Tunable biomaterials from synthetic, sequence-controlled polymers

Mariah J. Austin and Adrianne M. Rosales *
McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712, USA. E-mail: arosales@che.utexas.edu

Received 29th September 2018 , Accepted 21st December 2018

First published on 26th December 2018

Polymeric biomaterials have many applications including therapeutic delivery vehicles, medical implants and devices, and tissue engineering scaffolds. Both naturally-derived and synthetic materials have successfully been used for these applications in the clinic. However, the increasing complexity of these applications requires materials with advanced properties, especially customizable or tunable materials with bioactivity. To address this issue, there have been recent efforts to better recapitulate the properties of natural materials using synthetic biomaterials composed of sequence-controlled polymers. Sequence control mimics the primary structure found in biopolymers, and in many cases, provides an extra handle for functionality in synthetic polymers. Here, we first review the advances in synthetic methods that have enabled sequence-controlled biomaterials on a relevant scale, and discuss strategies for choosing functional sequences from a biomaterials engineering context. Then, we highlight several recent studies that show strong impact of sequence control on biomaterial properties, including in vitro and in vivo behavior, in the areas of hydrogels, therapeutic materials, and novel applications such as molecular barcodes for medical devices. The role of sequence control in biomaterials properties is an emerging research area, and there remain many opportunities for investigation. Further study of this topic may significantly advance our understanding of bioactive or smart materials, as well as contribute design rules to guide the development of synthetic biomaterials for future applications in tissue engineering and regenerative medicine.

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Mariah J. Austin

Mariah Austin joined Professor Adrianne Rosales’ lab at the University of Texas at Austin in the fall of 2017 as a graduate research assistant pursuing a Ph.D. in Chemical Engineering. She received her B.S. in Chemical Engineering from the University of New Mexico. Her research interests include developing novel polymeric materials for biomaterial applications and her Ph.D. project focuses on investigating synthetic macromolecules aimed at enhancing substrate-specificity to enzymes implicated in various diseases.

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Adrianne M. Rosales

Adrianne Rosales is an Assistant Professor in the McKetta Department of Chemical Engineering at the University of Texas at Austin. She obtained her Ph.D. in Chemical Engineering from the University of California, Berkeley, where she studied sequence effects of biomimetic materials on polymer properties. Prof. Rosales then served as an NIH NRSA Postdoctoral Fellow and a Burroughs Wellcome Fund PDEP fellow at the University of Colorado Boulder in the field of biomaterials. She is the recipient of the BWF Career Award at the Scientific Interface, and her research interests include molecular design of biomimetic materials, dynamic polymers for cell culture, and advanced materials for biomedical applications.

1 Introduction

Polymeric biomaterials represent a large class of materials that interface with biological systems either in vivo or in vitro. Currently, biomaterials applications span diagnostic, therapeutic, and regenerative purposes, as well as in vitro disease modeling.1–5 While there are common themes among the requirements for these materials,6i.e. high biocompatibility, ease of processing, and stability through implementation, the properties which must be tuned for each individual application are far more extensive. This demand is what has evolutionarily driven biological systems to develop a high level of complexity. While naturally derived materials have found some success in the clinic,7 they also have key limitations, such as their susceptibility to react or degrade with other biologics, their difficulty of synthesis at large scales, and restricted diversity of building blocks for customization.2 In addition, in vivo use of natural materials requires careful consideration of immunocompatibility. For these reasons, synthetic polymers are attractive for expanding the repertoire of biomaterials. The ultimate challenge with utilizing synthetic approaches is the gap in complexity achieved as compared to naturally derived materials.

Biomolecule complexity stems from a hierarchical structure, and many efforts have recapitulated this structure in synthetic materials, especially at the meso- and macroscale.8 However, structure and function also arise from the precise sequence of building blocks in natural biopolymers, and even short fragments of these molecules can retain function (e.g., the RGD cell adhesion domain of fibronectin protein9 or the protein transduction domain of the TAT protein in HIV-110). Replicating monomer-level structure in synthetic polymers may preserve bioactivity while leveraging the features of abiotic backbones, such as enhanced biostability or decreased immunogenicity. In addition, previous work in synthetic polymers indicates sequence control may be used as a handle to tune polymer properties without altering composition,11 which is especially attractive for modifying clinically approved polymers. For these reasons and many others, synthetic methods to improve production of sequence-controlled polymers has long been an area of interest in polymer chemistry.12 Recent progress in these synthesis methods have enabled applications for sequence-controlled synthetic biomaterials, and many works have shown an impact of sequence control on both in vitro and in vivo results.

To highlight this emerging area of research, we here summarize contributions to the biomaterials field using sequence-controlled synthetic platforms. We first summarize synthetic methods of sequence control and challenges in sequence selection from the perspective of biomaterials engineering. We then provide a compilation of current reports on biomaterials properties investigated using sequence control, as well as functional results in both in vitro and in vivo systems. In this mini-review, we aim to highlight the impact of sequence-controlled polymers in the context of biomaterials applications, as well as identify areas of future growth in the field.

