A Novel Poly (vinylidene fluoride)-Based 4-Miktoarm Star Terpolymer

Mar 15, 2018 - Yogesh Patil, Panayiotis Bilalis, George Polymeropoulos, Sarah Almahdali, Nikos Hadjichristidis, and Valentin Rodionov*. KAUST Catalysi...
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Communication Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

A Novel Poly(vinylidene fluoride)-Based 4‑Miktoarm Star Terpolymer: Synthesis and Self-Assembly Yogesh Patil, Panayiotis Bilalis, George Polymeropoulos, Sarah Almahdali, Nikos Hadjichristidis, and Valentin Rodionov* KAUST Catalysis Center and Division of Physical Sciences and Engineering, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Kingdom of Saudi Arabia S Supporting Information *

first time the synthesis and morphological characterization of a well-defined star polymer featuring a combination of hydrophobic PVDF and hydrophilic poly(ethylene glycol) (PEG) arms. We examine the self-assembly of this amphiphilic heteroarm (or miktoarm)16 polymer in aqueous and organic media. Finally, we explore the use of the PVDF star polymer assemblies for encapsulation of small hydrophobic molecules in water. The most common and useful reversible deactivation radical polymerization protocols, atom-transfer radical polymerization (ATRP) and nitroxide-mediated polymerization, cannot be applied to 1,1-difluoroethylene due to the highly reactive nature of the primary propagating radical. Methods involving degenerate chain transfer, such as macromolecular design via the interchange of xanthates (MADIX)17 and iodine transfer radical polymerization (ITP),18 fare better and have been used previously for the synthesis of block copolymers of PVDF.19−21 We reasoned that most bromide ATRP initiators could be seen as masked ITP initiators, since Br → I substitution is in most cases a trivial transformation. Thus, a polymer synthesized via ATRP could potentially be extended using ITP. For the proof of concept synthesis described here, we decided to combine this ATRP-ITP chain extension strategy with copper-catalyzed azide−alkyne cycloaddition (CuAAC).22,23 To synthesize our star polymers, we began with 2, a bifunctional ATRP initiator featuring the N3(C3)N3 “clickable” moiety (Scheme 1). ATRP of styrene initiated by 2 yielded linear polystyrene P3 with the 1,3-diazide functionality positioned in the middle of the chain. The terminal benzylic Br groups of P3 could be easily converted to I through a reaction with NaI, yielding the ITP macroinitiator P4. This initiator was used for polymerization of vinylidene fluoride, yielding the functional (PVDF-b-PS)2 triblock copolymer P5. Size exclusion chromatography (SEC) data were collected after each of the reactions. The peak belonging to the macroinitiator P4 smoothly disappeared after the ITP step (Table 1 and Figures S10 and S11). FT-IR analysis (Figure 1 and Figure S14) confirmed the preservation of the azides after each step. It is worth highlighting the narrow Đ of P5, which validated our

ABSTRACT: A well-defined amphiphilic miktoarm polymer incorporating poly(vinylidene fluoride) (PVDF), polystyrene (PS), and poly(ethylene glycol) (PEG) blocks was synthesized via a combination of atom-transfer radical polymerization (ATRP), iodine transfer radical polymerization (ITP), and copper-catalyzed azide−alkyne cycloaddition (CuAAC). Morphology and self-assembly of this star polymer were examined in organic solvents and in water. The aggregates formed in water were found to possess unusual frustrated topology due to immiscibility of PS and PVDF. The polymer was evaluated for transport of small hydrophobic molecules in water. KEYWORDS: poly(vinylidene fluoride), miktoarm star polymer, iodine transfer polymerization, CuAAC, self-assembly

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luoropolymers are gold standard materials in biomedicine and bioengineering due to their extreme chemical inertness, combination of hydrophobicity and oleophobicity, and distinctive mechanical properties.1 The unique selfassembly, phase separation, and dispersion properties of fluorocarbons and fluoropolymers in both aqueous and organic media make these attractive building blocks for nanoscale drug delivery vehicles. A variety of experimental systems of this kind have been described.2−4 However, intrinsic chemical inertness of highly fluorinated compounds limits the structural diversity of the molecular and supramolecular architectures that have been explored for their applications in drug delivery and controlled release. Poly(vinylidene fluoride) (PVDF) is a partially fluorinated polymer with critical surface tension of wetting halfway between those of poly(tetrafluoroethylene) and polyethylene.5 PVDF and its composites have found use in sutures,6,7 surgical meshes,8,9 and contact lens coatings10 and have been explored as membranes for topical drug release.11,12 However, the dispersive capabilities of well-defined soluble PVDF-based polymers and their biomedical applications remain to be investigated. One of the reasons for this is relative paucity of strategies for controlled and efficient synthesis of PVDF-based complex macromolecular architectures. While a few welldefined amphiphilic block copolymers of PVDF have been described,13−15 complex branched PVDF-based macromolecules have so far eluded capture despite their potential usefulness and interesting properties. Here, we report for the © XXXX American Chemical Society

