-5)-b-(PEO 2.5k)]4 in water and in D2O. Samples were studied at native pH and pH 12. The native pH values of [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(L-tyrosine3-4)-b-(PEO 5k)]4 were 7.74 and 6.90 respectively. For the basic solutions, pH was adjusted to 12 using 1M NaOH solution. The [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 conjugate was found to be more soluble at pH 12 than at native pH.

Fluorescence assays The critical aggregation concentrations (cac) of [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(Ltyrosine3-4)-b-(PEO 5k)]4 were determined using fluorescence spectroscopy. Fluorescence spectra were recorded with a Varian Cary Eclipse fluorescence spectrometer with samples in 4 mm inner width quartz cuvettes. 8-anilionaphthalene-1-sulfonic acid (ANS) was used to probe the aggregation its fluorescence is sensitive to hydrophobic environments making it suitable to locate the cac.53–57 ANS assays were performed using 66.8µM ANS solution to solubilise the telechelic conjugates. Fluorescence spectra were recorded between 400-650 nm wavelength (λex = 356nm). The possibility of dityrosine formation for [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(L-tyrosine3-4)b-(PEO 5k)]4 at native and alkaline pH was studied by fluorescence methods, using a Varian Cary Eclipse fluorescence spectrophotometer. The fluorescence was measured for water and for 0.1 wt% of [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(L-tyrosine3-4)-b-(PEO 5k)]4 at native pH and pH 12. Samples were measured in a 1.0 cm path-length quartz cuvette, and the slit width was 5 nm. Emission spectra were recorded at λex=320 nm from 340 to 600 nm. A total of 5 averages were


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taken, and the background was subtracted. Excitation spectra were also measured at 410 nm, from 250 nm to 410 nm. The data was averaged over five repeats, and the background was subtracted.

Circular Dichroism (CD) CD spectra were recorded using a Chirascan spectropolarimeter (Applied Photophysics, UK) in the wavelength range 180 – 260 nm. The samples, 0.025 wt% in D2O, were pipetted into a 1 mm path length bottle, with absorbance less than 2 at any point being reported. Measurements were recorded with a 0.5 nm bandwidth, 1 nm step and 1 s collection time per point. Spectra were measured at temperature intervals between 10 - 70 oC, with samples being allowed to adjust at each temperature for 5 minutes. The CD signal for the background was subtracted from the CD signal of the sample, and molar ellipticity was calculated.

Fourier Transform Infrared Spectroscopy (FTIR) Studies of Self-Assembly Spectra were recorded using a Thermo Scientific Nicolet iS5 equipped with a DTGS detector, with a PEARL liquid cell (the sample was contained between fixed CaF2 plates). An amount 80 µl of 1 wt% sample dissolved in D2O were prepared and added into the liquid cell. Spectra were scanned 128 times over the range of 900 − 4000 cm-1

Cryogenic Transmission Electronic Microscopy (Cryo-TEM) Vitrified specimens were prepared using an automated FEI Vitrobot device using Quantifoil 3.5/1 holey carbon copper grids with a hole size of 3.5 μm. Prior to use, grids were plasma cleaned using a Gatan Solarus 9500 plasma cleaner and then transferred into an environmental chamber of a FEI Vitrobot at room temperature and 100% humidity. Thereafter sample solution was applied onto the grid, and it was blotted twice for 5 s and then vitrified in a 1/1 mixture of liquid ethane and propane at a temperature of −180 °C. The grids with vitriﬁed sample soluRon were maintained at liquid nitrogen temperature and then cryo-transferred to the microscope. Imaging was carried out using a field emission cryo-electron microscope (JEOL JEM-3200FSC) operating at 200 kV. Images were taken 11 ACS Paragon Plus Environment


in bright field mode and using zero loss energy filtering (Ω type) with a slit width of 20 eV. Micrographs were recorded using a Gatan Ultrascan 4000 CCD camera. Specimen temperature was maintained at −187 °C during the imaging.