2 Synthesis and selection of sequence-controlled biomaterials

While biological systems are innately designed to produce sequence-defined matter (i.e., exactly monodisperse with respect to sequence and chain length, as contrasted by statistical sequence control), duplicating this level of control in synthetic systems continues to be a major challenge in polymer chemistry.13 Namely, researchers must identify avenues to achieve the scalability of traditional polymer synthesis without sacrificing the sequence complexity required for biological function.14 In the scope of synthetic approaches for sequence control, strategies include controlled polymerization techniques, consisting of both chain and step-growth mechanisms, iterative approaches in which each monomer unit is added sequentially, and bio-inspired and assisted methodologies like molecular imprinting and templated syntheses (Fig. 1). In addition, some more recent approaches blend multiple strategies, such as employing a controlled polymerization strategy followed by sequence selective post-polymerization functionalization.15 The remarkable strides accomplished in the synthesis of sequence-controlled polymers are well compiled in many comprehensive, detailed reviews.12,16–19 Here, we provide a brief digest of these methodologies as context for the use of precision polymers in biomaterials applications.
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Fig. 1 Survey of synthetic methods for incorporating sequence control into synthetic polymers. Chain-growth, multicomponent, ribosomal translation, and molecular imprinting depictions reprinted with permission from ref. 29, 35, 56*, and 61. Copyright 2007, 2015, 2008, and 2017 (respectively) American Chemical Society. Step-growth illustration reprinted with permission from Springer Nature: Science China Chemistry, ref. 19, 2015. Exponential growth and DNA-templated images adapted by permission from Springer Nature: Nature Chemistry, ref. 46 and 55, 2015 and 2013, respectively. Solid-phase supports figure reprinted from ref. 154 with permission from Elsevier. Support-free image reproduced from ref. 43 with permission from The Royal Society of Chemistry.

2.1 Sequence control with controlled polymerizations

Traditional methods of polymerization are attractive for rapid synthesis on a large scale, and recent developments include increasing levels of sequence control for polymers relevant to biomaterials. Compared to chain growth polymerizations, step-growth mechanisms offer less control over molecular weight and distribution, but readily allow for alternating or repeating sequence architectures.20 For example, in acyclic diene metathesis (ADMET) polymerization, step-growth condensation of α–ω dienes have allowed for synthesis of high molecular weight, biodegradable polyesters with precisely placed esters at every nth monomer.21 In a similar manner, “click” step-growth polymerizations have been demonstrated to proceed in high yield,22,23 in which short, sequence-controlled oligomers are polymerized by click reactions such as copper-catalyzed azide–alkyne cycloaddition (CuAAC) for generation of materials as 1D bioarrays.24 Another technique, segmer assembly polymerization (SAP) has also found good utility in the synthesis of sequence-controlled biodegradable polyesters.25 SAP first programs sequence into short reactive monomer chains, then polymerizes these segments into longer repeating sequences via multiple step-growth mechanisms, including cross-metathesis polymerization.26

In contrast, chain growth polymerizations may offer sequence diversity beyond repeating structures while better controlling chain length, composition, and architecture, but highly reactive propagating species can limit precise placement of specific monomers and introduce dispersity.13,27 Approaches utilized to modulate monomer incorporation include exploiting monomers with significantly different reactivity ratios28 or performing time-controlled addition of monomers.29–31 For example, oxanorbornene copolymers with various cationic and hydrophobic densities (but same overall content) were synthesized via ring-opening metathesis polymerization (ROMP) for use as intracellular delivery materials;32,33 in addition, ring opening polymerization was used to introduce gradient sequences into thermogelling block copolymers.34 While some disparity remains between chain-growth mechanisms and complete sequence definition, these advancements have elucidated important information on the role of sequence in properties and function and may provide the platform for scalability when used in conjunction with methods capable of finer sequence tuning.

An exciting extension to controlled polymerizations is multicomponent reactions (MCRs), which combine three or more starting components in a one-pot polymerization. MCRs, including the Passerini reaction (three components), the Ugi reaction (four components), the Hantzsch and Biginelli reactions (three-components) and copper-based click MCRs, further extend the functional diversity possible in sequenced chains.35 Multicomponent reactions have also been combined with other controlled polymerization mechanisms. For example, Wu et al. recently worked to mimic nature's two stage strategy in protein synthesis by preparing precisely fabricated polymer precursors using reversible addition–fragmentation chain transfer (RAFT) polymerization, followed by combinatorial synthesis via the Biginelli reaction to generate diverse polymers with known sequence in a high-throughput manner.15

2.2 Sequence control with iterative strategies

To fully achieve the exact sequence definition prescribed by nature, no synthetic method has been more successful than multistep growth polymerization. These iterative strategies introduce only one monomer at a time to yield a monodisperse sequenced chain; however, this strategy has obvious limitations including long synthesis timescales, steep reagent requirements, and low yields for long sequences.36 While solid-phase strategies have been invaluable to the biomaterials field and have expanded to include non-natural backbones, there still exists a need to bridge the sequence-definition capabilities offered by this method with the scale and throughput exhibited in traditional polymer synthesis.

Efforts toward scalability include the development of protecting group-free syntheses utilizing multiple monomers which are not able to self-polymerize, or the use of commercially available reagents in a sub-monomer synthesis, such as that developed for polypeptoids.37,38 Improvements targeting scalability and efficiency via reactant accessibility make use of liquid-phase supports that are soluble in the reaction solvent. This method relies on chemoselective monomer reactions and precipitation of the support, but benefits from real-time monitoring of synthesis conditions using solution-phase analytical techniques.39,40 Fluorous tags are a popular purification handle wherein chains are grown on perfluorocarbon-modified silica, allowing for selective separation of excess reagents by employing fluorophobic solvent washes. This method has been demonstrated to be adaptable to a wide range of monomers,41,42 and one example demonstrated application to high throughput synthesis of sequence-defined hydroxyproline-based oligomers for nucleic acid delivery. Perhaps the most effective method for increasing product scale in iterative syntheses is to remove the support altogether.43,44 Without the support, controlled polymerization relies on protecting groups or some other method of ensuring only one monomer adds at a time, and the sequences must be symmetrical because growth is happening in both directions. While the scalability is increased by reducing the hindrance from the supports, the ease of purification is lost, reintroducing time-and labor-intensive steps to these syntheses.

