Chapter 1: Click Chemistry in Polymer Science

The impact of click chemistry has been clearly illustrated by the fact that the Nobel prize in chemistry 2022 was awarded to K. Barry Sharpless, Morten Meldal and Carolyn R. Bertozzi for “the development of click chemistry and biorthogonal chemistry”. Since the introduction of the concept of “click chemistry” by Sharpless and coworkers, click chemistry has taken a huge flight and has found application in a wide range of domains, from the biomedical realm to materials science.

This chapter will give a brief introduction to click chemistry and specifically review click reactions that are employed in (i) polymer functionalization with biological motifs, (ii) amphiphilic block polymer synthesis and (iii) polymer-based supramolecular systems. We will demonstrate the versatility and applicability of this type of reaction in the preparation of macromolecular systems with potential in biological applications. We will restrict our review to the chemical approaches followed and will not go in depth regarding the biological assessment. We will thereby focus on cycloaddition reactions and thiol-based click reactions, as these are the most commonly used reactions in this field of science.

In 2001, Sharpless and coworkers introduced the concept of “click chemistry”. They defined this class of reactions as follows: “The reaction must be modular, wide in scope, give very high yields, generate only inoffensive byproducts that can be removed by nonchromatographic methods, and be stereospecific. The required process characteristics include simple reaction conditions, readily available starting materials and reagents, the use of no solvent or a solvent that is benign (such as water) or easily removed, and simple product isolation.”¹ The copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction between alkynes and azides, which was independently developed by Meldal² and Sharpless³ in 2002 is one of the most popular click reactions that fits this definition, and which is derived from the uncatalyzed 1,3-dipolar cycloaddition between azides and alkynes that was developed by Rolf Huisgen.^4,5 Sharpless and Meldal modified the Huisgen cycloaddition reaction by introducing a copper catalyst, which resulted in an accelerated reaction and an improved selectivity to produce only one regioisomer instead of two. Thereafter, in 2002, Sharpless and coworkers published their first study on the copper catalyzed synthesis of 1,4-disubstituted 1,2,3-triazoles via an in situ prepared Cu(i) catalyst from Cu(ii) in the presence of ascorbic acid/sodium ascorbate.³ In the same year, Meldal and coworkers utilized this approach and demonstrated the copper catalyzed peptide conjugation via a 1,2,3-triazole link on a solid phase, highlighting the potential of CuAAC for applications in biochemistry.² In the years after, the CuAAC reaction has been applied in numerous technologies, as it is a rapid, stereoselective and site specific reaction that can tolerate a wide variety of environmental conditions.

The group of Carolyn Bertozzi exploited the concept of bioorthogonal chemistry, a set of reactions that don’t interfere or interact with biochemical processes, and developed it further to be performed safely in living cells.^6,7 An important reaction in this regard is the Staudinger ligation, modified from the Staudinger reaction,⁸ which occurs between azide and phosphine moieties to produce an amine via an aza-ylide intermediate.⁸ The spontaneous hydrolysis of the aza-ylide product in water traditionally limits the applicability of the Staudinger reaction in biological settings. However, in 2000, Carolyn Bertozzi developed the Staudinger ligation by introducing electrophilic traps (e.g. methyl esters) to the phosphine structures, which, in turn, result in an intramolecular cyclization of the nucleophilic aza-ylide and immediate production of stable amide bonds.⁹ The advancement of the Staudinger ligation by the group of Bertozzi has been shown for the improvement of cell surface engineering.^10,11 Later on, this particular Staudinger ligation was named the Bertozzi–Staudinger ligation and is considered to be one of the most important biorthogonal reactions that can be performed safely in biological systems and living animals.

An important property of CuAAC is its biorthogonality, as the reaction between azides and alkynes is highly specific and does not impact any other ongoing (bio)chemical reactions which, when viewed in the context of applications in the field of life sciences, is crucial. However, the possible toxicity of copper still limits its usage. Thereafter, in 2004, an important biorthogonal copper-free click reaction was developed by Bertozzi’s group, which is the strain-promoted azide alkyne cycloaddition reaction (SPAAC).¹² The SPAAC reaction requires strained alkyne molecules (i.e. cyclooctynes), which readily react with azides and eliminate the need for metal catalysts (i.e. copper catalysts as in CuAAC).¹³ The SPAAC reaction can be performed safely in living systems. In an inspiring example, it was used to fluorescently label zebrafish embryos and visualize their development.¹⁴ 

Alternative biorthogonal reactions that can be applied in living systems (in addition to SPAAC and Staudinger ligation) have been demonstrated.^15,16 One example is the inverse-electron-demand Diels–Alder (IEDDA) reaction between s-tetrazine and trans-cycloocytene derivatives, which was firstly shown by Joseph M. Fox et al.¹⁷ IEDDA also does not require any catalyst and can be performed in a wide range of solvents (e.g. organic solvent, water). Scott Hilderbrand and coworkers demonstrated its utility in fluorescently labeling living cells and subsequent in vitro imaging.¹⁸ 