X-ray diffraction (XRD) Measurements were performed on a stalk prepared by suspending a 3 wt% solution of conjugate between two wax coated capillaries. After drying, the wax capillaries were separated leaving the stalk on the end of one capillary. Diffraction was measured using an Oxford Diffraction Gemini Ultra instrument. Stalks were mounted vertically onto a four axis goniometer. The sample-detector distance was 44 mm, and the X-ray wavelength was λ = 1.54 Å. The wavenumber scale (q = 4π sin θ/λ where 2θ is the scattering angle) was geometrically calculated. The detector was a Sapphire CCD.

Small-angle X-ray scattering Collection of solution small-angle X-ray scattering (SAXS) data was performed on the bioSAXS beamlines B21, at Diamond Light Source, Harwell, United Kingdom and BM29, ESRF, Grenoble, France. Solutions of 1 wt% [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(L-tyrosine3-4)-b-(PEO 5K)]4 at native pH and pH 12 were loaded into PCR tubes in an automated sample changer. Samples (30 µl) were then delivered into a temperature controlled quartz capillary and exposed for 15 s, collecting 18 frames at 20 oC. Data was collected using a Pilatus Dectris 2M detector. Background was manually subtracted using ScÅtter.58 Form factor modelling was done using SASfit.59 At the ESRF, solutions of (PEO 2.5k)4 and (PEO 5k)4 at native pH were loaded into PCR tubes in an automated sample changer. Samples of 30 ul were then delivered into a temperature controlled quartz capillary and exposed for 3 s, collecting 10 frames at 20 oC. Data was collected using a Pilatus Dectris 1M detector. The sample detector distance was 2.89 m. Form factor modelling was done using SASfit.59


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Cytotoxicity Studies The cytotoxicity of both the [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(L-tyrosine3-4)-b-(PEO 5k)]4 conjugates was examined. In vitro cell culture was conducted using 161Br (ECACC), a human skin fibroblast cell line. Cells were cultured in EMEM, with 2 mM glutamine, enriched with 15% fetal bovine serum (FBS) and 1% non-essential amino acids (NEAA). Cells were maintained in a humidified atmosphere at 37 oC, 5% CO2. Potential cytotoxicity effects were examined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay. The polymers [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(Ltyrosine3-4)-b-(PEO 5k)]4 were dissolved in complete medium and 2% DMSO, and sterile filtered. Cells were seeded into a 96-well plate at 4x104 cells/mL and allowed to adhere for 24 hours in 100 µL complete medium. After this, 100 µL of either complete media and/or peptide solution was added, to give either control solution (complete media, 1% DMSO), and solutions containing 5 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.1 mg/mL and 0.05 mg/mL peptide (1% DMSO). Cells were incubated for 67 hours. After this, 20 µL MTT (5 mg/ mL, in PBS) was added to each well plate and allowed to incubate for 5 hours. After a further 5 hours (72 hours total) the solution was removed from the wells and replaced with 100 µL DMSO per well, which dissolves the formazan crystals. Plates were incubated for 30 minutes, and then analysed using a UV microplate reader (λ = 570 nm). Results are reported as a % cell viability compared to control (untreated) values. In comparing results, the ANOVA and Bonferoni post hoc tests were applied.

Results and Discussions A. Synthesis of the copolymers Two four-arm star conjugates with a PEO core and tyrosine end blocks were prepared by living NCA polymerization. The molecular characteristics of the polymers are shown in Table 1.



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Table 1. Molecular Characteristics of the Synthesized Polymers Polymer

Total Mn PEO Star x 10-3 a

Mn Copolymer x 10-3 a

Stoichiometric Mn x 10-3 b

Total Tyr units a,c

[poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 [poly(L-tyrosine3-4)-b-(PEO 5k)]4

9.95

12.85

12.6

19.9

22.5

22.3

18

1.15

16

15

1.09

a

Obtained by SEC-TALLS (two-angle laser light scattering) at 60 oC in DMF with 0.1N LiBr. From stoichiometric amounts. c Obtained by 1H NMR. b

