University of Groningen DNA-based drug carriers and dynamic proteoids with tunable properties Liu, Yun

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DNA-based drug carriers and dynamic proteoids with tunable properties Liu, Yun

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Publication date: 2017

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Liu, Y. (2017). DNA-based drug carriers and dynamic proteoids with tunable properties. University of Groningen.

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DNA-based drug carriers and dynamic

proteoids with tunable properties

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The work described in this thesis was carried out at the Stratingh Institute for Chemistry, University of Groningen, The Netherlands.

This work was financially supported by the Chinese Scholarship Council.

Printed by: Ipskamp Printing, Enschede, The Netherlands

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DNA-based drug carriers and

dynamic proteoids with tunable

properties

PhD thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the Rector Magnificus Prof. E. Sterken

and in accordance with

the decision by the College of Deans. This thesis will be defended in public on

Friday 10 March 2017 at 12.45 hours

by

Yun Liu

born on 16 June 1987 in Henan, China

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Supervisors Prof. A. K. H. Hirsch Prof. A. J. Minnaard Assessment committee Prof. K. U. Loos Prof. S. Otto Prof. N. Katsonis

ISBN: _{978-90-367-9509-8 (printed version)} 978-90-367-9508-1 (electronic version)

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To

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

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Chapter 1

Nucleic Acid Amphiphiles as Drug Carriers

This chapter gives an introduction on the development of nucleic acid amphiphiles as drug carriers, including their synthetic approaches, controllable self-assembly behavior, and modification methods.

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1.1 Introduction

Drug discovery is a challenging and time-consuming undertaking. Over the past decades, new technologies in drug discovery have emerged, including combinatorial chemistry,[1] structure-based drug design,[2] high-throughput screening,[3] fragment-based drug design[4] and ultrahigh-throughput screening.[5] With these methodologies, numerous pharmacologically interesting molecules have been created and identified. Increasingly, people focus on optimizing their properties from already in the early stage of drug development. Despite this, it is surprising that more than 40% of approved drugs on the market and about 90% of drug candidates in the discovery pipeline are poorly soluble in water.[6]

Poor water solubility is undesirable as it slows down the absorption process, thereby leading to inadequate and variable bioavailability. Increasing the dosage is necessary to achieve the required concentration in the systemic circulation at which the desired therapeutic response may take place.[7] In most cases, this causes toxicity issues and adverse effects. Therefore, research efforts have been focused on establishing drug delivery approaches and numerous delivery techniques have been reported and developed (Figure 1).[8]

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Among all approaches currently available, nanomedicine has emerged as a particularly active field of research. Nanomedicine is the application of nanotechnology to medicine.[9] One of its applications is to utilize nanoparticles as drug-delivery vehicles. These nanocarriers generally range from 1 to 1000 nm in diameter. Typically, the drug is dissolved, entrapped, adsorbed, attached or encapsulated into or onto nanocarriers. Based on their material composition, nanocarriers are divided into three types (Figure 2): bio-inspired nanocarriers,[10] inorganic nanocarriers[11] and synthetic polymeric nanocarriers.[12] The increasing research interest in synthetic polymers for drug delivery is due to their diversity, which is attributed to the diversity of synthetic polymers. It endows the resulting nanocarriers with multiple capacities, such as stimuli-responsiveness (pH,[13] temperature,[14] light,[15] etc.), bioavailabity,[16] and self-assembly.[17] According to their structural features, synthetic polymeric nanocarriers can be classified into dendrimers,[18] polymer–drug conjugates[19] and self-assembled nanocarriers.[20]

Figure 2. Categories of nanocarriers.

Molecular self-assembly is omnipresent in nature and plays an essential role during the formation of many biological structures.[21] Furthermore, it is an important approach to create well-defined and controllable nanostructures, a concept referred to as bottom-up construction.[22] Molecular self-assembly allows the fabrication of

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different types of organized nanostructures from simple building blocks. The whole process is driven by non-covalent interactions, such as hydrogen bonding, electrostatic, van der Waals and hydrophobic interactions, does not require any external input and depends on the inherent nature of molecules themselves. Numerous types of molecules and materials have been utilized to fabricate self-assembled nanocarriers, resulting in nanocarriers with diverse nanostructures and properties for various medical conditions and drugs. Because of their diversity and infinite potential, self-assembled nanocarriers have drawn great research attention over the past decades. Nanocarriers are formed through self-assembly of various molecules, including nanospheres,[23] nanocapsules,[24] micelles,[25] polymersomes,[26] nanogels,[27] nanofibers (Figure 3).[28]Desired structures of the resulting nanocarriers can be tuned by designing and synthesizing the building blocks at a molecular level.

1.2 Self-assembled DNA nanocarriers

DNA is a material that can self-assemble into double helices through Watson-Crick base pairing, and the structure is stabilized by non-covalent interactions, including hydrogen-bonding, π−π-stacking and hydrophobic interactions. The strict base-pairing principles enable DNA to construct various types of well-ordered nanostructures (from 1D to 3D structures) for a range of medical applications.[29]

Structural DNA nanotechnology[30] was pioneered around three decades ago by Seeman, who performed research with DNA junctions and lattices.[31] Since then, a rapid growth and development has been witnessed in this field. The key contribution to this research area is the invention of DNA origami in 2006,[32] which is the nanoscale folding of DNA used to create two- and three-dimensional nanostructures through Watson-Crick base pairing. To date, a variety of DNA nanostructures have been successfully constructed and applied as targeted drug carriers. The inherent properties of DNA endow these nanocarriers with improved biocompatibility and biodegradability. Based on the molecular composition of DNA nanocarriers, they are grouped into pristine DNA nanocarriers and hybrid DNA nanocarriers.