Recent literature has demonstrated the broad utility of click reactions in inorganic nanoparticle surface modification,^19–21 liposome functionalization^22,23 and polymeric vesicles labelling.^24,25 For instance, surface labeling of polymersomes without compromising their morphological integrity was demonstrated by the group of van Hest.²⁶ Other examples by Meldal and coworkers demonstrated chemical modification of polymeric membranes using the CuAAC reaction,²³ further highlighting the versatility of click chemistry.

Click chemistry nowadays encompasses a diverse set of reactions which are robust and modular.¹ In addition to cycloaddition reactions, which are amongst the first examples, nucleophilic ring-opening reactions of strained heterocyclics (epoxides, aziridines, etc.), non-aldol type carbonyl reactions (urea, thiourea, hydrazone, etc. formations) and thiol–ene reactions are classified as click reactions.^27,28 Here we will specifically highlight the click reactions that are most commonly used in polymer science, i.e. the cycloaddition reactions and reactions involving thiols.

Cycloaddition reactions, as the name implies, produce a new cyclic adduct from two π-bonded molecules. Diels–Alder and 1,3-dipolar cycloaddition reactions are well-known examples.²⁹ In comparison to the copper-catalyzed azide–alkyne 1,3-cycloaddition (CuAAC) reaction, which results in only the 1,4-triazole product (Figure 1.1A), the thermal Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes generates both 1,2-triazole and 1,4-triazole products (Figure 1.1B). Interestingly, cycloaddition reactions resulting in 1,5-triazole products have also been demonstrated, by using a ruthenium catalyst instead of a copper catalyst, leading to the ruthenium-catalyzed azide–alkyne cycloaddition (RuAAC) reaction (Figure 1.1C).^30,31 In this case, the reaction proceeds via oxidative coupling of azides and alkynes to form a ruthenacycle intermediate, introducing regioselectivity. An alternative highly effective azide–alkyne cycloaddition reaction is the copper‐free strain‐promoted azide‐alkyne 1,3‐cycloaddition (SPAAC) reaction (Figure 1.1D), which was pioneered by Bertozzi et al.^12,32 and offers a high degree of reactivity and selectivity due to the cycloalkyne ring strain. Since no metal catalyst is required, there is no need for removing metal contamination, which could otherwise be an issue in a range of different fields, such as the semi-conductor industry and life science applications. This is especially relevant for living cell related applications, which are not well compatible with copper(i) owing to its potential toxic effect.³³ For that reason, SPAAC reactions have become one of the most leading technologies in biological applications. Another interesting metal-free cycloaddition click reaction is the strain-promoted alkyne nitrone cycloaddition reaction (SPANC) (Figure 1.1E), which involves reaction of cyclooctynes with 1,3 nitrones, yielding N-alkylated isooxazolines.³⁴ Although SPANC reactions offer rapid kinetics, even faster than SPAAC reactions, their applications have remained limited so far, as isooxazoline products are rather unstable, and could undergo unwanted rearrangements in complex biological media. In polymer conjugation reactions, mainly the cycloaddition reactions CuAAC and SPAAC have been employed.

Thiol-based click reactions mainly involve thiol–ene and thiol–yne reactions. Generally, thiol–ene reactions comprise hydrothiolation of carbon–carbon double bonds, via either radical or nucleophilic addition (i.e. Michael addition) pathways (Figure 1.2A).^35,36 Typically, radical or Lewis catalysts are used, allowing radical or nucleophilic pathways, respectively. Thiol–ene reactions can be performed in a wide range of solvents (i.e. protic and aprotic) under moderate temperatures. Furthermore, various alkenes (i.e. non-activated, or those comprising terminal or embedded enes), and a range of thiols, either substituted or not substituted, can be employed; their choice plays a key role in determining the rate of the reaction.³⁵ 

Thiol–yne reactions on the other hand involve alkynes and thiols, and are two-step reactions; the first step yields a vinyl sulfide which, in a second step, reacts with a second thiol to produce 1,2-disubstituted products (Figure 1.2B).³⁷ Thiol based click reactions have been well-reviewed.^38–40 Because of their robustness and versatility in reagents, they have found widespread application in the macromolecular sciences.

