Supramolecular for biomedical applications

Berg, A. I.∗, supervisor: Prof. dr. K.U. Loos†

∗ s2408759, [email protected] † Department of Chemistry, University of Groningen.

Abstract: Supramolecular polymers are promising candidates for the use in biomedical applications. The noncovalent interactions that characterize these systems create possibilities for dynamic reversibility and stimuli responsiveness. The systems can be easily functionalized, allowing for increased targeting and bioactivity of the materials. Additionally, the reversible interactions present in these systems allow for a bottom up approach for synthesizing and controlling the size and shape of the supramolecular materials. In this review the use of supramolecular polymers in biomedical applications, such as drug deliver systems, , and self healing materials are discussed. Typical polymer systems described con- sist of benzene-1,3,5-tricarboxamide (BTA), ureido-pyrimidone (UPy) or host-guest interactions.

Contents spans multiple length scales; from fibers to monolayers, vesi- 1 Introduction1 cles, gels, and membranes. The field of supramolecular polymers based on controlling 2 Background supramolecular poly- noncovalent interactions between monomers mers2 and the process of self assembly to form 2.1 Interactions and stability...2 well defined structures.1 A good definition 2.2 Supramolecular polymerization4 of supramolecular polymers was proposed by Brunsveld et al.: ”Supramolecular polymers 3 Applications4 are defined as polymeric arrays of monomeric 3.1 Drug delivery...... 4 units that are brought together by reversible 3.1.1 BTA...... 4 and highly directional secondary interactions, 3.1.2 UPy...... 6 resulting in polymeric properties in dilute 3.1.3 Host-guest systems...7 and concentrated solution as well as in the 3.2 Tissue engineering...... 9 bulk. The directionality and strength of the 3.3 Self healing materials...... 10 supramolecular bonding are important fea- 4 Summary and outlook 11 tures of systems that can be regarded as polymers and that behave according to well- 5 Acknowledgments 12 established theories of polymer physics.”4 Applications of supramolecular polymers References 12 are in light harvesting systems5, liquid crys- talline materials3, block assem- 1 Introduction blies3, organogels1, and biomelecular materi- als1. Here the focus will be on the biomedical Supramolecular chemistry emerged as a field applications of supramolecular polymers. in the not too distant past. The work of Lehn Over the last couple of years the interest started supramolecular chemistry as a field for the development and application of smart in 1978 when he connected it to functional drug delivery systems has increased and it is structures.1 Since then the field developed fur- increasing still. With growing amounts of peo- ther and is now an established sub-dicipline of ple suffering from degenerative diseases, the chemistry. In 1990 Fouquey et al. designed focus is on the development of systems that and synthesized the first example of a linear will increase the quality of life of patients.6 supramolecular polymer.2,3 However, the application of supramolecular