Structural DNA nanotechnology allows scientists to engineer pristine DNAs into nanocarriers with well-defined structures, such as cubic, tetrahedral, octahedral, buckyball, icosahedra and origami.[33] This topic has been recently reviewed and will not be covered in detail in this chapter.[34] The assembled nanocarriers have several advantages, including: (1) programmable and well-ordered structure, (2) high loading capacity, (3) good structural stability in a physiological environment. Aside from pristine DNA, DNA-polymer or -lipid hybrid macromolecules are also exploited as

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building blocks to construct self-assembled DNA nanocarriers and will be discussed in section 1.3.

Figure 3. Schematic representations of various polymeric nanocarriers.

1.3 Amphiphilic DNA nanocarriers

The self-assembly of DNA-polymer or -lipid conjugates generates various nanostructures such as hydrogels, micelles,[35] vesicles and tubes.[36] Hydrogels formed from DNA hybrid materials have been reviewed elsewhere.[37] In this chapter, the focus is on DNA amphiphiles self-assembling into micelles, tubes and vesicles are focused (Figure 4), especially micelles.

Nucleic acid amphiphiles consist of a hydrophilic DNA moiety and a hydrophobic segment. They are synthesized and used as amphiphilic DNA nanocarriers by self-assembling into micelles, tubes or vesicles. In aqueous solution, these structures are stabilized by both non-covalent and hydrophobic interactions from the DNA and hydrophobic moieties, respectively. For micelles and tubes, DNAs are exposed to the aqueous environment, so they have a hydrophobic interior and a hydrophilic external corona. Therefore, hydrophobic drugs can be encapsulated inside the core. In contrast, vesicles are composed of an amphiphilic bilayer, such that both the interior and exterior are hydrophilic, with a hydrophobic layer in between. The latter is suitable for insoluble drugs, while hydrophilic drugs can be encapsulated in the hydrophilic interior. As a result, vesicles are capable of delivering both hydrophobic drugs and

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hydrophilic drugs. Given that the drug interacts with the drug carrier through non-covalent interactions, there is no need for covalent modification of the drug.

Figure 4. Schematic representation of the self-assembly of DNA amphiphiles into

micelles, vesicle and tubes.

Owing to their small size and use of biocompatible DNA as a component, these nanocarriers feature advantages including: (1) high loading capacity attributed to large surface area to volume ratio; (2) improved biocompatibility by reducing the dose; (3) passive targeting to and drug accumulation within tumors because of the enhanced permeability and retention (EPR)[38] effect of the vasculature; (4) ease of manufacture; (5) compatibility with a wide range of drugs; (6) automated synthesis; (7) active targeting to specific sites and controllable activation of drugs given their responsiveness to internal or external stimuli; (8) active targeting to specific sites through ligand-receptor-mediated drug targeting.[39] Furthermore, these advantages can be of benefit to almost all drug-administration routes, namerly parenteral, oral, nasal and ocular administrations.

In some diseases, especially tumors, there is an overexpression of cell surface tumor-associated receptors on the cell surface that are found at low levels in normal tissues. In this case, by functionalizing the surface of nanocarriers with specific ligands for these receptors, an increased accumulation and selective cellular uptake could be expected. As a result, the therapeutic efficacy of the drug is improved

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leading to a lower dose. The following ligands have been investigated in various drug-delivery systems: carbohydrates,[40] monoclonal antibodies,[41] folate,[42] homing peptides,[43]targeting proteins[44] and aptamers.[45]

1.3.1 Synthesis of DNA-polymer or -lipid conjugates

DNA-polymer conjugates were first synthesized in the 1980s by grafting DNA onto a poly((L-lysine) backbone.[46] Since then, various synthetic methods have been

exploited to obtain DNA-polymer (including hydrophobic and hydrophilic polymers) or -lipid conjugates, using chemical synthesis and molecular biology techniques (Figure 5). It is noteworthy that these synthetic methods can also be used for the surface modification and functionalization of the nanocarriers.

Figure 5. Methods explored for chemical synthesis of DNA hybrid materials.

Through chemical synthesis, designed and specific DNA sequences can be obtained. Generally, three types of strategies are reported for chemical synthesis: (1) Solution-phase synthesis. DNA and polymer are independently synthesized through standard methods, followed by coupling them in solution. The advantage of this strategy is the ease of functionalization and diversity allowed for the polymer moiety. However, it is not an easy task to find the proper solvent for the reaction. Poor solubility of hydrophobic polymers in aqueous media generally leads to low yields. In

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contrast, hydrophilic polymers can be coupled relatively easily to DNA given that both are sufficiently soluble in aqueous media. Different conjugation reactions have been used for the coupling in aqueous solution, including click chemistry,[47] Michael addition[48] and the formation of disulfide[49] and amide bonds (Figure 5a).[50] (2) Polymer-linked phosphoramidites or lipid-modified nucleobases are used for solid-phase synthesis (Figure 5b).[51] The essential step of this method is the functionalization of the polymer or the modification of the nucleobase with a polymer/lipid. As a result, the product can be directly applied in standard solid-phase synthesis of DNA. For instance, Herrmann and coworkers designed and synthesized a family of lipid-modifed DNA 12-mers by using dodec-1-yne-modified uracil. The hydrophobicity of the lipid-modified DNAs is tuned by adjusting the number and position of the modified uracils.[52] Since the functionalized nucleobases can be attached in a sequence-defined manner, the key benefit of this method is the tunability. A drawback might be the poor solubility of the functionalized polymer or modified nucleobases. (3) DNA with a polymerization initiator is synthesized through solid-phase synthesis (Figure 5c), followed by polymerization in solution after cleavage or on the solid support prior to cleavage. In this method, the issue of solubility is circumvented. The former requires the initiator to be stable in solution, while the resultant DNA-polymer conjugates need to survive the cleavage step in the

latter case. For example, Das and coworkers prepared

DNA-b-poly[oligo(ethyleneoxide) methacrylate] (POEOMA) both on solid support and in solution after cleavage of DNA. DNA-b-poly(benzyl methacrylate) (PBnMA) was also synthesized after cleavage.[53] Importantly, a phosphoramidite containing a bromoisobutyryl atom-transfer radical polymerization (ATRP) initiator capable of tolerating typical DNA deprotection conditions is necessary for solution-phase initiated polymerization.