In this section, cycloaddition and thiol-based click reactions that are exploited in the synthesis of polymer conjugates, and the formation and modification of polymeric supramolecular assemblies will be reviewed. We will specifically focus our attention on polymer systems that can be applied in drug delivery. Due to the vast amount of literature available we do not strive to be comprehensive, but limit ourselves mainly to illustrative examples from recent literature. For synthetic protocols related to CuAAC applications in polymer and supramolecular chemistry the reader is referred to Chapter 2.6 of Science of Synthesis.⁴¹ 

Click chemistry has been widely used for polymer functionalization and as such has become a significant line of research in the area of drug delivery. Polymeric drug delivery systems have attracted much attention due to their high application potential, which is facilitated by their remarkable capacity to achieve high drug loading efficiency, whilst being able to precisely target and release their cargo at specific locations.⁴² Through covalent conjugation of drugs to polymers, a high-degree of control over drug loading efficiency can be achieved. Since the pioneering work by Ringsdorf,⁴³ many examples have been reported of drug-loaded macromolecular carrier systems. Because of the selectivity and effectiveness of click reactions, it has been a logical development that this conjugation chemistry has also become widely applied in this field of science.

Click chemistry provides a versatile toolbox to design polymer drug conjugates, and examples of this are numerous.^44–47 For instance, research by Yu and coworkers illustrated the preparation of polylactic acid grafted doxorubicin (PLA-g-dox) for cancer treatment.^48,49 First, acetylene functionalized PLA was synthesized by ring opening polymerization using acetylene functionalized lactide in addition to l-lactide. Thereafter, an azide functionalized aldehyde was chosen to be clicked to the polymer chain, as shown in Figure 1.3. The produced aldehyde-grafted PLA was subsequently conjugated to the amine-group of the drug dox, via an acid-labile imine linker. This acid-sensitive linker between dox and the polymer enabled controlled release of the drug in the more acidic tumor micro-environment.

Another example by Chen and coworkers demonstrated the suitability of click chemistry for the preparation of anti-cancer drug grafted polymers.⁵⁰ In this study, methacryloyloxyethyl phosphorylcholine (MPC) and trimethylsilyl protected propargyl methacrylate were polymerized by the Cu(i) catalyzed atom transfer radical polymerization (ATRP). After deprotection of the alkyne side chains, azide functionalized camptothecin, a natural alkaloid, was grafted to the polymer chains via the CuAAC reaction (Figure 1.4). It is worth mentioning that chemical modifications for adding clickable handles to drugs may have an impact on their activity unless the linker is completely removed when the drug is released. Additionally, the linker itself has an effect on drug release kinetics; however, this aspect is not covered in this chapter. Here, click chemistry allowed for high drug loading efficiency (5–15 wt%) while ATRP showed exceptional monomer conversion (>98%). Moreover, the utility of copper as a catalyst for both the controlled radical polymerization and click reaction allowed the one-pot multistep synthesis (Figure 1.4) of polymer–drug conjugates without additional purification.

Xu and coworkers synthesized a polymer backbone with mannose acrylamide and 1-(azidomethyl)-4-vinylbenzene by reversible addition–fragmentation chain transfer (RAFT) polymerization. The azide groups in the side chain were conjugated with the alkyne functionalized drug bufalin and an alkyne-bearing fluorescent agent via CuAAC; this allowed the simultaneous conjugation of the drug and the imaging probe.⁵¹ Another study by Sun et al. took advantage of the orthogonality of the thiol–ene and azide–alkyne cycloaddition click reactions and utilized both reactions to modify polymers with different functionalities.⁵² In this study, polylactic acid bearing both allyl- and alkyne grafts was synthesized from allyl-functionalized lactide (ALLA) and acetylenyl-functionalized lactide (ACLA). The alkyne bond was used to conjugate sulfobetaine and a fluorescent compound Cy5.5 via CuAAC, the drugs paclitaxel and gemcitabine were loaded via thiol–ene click reaction (Figure 1.5). A high yield of drug loading on the polymer–drug conjugate (6.5% paclitaxel and 17.7% gemcitabine by weight) was reached. This approach for co-delivery of the drugs by producing multifunctional polymer–drug conjugates was further explored by the authors.⁵³ These studies show that click reactions facilitate the synthesis of polymer–drug conjugates by reducing synthesis steps and enabling multifunctional grafting.

Other examples demonstrated the ability to conjugate biomolecules, such as peptides, to polymers via click reactions, creating the so-called bioconjugates.⁵⁴ For instance, Canalle and coworkers designed a synthetic polymer–peptide conjugate by employing an azide-functional methacrylate polymer and a gramicidin S decapeptide equipped with a (strained) alkyne.⁵⁵ They synthesized the bioconjugates using two different click reactions, CuAAC and SPAAC, in order to compare the outcome. Although SPAAC is considered a better alternative to CuAAC, as it prevents the utility of copper, the authors found that the steric bulkiness of the dibenzocyclooctyne (DBCO) group was not favorable and led to a diminished degree of grafting. These results demonstrate that the size of the conjugation group is important especially when bulky molecules are employed.