The number of tyrosine units obtained by 1H NMR as well as SEC-TALLS analysis for each copolymer was identical, i.e. 18 total units for [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and 13 for [poly(L-tyrosine4)-b(PEO 5k)]4. The SEC analysis showed that the copolymers exhibit a high degree of molecular and compositional homogeneity. Although the number of tyrosine units per arm is low, the polymers were observed to be aggregated in the solvent used for SEC analysis and it was necessary to use very low concentrations for the SEC analysis to disrupt the aggregation. The SEC analysis as well 1H NMR spectra are shown in Figures S1-S4, supplementary material. From the 1H NMR spectra (Figure S1 and S2) it is obvious that the tyrosine units have been effectively deprotected, shown by the complete absence of any peak at 1.45 ppm, where the peak due to the 9 protons of the tert-butyl protective group would appear.


Ða

Total Tyr b units 15

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250

Intensity / A.U.

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[poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 [poly(L-tyrosine3-4)-b-(PEO 5k)]4

200 150 100 0.0035%

50

0.019 wt%

0 -3.5

-3.0

-2.5

-2.0

-1.5

-1.0

log [c / wt%]

Figure 1 | Critical aggregation concentrations for the two star polymers at native pH.

To examine whether there is a critical concentration for aggregation of the telechelic polymers, a fluorescence assay using 8-anilinonaphthalene-1-sulfonic acid (ANS) was performed.

ANS

fluorescence is dependent on hydrophobic environment,53–57 thus giving information on whether a polymer is aggregated or monomeric at a given concentration. Figure 1 shows fluorescence intensity against concentration. Based on clear discontinuities in slopes, the cac for [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 was found to be 0.0035 wt% whereas for [poly(L-tyrosine3-4)-b-(PEO 5k)]4 it was found to be considerably higher at 0.019 wt%. This could be explained by [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 being relatively more hydrophobic, as the molecule has a shorter PEO chain and more units of tyrosine than [poly(L-tyrosine3-4)-b-(PEO 5k)]4 .



Figure 2 |Temperature-dependent CD spectra. a) 0.025% [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4, native pH 7.8 b) 0.025% [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4, pH 12 c) 0.025% [poly(L-tyrosine3-4)-b-(PEO 5k)]4, native pH 6.9 d) 0.025% [poly(L-tyrosine3-4)-b-(PEO 5k)]4.

The secondary structure of the peptide end blocks was examined at a concentration above the cac using a combination of circular dichroism (CD) and FTIR spectroscopies. Figure 2 shows the CD spectra. The temperature dependence of the CD data was examined in the range 10-70 oC and there were no significant changes in molar ellipticity upon heating for either [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 or [poly(L-tyrosine3-4)-b-(PEO 5k)]4 at native pH or pH 12. For [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4, at native pH (pH 7.88), the spectra (Figure 2a) show minima at 195 nm and 218 nm. The latter is ascribed to b-sheet structure, consistent with the FTIR spectra. When adjusted to pH 12 (Figure 2b), there is reduced molar ellipticity indicating a loss of structure, although there is similarity in the shape of spectra compared to those measured at native pH. For [poly(L-tyrosine3-4)b-(PEO 5k)]4 at native pH, the spectra have maxima at 205 nm and 228 nm. The latter feature is due 16 ACS Paragon Plus Environment