Iterative exponential growth (IEG) is another approach aimed at boosting scalability of both yield and chain length. IEG uses separate, orthogonal deprotection of two different monomers before combining them and growing the chains exponentially at each step to yield repeating or periodic sequences.45 In IEG+, the monomer unit is suitable for chemoselective substitution and subsequent functionalization, allowing for specific sidechain placement in each iteration.46 Further advancements have improved the synthesis time and expanded the diversity of protecting groups,47,48 which will ease biomaterials application.

2.3 Sequence control with templating approaches

With inspiration drawn largely from nature's ability for sequence precision, it is rational to mimic the templating procedure by which living organisms produce natural biopolymers.49–51 Specifically, generating DNA-templated platforms can be done by modifying polymerases or by introducing new functional groups to the nucleic acid bases of the templating strand. While both methods increase chemical diversity, synthetic efficiency is often hindered by disruption of the specific recognition interaction between polymerases and the monomers targeted.50 Artificial base pairs have also been investigated;52 however, polymerase selectivity is limited, suggesting the need for synergistic adaptations to both the chain functional groups and the enzymes carrying out the polymerization. Indeed, shifting to enzyme-free templated strategies greatly extends the backbone (e.g., peptide nucleic acids) and base chemistry (e.g., threose and glycerol) available, but requires means of enacting hybridization between the template and strand to be synthesized to produce chains in reasonable yield.49 Other approaches include reprogramming of codons to make ester-bonded rather than amide-bonded polymer backbones by mRNA translation53,54 and translating nucleotide sequences in DNA to a polymer using artificial tRNAs.55 Many other novel strategies to mimic the templating processes carried out in nature have been investigated, but most products still resemble peptides or nucleic acids very closely.56

Recent work has also extended templated synthesis beyond biological templates to produce non-natural polymers. Ida et al. developed template initiators which are selectively recognizable by monomers for cationic and radical polymerization, a platform which could be further developed to select for each monomer individually.57,58 Supramolecular interactions between monomers can also be employed as sequence control methods and are described in other reviews.49 Similarly, molecular imprinting uses functional groups bound to a template (usually a biomolecule), which are then polymerized into place. Removal of the template leads to a cavity with the shape and functionality specifically susceptible to compounds matching the template.59 Molecular imprinting has been investigated for the fabrication of hydrogel-encapsulated particles with recognitive ability for proteins such as serotonin,60 but challenges remain with respect to protein template cost, availability, and competitive binders.61

2.4 Choosing functional sequences

When it comes to designing materials for specific functions, selecting a sequence is often as much of a challenge as synthesis. Several methods exist to aid peptide sequence selection, but the translation to synthetic analogues is not direct. Brummelhuis, Wilke, and Börner recently compiled methods of identifying functional peptide sequences in pursuit of extension to synthetic precision polymers in a comprehensive review.62 They identified three main routes to determining peptide sequences: “bioabstraction” (translating essential sequence components to synthetic platforms), combinatorial assisted design, and rational de novo design (Fig. 2). We highlight these approaches specifically for synthetic sequence-controlled biomaterials here.
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Fig. 2 Concept drawing of biomaterial design using sequence-controlled polymers. Sequence control may be obtained by mimicking biological molecules or tuning microstructure with controlled polymerizations. To enhance these approaches, selection of appropriate sequences may be aided by methods including bioabstraction, combinatorial screening, and rational design.

“Bioabstraction”, or mimicking minimal functional sequences in proteins or DNA, is a relatively straightforward method of sequence selection that has been applied to biomaterials composed of specific peptidomimetics and statistical copolymers alike. This approach has been widely adopted for fabricating synthetic analogues of biologically relevant molecules like antimicrobial peptides (AMPs),63–66 cell penetrating peptides (CPPs),67 host defense peptides (HDPs),68 and others.69 For peptidomimetics, modifications to the fundamental α-peptide structure often preserves primary level sequence definition and functional side chain chemistry but enhances some feature (e.g., stability/resistance or bioavailability) with backbone changes. In some cases, “bioabstraction” emulates the secondary and even tertiary structure necessary for function, yielding synthetic “foldamers”.70 One example of direct bioactive sequence translation to a synthetic analogue is oligourea and oligocarbamate mimics of the trans-activation response (TAR) binding portion of HIV-1 Tat protein.71 The non-natural oligomers displayed sidechains exactly analogous to amino acids 48–57 in the protein, and were found to suppress HIV-1 gene expression in vivo due to competitive inhibition of the Tat-TAR interaction. With regard to statistical copolymers, exact sequence definition is not preserved, yet a controlled distribution of functional groups can retain or even enhance function. Examples include copolymers of ternary Nylon-3 containing polar (but uncharged), hydrophobic, and cationic units to mimic HDPs,72 as well as copolymers with cationic and hydrophobic groups to mimic cell penetrating sequences.32,33,73–76

Direct sequence translation does not always preserve function, and even with just twenty natural amino acids, there is an overwhelming number of sequence permutations possible, an issue that only grows with the inclusion of non-natural monomers. Thus, to find functional synthetic sequences, combinatorial synthesis has assisted in rapid selection of target polymers from libraries of compounds. In particular, combinatorial libraries have been generated by SPOT microarrays77,78 and split-and-mix libraries79–82 for non-natural backbones. Combinatorial libraries of polypeptoids, or N-substituted glycines, have especially had success in identifying functional sequences for gene delivery or molecular recognition.83,84 However, an ongoing challenge for synthetic substrates is sequencing after a hit compound has been identified. While the sequence is known for some libraries (e.g., SPOT), others usually require a method such as Edman degradation, which does not apply for N-alkylated compounds, or tandem mass spectrometry, which relies on straightforward fragmentation patterns to identify sequence.85 Extension of novel combinatorial approaches to other synthetic systems will increase sequence discovery for functional biomaterials.