Application of molecular-biology techniques offers another way for fabrication of DNA hybrid materials. The limitation of the length of DNA imposed by solid-phase synthesis can be circumvented, by utilizing various molecular-biology techniques, enabling the synthesis of conjugates with thousands of base pairs in the DNA moiety. Such techniques include the polymerase chain reaction (PCR)[54] and enzymatic restriction and ligation.[55]

1.3.2 Controllable self-assembly of amphiphilic DNAs

When amphiphilic DNAs are dissolved in aqueous media, self-assembly takes place to form in principle various well-organized nanostructures. In practice, mainly micelles are observed. The first formation of vesicles was reported in 2007.[56] By

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controlling the self-assembly of the materials, the function will be improved and the scope of potential applications expanded. Control of self-assembly is achieved in various ways.

Figure 6. Schematic representation of hybridization of DNA-b-polypropylene oxide

(PPO) micelles with various DNA molecules. (a) Base pairing with a short complementary DNA (cDNA) yields micelles. (b) Hybridization with long cDNA templates results in nanorods consisting of two double helices aligned in parallel.[58]

The first strategy is to adjust the chemical composition of the building block. Self-assembly of amphiphilic DNAs is driven by hydrophobic interactions from the polymer moiety, and the well-ordered nanostructures are stabilized by both hydrophobic interactions and the hydrophilic layer. As a result, the chemical composition has a great influence on the resultant structures. By altering the hydrophobic to hydrophilic ratio or the position of hydrophobic moieties, properties of nanostructures formed (size, shape etc.) are tunable. As mentioned in section 1.3.1, Herrmann and coworkers synthesized 12-mer lipid-modified DNAs with different numbers and positions of lipid chains. When increasing the numbers of lipid chains from two to four, the hydrophobicity is enhanced, leading to a decrement in both size

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and critical micelle concentration (CMC).[51] Besides the hydrophobic moiety, adjusting the length of the DNA segment is also used to control self-assembly.[57]

One of the key advantages of amphiphilic DNAs is their capacity of hybridization with complementary DNAs (cDNAs) even after self-assembly. This ability can be used for functionalization (see section 1.3.3) to control self-assembly. Herrmann and coworkers reported for instance that upon addition of the cDNAs, micelles were switched into nanorods (Figure 6).[58]

Other ways to control self-assembly include application of enzymatic reactions[59] and change of environmental conditions, such as pH,[61] metal ions [50]and so on.

1.3.3 Modifications of amphiphilic DNAs

Further modification can be performed on the surface of nanocarriers, which corresponds to the DNA moiety. The modification can be accomplished through chemical or physical methods. The former consists in coupling functional groups during the synthesis of amphiphilic DNA (see section 1.3.1). Physical modification is achieved by hybridization of amphiphilic DNAs with cDNAs bearing the desired functional groups. The main advantage of physical modification is the reversibility. Surface modifications are employed for two main purposes: (1) to improve the biocompatibility and reduce the immunogenicity by functionalization with polyethylene glycol (PEG).[61] This can be easily realized through hybridization with PEG-cDNA strands, while the PEG-cDNA moiety also can be replaced through hybridization by adding pristine DNAs. (2) to functionalize the nanocarriers with targeting and cell-penetrating groups, such as carbohydrates, antibodies, folate, homing peptides, targeting proteins, aptamers or cell-penetrating peptides through hybridization.[62]Herrmann and coworkers reported that DNA-b-PPO micelles were successfully functionalized with folate-modified cDNAs through hybridization. This material was used as a drug carrier of doxorubicin. Cell-culture experiments showed that cell uptake is related to the density of folate on the surface, and cancer cells were efficiently killed (Figure 7).[63]

It should be pointed out that hybridization offers a potential way for multifunctionalization of DNA nanocarriers. Since cDNAs bearing different functional groups can be used for hybridization, by adding several types of modified cDNAs at once and controlling their ratios, multifunctionalization can be accomplished simultaneously in one system (Figure 8). This method is more convenient, rational, flexible and controllable compared with other methods for surface modification of drug carriers. A possible disadvantage of this method may be that it might change the self-assembly of the resultant system. One conceivable

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solution is to stabilize the micelle structures by crosslinking before hybridization. On the other hand, the multifunctionalization strategy can also be applied to multifunctionalize the surface of inorganic nanoparticles, which are rigid nanocarriers. In brief, careful design and intensive research are necessary to develop an efficient multifunctional delivery system.

Figure 7. Schematic representation of the drug delivery system based on DNA-b-PPO

micelles. (a) The micelles are functionalized through hybridization with folate (red dots) modified cDNA. (b) The anticancer drug doxorubicin (green dots) is loaded into micelles. [63]

Figure 8. Schematic representation of the drug-delivery system based on

lipid-modified DNA micelles. (a) The micelles are functionalized with polyethylene glycol (PEG) and targeting groups at once through hybridizing with modified cDNA. (b) The hydrophobic drug is loaded into the micelles.

1.4 Conclusions and outlook

In this chapter, self-assembled DNA nanocarriers especially amphiphilic DNA nanocarriers are introduced and discussed, including their synthesis, controllable self-assembly and surface functionalization. The nanocarriers obtained can be readily

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decorated with functional groups to target specific sites of the body, elongate the circulation time, respond to internal or external stimuli, penetrate the cell membrane, reduce immunogenicity and incorporate drugs. Both chemical modification and hybridization with cDNAs are commonly employed for functionalization. Recent progress demonstrates the potentially wide usage of the controllable and targeted drug carriers in the field of nanomedicine.