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to tyrosine absorbance,11,40 similar features having been observed for Tyr5-PEO6k-Tyr5 and Tyr2PEO1.5k-Tyr2.40 This masks any signal from secondary structure. When adjusted to pH 12 (Figure 2d), [poly(L-tyrosine3-4)-b-(PEO 5k)]4 shows reduced molar ellipticity implying loss of secondary structure. Moreover we looked at the reversibility of the CD spectra through pH adjustment (figure S7) and found that the conjugates have the ability to readjust their conformation upon lowering pH back to native conditions. This indicates that increasing pH to 12 does not cause hydrolysis of the conjugates. The FTIR spectra measured for the two polymers at native pH (Figure 3) show a strong peak at 1515 cm-1 which is assigned to the Tyr-OH side chain residue.60 Most informatively, the amide I’ region provides information about C=O deformation modes, meaning secondary structure can be determined. For comparison, spectra of the precursor molecules, (PEO 2.5k)4 and (PEO 5k)4 star polymers were also examined. The spectra for both [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(Ltyrosine3-4)-b-(PEO 5k)]4. at native pH show some similar features. Namely, a peak at 1629 cm-1, indicating the presence of β-sheet structure. This peak is less defined for the [poly(L-tyrosine3-4)-b(PEO 5k)]4 conjugate.. Additionally there is a peak at 1612 cm-1 which can be associated with tyrosine deformation modes.61At pH 12, [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 has a broad peak, with a value of 1630 cm-1. The FTIR spectrum measured for [poly(L-tyrosine3-4)-b-(PEO 5k)]4 at pH 12 has no defined peak across the amide I’ region, there is a broad and weak feature between 1640-1650 cm-1 suggesting that the peptide is disordered.62 Both samples at native pH show a peak near 3400 cm-1 which is consistent with –OH stretch deformations of hydrogen bonded carbonyl groups in PEO.63,64 All samples show peaks around 1455 cm-1 and 1474 cm-1 which are assigned to backbone –CH2 deformation PEO modes.



Figure 3 | FTIR spectra for 1 wt % samples. a) Spectra are normalised to the absorbance at 1900cm-1. b) -1 spectra normalised to the absorbance at 1730cm

X-ray fibre diffraction was used to investigate the structure (Figure S8 and S9 ). The fibre X-ray diffraction patterns were found to be isotropic and were reduced to one-dimensional form. All the samples show peaks at around 4.6 and 3.8 Å consistent with a high degree of PEO crystallinity.40 These peaks overlap with those from β-sheet structures and it is therefore not possible to deconvolute PEO crystallinity and β-sheet formation in these samples.11,40 The assignment of these peaks to PEO crystallinity was confirmed since the same features were observed in the XRD patterns of the PEO star precursors. Previously, tyrosine end groups have been shown to affect PEO unit cell dimensions, in particular for Tyr5-6kPEO-Tyr5.40 For the star polymers there is a decrease in crystallinity between [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 at native pH and pH 12. A slight decrease in crystallinity is also observed at pH 12 in the x-ray diffraction patterns obtained for [poly(L-tyrosine34)-b-(PEO

5k)]4.

In order to examine the possibility of di-tyrosine formation under alkaline condition at pH 12, fluorescence spectroscopy was performed. Figure 4 shows the emission spectra obtained for λex = 320 nm. A peak close to 400 nm is associated with dityrosine formation.65–67 At pH 12, spectra for both poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and for [poly(L-tyrosine3-4)-b-(PEO 5k)]4 show a peak at 18 ACS Paragon Plus Environment

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Biomacromolecules

approximately 400 nm. At native pH of 7.8 and 6.9 respectively, this peak is absent. This is expected due to the fact that the tyrosine hydroxyl unit is present at native pH as opposed to the phenolate form present at high pH.65–67 The excitation spectra (Figure 5) for both poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and for [poly(L-tyrosine3-4)-b-(PEO 5k)]4 show a red shift of the 280 nm peak at native pH to 320 nm at pH 12, also consistent with di-tyrosine formation.66,67 Contrary to other reports, the intensity in the excitation spectrum is lower at pH 12 than at the native pH.67

Figure 4 |Fluorescence emission spectra from 0.1 wt % solutions of the conjugates (λex = 320).

Figure 5 |Fluorescence excitation spectra of 0.1 wt % solutions of the conjugates (λem = 410).



