A Library of Multifunctional Polyesters with “Peptide-Like” Pendant

Communication pubs.acs.org/Biomac

A Library of Multifunctional Polyesters with “Peptide-Like” Pendant Functional Groups Sachin Gokhale,† Ying Xu,† and Abraham Joy* Department of Polymer Science, The University of Akron, Akron, Ohio 44325, United States S Supporting Information *

ABSTRACT: The synthesis and characterization of a library of modular multifunctional polyesters with pendant functional groups are described. The polyesters were synthesized at room temperature by carbodiimide-mediated polymerization of pendant functionalized diols and succinic acid. The pendant groups are designed to mimic the side chains of peptides, and it is shown that the physical properties of the polyesters can be modulated over a wide range by the choice of pendant groups. We also show that the pendant groups can be orthogonally functionalized with ligands such as fluorophores, poly(ethylene glycol) (PEG) or Arg-Gly-Asp (RGD).

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INTRODUCTION The design of biomaterials has seen an evolution from firstgeneration inert materials such as stainless steel and poly(methyl methacrylate) to current biomaterials that interface with the body.1 Subsequent to the development of inert materials, degradable polymers such as polyesters, polyanhydrides, and polycarbonates were developed.2 In addition to this requirement for biodegradability, there is a crucial need for biomaterials that are appropriately functionalized to enable interaction with and integration into the cellular environment.2a,3 Natural materials such as collagen provide optimum cell−extracellular matrix (ECM) interactions and oftentimes outperform synthetic materials in biomedical applications.4 However, due to their advantages of reproducibility and scale up, well-designed synthetic polymers that provide optimal biological cues will find increasing applications in tissue engineering. It is this design challenge that we sought to address in the present work. Our goal was to design a hydrophilic degradable polyester system wherein multiple pendant functional groups could be incorporated in a modular fashion. The pendant groups were chosen to mimic the functional repertoire of peptides and to provide orthogonal functional groups. It was hypothesized that similar to peptoids and peptides, the pendant groups would modulate the physical and biological properties of the polyesters. The repeating unit of the polyester was designed to have an appreciably high level of hydrophilicity in order to improve interactions with the cellular environment and enable cells to ‘sense’ the functional groups on the polyester. In this communication, we report the synthesis, characterization and properties of this novel class of biodegradable multivalent polyesters with “peptide-like” pendant functional groups. Nondegradable polymers with a diverse variety of multifunctional pendant groups have been synthesized.5 However, in © 2013 American Chemical Society

several biomedical applications, nondegradable systems are not the preferred choice. Degradable polymers such as poly(lactic acid) and polycaprolactone are commonly used in many tissue engineering applications, but they lack appropriate functional groups to enable interactions within a biological environment.6 Several recent reports related to the design of polyesters, polyesteramides, and polyurethanes have addressed this lack of functionality.7 Hydroxyl-functionalized polyesters have been prepared by polymerization of diepoxides with diacids or by the polymerization of protected tartaric acids with diols.8 Several functionalized polyesters have been prepared via ring-opening polymerization of functionalized cyclic lactones.9 Functionalized polyesteramides have also been synthesized, and their post-polymerization modifications have been demonstrated.10 The inspiration for our current work stems from peptoids and peptide nucleic acids, wherein the repeat structure is shorter and has several nitrogen and oxygen atoms in the structure, which increase their hydrophilicity.11

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RESULTS AND DISCUSSION We designed a functionalized diol that is structurally similar to the repeating unit in peptide nucleic acids. The functionalized diol was prepared by the reaction of diethanolamine with an ester derivative of the required pendant functional unit (Scheme 1). Alternatively, this functionalized diol was also synthesized by coupling of the carbodiimide (EDC)-activated acid with the tert-butyldimethylsilyl (TBDMS)-protected diethanolamine (Supporting Information). As demonstrated by Stupp, and recently by Meyer, carbodiimide-mediated coupling of diols and diacids (or Received: May 14, 2013 Revised: June 21, 2013 Published: June 24, 2013 2489