So far, structural DNA nanotechnology, and in particular drug-delivery research, remain in their early stages. Amphiphilic DNAs have shown outstanding performance as future drug carriers and need to be extensively studied to gain a better understanding enabling applications. To the best of our knowledge, no clinical trials are in progress yet. Despite of the rapid growth and development over the past decades, there are still several key issues that need to be solved prior to any medical or clinical applications. First, like all other micellar systems, their instability upon exposure to the cellular environment remains a major concern for drug delivery. Incorporating cross-linkers may prevent disassociation and might be a solution.[64] Second, the mechanism for cell entry remains unclear. Although ready cell uptake of amphiphilic DNA nanocarriers has been observed, unraveling the underlying mechanism will help to improve the delivery efficiency. Important questions to be addressed are: how do key factors such as size and shape influence cell entry. Third, safety issues, such as their intracellular route and fate, the immune response due to the high local DNA concentration and the relationship between the intracellular behavior and their physical/chemical properties, shape and size need to be investigated. Even if DNA is more biocompatible and biodegradable than other materials, the human body is so complex that DNA may still have biological effects on the host, which have not been anticipated during the design of the system. Without enough data and careful analysis, overestimates of the functionality and misleading prediction of the clinical effects might occur. Finally, method and cost of the large-scale production have always been a major challenge for synthetic DNA nanocarriers due to low yields. The synthesis of starting materials and purification on large scale are more expensive than for other materials such as polysaccharides and poly-amino acids. As this is a constantly growing and developing research field, the production needs to be scaled up, leading to a lower cost in the future. A few efforts have been made to address these problems. For instance, the production of pure DNA origami has been scaled up to gram scale from micro-/milligram scale.[65]

Taking into consideration all the advantages and disadvantages, self-assembled DNA nanocarriers are holding a great promise to be employed in nanomedicine. We anticipate multiple in vivo applications in the near future.

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1.5 Outline of this thesis

Chapter 2 gives an introduction on the development of constitutionally dynamic covalent analogues of nucleic acids (DyNAs), polysaccharides (glycodynamers) and proteins (dynamic proteoids) as novel functional biomaterials, including their synthetic approaches and various applications.

In chapter 3, we report on the design and solid-phase synthesis of three types of lipid-modified DNAs. Their self-assembled micelles were ultilized as solubilizers of the insoluble photosensitizer Foscan used in photodynamic therapy. High drug loading capacities were achieved while not influence the biological activity of the active pharmaceutical ingredient.

In chapter 4, describes the use of lipid-modified DNA that forms micelles at comparatively low critical micelle concentration to render budesonide, a hydrophobic glucocorticoid with high anti-inflammatory activity, water soluble with a high loading capacity. The inhibition of interleukin-8 release showed that the new delivery system retains the inhibitory activity in cell-based assays.

In chapter 5, describes the design and synthesis of a range of dynamic proteoids through polycondensation of different types of amino acid hydrazides with a nonbiological dialdehyde. The polymerization is driven by the self-organization/folding of the generated polymers. It demonstrates that the side chains of the amino acid hydrazides have a strong influence on the rates of polymerization, structures and dynamic properties of the resulting biodynamers.

In chapter 6, we report on the design and synthesis of dynamic proteoids through polycondensation of different types of amino acid and dipeptide hydrazides with a nonbiological aromatic dialdehyde and a biological aliphatic dialdehyde. By using the biological dialdehyde, biocompatibility of the resulting biodynamers was improved. Further study demonstrated that the aromatic group in the side chain of the amino acid plays an essential role through π−π-stacking interactions, and the hydroxyl group has a less important effect and stabilizes the architectures formed via hydrogen bonds, whereas a high density of positive charges hinders the generation of biodynamers owing to the electrostatic repulsions.

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Chapter 2

Molecular Biodynamers: Dynamic Covalent Analogues of

Biopolymers

This chapter gives an introduction on the development of constitutionally dynamic covalent analogues of nucleic acids (DyNAs), polysaccharides (glycodynamers) and proteins (dynamic proteoids) as novel functional biomaterials.

This chapter is adapted from the original publication:

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2.1 From constitutional dynamic chemistry to dynamers

Importing the concept of constitutional dynamics from supramolecular chemistry into molecular chemistry through the use of reversible covalent bonds instead of supramolecular non-covalent interactions opens up novel perspectives to chemistry and leads to the emergence of constitutional dynamic chemistry (CDC), which leads toward adaptive chemistry.[1] CDC encompasses both dynamic covalent chemistry (DCC) and dynamic non-covalent chemistry (DNCC). It takes advantage of the lability of reversible covalent bonds formed by reversible chemical reactions or of non-covalent interactions between molecular recognition groups to generate constitutional molecular or supramolecular diversity within constitutional dynamic libraries (CDLs) of chemical species of either molecular or supramolecular type. By contrast, “static” molecular chemistry makes use of non-reversible covalent bonds to synthesize constitutionally stable chemical substances.[1] CDC features the formation of reversible connections, either reversible covalent bonds or non-covalent interactions, which are implemented to respectively link the subunits of a molecular or supramolecular entity in chemical systems. The resulting CDLs consist of entities that can undergo continuous constitutional changes/adaptations through incorporation/decorporation or reshuffling of components in response to physical or chemical, internal or external stimuli. As a consequence, the resulting systems are chemically diverse, dynamic and adaptable at both molecular and supramolecular levels, providing new possibilities and tools for the screening of bio-active compounds, exploitation of receptors or substrates driven by molecular recognition, and fabrication of constitutionally dynamic materials.[1a, 2]