Special Issue: Click Chemistry for Medicine and Biology Received: Revised: Accepted: Published: A

January 3, 2018 March 1, 2018 March 15, 2018 March 15, 2018 DOI: 10.1021/acs.molpharmaceut.8b00010 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Scheme 1. Synthesis of (PVDF-b-PS)2(PEG)2 Miktoarm Star Polymer P6

Table 1. Fundamental Characterization Data for Polymers P3−P6 polymera

entry 1 2 3 4

P3, P4, P5, P6,

(Br-St52)2 (I-St52)2 (I-PVDF32-b-PS52)2 (PVDF32-b-PS52)2(PEG113)2

Mn, Daa

Mnb

Mwb

Đb

Dh, nmc

PDIc

11000 11000 16000 27000

12000 12000 18000 37000

13000 13000 24000 45000

1.09 1.10 1.33 1.21

89 99

0.121 0.140

a Degree of polymerization and Mn estimated from 1H NMR. bDetermined by SEC analysis collected in DMF and calibrated against linear PS standards; the refractive index detector was used. cData obtained using multiangle DLS; Dh is the hydrodynamic diameter obtained for 1 wt % DCM solution.

reactivity is the complex nature of the Cu catalyst, which incorporates at least two Cu centers and multiple bound alkynes in its activated state. This feature of CuAAC is extremely valuable here, as it obviates the necessity of separating the four-arm miktoarm star from the incompletely reacted three-arm intermediate, and a large excess of PEGalkyne is not required. The reaction was monitored by FT-IR through the disappearance of the characteristic azide band at 2095 cm−1 (Figure 1 and Figure S15). Conversion was further confirmed by 1H NMR (Figure S6). SEC analysis of the terpolymer star showed a lower apparent Đ value than that obtained for linear P5 (Table 1, entries 3 and 4, and Figure S11). Such a decrease in SEC-estimated Đ is often observed in the transition between linear and branched macromolecules, as branching reduces the hydrodynamic volume of the polymer and profoundly impacts the apparent size distribution of the species.25 The mutually immiscible hydrophilic, lipophilic, and lipophobic PVDF blocks should lead to phase separation and selfassembly in solution. We explored the morphologies of block copolymers P5 and P6 in different solvent systems using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Additionally, differential scanning calorimetry (DSC) data were also collected for star terpolymer P6 to investigate the thermal properties and miscibility of the constituent blocks. DLS data were collected for P5 and P6 in DCM at 20 °C initially at a fixed measurement angle of 173° (Figures S17 and S18), and then using a multiangle instrument at 60°, 90°, and 120° (Table S1 and Figures S20−S25). The measurements performed using both instruments indicated that DCM was a selective solvent, resulting in aggregates that likely formed due to the presence of PVDF blocks. The hydrodynamic diameter

Figure 1. FT-IR spectra of P4, P5, and P6. The dotted red line highlights the position of the characteristic −N3 peak (2095 cm−1).

choice of ATRP-ITP chain extension strategy for the synthesis of a PVDF-based block copolymer. To confirm the symmetrical nature of the (PVDF-b-PS)2 copolymer P5 we subjected it to alkaline hydrolysis to cleave the central ester bonds. A comparison of the Mn values before (MnSEC = 24000 Da) and after (MnSEC = 11500 Da) hydrolysis, as well as the consistency of the Đ values (Figure S12), suggests that there were few drawbacks to using a bifunctional initiator strategy, and both arms grew simultaneously during both ATRP and ITP steps. P5 was coupled with alkyne-terminated PEG (Mn = 5000 Da) using CuAAC to yield the amphiphilic (PVDF-bPS)2(PEG)2 miktoarm star polymer P6. Due to the peculiarity of the mechanism of CuAAC first discovered by Finn and coworkers,24 both azides of conformationally biased N3(C3)N3 moieties react simultaneously, and the corresponding monotriazole is never an intermediate. The reason for this unusual B