1 polymers in the field of biomedical applica- on discussing the different bonding motifs that tions has only received limited attention.7 An are characteristic for supramolecular polymers important and growing area is to develop (fig.1); hydrogen bonding, metal ligand inter- drug delivery systems that respond to intrin- actions, π −π stacking and host-guest interac- sic stimuli characteristic of the target site, tions. All these bonding motifs are noncova- improving the selective targeting and deliv- lent and they allow for the dynamic interac- ery of drugs. This is where applications for tions and reversibility observed in supramolec- supramolecular polymers lie.7,8 Examples of ular systems.1,6,16,18,19 Additionally these in- some supramolecular polymers that are al- teractions allow for stimuli responsiveness. ready being used in medicine are peptide am- Where an external stimulus, for example a phiphiles and ureido-pyrimidone (UPy) mate- change in pH, can trigger structural transi- rials.7 tions of the supramolecular assembly18. Supramolecular polymers offer interesting Hydrogen bonds have tunable directionality applications in the biomedical field, due to and diversity, especially in the case of mul- the noncovalent interactions that character- tiple hydrogen bonding moieties. This motif ize these systems. Reversibility of the in- offers increased stability to the supramolec- teractions and stimuli responsiveness allows ular structure. Hydrogen bonds are formed for applications in drug delivery systems, tis- by dipole-dipole interactions between an elec- sue engineering and self healing materials. tronegative atom, such as oxygen, nitrogen, or This critical review discusses the important a halogen, and a hydrogen bonded to one of bonding motifs in supramolecular polymers such electronegative atoms (fig.1a). 20 These and the basis of supramolecular polymeriza- bonds are typically sensitive to pH9, which tion. Then various biomedical applications makes them suitable for applications in stim- of supramolecular polymers will be discussed. uli responsive systems. The review will be concluded by a summary π − π interactions are one of the im- and critical outlook on the field. portant noncovalent interactions, they arise from overlapping p-orbitals between conju- 2 Background supramolecu- gated molecules (fig.1c). These interactions are relatively weak and non-directional com- lar polymers pared to the other bonding motifs described.9 Different types of architectures can be found Host guest interactions such as those de- for supramolecular polymers: supramolecu- picted in fig.1d, show specific recognition lar homopolymers, alternating , and strong binding between the host and the 9 and block copolymers9; similar to their co- guest molecules . Actually often a combi- valent counterparts. Supramolecular poly- nation of multiple interactions are used in mers of these architectures can form dif- these systems: hydrophobic (or hydrophilic) ferent kinds of structures: stacks10,11, mi- effects, electrostatic and van der Waals inter- 20 celles12,13, polymerosomes14,15, dendrimers16, actions, hydrogen bonding and ionic bonds. and 3D crosslinked polymer networks9,17. The shape and size of the host and guest are These structures are kept together through complementary, such that the guest has the noncovalent interactions and will self assem- right properties to be able to bind the host 20 ble under specific conditions. This section molecule . will discuss the background of supramolecu- Metal ligand interactions are responsive to lar polymerization; the interactions, stability, redox reactions and can show photophysical and supramolecular polymerization. properties (from the metal ion and ligands).9 A reason for the interest in these interactions 2.1 Interactions and stability in supramolecular polymers is the combina- tion of properties from the organic polymer Small building blocks can interact trough non- and those of the metal ions (fig.1b); magnetic, covalent interactions to form materials on catalytic, electronic and also optical proper- multiple length scales. This section will focus ties20. Because of the wide variety of metal

2 Figure 1: A collection of bonding motifs for supramolecular polymers. a) H-bonding, b) metal coordination, c) π − π interactions, d) different host guest systems: α-Cyclodextrin, 24-crown-8, Cucurbit[8]uril, Calix[5]arene, Pillar[5]arene. Image reproduced from Dong et al.9 ions and ligands, properties such as the bind- allowing for the formation of supramolecular ing strength, directionality, solubility, and re- polymers.20 20 versibility can be tuned for these systems. Compared to covalent interactions, the use Incorporating multiple bonding motifs al- of noncovalent interactions in polymer sys- lows for better control of the supramolec- tems holds several advantages. The synthe- ular polymerization, giving rise to well de- sis is often easier, since only the smaller com- fined structures. The motifs described here ponents need to be synthesized, avoiding dif- are mainly used for tuning the supramolecu- ficult multi step synthesis, while the bigger lar polymers.20 In order to assure higher sta- system will form through dynamic self as- bility and strength of the polymers multiple sembly. The dynamic self assembly allows bonding motifs should be combined, i.e. hy- for a bottom up approach in which the size drogen bonding and π − π stacking. Ideally and shape of supramolecular materials can be more motifs combined result in higher stabil- controlled. Additionally the synthesis is of- ity.9 For example; one is far ten environmentally friendly and cost effec- from strong enough to create a supramolecular tive, since it can be performed under ambient polymer. However, creating arrays with mul- conditions.15,18 The noncovalent interactions tiple hydrogen bonding moieties will increase and stimuli responsiveness lead to degradable the strength and directionality of the bonds, polymer backbones9. These properties com-

3 bined make supramolecular (polymeric) mate- find the lowest energy structure.21 For an ex- rials very interesting for applications as smart tended overview of the theory and mecha- supramolecular devices and functional mate- nism behind supramolecular polymerization, rials.9 the reader is referred to De Greef et al.19