Cryo-TEM was used to image the self-assembled nanostructures and SAXS was used to probe the nanostructure more quantitatively (providing information on average aggregate dimensions). The ANS fluorescence assays confirm that aggregation occurs above a critical concentration for the conjugates and cryo-TEM and SAXS were performed to probe self-assembly at concentration concentration above the corresponding cac. Representative cryo-TEM images are shown in Figure 6 with additional images (including less common structures) provided in Figure S10 For comparison, cryo-TEM images for the precursor molecules (PEO 2.5k)4 and (PEO 5k)4 were also obtained. For [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 at native pH (Figure 6a), there was a mixture of large spherical objects (possibly vesicles) with an average size of 31 nm and short clusters of fibres. Interestingly, cryo-TEM for the same conjugate in a pH 12 solution (Figure 6b) shows much less assembly, showing that adjusting the pH disrupts the self-assembly of [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4. The cryo-TEM images for the precursor molecule (PEO 2.5k)4 show very little evidence for self-assembly with only occasional clusters observed (Figure 6c). The SAXS data (vide infra) confirms that essentially all of the precursors are unaggregated. The [poly(L-tyrosine3-4)-b-(PEO 5k)]4 sample at native pH (Figure 6d) shows a mixture of fibrillar structures and small spherical structures. At pH 12 (Figure 6e) the self-assembled nanostructure is a mixture of long straight fibrils and smaller spherical nanostructures with an average diameter of 15 nm. Straight fibres have been previously observed with linear conjugates of aromatic peptides with linear PEG (PEO), i.e. Phe4-PEO5k68 and Tyr5-PEO6kTyr5.40 The precursor molecule (PEO 5k)4 show very little self-assembly with only a few large clusters (Figure 6f). Based on the cryo-TEM images we summarize the self-assembled structures in Scheme 2. The major population of the star polymer assemble into large clusters, whereas a minor population assemble into fibril structures. This can be seen as there are more cluster like assemblies in the cryoTEM images than fibrils. The presence of a small population of fibrils is consistent with the FTIR spectra which show the presence of some β-sheet structure. The cryo-TEM images indicate that the precursor molecules do not assemble into any defined structure and remain primarily as monomers in solution. 20 ACS Paragon Plus Environment

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Figure 6 |Cryo-TEM images of 1 wt % solutions of conjugates. a) [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 (native pH). b) [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 (pH 12) c) (PEO 2.5k)4 d) [poly(L-tyrosine3-4)-b-(PEO 5k)]4 (native pH) e) [poly(L-tyrosine3-4)-b-(PEO 5k)]4 (pH 12), f) (PEO 5k)4



Scheme 2 | Schematic of proposed aggregation of the PEO conjugates.

To further probe the self-assembled structures of the PEO conjugates, small-angle X-ray scattering measurements were performed. The measured data and corresponding fits are shown in Figure 7. SAXS data for both the pH adjusted and native pH polymers was fitted to a general mass fractal Gaussian form factor model to describe the aggregated structures along with a Gaussian coil form factor to account for the presence of monomers in solution. This model describes the data very well. The SAXS data for the precursor polymers was fitted to a generalised Gaussian coil model consistent with cryo-TEM which shows a lack of defined aggregates, indicating the presence of monomers in solution. The parameters obtained from the SAXS fits are listed in Table 2 and 3. Kratky plots (Figure S11) were also used also used to provide additional information on the conformation of the PEO star precursors (it is not appropriate to perform Kratky analysis for aggregated molecules). These plots confirm the branched nature of the PEO stars with extended arm conformations.


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Figure 7 | SAXS Profiles for 1 wt% conjugates dissolved in water. a) SAXS profiles for [poly(L-tyrosine4-5)-b(PEO 2.5k)]4 and b) SAXS profiles for [poly(L-tyrosine3-4)-b-(PEO 5k)]4 . Data is multiplied by factors of 10 for ease of viewing, and only every third data point is shown.

3

The radius of gyration (Rg) of the star polymers was calculated using

.69,70 The Rg

values for (PEO 2.5k)4 and (PEO 5k)4 were calculated to be 1.36 and 1.93 nm respectively. The fits show radii of gyration of 3.25 nm and 5.80 nm respectively which indicates significant swelling. The pH adjusted [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 shows the signature of scattering from unswollen (ideal) Gaussian coils with Rg=5.59 nm.