DOI: 10.1021/acs.molpharmaceut.8b00010 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Communication

Molecular Pharmaceutics of the aggregates of linear triblock P5 was ∼90 nm, and it increased slightly to ∼100 nm after the coupling with PEGalkyne which yielded the star polymer P6. The introduction of the PEG arms also allowed P6 to disperse well in aqueous media. DLS analysis of P6 in water indicated ∼150 nm micellar aggregates at 20 °C (Figure 2A and Table S1 and Figures S26−

we observed primarily the most electron-dense constituent block, PVDF. Additional dry-state TEM images were collected for a sample of P6 prepared from aqueous solution (Figure 2C and Figure S32). The PEG block was preferentially stained using phosphotungstic acid. The images in this mode showed roughly spherical aggregates ∼100 nm in diameter. The PEG block appeared as a light gray corona due to the stain, and the PVDF/ PS blocks appeared darker gray as their relatively high electron density was superimposed with that of stained PEG. It was possible to observe distinct phase separation between the PEG corona and PVDF/PS core. Cryo-TEM images of P6 in water (Figure 2C and Figures S33−S34) revealed aggregates similar in size to those seen in the dry state. The size distribution was narrow, and in close agreement with DLS results (Figure S34). The PEG block is extremely hydrophilic and must form diffuse coronas around the aggregates. Since we observed sharply defined boundaries instead, and the images were obtained with no staining, we concluded that this mode of imaging selectively revealed only the hydrophobic cores of the polymer micelles. Phase separation between more electron-dense center and lighter periphery was visually apparent. The boundary between the phases was sharp and well-defined. We hypothesize that the inner electron-denser phase is primarily PVDF. An even more striking feature of the aggregates is their “wrinkled”, frustrated topology. Ordinarily, soft hydrophobic objects assume smooth spheroid shape in water to minimize the surface area and the thermodynamically unfavorable interaction with the solvent.27 We believe that phase separation and imperfect packing between the PVDF and PS blocks could be the thermodynamic driving force for the formation of “wrinkles” on the hydrophobic cores of the micelles of P6. Further evidence for phase separation in the aggregates of P6 was obtained from DSC experiments. The heat flow curve (Figure S16) clearly reveals the PVDF and PEG melting points (Tm = 140 and 39 °C, respectively) and PS segment glass transition temperature (Tg = 80 °C), indicating that the three constituent blocks are separated and behave as independent phases. We are considering the application of amphiphilic copolymer architectures like P6 for the encapsulation and transport of small hydrophobic molecules in water. As a proof of concept, we examined the ability of P6 to transport Nile Red (NR).28 Nile Red is a water-insoluble solvatochromic dye that has previously been used as a model drug when exploring drug delivery. Its incorporation into a hydrophobic environment can be easily detected by the coloration of the solution and by fluorescence. In polar solvents such as water, NR is only weakly fluorescent. Pink micellar solutions of NR@P6 could be readily obtained either by gentle sonication of solid NR powder with a 1 wt % aqueous dispersion of P6 or by adding 1 μL of 1 mM solution of NR in acetone to 1 mL of aqueous dispersion of P6, followed by bubbling of N2 through the solution to remove the acetone. The solutions of NR@P6 thus obtained were filtered through 0.45 μm nylon membranes to remove any undissolved NR. Attempting either of the two NR dissolution protocols with either pure water or a 1 wt % aqueous solution of PEG5000 resulted in colorless solutions that contained trace amounts of NR and did not fluoresce. The fluorescence emission spectrum (λexc = 515 nm) of NR@P6 exhibited a λmax at 603 nm, suggesting a local solvent environment with a polarity comparable to that of DCM (Figure 3). While the

Figure 2. (A) Intensity DLS plots collected for P6 in DCM and water; each plot is averaged from the three or more sets of data. (B) Dry-state TEM image of a P6 sample prepared from DCM. (C) Dry-state and cryo-TEM images of a P6 sample in water; the scale bar is applicable to both sections of the image. The PEG block was preferentially stained with phosphotungstic acid for the dry-state TEM. The corresponding schematic images illustrate the aggregation due to selective solvent interactions. Polymer blocks are color-coded purple (PS), orange (PVDF), and green (PEG). The dotted line indicates the electron-dense regions visible in the TEM images.