2.2 Supramolecular polymerization 3 Applications

Multifunctionalized molecules can form Supramolecular polymers can have the same supramolecular polymers through reversible mechanical properties as covalent polymers. interactions (see section 2.1). Obtaining a However, they show great capacity for pro- high degree of polymerization depends on cessability due to their dynamic nature.3 This several factors: the association constant dynamic behavior and modularity of the sys- between the monomers, the purity of the tems allows for easy, noncovalent synthe- material, and the concentration of the so- sis.7 The following properties of supramolec- 9 lution . Cooperative self assembly between ular polymers are of key importance for monomers, and complexes, allows for the biomedical applications: good biocompati- generation of well-defined and highly ordered bility and bioactivity, low cytotoxicity, spe- 9 structures. cific targeting, aqueous compatibility, and In supramolecular systems competition ex- stimuli responsiveness.9,22,23 The functional ists between inter- and intramolecular poly- supramolecular polymers described show spe- merization reactions. Intermolecular reac- cific functions and are able to dynamically tions will result in polymers of high molecu- adapt to the environment through exter- lar weight, whereas intramolecular polymer- nal stimuli. These properties offer a good ization will result in low molecular weight platform for design of smart supramolecular complexes. Effective molarity (eq.1) is de- polymeric materials that combine dynamics fined as the ratio between the intramolecu- and molecular order to realize multiple func- lar and intermolecular equilibrium constants tions.9 In this section various applications of (Kintra and Kinter respectively). supramolecular polymers in drug delivery sys- tems, tissue engineering, and self healing ma- K EM = intra (1) terials will be discussed. Kinter

−1 Here Kinter has units of M , while Kintra 3.1 Drug delivery is dimensionless, giving EM units of M. In this section benzene-1,3,5-tricarboxamide Bifunctionalized monomers can either un- (BTA), ureido-pyrimidone (UPy), and host- dergo cyclization or linear polymerization, the guest interactions and their applications in EM represents a critical concentration above drug delivery systems will be discussed. The which linear polymerization is favored over important bonding motifs for these systems 19 cyclization. Note, bonding within the same are displayed in fig.1 a, c, and d. complex of multiple monomers is considered to be intramolecular polymerization, therefor 3.1.1 BTA leading to lower molecular weight materials. Whereas covalent polymerization reactions The BTA monomers can dynamically self as- (formation of covalent bonds) are often un- semble into one dimensional supramolecular der kinetic control, supramolecular polymer- fibers in water. These fibers are stabilized ization is under thermodynamic control.19,21 by the formation of three hydrogen bonds be- In a system under thermodynamic control, the tween the amide groups, resulting in a he- components are mixed and the final product lical lateral ordering of the monomers (fig. that is formed is the system with the most 2). Additionally, the structure is stabilized by stable ground state. This requires that all re- π − π interactions.4,7,10,11,24–27 The BTA moi- actions are reversible under the reaction con- eties can be functionalized with amphiphilic ditions, leading to a dynamic system that can chains to obtain higher functionality. There is