A third route to sequence selection is rational design. Rational design is still an ongoing and large challenge in protein engineering due to the complexity of interactions in biological macromolecules.86 Modeling some of these interactions could be simplified in a synthetic analogue with higher regularity and predictability. However, limited endeavors toward de novo and in silico design strategies have been reported thus far.87 Representative examples of the potential of these methods include monomer sequence properties in hydrogel applications modeled by full-atomistic molecular dynamics simulations88,89 and a coarse-grain approach used to study drug–polymer interactions for oral drug delivery.90 One long standing approach has been to model polymers as hydrophobic/hydrophilic sequence patterns to yield folding86,91,92 or functional synthetic molecules. It is clear the evolution of sequence-controlled biomaterials will require a collective effort across multiple platforms of sequence selection methods, as has been pursued for natural biopolymers, to achieve its full potential with regard to function.

3 Tuning biomaterial properties with monomer sequence

Biomaterials applications in tissue engineering, regenerative medicine, and therapeutic delivery often entail incorporation of biodegradable polymers or polymeric hydrogels. In therapeutic delivery, these engineered systems can lead to sustained release profiles with tunable kinetics, while in regenerative medicine, hydrogels or biodegradable polymers may address long-term biocompatibility issues for some permanent implants.93 Given that biomaterial properties are closely tied to therapeutic efficacy (e.g., delivered drug dose or replacement tissue deposition), extensive work has been devoted to designing polymer systems with controlled degradation kinetics, porosity, and mechanics. Toward this end, monomer sequence is an attractive handle to tune these properties in heterogeneous copolymers without altering established chemistries.

3.1 Controlled degradation and release

Biodegradability is readily achieved via incorporation of hydrolytically or enzymatically degradable monomers but still presents a complex engineering problem as assessment of each polymer's absorption, distribution, metabolism, and excretion (ADME) is crucial.94 One of the most studied biodegradable copolymers is poly(lactic-co-glycolic acid) (PLGA). PLGA's popularity stems from its established clinical approval, availability from renewable resources, tunable degradation characteristics, and potential for demonstrating sustained release behavior.95,96 Meyer and coworkers identified PLGAs as ideal candidates for employing sequence control tools in polymer synthesis to investigate degradation in vitro and in vivo.97–102 Hydrolysis studies compared microparticles composed of random copolymers to those made via SAP with alternating lactic and glycolic acid units.98 The alternating sequences showed a deviation from the exponential decay in molecular weight demonstrated for the random sequences. The initial burst release was reduced, both in time and weight loss, for sequence-controlled structures, followed by a linear degradation profile exhibited consistently for samples spanning a 10 kDa range of initial molecular weight. The overall rate of hydrolysis was also slower for polymers following the alternating sequence with only a 47% molecular weight decrease after 28 days, as compared to the 71% drop observed for the random sequence. This observation, in conjunction with differential scanning calorimetry (DSC) traces, provided evidence that neighboring glycolic acid units cleave significantly faster than the glycolic acid–lactic acid site, followed by neighboring lactic acid units, in agreement with previous studies on copolymer composition.103 Thus, alternating PLGA copolymers that contained a higher fraction of glycolic acid–lactic acid sites degraded slower compared to random PLGA sequences. The implications of this first-level comparison were that monomer sequence has a significant impact on hydrolysis and harnessing the power of sequence definition may be a tool for tuning degradation time over the span of at least days without altering the chemistry of the system.

Subsequent research on sequence-controlled PLGAs extended the variety of sequences tested to analyze the influence of stereospecificity and block length in conjunction with sequence effects on the controlled release of a model compound, rhodamine-B, from polymer microparticles.101 A correlation was noted between release profile and hydrolytic degradation, in that sequence-controlled copolymers degrade at a slower rate than their random counterparts and release encapsulated cargo according to the same trend. Further study highlighted the role that monomer sequence plays in acidic microclimates as a result of localized degradation sites.99 Namely, low pH regions arose from acidic hydrolysis byproducts in PLGA microspheres, and the spatial distributions of these regions was visualized using radiometric two-photon microscopy (Fig. 3a). Microspheres with significantly different acid distributions were subcutaneously injected in mice to analyze the in vivo foreign body response. In agreement with previous findings, glycolic acid-rich blocks degraded more quickly than lactic acid-rich blocks, leading to acidic byproducts that further accelerated hydrolysis. In addition, variations in monomer sequence led to distinct swelling behavior for the PLGA microspheres, which in turn affected localized pH climates. As observed in vitro, particles with sequenced chains degraded slower in vivo in comparison to random chains, which led to a diminished foreign-body response, likely in part due to milder pH environments. These data also have important implications for enhancing the stability of pH-sensitive cargo using sequence-controlled microparticles.

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Fig. 3 Monomer distribution effects on degradation, swelling and erosion of PLGA microparticles with 50[thin space (1/6-em)]:[thin space (1/6-em)]50 L[thin space (1/6-em)]:[thin space (1/6-em)]G content. (A) Acidic microenvironment quantification comparing random and sequenced PLGAs showing more mild pH as a result of slowed, constant degradation rate for alternating sequences and the diminished foreign body response when implementing the particles in vivo. Reprinted from ref. 99 with permission from Elsevier. (B, D) Swelling % and (C, D) erosion half-life (measured by mass loss) correlation to decreasing glycolic acid content and block length as a result of hydrolytic susceptibility. Terminology: Random-PDLGA-50 (ROP, racemic lactic acid, long L- and G-blocks), PLLGA-50 (ROP, L-lactic acid, long L- and G-blocks), R-SAP (SAP, stereopure lactic acid, short L-and G-blocks). Sequenced-polyGLG, (stereopure), polyGLLG (stereopure), polyLracG (racemic), and polyLG (stereopure). Adapted from ref. 106 with permission from Elsevier.