The implementation of CDC specifically in polymer science, leads to the generation of constitutionally dynamic polymers or “dynamers” (Figure 1), at both molecular and supramolecular levels through DCC and DNCC, respectively.[3] Dynamers are defined as polymers in which the monomers are connected through reversible covalent bonds or non-covalent interactions. By virtue of the properties of reversible linkages and core groups, dynamers possess both dynamic and adaptive features, and may undergo spontaneous and continuous constitutional modifications via assembly/disassembly and exchange of their components in response to internal or external stimuli. Compared with constitutionally static polymers, dynamers behave as smart polymers with novel features such as self-healing, stimuli-responsiveness, tunable mechanical and optical properties.[3b, 3c]

According to the type of reversible connection, dynamers can be subdivided into three categories (Figure 1): (1) molecular dynamers: covalent equilibrium polymers,

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generated by polymerization through the construction of reversible covalent bonds including Diels–Alder linkages, imines, acylhydrazones, oximes, boronate esters, and disulfides;[4] (2) supramolecular dynamers: non-covalent reversible polymers, produced by poly-association of ditopic static monomers via formation of non-covalent bonds such as hydrogen bonding, π−π-stacking, electrostatic interactions, metal ion coordination, host–guest recognition and van der Waals forces; [4a, 5]

(3) double dynamers: polymers with constitutionally dynamic properties at both molecular and supramolecular levels, fabricated through a combination of reversible covalent bonds with non-covalent interactions.[4a, 6] In particular, molecular dynamers are currently receiving extensive research attention. The use of DCC in equilibrium polymerization provides a new methodology for polymer synthesis.

Figure 1. Generation of molecular and supramolecular dynamers through

constitutional dynamic chemistry.

2.2 Molecular biodynamers: molecular/covalent dynamers with

biologically relevant monomers

Biopolymers or biomacromolecules, are polymeric molecules created by living organisms. Owing to their mode of generation, their molecular constitution and well-defined 3D structure, they exhibit various functions and biocompatibility. Based on the type of basic building block, they are grouped into three categories: nucleic

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acids, polysaccharides and proteins. Extending the principles of dynamers into the field of biopolymers leads to the definition of biodynamers, that is, dynamers implementing biorelevant residues.[3b] Biodynamers are prepared by reversible covalent polymerization or non-covalent poly-association. As a result, biodynamers are constitutionally dynamic analogues of biopolymers at both molecular and supramolecular levels and hold the ability to combine biofunctionality (recognition, catalysis) of biopolymers with the adaptive feature of dynamers leading to synergistic properties. By analogy to the classification of dynamers, biodynamers can be divided into molecular, supramolecular and double biodynamers.

In contrast to naturally occurring biopolymers or static analogues of biopolymers, molecular biodynamers are based on biorelevant monomers connected by reversible linkages. As consequence of the inherent dynamic properties of DCC, molecular biodynamers are capable of reorganizing their components, modifying their sequence or adapting their length in response to various physical or chemical factors even after polymerization. Therefore, unlike either static biopolymers featuring structural stability and unity owing to their irreversible covalent bonds, or supramolecular biodynamers displaying chemical lability and diversity resulting from their comparatively fragile non-covalent interactions, molecular biodynamers display an attractive balance by taking advantage of reversible covalent bonds. As a result, molecular biodynamers exhibit an optimal combination of relative structural stability and lability with comparable chemical unity and diversity. More specifically, the inherent nature of biorelevant constituents and reversible covalent bonds may confer to molecular biodynamers biocompatible, biodegradable, biofunctional, changeable, tunable, controllable, self-healing and stimuli-responsive properties.

As biofunctionalities of nucleic acids, polysaccharides and proteins rely on their highly-ordered assembled 3D structures,[7] mimicking or modifying biopolymers also provides novel tools to unravel the correlation between biofunctionality and structure of biopolymers. In view of all these considerations, the development of novel molecular biodynamers as adaptive and functional biomaterials is presently receiving considerable attention. Accordingly, the construction of molecular biodynamers, through the incorporation of nucleobase-, carbohydrate- or amino-acid-derived moieties, gives rise to the formation of covalent dynamic analogues of nucleic acids (DyNAs), polysaccharides (glycodynamers) or proteins (dynamic proteoids), respectively.[3b] These molecular biodynamers are created by reversible polymerization in aqueous media under mild conditions, which resemble the physiological environment for future application as smart biomaterials.

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In this chapter, we will give a brief review of recent work on molecular biodynamers, namely the fabrication of DyNAs, glycodynamers and dynamic proteoids.

2.3 Molecular biodynamers: DyNAs, glycodynamers and dynamic

proteoids

2.3.1 DyNAs: dynamic analogues of nucleic acids

DyNAs, with ribose- or non-ribose-backbones, can be classified into main-chain- and side-chain-dynamic categories. The former are made by reversible polymerization of nucleobase-derived monomers, while the latter are prepared by reversibly grafting nucleobase residues through DCC (Figure 2). Hence, their constitution and properties are adaptable under given conditions in response to driving forces such as self-folding into stable secondary or tertiary architectures, substrate binding or addition of target entities, including complementary DNAs (DNA-templated reversible polymerization[8]) or non-DNA targets.[9]

Figure 2. Generation (a) of main-chain DyNAs and (b) side-chain DyNAs.

DNA-templated reversible polymerization of nucleobase-modified ditopic monomers allows for the synthesis of main-chain DyNAs (Figure 3a). As DNA template, the complementary DNA acts as catalyst during equilibrium polymerization to facilitate the reaction through specific Watson−Crick base-pairing interactions (DNA hybridization). Therefore, reversible polymerization cannot take place in the absence of DNA template, and the change of DNA template leads to the fabrication of compounds with a different sequence. In other words, DNA-templated reversible synthesis of DyNAs displays sequence-specificity and –selectivity such that only sequence-matched DyNAs are generated and amplified.[8b] Lynn and coworkers

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pioneered DNA-templated synthesis of main-chain DyNAs with ribose backbones. They accomplished DNA-templated reversible polycondensation of synthetic mono-, di-, tetra-nucleotides to produce octamer DyNAs with ribose main chains through formation of reversible imine bonds in aqueous media, affording stable products in high yield (~80%) after reductive amination.[10] With this methodology, even sequence defined DyNAs of main-chain-dynamic type with 33 nucleotides were synthsized.[11] Furthermore, similar polymerization was achieved by using solid-supported DNA templates in high yield (~90%).[12] The solid-supported templates can be conveniently prepared by automated solid-phase DNA synthesis and repeatedly utilized for catalysis and purification of products, which saves time and effort for the synthesis of DyNAs.