4 hydrogen bonded (fig.2). They specifically looked into the use of this supramolecular sys- tem as a dual delivery system, with siRNA as one of the targeted drugs to deliver. The siRNA can be electrostatically bound to the fiber exterior, allowing it to pass cellular mem- branes without degrading. While a small or- Figure 2: A schematic representation of the self as- ganic, hydrophobic drug can bind to the in- sembly of benzene-1,3,5-tricarboxamide (BTA), form- ner, hydrophobic pockets of the fiber (fig.3). ing helical stacks that are stabilized by the formation They showed that the charge of the monomers of three hydrogen bonds. Image reproduced from Can- tekin et al.24 determines renal uptake, cell binding, cyto- toxicity etc. Mixing of monomers allows for control over the bioactivity of the carrier. The a delicate balance between the sizes of the hy- internalization pathway for this carrier system drophobic spacer and the hydrophilic units in is not known yet, although it is important to the BTA derivate that influences the self as- find out how the polymers will be internalized 26 sembly process. Hydrophobic units of eleven in order to know the full potential of these ma- or twelve atoms result in the formation of sta- terials as future drug delivery systems. For ex- ble fibers, changing the lenght of the spacer ample, there are indications that the key lim- further influences the aggregate size and ther- iting factor for cellular drug delivery systems 26 mal stability of the complex. This section is an inability to escape lysosomes, when inter- will discuss the use of BTA moieties in the nalization pathway is through endocytosis.7 design of drug delivery systems. Another factor of the delivery process that Albertazzi et al.11 reported a supramolec- is still unknown is whether the polymers will ular system based on BTA, that dynamically remain aggregated or depolymerize after suc- self assembles in water into a fiber. They in- cessful binding and internalization into cells.7 vestigated the effect of a multivalent binder to It is important that further research will be the ordering of functionalized monomer units performed on this subject, since it might affect in the supramolecular polymer. Results show the delivery of drugs and/or the metabolism that the monomer distribution could be con- of the carrier. The authors conclude that trolled by the multivalent binder, allowing this their supramolecular BTA fibers show promis- to be a viable scaffold for different multiva- ing applications as dual drug delivery systems. lent binders by tuning of the monomers. This However, cytotoxicity and bioactivity need to flexibility could be very useful in drug delivery be optimized by optimizing the charge density systems that need to deliver multivalent drugs along the fiber. A balance has to be found be- such as DNA or RNA strands. tween the toxicity and cell binding.7 The use of self assembled supramolecular BTA fibers as a mechanism for drug de- In 2015 Albertazzi et al.27 reported a new livery systems was also reported by Bakker type of a stimuli-responsive supramolecular et al.7 Supramolecular polymer fibers from polymer from PEGylated BTA monomers. PEGylated BTA monomers were proposed for Co-assembly of two different types of intracellular drug delivery systems. These monomers (neutral, non-functionalized BTA fibers have a hydrophobic core and a hy- monomers and cationic, amine functionalized drophilic exterior, allowing for a dual drug de- BTA monomers) resulted in the formation livery system; two different types of drugs in of a supramolecular fiber with a random one carrier. The properties of the supramolec- distribution of monomers. The cationic ular polymer fibers could be controlled by monomer species were able to bind to nu- mixing differently functionalized monomers. cleic acid through electrostatic interactions. The important noncovalent interactions in Upon binding with a DNA-RNA strand, this system are hydrogen bond formation, the monomer sequence was altered, and electrostatic and hydrophobic interactions. could therefore be controlled. Addition of The molecules are helically stacked and triple the RNAse enzyme removed the DNA-RNA

5 Figure 3: BTA fiber complexing with siRNA at the exterior through electrostatic interactions, and encapsulation of hydrophobic nile red molecules in the hydrophobic core. Image reproduced from Bakker et al.7 strand, which resulted in a restoration of hydrogel was shown to be sensitive to pH the random monomer distribution in the and underwent a sol-gel transition at a pH fiber (fig.4). These results were in good above 8.5. This property could be used for agreement with their previous research on transport through a catheter to the target tis- this type of system.11 This research is the sue; the transporting solution has a pH higher first step towards complex systems in which than 8.5, upon contact with the tissue a gel is the polymer can respond to multiple stimuli, formed. The hydrogels form fibers in aque- involving both positive and negative feedback ous solutions and they crosslinked to form loops, which mimics the complex cell signal- supramolecular hydrogel networks. The sol- ing of real biological systems. These results gel transition was observed to be very fast. show a very important step in the design and It was proposed that the switching is caused development of more complex supramolecular by breaking the cross-linking between fibers, polymer systems that resemble complex instead of complete depolymerization. Their pathways of real biological systems. This hydrogel was tested to be nontoxic to cells in makes it a very important and interesting both the neutral and basic solutions. One field of research, to use this complexity of the proposed applications for this hydro- to further improve drug delivery systems gel could be drug delivery to the heart. The and tissue engineering applications of these storage modulus of the described hydrogel supramolecular polymeric systems. matches the mechanical properties of a (rat) heart, and the material showed self healing 3.1.2 UPy properties, indicating it could withstand the high shear of the contracting heart muscle. Ureido-pyrimidone (UPy) molecules have a They showed the incorporation and release of quadruple hydrogen bonding motif and are a growth factor and a size-dependent release suitable for use in supramolecular polymers profile was observed, 100% of the growth fac- because of the stability offered by the hydro- tor was released after 7 days. For this study gen bonds. Two UPy monomers will dimer- both in vitro and in vivo tests were performed. ize upon formation of four hydrogen bonds. A similar system as described above, UPy- Subsequently these dimers stack through π − PEG hydrogel was reported by Dankers et π and van der Waals interactions, forming al.31 However, this system does not use pH as supramolecular polymer fibers (fig.5). These a trigger for a sol-gel transition, in this case molecules are very suitable for use in bioactive exertion of pressure allowed the hydrogel to materials, due to their low processing temper- flow though a needle. The application for this ature, self assembly in water, favorable degra- system is proposed to be in drug delivery to dation and overall biocompatibility.28,29 the kidneys. The hydrogel was inserted under Bastings et al.17 showed the use of ureido- the renal cortex, from there the drugs can be pyrimidone modified PEG hydrogels for drug released from the hydrogel over the course of delivery to organs with high blood flow. This several days. In earlier research Dankers et