Other studies have also investigated the influence of sequence on degradation to yield uniform degradation products. SAP techniques were applied to synthesize copolymers of lactic, glycolic, and caprolactic acid, as caprolactic acid is another commonly employed monomer for biomaterials applications.100 Precisely placed acid-labile acetal units were incorporated into both linear and star-shaped polymer chains by adding small amounts of aldehyde during the living cationic polymerization of a vinyl ether.104 The time intervals on which the aldehyde was added allowed for prescribed degradation points to be incorporated in the chains and gave predictable, narrowly dispersed degradation products. Hartmann and coworkers applied their solid phase synthesis of oligo(amidoamine)s using Fmoc protecting groups to make uniformly spaced enzyme degradable linkages with defined end groups.105 The oligomers were then combined using CuAAC-mediated polymerization to produce molecular weights reaching above 20 kDa with predetermined density and spacing of cleavage sites for selective degradation. The ability to precisely designate degradation sites within polymer backbones as illustrated in these studies provides valuable control over fragment size and distribution, which are key factors for clearance from the body.

3.2 Gelation, swelling and mechanics

Given the complex and specific criteria hydrogels must answer to for successful translational application, sequence may be a key component in the evolution of designer hydrogels as indicated by both simulation and experimental results reported thus far. Molecular modeling studies conducted on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate) hydrogels characterized their mechanical and transport properties at various hydration levels as a function of monomer sequence.88,89 Comparisons between random and blocky copolymers demonstrated lower chain density for the blocky chains, and non-uniform distribution of water and other guest molecules according to molecular interaction with the monomers. Compressive deformation simulations revealed higher stress levels in the random sequences, which decreased at 10% hydration, in contrast to the blocky sequences, which maintained consistent stress behavior between the dry and hydrated states. It is believed the random sequence is more readily able to structurally rearrange in the presence of water molecules, but the discrepancies in properties like compressive stress response and molecular transport between the sequence types diminish as hydration percentage and degree of polymerization increase.

In an experimental report, Meyer and colleagues explicitly studied the effect of their PLGA monomer sequences with 50[thin space (1/6-em)]:[thin space (1/6-em)]50 L[thin space (1/6-em)]:[thin space (1/6-em)]G content on matrix properties.106In vitro monitoring of swelling, degradation, and erosion of compressed polymer pellets revealed that both degree of swelling and rate of erosion scale positively with the faster cleaving glycol–glycol linkages (Fig. 3b–d). For random PLGA sequences (PDLGA-50, R-SAP, PLLGA-50), the matrices swelled by 107% as compared to only 6% for the alternating PLGA sequences (poly GLG, poly GLLG, poly LracG, poly LG – Fig. 3b). In addition, the random PLGA sequences eroded with a half-life over 3–4 weeks, while the alternating sequences required a half-life of erosion greater than 10 weeks (Fig. 3c). Hydrated polymer films of poly(L-lactide-co-ε-caprolactone) (PLCL) biomedical elastomers also showed chain microstructure effects on mechanical properties.107 In statistical PLCL copolymers of 70% lactide content, sequence was varied from blocky to random, and the random sequences showed increased strength-related properties as compared to the random sequences. Similarly, degradation of polymer films occurred more slowly for blocky PLCL sequences.107,108 These differences were attributed to the crystalline microdomains in the blocky sequences, which hindered water penetration and rendered the mechanics brittle as degradation progressed.

Sequence control in statistical copolymers has also informed gelation parameters for thermoresponsive hydrogels, indicating that random sequences form less stable hydrogels or no hydrogels in comparison to blocky sequences.109 To more explicitly probe sequence effects, Yu et al. synthesized PLGA-PEG-PLGA block copolymers and varied the sequence of the lactic acid and glycolic acid units in the PLGA block from tapered (glycolide units close to PEG block) to inverse tapered (lactide units close to PEG block).34 The inverse tapered sequence gelled at lower temperatures (20 °C) compared to the tapered sequences (37 °C). In another study, Petit et al. varied the end group of PCLA-PEG-PCLA thermoresponsive hydrogels.110 When the polymers were capped with a propionyl group, the hydrogels exhibited an increased maximum storage modulus (Gmax = 600 ± 200 Pa) compared to those capped with an acetyl group (Gmax = 200 ± 100 Pa). The authors speculated the effect arose from increased hydrophobic interactions between the polymer chains, which stabilized hydrogel formation. End groups also had a similar effect on degradation time in related polymer systems, where the propionyl group led to longer degradation times (∼275–290 days) compared to the acetyl group (110–150 days) at 37 °C. These studies indicate that even one monomer difference can have drastic effects on hydrogel properties, a result that has also been seen in protein-based hydrogels.111 As gelation and mechanics have important implications for in vivo hydrogel injectability, it is expected that sequence-controlled polymers will play an increasing role in hydrogel construction as the scalability of synthesis methods continues to improve.

4 Mimicking protein function with biomaterials

A main driving force in the progression of advanced biomaterials is replicating the exquisite efficiency and specificity demonstrated in nature's polymers. Biomaterial-based therapies can mitigate protein dysfunction in cases of disease, often using a more cost-effective or biologically stable platform. Sequence definition is innately a design criteria that must be fulfilled when attempting to mimic proteins, as well as structural control of the biomimetic material. In fact, synthetic molecules designed to replicate the conformational order of proteins are termed foldamers, and considerable research has been focused on their development.70,112 Here, we highlight studies that illustrate the capacity of protein mimetics as biomaterials in three areas: therapeutic foldamers, intracellular delivery materials, and cryopreservative polymers.