In addition to polymer–drug conjugates, supramolecular polymeric structures with hydrophobic compartments also provide a platform for carrying cargos such as drugs, enzymes and catalysts. For such systems, an important initial step is to synthesize their building units, namely amphiphilic building blocks.⁵⁶ Click chemistry represents an efficient approach to synthesize such building blocks as it provides the necessary chemistry to connect two polymers with different polarity that could not necessarily be prepared via the same chemical polymerization route. Because of their benign conditions and high efficiency, click reactions offer the ability to conjugate homopolymers together, leading to copolymers with narrow dispersity, which is an important aspect for defined self-assembly.^57–59 Combining linear polymers is generally achieved via either CuAAC or SPAAC reactions. The latter is of particular interest as it can be performed at ambient temperatures, without the need for catalysts, minimizing the chance of side reactions and facilitating polymer purification.⁶⁰ In order for conjugation of the premade homopolymers to be achieved, they have to be functionalized with the click moieties. For instance, acetylene-functionalized polymers can be synthesized via controlled radical polymerizations (e.g. ATRP) by using protected acetylene containing initiators, followed by deprotection after polymerization is completed. In this respect, click chemistry is highly compatible with controlled radical polymerization methods as these allow the usage of both clickable initiators and monomers.⁵⁸ Another approach is post-polymerization that includes the conversion of a terminal halide (e.g. bromide generally originates from the ATRP process) into an azide end group by a substitution reaction with sodium azide (NaN₃).⁵⁸ Click chemistry was furthermore shown to be applicable for the synthesis of ABA-^61,62 and ABC-type triblock copolymers.⁶³ Although the click chemistry approach to synthesizing copolymers requires an additional functionalization step of the polymers, it is highly versatile and modular, thereby greatly extending the application potential of block copolymers.

An example by Zhang and coworkers demonstrated the synthesis of a polymer that contained disulfide bonds in the backbone (PSS), by utilizing dipropargyl 3,3′-dithiodipropionate and 2,2-bis(azidomethyl)propane-1,3-diol as monomers via click reaction (Figure 1.6).⁶⁴ Thereafter, they used the CuAAC reaction to convert the initially synthesized polymer to its triblock counterpart PEG-b-PSS-b-PEG, by using azido-poly(ethylene glycol) (N₃-PEG). This reaction was conducted in N,N-dimethylformamide (DMF) at 60 °C; under these conditions the disulfide bonds were stable. The availability of hydroxyl groups on the PSS block made it possible to attach methotrexate, a drug, through ester bond formation. The mild conditions of the click reaction allowed the successful synthesis of these sensitive disulfide bond-containing block polymers.

In addition to the synthesis of linear amphiphilic block polymers, click chemistry has been used to synthesize copolymers with diverse shapes.⁶⁵ For example, Ehe and coworkers synthesized star shaped building blocks using CuAAC.⁶⁶ This was performed by utilizing a star-shaped alkyne-terminated poly(ɛ-caprolactone) and subsequently conjugating it to an azido-poly(ethylene glycol) (N₃-PEG) and poly(2-ethyl-2-oxazoline) azide (PEtO_x–N₃) as hydrophilic parts, allowing the synthesis of star-shaped block copolymers. Other studies by Laurent and Grayson showed the efficient synthesis of cyclic polymers⁶⁷ and cyclic amphiphilic polymers^68, via the CuAAC reaction. Additionally, Curole and coworkers designed amphiphilic comb polymers by employing alkyne-terminated PEG and alkanedithiols, which were reacted via thiol–yne click chemistry to induce intramolecular cyclization.⁶⁹ Furthermore, dendritic polymers^70,71 and polymer brushes^72,73 were synthesized, highlighting the broad applicability of click-chemistry in the construction of amphiphilic copolymers with controlled architectures.

Supramolecular chemistry is a field that studies the assembly of higher-order molecular structures via non-covalent interactions.⁷⁴ Although click chemistry focuses on the construction of covalent bonds, its impact on the progression of the field of supramolecular chemistry is rather substantial, as it offers a methodology that allows a facile functionalization of polymeric building blocks in a specific and mild manner.^75–78 In this section, we will focus on supramolecular systems generated from polymer building blocks, micelles and polymersomes in particular. We will discuss how click chemistry proves to be a useful tool for widening the application window of such systems.

Micelles are supramolecular structures obtained by the self-assembly of amphiphilic molecules. Generally, polymeric micelles have a hydrophobic core and hydrophilic shell, and acquire a size ranging between 30 nm and 100 nm. Due to their structural features, they are widely applied as drug delivery systems for the transport of hydrophobic pharmaceuticals.²⁴ Drugs can be either physically entrapped in- or covalently attached to- the micellar system. For the latter approach, click chemistry provides opportunities to modify micelles in order to optimize their performance by either increasing drug loading efficiency and facilitating drug release.