S28). The observed increase in size could be expected, as water is an excellent solvent for PEG. Under all the conditions surveyed with the multiangle DLS instrument (P5 and P6 in DCM and P6 in water), the observed hydrodynamic diameter of the aggregates was similar for measurements collected at all angles, suggesting that their shape was roughly spherical.26 TEM images were collected for dry samples of P5 (Figure S29) and P6 (Figure 2B and Figure S30) prepared from DCM solutions. In general agreement with multiangle DLS measurements, these TEM images revealed primarily spherical aggregates. The TEM images of P5 showed compact mostly crystalline domains surrounded by diffuse amorphous regions. Dark electron-dense domains could also be observed in the image of P6, although no diffuse halo could be observed around these. The average size of the electron-dense domains was ∼20 nm for both P5 and P6, which was significantly smaller than the aggregate size estimated for either polymer using DLS. As the polymer samples have not been stained, we hypothesized that C

DOI: 10.1021/acs.molpharmaceut.8b00010 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

fluorinated polymers, as well as to gain additional insights into their morphology, self-assembly, and dispersive properties, are currently underway in our laboratories.

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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/acs.molpharmaceut.8b00010. Additional details of experiments and material characterization including additional TEM images, SEC traces, DLS and DSC data, and 1H, 19F, and 13C NMR, FT-IR, and UV spectra (PDF)

Figure 3. Fluorescence emission spectra (λexc = 515 nm; emission intensity normalized) of solvatochromic dye Nile Red (NR) in organic solvents (1 μM) and in water solubilized by star polymer P6 (which is not fluorescent itself).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +966-128084592.

values of λmax were similar for NR@P6 and NR in DCM, the emission peak for NR@P6 was broader by ∼20 nm on the blue edge at normalized peak half-height. The considerable degree and the direction of this broadening suggest that it is caused by solvatochromism of NR rather than by the difference in the vibrational transitions of NR in DCM and inside the polymer micelles. We hypothesize that some of the NR molecules are exposed primarily to PEG and PS blocks, while others penetrate the more hydrophobic PVDF domains. We obtained DLS data for the solutions of P6 used for the NR solubilization, as well as for NR@P6 (Figure S19). The micellar aggregates were uniform and stable both before and after the addition of NR. There was no detectable change in the size distribution at different concentrations of P6, and the addition of the hydrophobic dye payload did not induce aggregation of the micelles. Such induced aggregation is commonly observed for nonionic surfactants and highly hydrophobic payloads, including NR.29 We hypothesize that P6 is less prone to hydrophobe-induced aggregation due to the “wrinkled”, frustrated morphology of its hydrophobic domains in water. The hydrophobic payload is free to bind to the pockets created by imperfect packing of PVDF and PS blocks, thus filling up the space in the core of the micelle, making its shape closer to a perfect spheroid, and its solvent-exposed surface area smaller. Thus, P6 and related polymer architectures could be suitable for creating stable aqueous formulations of hydrophobic pharmaceuticals. In conclusion, we synthesized a novel PVDF-based amphiphilic miktoarm star polymer containing fluoropolymer PVDF, hydrophobic PS, and hydrophilic PEG blocks via a combination of ATRP, ITP, and CuAAC methodologies. The morphology and aggregation behavior of this polymer were investigated in organic solvents and in water. The constituent polymer blocks were found to phase-separate under all the conditions surveyed. The hydrophobic domains of the micellar aggregates formed in water had unusual “wrinkled” morphology due to the immiscibility of PVDF and PS blocks. Finally, we have demonstrated the use of this material for encapsulation and dispersion of a small hydrophobic molecule in aqueous media. The combination of ITP and CuAAC chemistries and judicious choice of multifunctional precursors provides easy access to a variety of otherwise-unattainable PVDF-based complex macromolecular architectures. Efforts to extend the combinatorial field of branched and hyperbranched semi-

ORCID

Panayiotis Bilalis: 0000-0002-5809-9643 George Polymeropoulos: 0000-0002-3352-0948 Nikos Hadjichristidis: 0000-0003-1442-1714 Valentin Rodionov: 0000-0002-9038-206X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the King Abdullah University of Science and Technology (KAUST) is acknowledged with thanks. The authors would like to thank Dr. Rachid Sougrat (KAUST Imaging and Characterization Core Lab) for his invaluable assistance with cryo-TEM.

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REFERENCES

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DOI: 10.1021/acs.molpharmaceut.8b00010 Mol. Pharmaceutics XXXX, XXX, XXX−XXX