4.1 Therapeutic foldamers

One class of molecules that mimics peptide structure and function and shows promising therapeutic potential is peptoids. Peptoids can retain sequence specificity via synthesis using stepwise, solid-phase methods. Their N-substitution confers resistance to proteolysis and decreased immunogenicity, and there are established sequence design rules to induce folding into a helical polyproline type-I-like structure.113 Barron and colleagues designed amphipathic, helical peptoid mimics of lung surfactants proteins B and C as a therapeutic alternative to animal-derived surfactant for acute lung injury (ALI) patients.114 In a rat model of lavage-induced ALI, the peptoid-based surfactant protein C mimic improved indicators of pulmonary gas exchange function (e.g., blood oxygenation and pH) and pulmonary function (e.g., shunt fraction) compared to the peptoid-based surfactant protein B mimic. The peptoid-based surfactant protein C mimic also performed as well as the animal-derived surfactant in some cases, indicating that these foldamers have potential as treatment for ALI. In another study of helical peptoids,115 27 diverse sequences were screened for antimicrobial activity against B. subtilis, E. coli, erythrocytes, and NIH 3T3 cells. The peptoids demonstrated good selectivity for the bacterial membranes over erythrocytes, and the best were further tested against 20 multi-drug resistant pathogens. The peptoid with the most broad antimicrobial activity compared favorably to two clinically-investigated antimicrobial peptides. Furthermore, in vivo testing in a mouse model of invasive S. aureus infection showed successful reduction of bacterial counts against saline controls. One downside of this peptoid is some negative activity on NIH 3T3 cells; however, further study showed that the addition of one cationic residue to the sequence improved the selectivity of antibacterial activity and decreased cytotoxicity to mammalian cells.116

Another class of molecules that aims to mimic protein structure is single chain polymeric nanoparticles (SCNPs),117–119 which have shown potential utility as in vivo imaging agents, drug delivery vehicles, and photodynamic therapy materials.120,121 Folding behavior or chain collapse to a nanoparticle has been accomplished by covalent linkages119,122 including click reactions,123 as well as non-covalent formation by self-assembly,124 metal complexation,125 and host–guest interactions.126 While controlled polymerization methods resulting in relatively low polydispersity are typically employed for these studies, precise sequence effects on SCNP assembly and function are less explored.123,127–131 One study examined the thermodynamic driving forces in the coil-to-globule transition of model polypeptoids for a protein-like (long stretches of hydrophobic and polar monomers) sequence and an alternating, repeating sequence.127 This study demonstrated that hydrophobic sequence patterning, separate from hydrogen bonding or chirality's influence, led to more compact globule formation in aqueous solution, which could aid in designing polymer micelles for drug delivery. Lutz and colleagues devoted significant efforts to guiding SCNP folding into unique shapes like loops129,130,132 and structured coils131 by installing positionable linkages in the polymer backbone to enact specific folding patterns. In recently published work, Cole et al. attempted to explore SCNPs more akin to real proteins by incorporating short peptide and depsipeptide sequences at localized points in the chain using an isocyanide-based multicomponent reaction approach.128 Although the syntheses proved to be challenging, hydrazino turns were demonstrated in addition to supramolecular folding, encouraging the field's trajectory towards sequence-definition in functional foldamers.

4.2 Intracellular delivery with synthetic cell-penetrating sequences

As biotechnology advances strategies such as gene therapy, genome editing, and monoclonal antibodies, the need for improved and efficient intracellular delivery vehicles grows more essential. While some proteins can cross the cellular membrane, issues such as synthetic complexity and stability limit their use as vehicles to protect these delicate cargos.67 To address these issues, significant research effort has revealed essential information on the structure of natural cell-penetrating peptides, allowing for incorporation of key features into synthetic polymer scaffolds. For example, Tew and coworkers explored the effect of sequence in synthetic protein transduction domains (PTDs) by varying degrees of segregation between guanidine residues and hydrophobic groups on polymers via ROMP with Grubbs’ third generation catalyst (Fig. 4a and b).75 Tradeoffs were encountered between internalization efficiency and cytotoxicity as a function of sequence segregation; the nonsegregated polymer was the most active but also the most cytotoxic to Jurkat T-cells and HEK293 T cells in vitro. However, valuable correlations were found between hydrophobic moiety spacing and cell interaction properties, and it was noted that the most strongly segregated (block) sequences buried the hydrophobic groups into a micellar core, thereby limiting cell membrane interaction and uptake (Fig. 4c). In a following study, blocky oxanorbonene copolymers were evaluated for influence of cationic and hydrophobic functional group density on intracellular green fluorescent protein delivery to three cell types in vitro.32 While it was found that high hydrophobic group content corresponded to increased internalization, it was also observed that various positive charge structures had little effect on endosomal uptake mechanisms.33 In a very recent study, Perrier and coworkers showed increased cellular uptake of guanidnium-functionalized acrylamide polymers over polyarginines in their systematic study on the impact of comonomer and monomer distribution.133 Key findings were also that increased hydrophobicity led to higher cellular internalization in both homo- and copolymers, but that monomer distribution impacted both level of uptake and mechanism of internalization. These fundamental studies elucidated the balance between cationic charge, hydrophobicity, and cargo association and their relationship to competing mechanisms of cellular internalization, as well as laid the foundation for further establishment of design rules to guide development of other delivery agents.74
image file: c8bm01215f-f4.tif
Fig. 4 Cell internalization as a function of hydrophobic residue segregation. (A) Monomers used in ROMP synthesis to install hydrophobic (phenyl, green) and hydrophilic (guanidine, blue, after Boc deprotection) units. (B) Representative polymeric isomers with varying degrees of hydrophobic segregation. (C) Polymer internalization results in Jurkat-T cells demonstrate that hydrophobic distribution influences uptake activity, with the homogeneous and gradient sequences having greater internalization than the blocky copolymer. Results hold both with (red) and without (purple) ATP depletion, which influences mechanism of uptake. Adapted with permission from ref. 75. Copyright 2014 American Chemical Society.