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(Tg), and modulus, are influenced by the pendant groups. Although there are prior examples of modulation of the physical properties of polyesters by varying the monomer ratio,13 the current polyesters cover a wider range of physical properties through the choice of various functional groups. For example, different pendant groups provided polyesters with a spectrum of surface energies, from hydrophobic polymers such as the phenylalanine mimic p(mPhe) (contact angle 87°) to hydrophilic polymers such as the serine mimic p(mSer) and the alanine mimic p(mAla) (contact angles of 22° and 27°, respectively). Copolymers of p(mPhe-co-mAla) and p(mPheco-mSer) show a correlation between contact angles and the amount of mPhe in the copolymer (Figure 2a). The pendant groups also influence the Tg, which varied from 4 °C for p(mGluBn) to 20 °C for p(mPhe). The Tg of p(mPhe-co-mGluBn) copolymers and the Tg of p(mAla-comPhe) copolymers increase linearly with an increase in the ratio of mPhe (Figure 2b). Interestingly, in the above examples the relationship between composition and Tg correlates well with theoretical predictions determined from the Fox equation.9d,14 This correlation is not observed in all copolymer compositions and deviation from the predicted value may indicate associative processes between the pendant functional groups or between the functional groups and the backbone. For example, the Tg of p(mSer-co-mPhe) copolymers deviate significantly from the theoretical values, indicating strong interactions when pendant groups such as hydroxyl groups are present. The plateau modulus of the homopolymers and copolymers is also influenced by the pendant group and varies from 0.2 MPa for p(mPhe0.73-co-mGluBn0.27) to 0.58 MPa for p(mAla). A linear correlation of the plateau modulus of the copolymer p(mPhe-co-mAsptBu) as a function of composition is observed (Figure S2 Supporting Information). Polyesters are hydrolytically degradable and preliminary degradation studies were carried out by incubation of p(mAla) in phosphate buffered saline (PBS, pH 7.4). SEC analysis showed a steady decrease in polymer molecular weight as a function of time (Figure S3, Supporting Information). The identity of the

Scheme 1. Synthesis of Polyesters with Pendant Functional Groups

hydroxyacids) is an efficient method for the synthesis of polyesters under mild conditions.12 In the current work, functionalized polyesters were synthesized via diisopropylcarbodiimide (DIC)-mediated coupling of a functionalized diol with succinic acid. As shown in Figure 1, a variety of polyesters containing diverse pendant groups have been synthesized using this methodology. Homo and statistical copolymers with two or more functional groups were obtained in high yield (60−70%) with relatively low polydispersity (1.3−2.2) by DIC mediated room temperature polymerization (chromatograms provided in Supporting Information, Figure S1). The low polydispersity is obtained by multiple precipitations, and such low PDIs in carbodiimide mediated step growth polymerizations have been reported by others.12b In this Communication, the nomenclature of the polyesters describes their similarity to the side chain of the corresponding amino acid or to the non-natural functional group. For example, p(mAla) refers to a polyester that is a mimic of alanine, and p(N3) refers to a polyester with an azide pendant group. In the case of a protected functional group, such as the polyester with N-tert-butoxycarbonyl protected lysine p(mLysBoc), the nomenclature describes the protected functional group (Figure 1). As characterized by NMR, the obtained polyester composition closely matched the feed ratio (Supporting Information). The number average molecular weight (Mn) and weight average molecular weight (Mw) of the polyesters were measured by size exclusion chromatography (SEC). Physical properties of the polyesters, such as contact angle, glass transition temperature

Figure 1. Representative examples of polyesters having mono-, di-, and trifunctional pendant groups. 2490

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Figure 2. (a) Variation of contact angels with copolymer composition. (b) Variation of Tg with polymer composition and its comparison to calculated values from Fox equation.

Figure 3. (a) Polyester tethered with AA and FITC. (b) UV absorbance spectrum of FITC−AA conjugated polyester (in water) and fluorescence spectrum of FITC−AA conjugated polyester (at 370 nm excitation) in DMSO−water (90:10). [AA: 338, 354, 370, 392, 462 (absorption peaks, nm); 396, 418 (emission peaks, nm); FITC: 494 nm (absorption); 524 nm (emission)].

cell mobility.16 We envisioned that cell adhesion could be modulated by tethering appropriate ligands. This was examined using a polyester with mAla, mAsp, and propargyl pendant groups in a ratio of 2:2:1. To improve adhesion of the polymer to glass substrates, 10% of the COOH groups of mAsp were functionalized with 2-phenylethylamine and is denoted as the base polymer (B). A short poly(ethylene glycol) (PEG) chain [CH3O−(CH2CH2O)2−CH2CH2NH2], which has been shown to decrease cell attachment, was covalently tethered to 20% of the COOH groups and is denoted as BP.17 The resulting functionalized polymer was spin coated on glass coverslips. Then the propargyl groups on the polymer surface were functionalized with the cell attachment peptide, N 3 − (CH2)5CONHGRGDSCO2H via Cu catalyzed azide−alkyne cycloaddition. The tripeptide Arg-Gly-Asp (RGD) has been shown to be a sufficient motif for enhancing cell attachment and proliferation.18 This process provided coverslips with both PEG and RGD and is denoted as BPR. Similarly, coverslips were made with either PEG (BP) or RGD (BR). The contact angles of spin coated polymer films on glass coverslips were influenced by the functionalization and varied as follows: base polymer (24.3°), base polymer with PEG (22.9°), base polymer with PEG and RGD (39.6°), base polymer with RGD (41.6°). The increase in contact angle with RGD is likely due to the addition of a five-carbon spacer and a triazole ring upon cycloaddition of the N3−(CH2)5CONHGRGDSCO2H moiety. These functionalized coverslips were plated with smooth muscle cells and the attachment and spreading were analyzed