6 Figure 4: a) Different monomers of the system: neutral (left), cationic, labeled monomers (center), and the multivalent DNA-RNA recruiter strand. b) The multivalent recruiter controls the monomer distribution in the polymer fiber, introduction of RNAse removes the recruiter and restores the random distribution of monomers throughout the fiber. Image reproduced from Albertazzi et al.27 al.32 reported these type of hydrogels also for could not bind to the cell membrane, nor be the use as intrarenal drug delivery systems. internalized. Result showed that the cationic They found that flexible, slow eroding hydro- species did not show cell cytotoxicity. Com- gels are feasible for long term drug delivery, bined with the good cellular uptake, it makes whereas the weaker, soft, and fast eroding gels these polymers very interesting for intracellu- were more suitable for short term de- lar drug delivery systems. These results are livery. promising for the application of this system in drug delivery. Bakker et al.29 reported the synthesis of UPy based functional supramolecular poly- 3.1.3 Host-guest systems mers. Functionality was achieved by mixing monomers with neutral and cationic moieties. Supramolecular amphiphiles are very good The polymers were functionalized with a UPy candidates for applications in drug deliv- end group. The monomers could self assemble ery systems, and are therefore actively in- into dimers through hydrogen bond formation vestigated. Working with host-guest in- and columnar stacks were then formed by π−π teractions, supramolecular amphiphilic poly- interactions between dimer moieties. The self mers can be developed that are highly func- assembly of this system was triggered by in- tionalizable. The noncovalent interactions jection into an aqueous medium. They ob- that are key for these systems allow for served that siRNA could bind to the cationic easy noncovalent synthesis and great stimuli- charges in the supramolecular polymer fiber. responsiveness.13,15,18,33 The siRNA complexes could be fabricated ei- Yu et al.8 reported a host-guest drug de- ther through a single or two step synthesis, livery system using a pillar[5]arene as the both resulting in a stable complex of polymer host, and a viologen salt as the guest. fiber with RNA (fig.6). In vitro testing was The pillar[5]arene was functionalized with a done with human kidney cells showing that PEG chain, containing a biotin end group the cationic polymer species could be internal- (P5-PEG-Biotin; hydrophilic). The biotin ized by the cell, whereas the neutral polymers molecule serves as the targeting group for re-

7 research shows great possible applications as novel drug delivery systems. A new diblock copolymer consisting of a hy- drophobic supramolecular- and a hydrophilic macromolecular polymer part was reported by the group of Ji et al.14. The supramolecu- lar polymer was based on host-guest interac- tions between a 32-crown-10 and a viologen salt moiety, and its length could be controlled by changing the concentration in the solu- tion. Depending on the length ratio between the two blocks, different types of assemblies were observed. For a small supramolecular block micelles with a hydrophobic core were observed. Whereas polymerosomes with a hy- drophilic interior and exterior were formed for a larger supramolecular block. Both these morphologies allow for the incorporation of Figure 5: a) Fourfold hydrogen bonding motif in the small drugs. The system showed controlled ureido-pyrimidone (UPy) dimer. b) Lateral stacking of release, and stimuli responsiveness to low pH. UPy dimers, leading to the formation of a nanofiber. Image reproduced from Dankers et al.30 This research made the first steps in the com- bination of supramolecular and traditional polymers with possible applications as drug delivery systems.14 ceptors, which are over-expressed in cancer 13 cells. A poly(carpolactone) molecule was ter- The group of Zhu and coworkers reported a stimuli responsive drug delivery system minated with the viologen group (PCL-C2V; hydrophobic). These two molecules form am- based on the self assembly of a supramolec- phiphilic supramolecular diblock copolymers ular amphiphilic polymer. The hydrophilic through host guest interactions. This copoly- part of the polymer consisted from PEGylated mer could subsequently self assemble in aque- calix[4]arene, the hydrophobic drug (chlorine ous media into polymerosomes (lysosome-like e6, Ce6) binds to the by a host- vesicles made from supramolecular polymers). guest interaction. This dimer could then self assemble into polymeric micelles, allowing the The hydrophobic PCL-C2V tails interact with each other, allowing for this structure to be complex to be internalized by cells (fig.8). formed. A small organic drug could be in- The system showed low cytotoxicity, indicat- serted into the solution to incorporate it in the ing it could safely be used as a drug delivery polymerosome during the self-assembly pro- system with long circulation times due to the cess. NAD(P)H reduced the viologen group enhanced permeability and retention (EPR) 33 after internalization, breaking the host-guest effect . This system was designed for photo- interactions and leading to the disassembly of dynamic therapy, yet the host molecules are the polymerosome and subsequent release of suitable for use with different types of guests, the drug (fig.7). They showed that this which also allows for applications in other system has excellent biocompatibility, while treatments. having low cytotoxicity, making it interest- A similar type of system was reported by ing for drug delivery systems. The target- Zhao et al.34 They report a self assembled ing ability of these drug delivery systems re- supramolecular drug delivery system based on duces the cytotoxicity to non-cancerous cells, host-guest interactions that could be used as while retaining the therapeutic efficacy of the a dual delivery system for both a drug and a drug toward the target cells. Further testing gene. γ-Cyclodextrin (γ-CD), functionalized should be performed before this system could with a cationic polymer acts in this system be used in clinical applications. However, this as the host. A hydrophobic anticancer drug