Similar to conjugating drugs to polymers (Section 1.3.1), click chemistry can be used to conjugate drugs to micelles via an approach of side chain functionalization of building blocks, before their assembly. For instance, Huynh and coworkers designed block copolymers made of (2-hydroxyethyl methacrylate) and poly[oligo-(ethylene glycol)methylether methacrylate] with acetylene and vinyl pendant groups, which were further reacted with 2-mercaptosuccinic acid and thioglycolic acid via thiol–yne and thiol–ene click chemistry, respectively.⁷⁹ The availability of the grafted carboxylic acids on the hydrophilic polymer allowed their complexation with cisplatin, a cancer drug, which is hydrophobic. The results showed that thiol–ene and thiol–yne reactions facilitated efficient grafting, which led to 87% drug loading efficiency. Interestingly, the combination of the drug and the polymers resulted in a conjugate with amphiphilic character, able to undergo self-assembly into a micelle-drug structure.

Another example by Zhang et al. demonstrated the synthesis of a polymer–drug conjugate by attaching directly the hydrophobic anticancer drug, camptothecin, as a pendant group via the CuAAC reaction.⁸⁰ Specifically, a polyphosphoester polymer with pendant alkyne groups was conjugated to a disulfide bond-containing linker with a terminal azide, resulting in a polymer–drug conjugate that underwent self-assembly into micelles. Such micelles preserved the drug in their structure until reaching a reducing environment (e.g. the cytoplasm of living cells). Similar approaches to construct redox-responsive drug–polymeric micelle conjugates have also been reported by other groups.^24,81 Due to the sensitivity of these linkers, they need to be installed using mild reaction conditions, and for this reason click chemistry was selected. The reaction conditions of CuAAC are benign and do not interfere with such disulfide bonds, leading to successful integration of diverse linkers to micellar structures.

In another example, Howe and coworkers demonstrated the concept of functionalization-induced micelle self-assembly by using click chemistry.⁸² In particular, they used a block copolymer composed of glycidyl methacrylate and poly(ethylene glycol) methyl ether methacrylate, of which the hydrophobicity was increased via post-polymerization modification with thiols such as naphthalenethiol, thiophenol, triphenylmethanethiol and pentafluorothiophenol. In this polymer design, the thiol-epoxide click reaction was conducted between the epoxide groups of the polymer and the four different thiol partners, resulting in an amphiphilic copolymer able to assemble into micelles.

Besides the functionalization of polymers to induce self-assembly, pre-assembled polymers can also be modified using click chemistry approaches. To this end, micelles have to be constructed in a way that results in accessible functional groups on their surface. For example, Li and coworkers designed micelles bearing alkynes by utilizing triblock amphiphilic polymers made of poly(ethylene glycol), carbonic ester with propargyl pendant group and l-lactide.⁸³ The presence of such propargyl groups in the hydrophilic block allowed them to be exposed on the micellar surface and, in turn, made them accessible for a click reaction to azide-functionalized hemoglobin via CuAAC. Using this approach, the authors demonstrated successful conjugation of hemoglobin to their micelles, resulting in conjugates that were applicable in biomedical applications. Another example by Doerflinger et al. demonstrated the preparation of polydiacetylene surface-functionalized micelles using alkyne-terminated PEG, which were conjugated to azido-biotin via CuAAC, facilitating the internalization of micelles by cancerous cells.⁸⁴ 

Cross-linking of micelles is a well-known strategy, used to enhance their stability. Such cross-linking can be either intra-micellar (within the same micellar structure) or inter-micellar (between different micellar populations). Cross-linking of the micellar core, in the presence of a drug, is generally performed to increase drug loading efficiency and minimize unwanted drug release. The examples are vast, and click chemistry has been one of the most applied chemistries herein in recent years.^24,76,85 For example, Wang et al. used thiol–ene chemistry to produce core cross-linkable micelles in presence of doxorubicin, which resulted in a two-fold increase in dox loading efficiency, compared to the non-crosslinked counter parts. Moreover, such micelle-drug constructs showed enhanced stability under physiological conditions.⁸⁶ Other types of click chemistry, such as azide–alkyne reactions,⁸⁷ were also used to conjugate drugs to the micellar core, generally through the utility of additional crosslinkers.