Figure 3. Schematic representation of the synthesis DyNAs with and without DNA

templates.

In comparison to DNA or RNA, peptide nucleic acids (PNAs) have peptide-like (non-ribose) backbones instead of ribose main chains. PNAs still hold the capacity to form stable double-helical structures with DNA, RNA, or themself in accord with Watson−Crick base-pairing rules.[13]

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reversible polymerization and the methodology of reductive amination to non-ribose peptide-like backbones leads to production of dynamic analogues of PNAs.[8c, 8d] DyNAs of both main-chain- and side-chain-dynamic types were efficiently fabricated (Figures 3a and 3b) through imine formation, and static products were obtained at high yields after reductive amination.[8c, 14] In addition to imine condensation, dynamic analogues of PNAs can also be generated by using other types of reversible covalent bonds, such as thioester formation.[15] Consistent with the conclusions of dynamic analogues of DNAs, DNA-templated synthesis of dynamic analogues of PNAs proceeds in a sequence-specific manner, resulting in sequence specificity and chain-length controllability.[14a] Thus, the use of complementary DNA as template not only provides a driving force for reversible polymerization through DNA base pairing, but also results in the sequence-directed synthesis of DyNAs.

In contrast to DNA-templated synthesis, it has been shown that the presence of polyanionic entities can also induce adaption in chain-length of DyNAs.[9] Constitutional modifications are, however, mainly driven by electrostatic forces between substrates and polyanionic targets instead of Watson−Crick base-pairing interactions (Figure 3c). Main-chain-dynamic types of DyNAs without ribose backbones were designed and synthezised through reversible polycondensation of dialdehydes with nucleobase-derived dihydrazides in aqueous media under mildly acidic conditions (Figures 4a and 4b). The formation of polyacylhydrazones was selected due to its synthetic accessibility. Furthermore, the resulting acylhydrazaones are doubly functional through reversible imine-bond formation and non-covalent hydrogen-bonding interactions via the amide groups. As a consequence, the resulting dynamic cationic polymers are able to optimize their constitution in response to pH, temperature or chemical additives to achieve tunability and stimuli-responsiveness even after polycondensation. More importantly, it was shown that anionic target species, such as inositol hexaphosphate (IHP), inositol tripyrophosphate (ITPP), polyaspartic acid and adenosine triphosphate (ATP) (Figure 4c), trigger modification of their chain length through electrostatic forces. Surface plasmon resonance (SPR) measurements indicated that high binding affinities were induced by electrostatic forces between DyNAs and anionic polynucleotides (Figure 4d).[9]

To conclude, these findings, from both DNA- and non-DNA-templated synthesis of DyNAs, reveal that the utilization of anionic entities (DNA or non-DNA) can initiate constitutional adaptions of DyNAs via specific or non-specific non-covalent interactions between building blocks and target molecules, and result in generation and amplification of the best adapted DyNAs. Nucleic acids, including both DNA and RNA, are essential biomacromolecules with biological functionalities, such as storage

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of genetic information (DNA) and translation of genetic code into proteins (RNA). Thus, DyNAs can provide in principle a novel methodology for designing and producing structural and functional biomimetics of nucleic acids, which can be used as biofunctional materials, for instance, in the areas of nucleic acid sensing and gene delivery.

Figure 4. (a) Ditopic cationic monomers used for polyacylhydrazone formation. (b)

Structures of generated polyacylhydrazones. (c) Structures of polyanionic targets. (d) Surface plasmon resonance (SPR) for binding of poly(1-3) and poly(1-4) to polyadenine at different pH values (“ ” pH = 4.5, “ ” pH = 5, “ ” pH = 6). Adapted with permission from ref 9. Copyright 2006 WILEY-VCH Verlag Gmbh & Co. KGaA, Weinheim.

2.3.2 Glycodynamers: dynamic analogues of polysaccharides

Saccharide recognition plays a key role in many biological processes, including cell-cell interactions and cell communication,[16] which makes carbohydrates attractive entities to create mimics of carbohydrate-based recognition processes. Given that carbohydrates are associated with numerous diseases, many attempts have

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been made to design and construct carbohydrate-based species for therapy and diagnosis of saccharide-associated diseases, such as tumors and chronic inflammation.[17] Application of DCC in glycoscience offers novel opportunities for this field.

CDLs of saccharides are generated by DCC at the molecular level and feature recombination of their components through reversible covalent bonds and amplification of specific compounds due to receptor-binding processes in response to the addition of target entities. Due to the inherent adaptive nature of dynamic saccharide libraries, such CDLs allow for target-driven and self-screening processes. Dynamic saccharide libraries were designed and generated through the formation of acylhydrazone[18] and disulfide bonds[19] in aqueous media at physiological pH. The CDLs obtained were applied for both rapid generation and efficient identification of ligands targeting lectin with enhanced inhibitory efficiency.