8 Figure 6: a) Chemical structure and schematic representation of the different functionalized UPy monomers. b) dimerization and subsequent polymerization of the UPy dimers in water, forming laterally stacked fibers. In the 1-step synthesis the siRNA is injected during the initiation of polymerization, for the 2-step synthesis the siRNA is injected after completion of polymerization. Image reproduced from Bakker et al.29

Figure 7: A schematic representation of the host-guest interaction between PCL-C2V and P5-PEG-Biotin, forming a amphiphilic supramolecular diblock copolymer. Subsequent self assembly of the PCL-C2V⊃P5-PEG-Biotin into polymerosomes allows for incorporation of a small organic drug, which can be released upon reduction of the viologen end groups. Image reproduced from Yu et al.8

(PTX) was loaded into the hydrophobic cav- polymeric materials. UPy-functionalized ity of the γ-CD. Together with plasmid DNA polymers are mainly used since they are (pDNA), these host-guest complexes could very suitable due to their quadruple hy- further self assemble into ’polyplexes’; posi- drogen bonding motifs (fig.5a). Ad- tively charged nanoparticles, see fig.9. These ditionally, they show low processing tem- complexes could be internalized through the peratures and result in mechanically sta- endocytosis pathway. The drug and pDNA ble and biocompatible materials.28,35 Combin- could then be released after a redox reaction ing UPy-functionalized polymers with UPy- on the host. Toxicity studies were performed functionalized will result in and showed high values of cytotoxicity to can- bioactive materials that could be used for cer cells when the drug was loaded. These re- tissue engineering.23 With these techniques sults show that this system could be a promis- bioartificial organs could be developed, im- ing candidate for anti cancer drug delivery sys- proving the quality of life and life expectancy tems. of people in need of donor organs.23 UPy-modified supramolecular polymers for 3.2 Tissue engineering cell free tissue engineering, specifically aimed for the use in vascular grafts were reported The formation of functional tissues from by Van Almen et al.35 They showed the de- supramolecular polymers requires bioactive velopment of mechanically stable grafts with