Inter-micellar crosslinking using click chemistry is mainly applied to conjugate micelles to each other to induce their aggregation and consequent stabilization under harsh conditions (e.g. biological conditions). This approach can be extended to cross-linking two micellar populations, each incorporating a certain drug, leading to increased efficacy. Mei et al. demonstrated this approach by using the CuAAC reaction between two different micelle populations; one functionalized with azides and the other with acetylenes.⁸⁸ This resulted in micellar clusters that retained their integrity in vivo. In addition, the SPAAC reaction was also used to induce micelle–micelle conjugation.⁸⁹ 

Besides the use of click reactions to functionalize micellar systems with active compounds and induce improved stability via crosslinking, click reactions can also be employed to trigger disassembly and subsequent drug release. This concept is referred to as click-to-release, which is a biorthogonal path for prodrug activation.^90–92 The click-to-release concept was applied on micelle–drug conjugates by Porte et al.; they functionalized the drug molecules with iminosydnone and integrated them in their micelles.⁹³ In these micelles, the strain-promoted iminosydnone–cycloalkyne cycloaddition (SPICC) reaction was triggered by addition of cyclooctyne derivatives leading to disassembly of the micelles and consequent drug release (Figure 1.7). Specifically, at concentrations of DBCO of 300 µM and higher micelle disassembly was effectively induced.

Polymersomes are polymeric vesicles, made of amphiphilic copolymers and have a bilayered hydrophobic membrane and an aqueous lumen. Due to the polymeric and chemical nature of polymersomes, they are mechanically stable, and can be tailor-made according to the desired applications. Owing to their amphiphilic nature, polymersomes can incorporate hydrophilic and hydrophobic cargos in their lumen and membrane, respectively.^94,95 Functionalization of polymersomes via click chemistry has been instrumental in increasing their stability, efficiency of cargo entrapment,⁹⁶ as well as gaining biocompatibility⁹⁷ and allowing their visualization using conventional confocal microscopy techniques.⁹⁸ Surface-decorated polymersomes can be prepared in two ways.⁹⁹ The first method includes the synthesis of end-group functionalized amphiphilic polymers, which undergo co-assembly with their unmodified counterparts. The other method requires the assembly of polymersomes with clickable active moieties such as azide and acetylene groups; after the completion of self-assembly, they should remain reactive for further modification.

A major risk in the case of post-assembly functionalization is that the presence of such reactive groups might influence the final supramolecular self-assembly structure. Generally speaking, for successful applications, the active groups should not affect the polymersome topology.^11,99 The integration of azides as functional groups is therefore ideal, due to their small size, linearity and stability; as such they often do not compromise the self-assembly process nor the final assembly. Due to this feature, next to its high degree of selectivity and efficiency, azide–alkyne click chemistry represents one of the most used methodologies for the conjugation of drugs to polymeric vesicles.^24,25,28,100

Utilizing the previously mentioned pre-assembly approach, Opsteen et al. synthesized the amphiphilic block copolymer polystyrene-block-poly(acrylic acid) (PS-b-PAA) via ATRP and then incorporated azides by substituting the end group, bromide in this case, with azidotrimethylsilane.²⁶ Self-assembly of these polymers resulted in polymersomes with functional azide groups on their surface. The authors showed the effective conjugation of alkyne-modified protein, an enhanced green fluorescent protein (GFP), allowing their visualization with confocal laser-scanning microscopy. This approach was extended to other polymersomes made of different building blocks such as poly(ethylene glycol)-block-polystyrene (PEG-b-PS), poly(ethylene glycol)-block-poly(D,l-lactic acid) (PEG-b-PDLLA) and polystyrene-b-poly(l-isocyanoalanine(2-thiophen-3-yl-ethyl)amide) (PS-PIAT), where other functional enzymes, such as horseradish peroxide¹⁰¹ and catalase¹⁰² were successfully attached on the polymersome surface, turning them into active nanoreactors.

Another example by Quadir and coworkers demonstrated the synthesis of azide-functionalized poly(ethylene glycol)-block-poly(γ-propargyl l-glutamate) (PEG-b-PPLG) block polymers, which were clicked to alkyne functionalized folic acid molecules.¹⁰³ Folic acid is known for its ability to specifically target ovarian cancer cells. Thereafter, a mixture of folic acid modified and unmodified copolymers were allowed to assemble into polymersomes. Such polymersomes were loaded with an anti-cancer drug, doxorubicin. Click chemistry-mediated conjugation of folic acid allowed these polymersomes to specifically target ovarian cancer cells. Following the same approach, Pangburn and coworkers used the CuAAC reaction to conjugate alkyne-terminated fibronectin mimetic peptide to azide-functionalized polymersomes to specifically target colon cancer cells.¹⁰⁴ The utility of click chemistry allowed efficient conjugation of such peptides to the polymersomes (up to 30 mol%).