On the other hand, DCC allows one to mimic, modify or (bio)functionalize polysaccharides through the generation of glycodynamers. As a consequence of the intrinsic dynamic features of DCC and the bio-activity of the carbohydrate-based components used, glycodynamers hold the potential to feature synergistic properties by combining adaptability with biofunctionality (molecular recognition), biodegradability and biocompatibility of carbohydrates and may thus find application in the field of biofunctional materials science. Through different synthetic approaches, one may envisage to create three types of glycodynamers (Figure 5): (1) glycosidic main-chain, resulting from either (a) polymerization of saccharide residues through reversible covalent reactions or (b) reversible conjugation of small molecules to a static glycosidic backbone; (2) glycosidic side-chain, in which saccharide residues are either (a) irreversibly attached to a dynamicbackbone or (b) reversibly appended on a static backbone; (3) glycodynamer containing both a dynamic backbone and reversible side chain(s).[20]

Glycodynamers with a dynamic glycosidic main-chain (type 1a) can be prepared by reversible covalent polymerization of ditopic saccharide residues (Figure 5a). As dynamic mimics of naturally occurring glycans, the resulting materials exhibit both adaptability and biorelevant properties. Oxime-bond formation, through reversible condensation of aldehyde and hydroxylamine monomers, is widely employed due to its inherent advantages: (1) efficient formation at mildly acidic pH; (2) higher stability against hydrolysis compared to corresponding imine; (3) stability in aqueous solution at physiological pH; (4) pH responsiveness.[21] Oxime polysaccharides fabricated through enzyme-triggered polycondensation both in moderately acidic (pH~5.5) and

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Figure 5. Schematic representation of the generation of different types of

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Figure 6. (a) Structures of ditopic monomers 5−7 and the termination agent 8. (b)

Generation of the glycodynamer poly9. (c) Generation of the glycodynamer poly(7-10). (d) Dynamic chain termination: equilibrium upon addition of 8 to poly(7-10). (e) 1H-NMR spectra of the exchange mixture obtained after addition of 8 to poly(7-10) after 15 min and 19 h. (f) pD dependence of the rate of exchange between poly(7-10) and 8. Adapted with permission from ref 20. Copyright 2008 Wiley Periodicals, Inc..

nearly physiological aqueous media (Figure 6) has been reported.[22] Galactose oxidase was utilized to selectively oxidize primary hydroxyl groups in the starting material and initiate the subsequent reversible polymerization of the monomers formed. Many enzymes, however, may lose their catalytic activity under the

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conditions required for the construction of reversible covalent bonds. Hence, reversible oxime polycondensation was performed without using enzymes.[20] Monomer 5 contains a protected aldehyde group and an amino-oxy group, which can be polymerized by in situ deprotection-polycondensation, leading to glycodynamer poly9. In contrast, the alternative copolymer poly(7-10) can be obtained by the addition of bisalkoxylamine 7 to a neutralized solution of deprotected dialdehyde 6 (Figure 6). The formation of glycodynamers was confirmed by using diffusion-ordered NMR spectroscopy (DOSY) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Furthermore, incorporation of

tert-butylhydroxylamine as a termination agent to the formed polymer solutions was

followed by 1H-NMR and DOSY-NMR spectroscopy. Based on integrations of 1

H-NMR spectra and diffusion coefficients from DOSY NMR spectra, half-lives for exchange at different pD values were obtained and the observation that polymer poly(7-10) shortens upon addition of tert-butylhydroxylamine (Figures 6d, e and f) demonstrated its dynamicity.

Glycodynamers with a static glycosidic backbone and dynamic side chains (type 1b) can be formed by reversible immobilization of functional species onto a static polysaccharide (Figure 5b), such as vanillin,[23] peptides,[24] flavouring, antimicrobial, antifungal or antitumoral small molecules.[25] The resulting glycodynamers are endowed with stimuli-responsiveness through the operation of reversible covalent bonds and present valuable properties of both components. In a given environment, specific chemical or physical stimuli can induce controlled release of the appended functional molecules. Thus, this type of glycodynamer provides a tool for the functional modification of saccharides, and can be synthesized as biodynameric films[25] or drug-delivery systems[24] with biofunctionality, biocompatibility, adaptability and stimuli-responsiveness.

The synthesis of glycodynamers of type 2a is conducted by equilibrium polymerization of monomers featuring irreversibly grafted saccharides (Figure 5c). The resulting glycodynamers consist of a dynamic backbone and glycosidic static side chains. In view of their structural diversity and synthetic accessibility, different carbohydrate-modified dihydrazides and dialdehydes were designed and synthezised as monomers for the formation of polyacylhydrazones and the investigation of this type of glycodynamers (Figure 7a).[26] A reversible polycondensation reaction in aqueous media under mildly acidic conditions afforded a series of glycodynamers with high molecular weights, which feature relevant biofunctional properties owing to their carbohydrate side chains (Figure 7b). Cryo-transmission-electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS) revealed the construction of

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cylindrical micelle-like and wormlike structures. Moreover, these dynamic glycopolymers displaed intense fluorescence (Figure 7c), which can be attributed to their tightly packed structures mediated by hydrophobic interactions of aromatic chromophores.[26a] Their dynamic properties were demonstrated by adding one equivalent of 14 to glycodynamer poly(10-13) and poly(12-13) and following monomer replacement through both 1H-NMR and fluorescence spectroscopy, because the incorporation of 14 to glycodynamer poly(10-13) and poly(12-13) induced changes in 1H NMR spectra and fluorescence properties (Figure 7d).[26b] In addition, the target-binding ability of these glycodynamers for peanut agglutinin was studied by SPR. Glycodynamers poly(11-14) and poly(12-14) showed enhanced affinity compared to their corresponding monomers and can be used as efficient ligands for peanut agglutinin (Figure 7e).[26b] Taking into account that exchange and replacement of monomers also induce the constitutional modification of the polyacylhydrazones, it provides a novel strategy for the preparation of adaptive carbohydrate-based biomaterials with controllable and tunable properties, such as fluorescence and affinity for a biological target.