9 ing or other bioactive materials that stimulate cellular processes. A supramolecular membrane formed from fibrous supramolecular polymers and human tubular cells was reported by Dankers et al.30 for the use in renal tissue engineering, es- pecially focusing on the renal epithelial tis- sue. By using supramolecular UPy modified polymers with urea groups, they created fi- brous membranes by electrospinning. As men- tioned before in section 3.1.2, the UPy dimers stack in the lateral direction and are stabi- lized by π − π interactions, and in this sys- tem also by additional hydrogen bonding be- tween the urea groups. Thin, monolayer thick- ness membranes could be created that resem- Figure 8: Schematic representation of the host-guest bled the natural extracellular matrix. Inser- interaction between the hydrophilic PEGylated cal- tion of kidney epithelial tubular cells resulted lix[4]arene and the hydrophobic drug (chlorin e6), and the subsequent self assembly into supramolecular poly- in the growth of monolayers of these cells af- meric micelles. Image reproduced from Tu et al.33 ter seven days of culturing period. The cells showed good cell viability and activity during the seven day tests they performed. Com- good mechanical properties. This report was a paring these results to conventional micro- proof of concept study showing that the use of porous membranes, the supramolecular mem- supramolecular UPy molecules provided a ma- branes were superior when concerning mono- terial that was mechanically stable and non- layer formation, cell viability, and cell activ- cell adhesive (no binding of unwanted cells) ity. When the extracellular matrix is mim- that could be applied in vascular tissue engi- icked by providing bioactive signals, the dif- neering. Other bioactive components could be ferentiated features and properties of normal incorporated in the polymer to facilitate cell epithelial kidney cells may be introduced into specific binding, allowing for the first stages the artificial membranes. These results show of tissue regeneration. Since the goal is that that the supramolecular UPy membranes offer after a while the biomaterial should be indis- good prospects for the use in epithelial kid- tinguishable from the real tissue, so specific ney tissue engineering, with applications in cells have to be able to bind to the material. bioartifical kidneys. These membranes might Dankers et al.28 used the quadruple hydro- be improved in functionality by incorporation gen bonded UPy moieties for bioactive ma- of bioactive materials that might further en- terials. The UPy-UPy association constant in hance the growth and differentiation of renal water is low. However, because of the polymer epithelial cells. film hydrophobic shielding occurs, resulting in strong but dynamic binding between the UPy 3.3 Self healing materials moieties. They observed that the supramolec- ular polymer could adapt its structure to the Most of the reported applications using hy- environment, and proposed that the polymers drogels are either in drug delivery or tissue could dynamically move over the surface, al- engineering. However interesting applications lowing to adjust for new cell binding. From could be thought of in, for example, the devel- in vivo experiments they found that the poly- opment of soft robotics and biomimetic pros- mer matrix influences the signaling cells of the theses.36 Tee et al. reported an composite surrounding tissue, more research is to be con- material from a supramolecular organic poly- ducted to these signaling processes. The bio- mer with embedded nickel micro-particles that functionality of the supramolecular polymer showed electrical and mechanical self healing matrix could be easily altered by introduc- properties.36 Additionally the material was

10 Figure 9: Schematic representation of the host-guest interaction between the γ-cyclodextrin (γ-CD) and the hydrophobic drug paclitaxel (PTX), complexation with DNA results in the formation of polymer/DNA polyplexes. Image reproduced from Zhao et al.34. found to be sensitive to both pressure and biomedical applications has been reviewed. A bending, making this material a good candi- background in supramolecular polymers; the date for the use in electronic skin. The sys- bonding motifs, stability, and supramolecu- tem showed both electrical and mechanical self lar polymerization was given. Subsequently healing. Electrical self healing was repeatable applications in drug delivery tissue engineer- with a 90% efficiency after just 15 seconds of ing, and self healing materials have been pre- contact at room temperature. The mechanical sented. self-healing was less successful at room tem- Upon injection into water, benzene-1,3,5- perature; 10 min of healing time would result tricarboxamide (BTA) monomers can dy- only in 41% healing, whereas full recovery was namically self assemble into one dimensional observed for the same healing time at 50◦C. It supramolecular fibers. These fibers could be was found that mechanical healing is impor- used for dual drug delivery systems. It was tant for electrical healing since contact time is shown that the monomer distribution in the important for conductive healing. It was pro- fiber could be controlled by the introduction posed that the healing mechanism is driven of a multivalent binder. The removal of this by the reformation of hydrogen bonds between binder resulted in the system reverting to the the cut surfaces.36 These results are very inter- original state with a random monomer distri- esting, showing applications in artificial skin. bution.27 This was the first step towards com- This type of material could possibly be used plex systems, that mimic cell signaling of real in making skin for prosthetics giving a more biological systems, in which the supramolec- lifelike look to the prosthetic. Electrical con- ular polymer could respond to multiple stim- ductivity would not necessary for applications uli that involve feedback loops. Making this a in prosthetics. However it might be interesting promising field of research, in which such com- for other applications, such as the suggested plex systems could be developed for further soft robotics36. improvement of drug delivery systems and a broader range of biomedical applications of 4 Summary and outlook supramolecular polymers. Ureido-pyrimidone (UPy) monomers can Supramolecular polymers are very interesting dimerize through the formation of hydrogen for the use in biomedical applications due to bonds, subsequently these dimers can stack, the noncovalent interactions that characterize forming supramolecular polymer fibers. Two these systems. These interactions allow for interesting cases of UPy hydrogels that have dynamic behavior through reversible interac- sol-gel transitions have been reviewed. The tions, stimuli responsiveness, and a bottom up most promising being a hydrogel which could approach for controlling the size and shape of be used for drug delivery to the heart. A gel supramolecular materials. was formed upon contact with the tissue, and The use of supramolecular polymers for was found to be able to withstand the high