Azide-decorated polymeric vesicles were employed by Wauters et al. to produce artificial antigen-presenting cells (aAPCs).¹⁰⁵ Specifically, DBCO-functionalized antibodies were clicked onto the surface of azide-functionalized spherical (Figure 1.8A) and tubular polymersomes. The authors showed unprecedented control over the density of the antibodies on the polymersome surface, which was reproducible across a library comprising more than 60 different polymersome morphologies. Similarly, Zhang and coworkers conjugated DBCO functionalized antibody αCD44 to azide-decorated polymersomes carrying maytansine, a nanotoxin, to target tumors.¹⁰⁶ Click chemistry in this instance resulted in control over the density of αCD44 and, in turn, its ratio to maytansine, which was important for an efficient anti-tumor effect.

Click chemistry to functionalize polymersomes is not limited to proteins and enzymes, but also other types of molecules such as fluorescent probes²⁶ and short synthetic polymers have been conjugated.¹⁰⁷ For example, Gillies et al. demonstrated the conjugation of alkyne terminated dendritic biocompatible polymers with azide-functionalized polymersomes (Figure 1.8B).^107,108 For this purpose, polymersomes were co-assembled using a combination of azide-modified and unmodified poly(butadiene)-block-poly(ethylene oxide) amphiphilic polymers. Meanwhile, the dendrons were designed to have an alkyne focal point for their conjugation to the polymersomes and peripheral amines for conjugation of bioactive molecules (mannose in this case).

Other examples explored the display of acetylene groups, instead of azides, on the polymersome surface to facilitate the conjugation of biomolecules.¹⁰⁹ For example, carbohydrate-decorated polymersomes were produced from amphiphilic glycopeptides, namely, benzyl-l-glutamate and propargylglycine N-carboxyanhydride.¹¹⁰ Utilizing the CuAAC reaction, azide functionalized galactose was conjugated to the polymer and displayed on the polymersome surface. The availability of carbohydrate groups was proven by interaction with a lectin which was known to bind strongly with galactose. Indeed, carbohydrate-lectin assays demonstrated the rapid kinetics of the interaction between galactose-modified polymersomes and free lectins, highlighting their potential in a biological context.

In addition to polymersome modification with solely one active moiety, Iyisan and coworkers generated multifunctional polymersomes, opening up new opportunities for the application of nanocarriers in drug delivery.¹¹¹ In order to do so, the authors used a combination of three different block copolymers, each with their hydrophilic segment modified, with either, methoxy-, azide-, or adamantane. The presence of the different active groups on the polymersome surface allowed the attachment of different moieties via different conjugation pathways. Specifically, the adamantane moieties enabled the polymersomes to conjugate with β-cyclodextrin modified Cy5 dye via non-covalent interactions. In addition, the available azide groups allowed the reaction between the polymersomes and the alkyne terminated protected amine, which was further activated to bind other molecules. A similar approach was demonstrated by Yassin et al., where they used three differently modified block copolymers (i.e., methoxy-, amine- and folate-) to produce polymersomes.¹¹² Unlike the previous example where post-assembly was used, pre-self-assembly was used, and folic acid was conjugated to the amphiphilic polymer via CuAAC prior to the polymersomes’ assembly. These polymersomes were loaded with doxorubicin, and the specific targeting to folate receptor bearing tumor cells was observed. These examples demonstrate the different approaches of how click chemistry can be combined with other chemistries to create multifunctional polymer systems.

Click chemistry has not only been used to modify the surface of polymersomes, but also their membrane. van Oers et al. demonstrated the immobilization of catalysts in polystyrene-based polymersome membranes, turning them into nanoreactors.^113,114 The authors used poly(ethylene glycol)-block-poly(styrene-co-4-vinylbenzyl azide) (PEG-b-P(S-co-4-VBA)) as a polymersome building block, which, upon coassembly into polymersomes with its non-functional counterpart, could tether alkyne functionalized catalysts in their membrane. The authors showed the immobilization of Cu(ii)-bis(4-phenyl-2-oxazoline) and l-proline catalysts in the polymersome membrane via CuAAC. In a recent study, a Cu(i)-tris(triazolylmethyl)amine catalyst was incorporated into the biodegradable polymersomes’ hydrophobic membrane via the CuAAC reaction and the prepared nanoreactors were shown to be potentially used in living cells.¹¹⁵ The polymersomes were constructed by the amphiphilic building blocks of poly(ethylene glycol)-block-poly(caprolactone-gradient-trimethylene carbonate) (PEG-b-P(CL-g-TMC)) and that of with azide-TMC in order to have azide functionality in the hydrophobic membrane which would react with the propargyl moiety bearing catalyst complex to produce the nanoreactors.