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Table 2| Parameters of SAXS fits for [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(L-tyrosine3-4)-b-(PEO 5K)]4 at native and adjusted pH. Fitting to a generalised Gaussian coil and mass fractal Gaussian model using SASfit.59 Here Rg is the radius of gyration, D is the dimension fractal, IG is intensity parameter, ν is the Flory exponent, N is a scaling factor and BG is the background. Generalised Gaussian coil

(PEO 2.5)4

Rg / nm

ν

IG

N

BG

3.25± 0.01

0.401 ±

5.60

5.07

0.56

13.2

5.43

0.54

0.003 (PEO 5K)4

5.80 ± 0.02

0.465 ± 0.002

Table 3| Parameters of SAXS fits for [poly(L-tyrosine4-5)-b-(PEO 2.5K)]4 and [poly(L-tyrosine3-4)-b-(PEO 5k)]4 at 59 pH 12. Fitting to a generalised Gaussian coil model and mass fractal gaussian model,using SASfit. Here Rg is the radius of gyration, D is the fractal dimension, ν is the Flory exponent, IMF and IG are intensity parameters, N is a scaling factor and BG is the background.

[poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 pH 7.89 (native)

Mass fractal Gaussian Rg / D IMF nm 28.3 2.38 26.1 ±0.19

Generalised Gaussian Coil Rg / nm IG N ν

[poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 pH 12

26.4 ± 0.69

2.22

9.34

11.5 ± 0.25

[poly(L-tyrosine3-4)-b-(PEO 5k)]4 pH 6.93 (native)

29.2 ± 0.24

1.90

4.68

[poly(L-tyrosine3-4)-b-(PEO 5k)]4 pH 12

34.4 ± 1.01

2.50

2.81

15.8 ± 6.63 x -7 10 5.59 ± 0.23

13.9 ±0.10

0.230 ± 0.002 0.344 ± 0.007 0.294 ± 0.001 0.538 ± 0.009

BG

3.70

1.28

3.54x10-4

1.69

0.73

6.56x10

-4

0.52

0.39

3.00x10

-5

0.52

0.14

9

1.15x10-3

To examine the cytocompatibility of the conjugates, the viability of [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(L-tyrosine3-4)-b-(PEO 5k)]4 was tested using 161Br skin fibroblast cells using the MTT assay (Figure 8). [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(L-tyrosine3-4)-b-(PEO 5k)]4 were shown to be tolerated by the cells up to a concentration of 0.5 mg/ml (above >83% viability, with non-significant difference between the measurements under these conditions and the negative control). Surprisingly, above this concentration there is significant decrease in cell proliferation. The cell viability in solution containing the precursor molecules was examined. It was found that there was significantly decreased cell viability at high concentrations for (PEO 5k)4, which displayed a 24 ACS Paragon Plus Environment

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significant reduction in viability at 5 mg/ml whereas (PEO 2.5k)4 showed a slight decrease in viability. This indicates that the decreased viability could be due to the PEO molar mass, as (PEO 5k)4 has a greater effect on cell viability than the (PEO 2.5k)4. This is in agreement with previous work, where high molecular mass PEG at high concentrations has previously been shown to display some toxicity.71 The precursor molecules have less effect on viability than the [poly(L-tyrosine4-5)-b-(PEO 2.5k)]4 and [poly(L-tyrosine3-4)-b-(PEO 5k)]4 , showing that the presence of the tyrosine does slightly reduce viability at higher concentrations. Interestingly, tyrosine alone at the same concentrations as in the conjugates (Figure S12) does not have an effect on viability, suggesting that the observed cytotoxicity is due to the oligomeric nature of the tyrosine block in the conjugates. It is noted that the concentrations tolerated by the cells are significantly higher than the critical aggregation concentrations.

Figure 8 | Cell Viability profiles for [poly(L-tyrosine4-5)-b-(PEO 2.5K)]4 and [poly(L-tyrosine3-4)-b-(PEO 5K)]4 error bars =SEM (where n=3). *= p

Self-Assembly of Telechelic Tyrosine End-Capped PEO Star Polymers

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