Numerous works have explored the advancement of solid-phase synthesized sequence-defined materials in nucleotide delivery applications as well.134–143 For example, Wagner and coworkers investigated 38 oligo(ethane amino) amides for structure–activity relationships,135 and found two key features that improved siRNA polyplex stability and transfection efficiency: (1) precise incorporation of lipid moieties at defined positions in the oligomer increased the pH specificity of polyplex lysis to endosomal pH, and (2) cysteine incorporation stabilized the polyplex via disulfide bonds. To test the best oligomers in vivo, the authors delivered polyplexes with EG5 (a cancer target) siRNA to Neuro2A tumor bearing mice and showed successful knockdown of EG5 mRNA 24 hours after injection.141 Another study incorporated redox-sensitive cleavage sites at precise locations between the lipophilic domain and the charged siRNA binding domain to increase siRNA release and decrease intracellular cytotoxicity.144 As another example, sequence-defined lipid-peptoid (or “lipitoid”) oligomers were shown to form spherical assemblies with siRNA in which the dimensions strongly depended on the ionic strength of the medium and the charge balance between the lipitoid and siRNA.145 A high silencing efficiency (>90%) was demonstrated in vitro for intermediately-sized nanoparticles, and the mechanism of internalization was shown to bypass the lysosome, unlike nanoparticles of other sizes. Finally, in a third example, sequence-defined oligothioetheramides (oligoTEAs, liquid phase stepwise synthesis) showed rapid and efficient cellular internalization without any charged residues at all, even outperforming a commonly used cell penetrating peptide in vitro.76 These studies and others have demonstrated the utility of finely tuning monomer sequence to control intracellular uptake efficiency while limiting cytotoxicity, and they have yielded insight to the various parameters that control cellular internalization.

4.3 Mimicking antifreeze proteins for cryopreservation

Synthetic macromolecular mimics of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are another emerging sector in the protein-mimetic field. Their common properties include ability to tune and modify ice growth by inhibiting ice recrystallization, controlling the morphology of ice crystals, and depressing the freezing point of their surroundings in a non-colligative manner.146 Understanding the mechanisms underlying these processes would be useful for designing materials for cryopreservation of cells and potentially complex tissues. Sequence-controlled polymers thus provide a valuable platform for investigating ice recrystallization inhibition (IRI), as they may bind to specific faces of ice crystals and modify their growth mechanisms.147 Toward this end, a library of peptoids was constructed containing hydroxyl, ether, and methyl functional groups, all of which have been used in antifreeze agents such as poly(vinyl alcohol) and glycerol.148 The peptoid library demonstrated depressed melting temperatures in excess of reduction expected from colligative effects, and one sequence with hydroxyl sidechains exhibited enhanced IRI as well. In another study, sequence control was utilized to study the IRI of regioregular alternating polyampholytes (polymers with both cationic and anionic side chains) with a stoichiometrically balanced number of charged groups.149 Comparison of the regioregular products to random sequences showed enhanced inhibition activity, but hydrophobic content and distribution required balance to maintain solubility. As with many similar applications, primary structure does not act independently in determining activity outcomes, but control of microstructure together with modulation of other macromolecular properties will be crucial for determining design rules and optimizing synthetic analogues to meet and exceed the criteria for biomaterials application.

5 Information-containing polymers

Sequence control lends itself well to applications such as molecular recognition and information storage, a topic less explored with biomaterials. As is theme throughout the field, nature has already optimized systems for both of these applications. When looking at DNA and RNA in the context of the information density and processing capability, there is much inspiration for sequence control of synthetic copolymers. Here we focus on a summary of the ways in which sequence specificity in biology has inspired materials science, and the two have combined for tangible advancements of biomaterials.

5.1 Sequenced polymers as molecular barcodes

Recent work has considered information storage for biomaterials applications using synthetic polymers.150 In fact, just a simple binary code of monomers exhibited sufficient properties for molecular-scale information storage. This idea was investigated by Lutz and coworkers who used distinct monomer units iteratively synthesized to represent 0/1 on a “readable” polymer.150–158 A variety of simple backbone chemistries have been identified, including polyesters, polyamides, and polyurethanes, which can be incorporated into solid-phase iterative syntheses to make sequence-defined polymeric codes. These sequences are then read using tandem mass spectrometry (MS/MS) by distinct spacing between fragmentation peaks according to the sequence in which they were built. In one study, this technology was adapted for biomaterials applications via use as abiotic barcodes on implanted biomedical devices (Fig. 5a). Synthetic molecular tags are especially promising candidates for this application as they are chemically and thermally robust, as well as biologically inert, meaning they will not disrupt or be disrupted by biological species during long-term implantation.158 The feasibility of this idea was examined in recent publications using polyurethane barcodes to tag intraocular implants157 and polymeric films studied in vivo.158 In both studies, taggants were easily sequenced after implementation in their respective devices and did not induce any inflammatory or cytotoxic response (Fig. 5b–d). Sequences used in all studies so far have been relatively short and simple, but would likely extend efficiently to polymers of higher complexity needed to realize this tool as a solution for anti-counterfeiting medical device applications.
image file: c8bm01215f-f5.tif
Fig. 5 Sequence-coded oligourethanes as abiotic taggants for material implants. (A) Experimental approach using hydrogen and methyl groups to generate a molecular barcode on oligourethanes incorporated into PVA films and monitored in vivo before removal and sequencing. (B) Tandem mass spectrum obtained after taggant extraction, post-explantation. (C) In vitro cytotoxicity assay with endothelial cells incubated with taggants. (D) Photograph of implantation sites on rats, two intramuscular (cuts 1 and 2) and two subcutaneous (cuts 3 and 4). Reprinted from ref. 158 with permission from John Wiley and Sons.