Finally, glycodynamers of type 2b (Figure 5d), with a static main chain and dynamic glycosidic side chains, are prepared by reversibly grafting saccharide residues to a linear or cyclic backbone. It offers novel tools for reversible post-polymerization modification of static polymers to achieve improved biocompatibility, combined biofunctionality and stimuli-responsiveness. In particular, the reversible modification of linear or cyclic functional polypeptide backbones give rise to the generation of dynamic analogues of glycopeptides, including both linear and cyclic types (for a review, see[27] ). For instance, cyclopeptide scaffolds for multivalent presentation of saccharides through the formation of oxime bonds were recently fabricated.[28] The multi-antigenic entities formed are composed of two types of analogues of tumor-associated carbohydrate antigens and an immunostimulating T-helper peptide acting as bioscaffold for carbohydrates, which can be used as synthetic vaccines capable of inducing potent and selective immune response against cancers. On another note, multiple presentation of glycosidic groups has been achieved through the self-assembly of grid-type metallosupramolecular architectures leading to octavalent entities that displayed selective binding of the mannose-functionalized derivative toward concanavallin A.[29]

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Figure 7. (a) Structures of the dialdehydes and dihydrazides. (b) Structures of

glycodynamers poly(10-13) and poly(12-13). (c) Photography of poly(11-13), poly(11-14), poly(12-13), poly(12-14) under UV irradiation (365 nm, left) and their emission spectra (right). (d) Evolution of the fluorescence of poly(12-13) after addition of 14 (left) and photography of poly(12-13) before and after monomer exchange with 14 (right). (e) Surface plasmon resonance (SPR) results of binding to peanut agglutinin.Reprinted with permission from ref 26b. Copyright 2010 American Chemical Society.

2.3.3 Dynamic proteoids: dynamic analogues of proteins

Various reversible reactions can be employed for the preparation of dynamic proteoids. Given that enzymes are capable of selectively catalyzing peptide synthesis under mild conditions, various dynamic systems based on positively or negatively charged peptides, were developed through the reversible enzymatic formation of

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amide bonds in aqueous media at physiological pH.[30] In the presence of oppositely charged polysaccharides, substantial increments in product yield were observed due to electrostatic interactions between peptides and templates. Reversible native chemical ligation reactions that selectively occur at N-(methyl)-cysteine residues in aqueous solution at physiological pH, afforded reversible proteoids in the absence of enzymes.[31] In this dynamic system, peptide fragments of the resulting product can undergo exchanges in the presence of dithiothreitol (DTT). Furthermore, disulfide bond formation is also widely used for the preparation of dynamic proteoids. Disulfides can be generated by autoxidation upon exposure to air and undergo rapid interchange in aqueous solution at physiological pH without influencing other functional groups. For instance, dynamic combinatorial systems consisting of two types of competitive peptide-functionalized compounds have been set up.[32] Under given conditions, two sets of self-replicating peptide-based macrocycles were created by selective incorporation of their favored building blocks into respective kinetically-controlled replicators.

Figure 8. (a) Structures of the dialdehyde 15 and amino acid hydrazides 16–25. (b)

Cyro-EM images of poly(15-18), poly(15-20), poly(15-21) and poly(15-24). (c) Schematic representation of dynamic proteoid generation. (d) Rate of polymerization: percentage of unreacted dialdehyde versus time. Adapted with permission from ref 33b. Copyright 2016 WILEY-VCH Verlag Gmbh & Co. KGaA, Weinheim.

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We generated dynamic proteoids using reversible C=N bond formation.[33] Polycondensation of a water-soluble amphiphilic dialdehyde 15 with various bifunctional amino acid hydrazides 16−25 in aqueous media (pD~5, Figure 8) using both imine and acylhydrazone formation, affords biodynamers with doubly covalent dynamicity. The dialdehyde features a tricyclic aromatic core to stabilize the resulting biodynamers through π−π-stacking interactions and a hexaglyme chain endowing the structures generated with water solubility. Under mildly acidic conditions, acylhydrazone formation proceeds readily and goes to completion, whereas imines are barely formed. It was found, however, that reversible polycondensation takes place in a nucleation-elongation (N-E) manner[34] and is driven by self-organization/folding of the dynamic proteoids formed through hydrophobic interactions between the dialdehyde core and the side chains of the amino acid hydrazides used.[33a] The architectures of the polymers were characterized by cryo-TEM, dynamic light scattering (DLS) and SANS, which revealed the generation of three types of nanostructures: globular nano-objects, nanorods and oligomers (Figures 8b and 8c).[33b] Furthermore, by studying their polycondensation and monomer exchange via 1

H-NMR spectroscopy, it became apparent that side chains of the amino acid hydrazides affect the rates of polymerization (Figure 8d), structure and dynamic properties of the resulting biodynamers. Given these findings, we concluded that:[33b] (1) aromatic rings (16, 17 and 18) speed up polymerization and stabilize biodynamers through π−π-stacking interactions to build globular nano-objects; (2) positively charged side chains (19 and 20) accelerate polymerization and give rod-shaped architectures, whereas negatively charged side chains block polymerization and produce oligomers; (3) the presence of hydroxyl groups (24 and 25) stabilizes the polymers and leads to globular nano-objects through hydrogen bonds; (4) electrostatic forces dominate the reversible polycondensention when two oppositely charged species are utilized, leading to equal incorporation of the monomers and to neutral dynamic proteoids; (5) when two amino acid hydrazides exist in a system, monomers with a faster rate of polymerization are preferably incorporated into the dynamic proteoids formed; (6) addition of an amino acid hydrazide with a faster rate of polymerization to an existing dynamic proteoid, leads to monomer replacement. Our findings set the stage for the rational design and production of various types of well-defined architectures and smart proteoid materials.

Dynamic proteoids combine the properties of all monomers, particularly the biocompatibility of the amino-acid-derived monomers with the adaptability from the reversible covalent bonds. Hence, such proteoid materials may be used in both biomedical and bioengineering fields. In addition, proteins play significant roles in