11 shear of the contracting heart muscle.17 This mainly been focused on the design and fabrica- is a breakthrough for drug delivery to organs tion of drug delivery systems. Mostly only ini- with high blood flow, since this hydrogel al- tial in vitro tests were performed, while only lows for prolonged retention of the drug. Due few report in vivo testing. This is an area that to their dynamic nature, UPy molecules are will need to be investigated further in order to also viable for applications in tissue engineer- give a better indication if a designed delivery ing, resulting in stable materials with good system is promising for clinical use. Addition- mechanical properties. These supramolecular ally, focus should be put on finding the in- polymers have shown their potential for vas- ternalization pathways for these drug delivery cular and renal tissue engineering. Thin fi- systems. Another important area is to bet- brous polymer membranes could be created ter understand the metabolism pathway for that resemble the biological extracellular ma- the carrier systems. The cytotoxicity of the trix. The introduction of cells to these mem- metabolites and possible unwanted accumula- branes resulted in the growth of monolayers tion of these in the cells. These topics are not of said cells with high cell viability and ac- always discussed in the current research, yet tivity.30 These membranes are important for they are very important for allowing these sys- renal tissue engineering, with possible appli- tems to take the next step towards commercial cations in bioartifical kidneys. biomedical applications. Supramolecular amphiphiles are interest- ing candidates for drug delivery systems. 5 Acknowledgments Supramolecular amphiphilic polymers were developed using host-guest interactions. Poly- I would gratefully like to acknowledge prof. meric lysosomes, micelles and other complexes dr. K.U. Loos for her supervision and help were formed, these could be incorporated with during this project. I would like to thank her small organic drugs. These systems could re- for fruitful discussions about the contents of act to external stimuli, allowing for the drugs this paper and for her insights and knowledge to be released. of the field. I would like to thank Lourens-Jan Supramolecular polymers have been shown Ugen for his technical help with the layout of to electrical and mechanical self healing, when this paper and for the fruitful discussions we embedded with nickel micro particles Pos- had during the process of writing. sible applications are in soft robotics and biomimicing prostheses.36 Combining the re- References search of tissue engineering and supramolecu- lar self healing materials might result in very (1) Stupp, S. I.; Palmer, L. C. Chemistry of interesting applications in the development of Materials 2014, 26, 507–518. self healing artificial skin for biomedical appli- (2) Fouquey, C.; Lehn, J.-M.; Levelut, A.- cations. Then skin grafts could be developed M. Advanced Materials 1990, 2, 254– to be used in (reconstructive) surgery, allow- 257. ing natural skin cells to regrow into skin, while the artificial skin will behave like normal skin (3) Aida, T.; Meijer, E. W.; Stupp, S. I. during the growth process. It might be diffi- Science 2012, 335, 813–817. cult to achieve this However, this might be a (4) Brunsveld, L; Folmer, B. J. B.; Meijer, very interesting direction to look into. E. W.; Sijbesma, R. P. Chemical Re- The field of drug delivery and tissue engi- views 2001, 101, 4071–4098. neering with respect to the use of BTA and (5) Zhang, D.; Liu, Y.; Fan, Y.; Yu, C.; UPy seems to be dominated by the groups of Zheng, Y.; Jin, H.; Fu, L.; Zhou, Y.; Dankers and Meijer. Their results for BTA Yan, D. Advanced Functional Materials and UPy seem very promising, so hopefully 2016, 26, 7652–7661. more research will be performed in this field (6) Webber, M. J.; Appel, E. A.; Meijer, to discover the full potential of these systems. E. W.; Langer, R. Nature Materials Most of the research conducted so far has 2015, 15, 13–26.

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