5.2 Recognition sequences: synthetic receptors and ligands

Molecular coding is also realized in the recognition motifs installed in cellular receptors and ligands. One area in which sequence-controlled materials have especially made an impact is the synthetic analogues of glycoproteins.159 These have been studied for their potential application as drug delivery components or biological sensors, as well as for contribution to fundamental understanding on the features underlying molecular recognition.160 Selectivity relies on factors including number, density, and spacing of ligands, which has led researchers to employ precision polymerization methods to synthetically modulate these properties in glycopolymers.161,162 Hartmann and colleagues dedicated multiple works to synthesizing highly-defined, carbohydrate functionalized oligo(amidoamine) segments via solid phase synthesis with various functionalization and segment combination chemistries.163–169 In particular, mannose units were incorporated into oligo(amidoamine) backbones and evaluated for their lectin binding affinity.163 Their investigation demonstrated a nonlinear correlation between number of ligands and binding affinity, suggesting that ligand spacing and overall scaffold chemical composition are synergistic properties that must be optimized to maximize lectin binding. The approach was extended to heteromultivalent glycomacromolecules, leading to observation of different binding mechanisms between glycopolymers presenting other functional groups,166 and to stimuli-responsive binders that controlled binding affinity upon photoisomerization.165 While these data contributed insights to binding mechanisms, solid phase synthesis limited the overall chain lengths, prompting further investigation toward achieving high molecular weight glycopolymers.167–169

Other instances of sequence-controlled glycopolymers include single-chain sugar arrays made by radical copolymerization of styrene via nitroxide-mediated polymerization (NMP) with time-controlled addition of N-protected maleimides able to later be selectively functionalized with carbohydrate units.170 Three distinct sugar modifications were installed on the backbone of single chains, which were demonstrated to maintain bioactivity by their ability to specifically recognize complementary proteins. In another study, concurrent enzymatic monomer transformation and RAFT were employed in a one-pot synthesis to make statistical glycopolymers, along with simulation of self-assembly properties in relevance to lectin binding.160 Enzyme-induced transesterification provided an efficient handle for integrated synthesis of blocky, statistical, and gradient copolymers. The blocky and gradient structures exhibited the best lectin-binding capability in experiments, a result explained by the lack of hydrophilic “hairs” on the micelles of statistical copolymers as illustrated by self-assembly simulation. Multiblock glycopolymers designed to inhibit a specific carbohydrate-bonding lectin (DC-SIGN) were made with single-electron transfer living radical polymerization for glycomonomers prepared by CuAAC.171 This approach enabled composition control of the various carbohydrate moieties and further enforced the dependence on both polymer sequence and structure.

6 Conclusions and outlook

The precise structure–property relationships of nature's biological materials have long been a source of inspiration for enhancing the tunability of synthetic polymers. Here, we sought to compile and review the translation of these efforts to applications specific to the biomaterials field, as these are especially susceptible to fine-tuned properties on a molecular scale. Challenges still remain in merging the exact sequence definition and high efficiency synthesis of biological molecules with the scalability of controlled polymerization methods; however, creative adaptations and fusions of traditional polymerization techniques, iterative syntheses, and templated mechanisms have made significant progress in reducing this synthesis gap to yield sequence-controlled synthetic polymers on scales relevant to biomaterials applications. While further progress is needed for large-scale clinical translation, the emerging reports detailed here of sequence impact on properties in vivo demonstrate that these materials already contribute insight to guide biomaterials design. In addition, further developing relationships between monomer sequence and function for these synthetic materials will bolster current methods of sequence selection highlighted here, including bioabstraction and combinatorial libraries.

Synthetic materials are attractive given the ability to modulate properties, and sequence control provides a handle for programming bioactivity. Sequence modifications were demonstrated to play a role in the degradation rates of biodegradable polymers and influence the material properties of hydrogel gelation and swelling, properties relevant for controlled-release and injectable drug delivery platforms. Further study involving regulation of hydrogel mechanical properties like stiffness and permeability using sequence-defined, conformationally distinct components would be useful for constructing extracellular matrix mimetic hydrogels without compositionally changing the polymers or chemistries employed. In addition to designing polymeric systems under sequence control, non-natural mimics of peptide and protein compounds have been reproduced and attuned by synthetic analogues. The breadth of investigation here spans a multitude of biomolecules from minimal functional sequences to complex foldamers meant to match the higher-order structure of proteins. Combining bioactivity regulation with programmed structure remains an obstacle, but continued contributions to understanding of functional domains and global polymer properties (e.g. hydrophobicity, charge, architecture, etc.) relevant to activity will aid in paving this direction. For example, artificial enzymes constructed by molecular imprinting techniques have demonstrated successful catalytic activity,172 and further modification of polymer sequence might provide insight for improving substrate selectivity to biomarkers implicated in diseases for the design of biosensors and therapeutic agents.

Finally, sequence control supplies synthetic polymers with information-containing capability as they can be designed as biologically relevant recognition motifs or engineered as molecular barcodes. Engineering further selectivity into such recognition motifs has promise for other biomaterials applications, such as the macroscale self-assembly of tissue engineered constructs173–175 or the tagging of single cell markers for personalized medicine.176 As advancements in MS sequencing methods continue to emerge, the complexity and capacity of these information-rich polymers is expected to increase in the direction of programmed polymers, thereby advancing both structure and function of synthetic biomaterials even closer to that of biological materials.

Conflicts of interest

There are no conflicts of interest to declare.


AMR gratefully acknowledges a Career Award at the Scientific Interface (#1015895) from the Burroughs Wellcome Fund. MJA gratefully acknowledges the University of Texas Engineering Doctoral Fellowship Endowment for their support.


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