pendant group is expected to influence the rate of degradation and such studies of correlating degradation rate with identity of pendant groups are currently underway. An important advantage of these polyesters is their ability to tag multiple ligands (dyes, drugs, growth factors, etc.) via orthogonal pendant functional groups. Tethering of various ligands is an effective method for presentation of imaging, therapeutic and signaling moieties.15 As a proof of concept, we aimed to functionalize 10% of the propargyl groups of a p(mNHBoc-co-propargyl) polyester with azido anthracene (AA). 1H NMR characterization of the conjugate showed that 9% of the propargyl groups were functionalized (Figure S4−S5, Supporting Information). This AA conjugate was also characterized by SEC analysis which showed the conjugate had UV absorption at 350 nm and a higher molecular weight, indicating successful AA tethering. Furthermore, IR characterization showed a decreased intensity of the CC stretching peak upon AA conjugation. Additionally, the azide stretch of azido anthracene was not present in the IR spectrum of the AA conjugate, indicating the absence of noncovalently adsorbed AA. Subsequently the amine groups of p(mNHBoc-copropargyl-AA) was deprotected and 1% of the amine groups were covalently functionalized with fluorescein isothiocyanate (FITC). As seen in Figure 3, the polyester exhibits the absorbance and fluorescence of both dyes, proving the orthogonal conjugation. Several biomaterials are being designed to direct cellular outcomes such as stem cell differentiation, proliferation, and 2491

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Figure 4. (a) Polyesters with PEG, RGD, or PEG+RGD used for smooth muscle cell spreading studies. (b) Cell area (over 50 cells) on glass coverslips (control), the base polyester (B), PEG conjugated polyester (BP), PEG and RGD conjugated polyester (BPR), and RGD conjugated polyester (BR). Images of cell spreading on the above samples: control (c); base polyester, B (d); BP (e); BPR (f); BR (g); scale bar = 25 μm (c−g). [Actin: red; cell bodies: green; nuclei: blue].

attachment of various growth factors, therapeutics, imaging agents, or other ligands and has potential utility in several applications where simultaneous presentation of multiple functional cues is necessary.

on the base polyester and compared to the same polyester functionalized with RGD or PEG or PEG+RGD. The results show that the base polymer induces high cell attachment and spreading relative to the glass control (Figure 4). The RGD functionalized polyester showed increased cell attachment and spreading relative to the base polymer. However, the lower than expected increase in cell spreading with the RGD functionalized polymer may be due to the already high cell attachment of the base polymer. A decrease in cell attachment was observed with the PEG derivatized polymer. The polymer with both PEG and RGD showed an intermediate response. It is interesting that although these polymers have low contact angles, they exhibit high cell attachment, which is similar to that seen with fibronectin or laminin. Usually synthetic polymers with low contact angles (e.g., HEMA, PEG) show decreased cell attachment, and those with high contact angles (e.g., polystyrene, PLGA) show higher cell attachment.19

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ASSOCIATED CONTENT

S Supporting Information *

Synthetic protocols for the synthesis of the monomers and polymers and their characterization is provided. Procedures for the post-polymerization functionalization, SEC and IR characterization of conjugates, degradation results, and contact angle measurements are also provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

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Author Contributions

CONCLUSION In summary, we have developed a versatile and modular polyester system that enables the efficient incorporation of multiple functional groups along the polymer. The polyesters were synthesized at room temperature by carbodiimidemediated polymerization in high yields. The polymerization is scalable to provide gram quantities of functionalized polyesters. The repeat unit of the polymer creates hydrophilic degradable polyesters with “peptide-like” pendant groups. Similar to a peptide scaffold, the physical properties, such as water solubility, surface energy, Tg, and modulus of the reported polyesters vary over a wide range and are modulated by the pendant groups. These polymers combine the breadth of properties (by choice of pendant groups) of acrylate type polymers and the degradable nature of conventional polyesters such as poly(lactic acid). In addition, these polymers have the functional group repertoire of peptides but with the scale-up and reproducibility of synthetic polymers. This multivalent “peptide-like” polyester platform will enable the covalent

†

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Matthew Becker and Erin Childers (Univ. of Akron, Polymer Science) for guidance with the cell biology experiments. The authors express their gratitude to The University of Akron for start-up funds.

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