Through the years, click chemistry has proven to be a valuable tool for diverse applications. This is due to its favorable characteristics such as versatility, site specificity and (bio)orthogonality. Various types of click reactions have ensued, and there are excellent reviews on the topic. We therefore have limited the focus of this chapter to cycloaddition reactions and thiol–ene/–yne reactions, and highlighted their impact on the progression of polymeric-based drug conjugates. First, we discussed how cycloaddition reactions can be used to functionalize polymers with drugs and biological motifs. In this regard, click reactions facilitate polymer modification and the synthesis of polymer–drug conjugates by providing mild reaction conditions as well as simple purification steps. Furthermore, they allow the drug loading efficiency in polymer–drug conjugates to be increased without affecting basic polymer features (e.g. molecular weight). Thus, click chemistry has increased grafting efficiency and promoted facile polymer–drug conjugate designs. Thereafter, we discussed how click reactions are used to facilitate the synthesis of amphiphilic block polymers. Here, the approach of click chemistry offers high purity and narrow polydispersity, which is important for the success of the self-assembly as well as homogeneity of the polymeric assemblies. It also allows the conjugation of building blocks that are constructed via incompatible polymerization mechanisms. The block copolymers are successfully synthesized by the covalent conjugation of two (or three) homopolymers with the help of click reactions by either clickable initiators or post-polymerization modification strategies to load clickable moieties. The compatibility of click chemistry with sensitive bonds (e.g. disulfide bonds) as well as controlled radical polymerization has increased the preference and therefore it has become a practical method for amphiphilic block polymer synthesis. Finally, the impact of click chemistry on supramolecular systems for life science applications was discussed. Importantly, click chemistry provides biorthogonality and allows facile modification in complex biological environments. The reaction conditions of specifically SPAAC (i.e. no catalyst, room temperature) are biocompatible and do not require purification. Furthermore, due to their small size, the active handles (i.e. azide and alkyne) do not significantly interfere with the self-assembly process of amphiphilic block copolymers so that they can be used in a post-assembly approach, being still accessible after either micelles or polymersomes are formed. In addition, click reactions can be used for crosslinking, leading to particles with increased drug loading efficiency and stability. In addition, the compatibility of click chemistry with other reactive groups such as disulfide bonds makes it possible to incorporate chemical features into the polymer assemblies that for example allow their controlled disassembly and release of their payloads at specific conditions. The versatile surface modification of micelles and polymersomes with click chemistry has been used for many different features, for example to increase biocompatibility, to achieve targeted delivery and in visualization (e.g. carbohydrates, folic acid, dyes). Again, the compatibility with other chemistries allows multi-functionalization of the surface of polymer assemblies. For these reasons it is clear that click chemistry has become one of the most used methodologies to functionalize and enhance the properties of polymers and their assemblies for application in the area of drug delivery.

aAPCs

Artificial antigen-presenting cells

ATRP

Atom transfer radical polymerization

CuAAC

Copper‐catalyzed azide‐alkyne 1,3‐cycloaddition

DBCO

Dibenzocyclooctyne

DMF

Dimethylformamide

Dox

Doxorubicin

GEM

Gemcitabine

GFP

Green fluorescent protein

IEDDA

Inverse-electron-demand Diels–Alder

MPC

Methacryloyloxyethyl phosphorylcholine

N₃-PEG

Azido-poly(ethylene glycol)

NaN₃

Sodium azide

PEG

Poly(ethylene glycol)

PEG-b-PDLLA

Poly(ethylene glycol)-block-poly(D,l-lactic acid)

PEG-b-PPLG

Poly(ethylene glycol)-block-poly(γ-propargyl l-glutamate)

PEG-b-PS

Poly(ethylene glycol)-block-polystyrene

PEG-b-P(CL-g-TMC)

Poly(ethylene glycol)-block-poly(caprolactone-gradient-trimethylene carbonate)

PEG-b-P(S-co-4-VBA)

Poly(ethylene glycol)-block-poly(styrene-co-4-vinylbenzyl azide)

PEtOx-N₃

Poly(2-ethyl-2-oxazoline)azide

PLA

Poly(lactic acid)

PLA-g-dox

Doxorubicin grafted poly(lactic acid)

PS-b-PAA

Polystyrene-block-poly(acrylic acid)

PS-b-PIAT

Polystyrene-block-poly(l-isocyanoalanine(2-thiophen-3-yl-ethyl)amide)

PTX

Paclitaxel

RAFT

Reversible addition–fragmentation chain transfer

RuAAC

Ruthenium-catalyzed azide–alkyne cycloaddition

SPAAC

Copper‐free strain‐promoted azide‐alkyne 1,3‐cycloaddition

SPANC

Strain-promoted alkyne nitrone cycloaddition reaction

SPICC

Strained-promoted iminosydnone-cycloalkyne cycloaddition

The authors acknowledge the support of the Advanced Research Center for Chemical Building Blocks, ARC CBBC, which is co-founded and co-financed by the Dutch Research Council (NWO) and the Netherlands Ministry of Economic Affairs and Climate Policy.